当前期刊: Applied and Environmental Microbiology Go to current issue    加入关注   
显示样式:        排序: 导出
我的关注
我的收藏
您暂时未登录!
登录
  • Emergence of a Plant Pathogen in Europe Associated with Multiple Intercontinental Introductions
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Landa, B. B., Castillo, A. I., Giampetruzzi, A., Kahn, A., Roman-Ecija, M., Velasco-Amo, M. P., Navas-Cortes, J. A., Marco-Noales, E., Barbe, S., Moralejo, E., Coletta-Filho, H. D., Saldarelli, P., Saponari, M., Almeida, R. P. P.

    Pathogen introductions have led to numerous disease outbreaks in naive regions of the globe. The plant pathogen Xylella fastidiosa has been associated with various recent epidemics in Europe affecting agricultural crops, such as almond, grapevine, and olive, but also endemic species occurring in natural forest landscapes and ornamental plants. We compared whole-genome sequences of X. fastidiosa subspecies multiplex from America and strains associated with recent outbreaks in southern Europe to infer their likely origins and paths of introduction within and between the two continents. Phylogenetic analyses indicated multiple introductions of X. fastidiosa subspecies multiplex into Italy, Spain, and France, most of which emerged from a clade with limited genetic diversity with a likely origin in California, USA. The limited genetic diversity observed in X. fastidiosa subspecies multiplex strains originating from California is likely due to the clade itself being an introduction from X. fastidiosa subspecies multiplex populations in the southeastern United States, where this subspecies is most likely endemic. Despite the genetic diversity found in some areas in Europe, there was no clear evidence of recombination occurring among introduced X. fastidiosa strains in Europe. Sequence type taxonomy, based on multilocus sequence typing (MLST), was shown, at least in one case, to not lead to monophyletic clades of this pathogen; whole-genome sequence data were more informative in resolving the history of introductions than MLST data. Although additional data are necessary to carefully tease out the paths of these recent dispersal events, our results indicate that whole-genome sequence data should be considered when developing management strategies for X. fastidiosa outbreaks. IMPORTANCE Xylella fastidiosa is an economically important plant-pathogenic bacterium that has emerged as a pathogen of global importance associated with a devastating epidemic in olive trees in Italy associated with X. fastidiosa subspecies pauca and other outbreaks in Europe, such as X. fastidiosa subspecies fastidiosa and X. fastidiosa subspecies multiplex in Spain and X. fastidiosa subspecies multiplex in France. We present evidence of multiple introductions of X. fastidiosa subspecies multiplex, likely from the United States, into Spain, Italy, and France. These introductions illustrate the risks associated with the commercial trade of plant material at global scales and the need to develop effective policy to limit the likelihood of pathogen pollution into naive regions. Our study demonstrates the need to utilize whole-genome sequence data to study X. fastidiosa introductions at outbreak stages, since a limited number of genetic markers does not provide sufficient phylogenetic resolution to determine dispersal paths or relationships among strains that are of biological and quarantine relevance.

    更新日期:2020-01-22
  • Impact of Classical Strain Improvement of Penicillium rubens on Amino Acid Metabolism during {beta}-Lactam Production
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Wu, M., Crismaru, C. G., Salo, O., Bovenberg, R. A. L., Driessen, A. J. M.

    To produce high levels of β-lactams, the filamentous fungus Penicillium rubens (previously named Penicillium chrysogenum) has been subjected to an extensive classical strain improvement (CSI) program during the last few decades. This has led to the accumulation of many mutations that were spread over the genome. Detailed analysis reveals that several mutations targeted genes that encode enzymes involved in amino acid metabolism, in particular biosynthesis of l-cysteine, one of the amino acids used for β-lactam production. To examine the impact of the mutations on enzyme function, the respective genes with and without the mutations were cloned and expressed in Escherichia coli, purified, and enzymatically analyzed. Mutations severely impaired the activities of a threonine and serine deaminase, and this inactivates metabolic pathways that compete for l-cysteine biosynthesis. Tryptophan synthase, which converts l-serine into l-tryptophan, was inactivated by a mutation, whereas a mutation in 5-aminolevulinate synthase, which utilizes glycine, was without an effect. Importantly, CSI caused increased expression levels of a set of genes directly involved in cysteine biosynthesis. These results suggest that CSI has resulted in improved cysteine biosynthesis by the inactivation of the enzymatic conversions that directly compete for resources with the cysteine biosynthetic pathway, consistent with the notion that cysteine is a key component during penicillin production. IMPORTANCE Penicillium rubens is an important industrial producer of β-lactam antibiotics. High levels of penicillin production were enforced through extensive mutagenesis during a classical strain improvement (CSI) program over 70 years. Several mutations targeted amino acid metabolism and resulted in enhanced l-cysteine biosynthesis. This work provides a molecular explanation for the interrelation between secondary metabolite production and amino acid metabolism and how classical strain improvement has resulted in improved production strains.

    更新日期:2020-01-22
  • Carriage and Subtypes of Foodborne Pathogens Identified in Wild Birds Residing near Agricultural Lands in California: a Repeated Cross-Sectional Study
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Navarro-Gonzalez, N., Wright, S., Aminabadi, P., Gwinn, A., Suslow, T. V., Jay-Russell, M. T.

    Current California agricultural practices strive to comanage food safety and habitat conservation on farmland. However, the ecology of foodborne pathogens in wild bird populations, especially those avian species residing in proximity to fresh produce production fields, is not fully understood. In this repeated cross-sectional study, avifauna within agricultural lands in California were sampled over 1 year. Feces, oral swabs, and foot/feather swabs were cultured for zoonotic Salmonella spp., Escherichia coli O157:H7, and non-O157 Shiga toxin-producing E. coli (STEC) and characterized by serotyping and pulsed-field gel electrophoresis. Of 60 avian species sampled, 8 species (13.3%, bird groups of sparrows, icterids, geese, wrens, and kinglets) were positive for at least one of these foodborne pathogens. At the individual bird level, the detection of foodborne pathogens was infrequent in feces (n = 583; 0.5% Salmonella, 0.34% E. coli O157:H7, and 0.5% non-O157 STEC) and in feet/feathers (n = 401; 0.5% non-O157 STEC), and it was absent from oral swabs (n = 353). Several subtypes of public health importance were identified, including Salmonella enterica serotype Newport, E. coli O157:H7, and STEC serogroups O103 and O26. In late summer and autumn, the same STEC subtype was episodically found in several individuals of the same and different avian species, suggesting a common source of contamination in the environment. Sympatric free-range cattle shared subtypes of STEC O26 and O163 with wild geese. A limited rate of positive detection in wild birds provides insights into broad risk profile for contamination considerations but cannot preclude or predict risk on an individual farm. IMPORTANCE The shedding dynamics of foodborne pathogens by wild birds on farmland are not well characterized. This yearlong study sampled wild birds for foodborne pathogens within agricultural lands in northern California. There was a low prevalence of Salmonella spp., Escherichia coli O157:H7, and non-O157 Shiga-toxin producing E. coli (prevalence, 0.34% to 0.50%) identified in bird populations in this study. However, pathogens of public health importance (such as Salmonella Newport, E. coli O157:H7, and STEC O103 and O26) were identified in fecal samples, and two birds carried STEC on their feet or feathers. Identical pathogen strains were shared episodically among birds and between wild geese and free-range cattle. This result suggests a common source of contamination in the environment and potential transmission between species. These findings can be used to assess the risk posed by bird intrusions in produce fields and enhance policy decisions toward the comanagement of food safety and farmland habitat conservation.

    更新日期:2020-01-22
  • Viral Attachment to Biotic and Abiotic Surfaces in Seawater
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Yamada, Y., Guillemette, R., Baudoux, A.-C., Patel, N., Azam, F.

    Viruses influence microbial community structure and biogeochemical cycles in marine environments. Viral attachment to nonhost surfaces could influence host viral infection rates; however, the prevalence of such viral attachment is not investigated quantitatively. We used coastal seawater viral assemblages and, as models, marine vibriophage (SIO-2) and enterobacteriophages (T2 and T4) to investigate their attachment to probable nonhost marine bacteria. We also studied viral attachment to colloids and other abiotic surfaces in seawater. Centrifugation experiments with bacterium-virus mixtures showed substantial viral loss in the supernatant presumably due to the viral attachment to bacteria. This attachment (0.04 to 24 viruses μm–2 [bacterial surface area]) varied with bacterium-virus combinations. Surprisingly, filtering seawater on 0.2-μm Anodisc or polycarbonate filters retained ~12 to 84% of viruses presumably attached to ≥0.2-μm-sized particles and/or the filter surface. Enzymatic digestion followed by epifluorescence and atomic force microscopy suggested that 7 to 25% of the total viruses were attached via β-glycosidic linkages. Furthermore, a substantial proportion (7 to 48%) of viruses became attached to model abiotic surfaces (polycarbonate, polypropylene, and glass), and this has significance for laboratory protocols as well as studies of virus ecology in particle-rich marine environments. Substantial attachment of viruses to nonhost surfaces could influence virus-driven biogeochemical cycles and microbial community structure. IMPORTANCE Viruses play important roles in altering microbial community structure and biogeochemical cycles in marine environments. Viral attachment to nonhost surfaces can influence host viral infection rates; however, the prevalence of viral attachment to nonhost surfaces and the ratio of attached viruses to total viruses are little known. We used coastal seawater viral assemblages and used marine vibriophage (SIO-2) and enterobacteriophages (T2 and T4) as models to investigate their attachment to abiotic and biotic surfaces in seawater. Viral attachment was observed on several surfaces, such as nonhost bacteria, polymers, filters, cover glasses, and tube surfaces. This study cautions against commonly used protocols that require viral incubation and seawater fractionation. More importantly, these results could influence virus-driven biogeochemical cycles and microbial community structure in the ocean.

    更新日期:2020-01-22
  • Fungal Seed Pathogens of Wild Chili Peppers Possess Multiple Mechanisms To Tolerate Capsaicinoids
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Adams, C. A., Zimmerman, K., Fenstermacher, K., Thompson, M. G., Skyrud, W., Behie, S., Pringle, A.

    The wild chili pepper Capsicum chacoense produces the spicy defense compounds known as capsaicinoids, including capsaicin and dihydrocapsaicin, which are antagonistic to the growth of fungal pathogens. Compared to other microbes, fungi isolated from infected seeds of C. chacoense possess much higher levels of tolerance of these spicy compounds, having their growth slowed but not entirely inhibited. Previous research has shown capsaicinoids inhibit microbes by disrupting ATP production by binding NADH dehydrogenase in the electron transport chain (ETC) and, thus, throttling oxidative phosphorylation (OXPHOS). Capsaicinoids may also disrupt cell membranes. Here, we investigate capsaicinoid tolerance in fungal seed pathogens isolated from C. chacoense. We selected 16 fungal isolates from four ascomycete genera (Alternaria, Colletotrichum, Fusarium, and Phomopsis). Using relative growth rate as a readout for tolerance, fungi were challenged with ETC inhibitors to infer whether fungi possess alternative respiratory enzymes and whether effects on the ETC fully explained inhibition by capsaicinoids. In all isolates, we found evidence for at least one alternative NADH dehydrogenase. In many isolates, we also found evidence for an alternative oxidase. These data suggest that wild-plant pathogens may be a rich source of alternative respiratory enzymes. We further demonstrate that these fungal isolates are capable of the breakdown of capsaicinoids. Finally, we determine that the OXPHOS theory may describe a weak primary mechanism by which dihydrocapsaicin, but not capsaicin, slows fungal growth. Our findings suggest that capsaicinoids likely disrupt membranes, in addition to energy poisoning, with implications for microbiology and human health. IMPORTANCE Plants make chemical compounds to protect themselves. For example, chili peppers produce the spicy compound capsaicin to inhibit pathogen damage and animal feeding. In humans, capsaicin binds to a membrane channel protein, creating the sensation of heat, while in microbes, capsaicin limits energy production by binding respiratory enzymes. However, some data suggest that capsaicin also disrupts membranes. Here, we studied fungal pathogens (Alternaria, Colletotrichum, Fusarium, and Phomopsis) isolated from a wild chili pepper, Capsicum chacoense. By measuring growth rates in the presence of antibiotics with known respiratory targets, we inferred that wild-plant pathogens might be rich in alternative respiratory enzymes. A zone of clearance around the colonies, as well as liquid chromatography-mass spectrometry data, further indicated that these fungi can break down capsaicin. Finally, the total inhibitory effect of capsaicin was not fully explained by its effect on respiratory enzymes. Our findings lend credence to studies proposing that capsaicin may disrupt cell membranes, with implications for microbiology, as well as human health.

    更新日期:2020-01-22
  • The Transcriptional Regulator HbxA Governs Development, Secondary Metabolism, and Virulence in Aspergillus fumigatus
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Satterlee, T., Nepal, B., Lorber, S., Puel, O., Calvo, A. M.

    Aspergillus fumigatus is the leading cause of invasive aspergillosis, which in immunocompromised patients results in a mortality rate as high as 90%. Earlier studies showed that HbxA is a global regulator in Aspergillus flavus affecting morphological development and secondary metabolism. Here, we determined its role in A. fumigatus, examining whether HbxA influences the regulation of asexual development, natural product biosynthesis, and virulence of this fungus. Our analysis demonstrated that removal of the hbxA gene caused a near-complete loss of conidial production in the mutant strain, as well as a slight reduction in colony growth. Other aspects of asexual development are affected, such as size and germination of conidia. Furthermore, we showed that in A. fumigatus, the loss of hbxA decreased the expression of the brlA central regulatory pathway involved in asexual development, as well as the expression of the "fluffy" genes flbB, flbD, and fluG. HbxA was also found to regulate secondary metabolism, affecting the biosynthesis of multiple natural products, including fumigaclavines, fumiquinazolines, and chaetominine. In addition, using a neutropenic mouse infection model, hbxA was found to negatively impact the virulence of A. fumigatus. IMPORTANCE The number of immunodepressed individuals is increasing, mainly due to the greater life expectancy in immunodepressed patients due to improvements in modern medical treatments. However, this population group is highly susceptible to invasive aspergillosis. This devastating illness, mainly caused by the fungus Aspergillus fumigatus, is associated with mortality rates reaching 90%. Treatment options for this disease are currently limited, and a better understanding of A. fumigatus genetic regulatory mechanisms is paramount for the design of new strategies to prevent or combat this infection. Our work provides new insight into the regulation of the development, metabolism, and virulence of this important opportunistic pathogen. The transcriptional regulatory gene hbxA has a profound effect on A. fumigatus biology, governing multiple aspects of conidial development. This is relevant since conidia are the main source of inoculum in Aspergillus infections. Importantly, hbxA also regulates the biosynthesis of secondary metabolites and the pathogenicity of this fungus.

    更新日期:2020-01-22
  • Multiple Megaplasmids Confer Extremely High Levels of Metal Tolerance in Alteromonas Strains
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Cusick, K. D., Polson, S. W., Duran, G., Hill, R. T.

    Alteromonas is a widely distributed genus of marine Gammaproteobacteria, with representatives shown to be key players in diverse processes, including biogeochemical cycling and biofouling of marine substrata. While Alteromonas spp. are early colonizers of copper-based antifouling paints on marine vessels, their mechanism of tolerance is poorly understood. PacBio whole-genome sequencing of Alteromonas macleodii strains CUKW and KCC02, isolated from Cu/Ni alloy test coupons submerged in oligotrophic coastal waters, indicated the presence of multiple megaplasmids (ca. 200 kb) in both. A pulsed-field gel electrophoresis method was developed and used to confirm the presence of multiple megaplasmids in these two strains; it was then used to screen additional Alteromonas strains for which little to no sequencing data exist. Plasmids were not detected in any of the other strains. Bioinformatic analysis of the CUKW and KCC02 plasmids identified numerous genes associated with metal resistance. Copper resistance orthologs from both the Escherichia coli Cue and Cus and Pseudomonas syringae Cop systems were present, at times as multiple copies. Metal growth assays in the presence of copper, cobalt, manganese, and zinc performed with 10 Alteromonas strains demonstrated the ability of CUKW and KCC02 to grow at metal concentrations inhibitory to all the other strains tested. This study reports multiple megaplasmids in Alteromonas strains. Bioinformatic analysis of the CUKW and KCC02 plasmids indicate that they harbor elements of the Tra system conjugation apparatus, although their type of mobility remains to be experimentally verified. IMPORTANCE Copper is commonly used as an antifouling agent on ship hulls. Alteromonas spp. are early colonizers of copper-based antifouling paint, but their mechanism of tolerance is poorly understood. Sequencing of A. macleodii strains isolated from copper test materials for marine ships indicated the presence of multiple megaplasmids. Plasmids serve as key vectors in horizontal gene transfer and confer traits such as metal resistance, detoxification, ecological interaction, and antibiotic resistance. Bioinformatic analysis identified many metal resistance genes and genes associated with mobility. Understanding the molecular mechanisms and capacity for gene transfer within marine biofilms provides a platform for the development of novel antifouling solutions targeting genes involved in copper tolerance and biofilm formation.

    更新日期:2020-01-22
  • In Situ Activation and Heterologous Production of a Cryptic Lantibiotic from an African Plant Ant-Derived Saccharopolyspora Species
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Vikeli, E., Widdick, D. A., Batey, S. F. D., Heine, D., Holmes, N. A., Bibb, M. J., Martins, D. J., Pierce, N. E., Hutchings, M. I., Wilkinson, B.

    Most clinical antibiotics are derived from actinomycete natural products discovered at least 60 years ago. However, the repeated rediscovery of known compounds led the pharmaceutical industry to largely discard microbial natural products (NPs) as a source of new chemical diversity. Recent advances in genome sequencing have revealed that these organisms have the potential to make many more NPs than previously thought. Approaches to unlock NP biosynthesis by genetic manipulation of strains, by the application of chemical genetics, or by microbial cocultivation have resulted in the identification of new antibacterial compounds. Concomitantly, intensive exploration of coevolved ecological niches, such as insect-microbe defensive symbioses, has revealed these to be a rich source of chemical novelty. Here, we report the new lanthipeptide antibiotic kyamicin, which was generated through the activation of a cryptic biosynthetic gene cluster identified by genome mining Saccharopolyspora species found in the obligate domatium-dwelling ant Tetraponera penzigi of the ant plant Vachellia drepanolobium. Transcriptional activation of this silent gene cluster was achieved by ectopic expression of a pathway-specific activator under the control of a constitutive promoter. Subsequently, a heterologous production platform was developed which enabled the purification of kyamicin for structural characterization and bioactivity determination. This strategy was also successful for the production of lantibiotics from other genera, paving the way for a synthetic heterologous expression platform for the discovery of lanthipeptides that are not detected under laboratory conditions or that are new to nature. IMPORTANCE The discovery of novel antibiotics to tackle the growing threat of antimicrobial resistance is impeded by difficulties in accessing the full biosynthetic potential of microorganisms. The development of new tools to unlock the biosynthesis of cryptic bacterial natural products will greatly increase the repertoire of natural product scaffolds. Here, we report a strategy for the ectopic expression of pathway-specific positive regulators that can be rapidly applied to activate the biosynthesis of cryptic lanthipeptide biosynthetic gene clusters. This allowed the discovery of a new lanthipeptide antibiotic directly from the native host and via heterologous expression.

    更新日期:2020-01-22
  • Anaerobic Degradation of Syringic Acid by an Adapted Strain of Rhodopseudomonas palustris
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Oshlag, J. Z., Ma, Y., Morse, K., Burger, B. T., Lemke, R. A., Karlen, S. D., Myers, K. S., Donohue, T. J., Noguera, D. R.

    While lignin represents a major fraction of the carbon in plant biomass, biological strategies to convert the components of this heterogeneous polymer into products of industrial and biotechnological value are lacking. Syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) is a by-product of lignin degradation, appearing in lignocellulosic hydrolysates, deconstructed lignin streams, and other agricultural products. Rhodopseudomonas palustris CGA009 is a known degrader of phenolic compounds under photoheterotrophic conditions via the benzoyl coenzyme A (CoA) degradation (BAD) pathway. However, R. palustris CGA009 is reported to be unable to metabolize meta-methoxylated phenolics, such as syringic acid. We isolated a strain of R. palustris (strain SA008.1.07), adapted from CGA009, which can grow on syringic acid under photoheterotrophic conditions, utilizing it as a sole source of organic carbon and reducing power. An SA008.1.07 mutant with an inactive benzoyl-CoA reductase structural gene was able to grow on syringic acid, demonstrating that the metabolism of this aromatic compound is not through the BAD pathway. Comparative gene expression analyses of SA008.1.07 implicated the involvement of products of the vanARB operon (rpa3619, rpa3620, rpa3621), which has been described as catalyzing aerobic aromatic ring demethylation in other bacteria, in anaerobic syringic acid degradation. In addition, experiments with a vanARB deletion mutant demonstrated the involvement of the vanARB operon in anaerobic syringic acid degradation. These observations provide new insights into the anaerobic degradation of meta-methoxylated and other aromatics by R. palustris. IMPORTANCE Lignin is the most abundant aromatic polymer on Earth and a resource that could eventually substitute for fossil fuels as a source of aromatic compounds for industrial and biotechnological applications. Engineering microorganisms for the production of aromatic-based biochemicals requires detailed knowledge of the metabolic pathways for the degradation of aromatics that are present in lignin. Our isolation and analysis of a Rhodopseudomonas palustris strain capable of syringic acid degradation reveal a previously unknown metabolic route for aromatic degradation in R. palustris. This study highlights several key features of this pathway and sets the stage for a more complete understanding of the microbial metabolic repertoire required to metabolize aromatic compounds from lignin and other renewable sources.

    更新日期:2020-01-22
  • Epidemiology of Salmonella enterica Serovar Dublin in Cattle and Humans in Denmark, 1996 to 2016: a Retrospective Whole-Genome-Based Study
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Kudirkiene, E., Sorensen, G., Torpdahl, M., de Knegt, L. V., Nielsen, L. R., Rattenborg, E., Ahmed, S., Olsen, J. E.

    Salmonella enterica serovar Dublin is a cattle-adapted S. enterica serovar causing both intestinal and systemic infection in its bovine host, and it is also a serious threat to human health. The present study aimed to determine the population structure of S. Dublin isolates obtained from Danish cattle herds and to investigate how cattle isolates relate to Danish human isolates, as well as to non-Danish human and bovine isolates. Phylogenetic analysis of 197 Danish cattle isolates from 1996 to 2016 identified three major clades corresponding to distinct geographical regions of cattle herds. Persistence of closely related isolates within the same herd and their circulation between epidemiologically linked herds for a period of more than 20 years were demonstrated. These findings suggest that a lack of internal biosecurity and, to some extent, also a lack of external biosecurity in the herds have played an important role in the long-term persistence of S. Dublin in Danish cattle herds in the period investigated. Global population analysis revealed that Danish cattle isolates clustered separately from bovine isolates from other countries, whereas human isolates were geographically spread. Resistance genes were not commonly demonstrated in Danish bovine isolates; only the isolates within one Danish clade were found to often harbor two plasmids of IncFII/IncFIB and IncN types, the latter plasmid carrying blaTEM-1, tetA, strA, and strB antibiotic resistance genes. IMPORTANCE S. Dublin causes economic losses in cattle production, and the bacterium is a public health concern. A surveillance and control program has been in place in Denmark since 2002 with the ultimate goal to eradicate S. Dublin from Danish cattle herds; however, a small proportion of herds have remained positive for many years. In this study, we demonstrate that herds with persistent infection often were infected with the same strain for many years, indicating that internal biosecurity has to be improved to curb the infection. Further, domestic cases of S. Dublin infection in humans were found to be caused both by Danish cattle isolates and by isolates acquired abroad. This study shows the strength of whole-genome sequencing to obtain detailed information on epidemiology of S. Dublin and allows us to suggest internal biosecurity as a main way to control this bacterium in Danish cattle herds.

    更新日期:2020-01-22
  • Short-Chain N-Acylhomoserine Lactone Quorum-Sensing Molecules Promote Periodontal Pathogens in In Vitro Oral Biofilms
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Muras, A., Mayer, C., Otero-Casal, P., Exterkate, R. A. M., Brandt, B. W., Crielaard, W., Otero, A., Krom, B. P.

    Acylhomoserine lactones (AHLs), the quorum-sensing (QS) signals produced by a range of Gram-negative bacteria, are involved in biofilm formation in many pathogenic and environmental bacteria. Nevertheless, the current paradigm excludes a role of AHLs in dental plaque formation, while other QS signals, such as AI-2 and autoinducer peptides, have been demonstrated to play an important role in biofilm formation and virulence-related gene expression in oral pathogens. In the present work, we have explored the effect of externally added AHLs on in vitro oral biofilm models for commensal, cariogenic, and periodontal dental plaque. While little effect on bacterial growth was observed, some AHLs specifically affected the lactic acid production and protease activity of the biofilms. Most importantly, the analysis of bacterial diversity in the biofilms showed that the addition of C6-homoserine lactone (C6-HSL) results in a shift toward a periodontal bacterial composition profile by increasing the relative presence of the orange-complex bacteria Peptostreptococcus and Prevotella. These results point to a relevant role of AHL-mediated QS in dental plaque formation and might be involved in the development of dysbiosis, the mechanism of which should be further investigated. This finding potentially opens new opportunities for the prevention or treatment of the periodontal disease. IMPORTANCE Dental plaque is omnipresent in healthy oral cavities and part of our commensal microbial colonization. At the same time, dental plaque is the cause of the most common human diseases, caries and gum disease. Dental plaque consists of billions of microbes attached to the surface of your teeth. Communication among these microbes is pivotal for development of these complex communities yet poorly studied in dental plaque. In the present study, we show that a specific communication molecule induces changes within the community related to the development of gum disease. This finding suggests that interfering with microbial communication may represent an interesting novel strategy to prevent gum disease that should be further investigated.

    更新日期:2020-01-22
  • Quantifying the Benefit of a Dedicated "Magnetoskeleton" in Bacterial Magnetotaxis by Live-Cell Motility Tracking and Soft Agar Swimming Assay
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Pfeiffer, D., Schüler, D.

    The alphaproteobacterium Magnetospirillum gryphiswaldense has the intriguing ability to navigate within magnetic fields, a behavior named magnetotaxis, governed by the formation of magnetosomes, intracellular membrane-enveloped crystals of magnetite. Magnetosomes are aligned in chains along the cell’s motility axis by a dedicated multipart cytoskeleton ("magnetoskeleton"); however, precise estimates of its significance for magnetotaxis have not been reported. Here, we estimated the alignment of strains deficient in various magnetoskeletal constituents by live-cell motility tracking within defined magnetic fields ranging from 50 μT (reflecting the geomagnetic field) up to 400 μT. Motility tracking revealed that mamY and mamK strains (which assemble mispositioned and fragmented chains, respectively) are partially impaired in magnetotaxis, with approximately equal contributions of both proteins. This impairment was reflected by a required magnetic field strength of 200 μT to achieve a similar degree of alignment as for the wild-type strain in a 50-μT magnetic field. In contrast, the mamJ strain, which predominantly forms clusters of magnetosomes, was only weakly aligned under any of the tested field conditions and could barely be distinguished from a nonmagnetic mutant. Most findings were corroborated by a soft agar swimming assay to analyze magnetotaxis based on the degree of distortion of swim halos formed in magnetic fields. Motility tracking further revealed that swimming speeds of M. gryphiswaldense are highest within the field strength equaling the geomagnetic field. In conclusion, magnetic properties and intracellular positioning of magnetosomes by a dedicated magnetoskeleton are required and optimized for bacterial magnetotaxis and most efficient locomotion within the geomagnetic field. IMPORTANCE In Magnetospirillum gryphiswaldense, magnetosomes are aligned in quasi-linear chains in a helical cell by a complex cytoskeletal network, including the actin-like MamK and adapter MamJ for magnetosome chain concatenation and segregation and MamY to position magnetosome chains along the shortest cellular axis of motility. Magnetosome chain positioning is assumed to be required for efficient magnetic navigation; however, the significance and contribution of all key constituents have not been quantified within defined and weak magnetic fields reflecting the geomagnetic field. Employing two different motility-based methods to consider the flagellum-mediated propulsion of cells, we depict individual benefits of all magnetoskeletal constituents for magnetotaxis. Whereas lack of mamJ resulted almost in an inability to align cells in weak magnetic fields, an approximately 4-fold-increased magnetic field strength was required to compensate for the loss of mamK or mamY. In summary, the magnetoskeleton and optimal positioning of magnetosome chains are required for efficient magnetotaxis.

    更新日期:2020-01-22
  • Structural and Functional Dynamics of Soil Microbes following Spruce Beetle Infestation
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Custer, G. F., van Diepen, L. T. A., Stump, W. L.

    As the range of bark beetles expands into new forests and woodlands, the need to understand their effects on multiple trophic levels becomes increasingly important. To date, much attention has been paid to the aboveground processes affected by bark beetle infestation, with a focus on photoautotrophs and ecosystem level processes. However, indirect effects of bark beetle on belowground processes, especially the structure and function of soil microbiota remains largely a black box. Our study examined the impacts of bark beetle-induced tree mortality on soil microbial community structure and function using high-throughput sequencing of the soil bacterial and fungal communities and measurements of extracellular enzyme activities. The results suggest bark beetle infestation affected edaphic conditions through increased soil water content, pH, electrical conductivity, and carbon/nitrogen ratio and altered bulk and rhizosphere soil microbial community structure and function. Finally, increased enzymatic activity suggests heightened microbial decomposition following bark beetle infestation. With this increase in enzymatic activity, nutrients trapped in organic substrates may become accessible to seedlings and potentially alter the trajectory of forest regeneration. Our results indicate the need for incorporation of microbial processes into ecosystem level models. IMPORTANCE Belowground impacts of bark beetle infestation have not been explored as thoroughly as their aboveground counterparts. In order to accurately model impacts of bark beetle-induced tree mortality on carbon and nutrient cycling and forest regeneration, the intricacies of soil microbial communities must be examined. In this study, we investigated the structure and function of soil bacterial and fungal communities following bark beetle infestation. Our results show bark beetle infestation to impact soil conditions, as well as soil microbial community structure and function.

    更新日期:2020-01-22
  • Control of Waterborne Human Viruses by Indigenous Bacteria and Protists Is Influenced by Temperature, Virus Type, and Microbial Species
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Olive, M., Gan, C., Carratala, A., Kohn, T.

    Human viruses are ubiquitous contaminants in surface waters, where they can persist over extended periods of time. Among the factors governing their environmental persistence, the control (removal or inactivation) by microorganisms remains poorly understood. Here, we determined the contribution of indigenous bacteria and protists to the decay of human viruses in surface waters. Incubation of echovirus 11 (E11) in freshwater from Lake Geneva and seawater from the Mediterranean Sea led to a 2.5-log10 reduction in the infectious virus concentration within 48 h at 22°C, whereas E11 was stable in sterile controls. The observed virus reduction was attributed to the action of both bacteria and protists in the biologically active matrices. The effect of microorganisms on viruses was temperature dependent, with a complete inhibition of microbial virus control in lake water at temperatures of ≤16°C. Among three protist isolates tested (Paraphysomonas sp., Uronema marinum, and Caecitellus paraparvulus), Caecitellus paraparvulus was particularly efficient at controlling E11 (2.1-log10 reduction over 4 days with an initial protist concentration of 103 cells ml–1). In addition, other viruses (human adenovirus type 2 and bacteriophage H6) exhibited different grazing kinetics than E11, indicating that the efficacy of antiviral action also depended on the type of virus. In conclusion, indigenous bacteria and protists in lake water and seawater can modulate the persistence of E11. These results pave the way for further research to understand how microorganisms control human viral pathogens in aquatic ecosystems and to exploit this process as a treatment solution to enhance microbial water safety. IMPORTANCE Waterborne human viruses can persist in the environment, causing a risk to human health over long periods of time. In this work, we demonstrate that in both freshwater and seawater environments, indigenous bacteria and protists can graze on waterborne viruses and thereby reduce their persistence. We furthermore demonstrate that the efficiency of the grazing process depends on temperature, virus type, and protist species. These findings may facilitate the design of biological methods for the disinfection of water and wastewater.

    更新日期:2020-01-22
  • Protective Effect of the Golden Staphyloxanthin Biosynthesis Pathway on Staphylococcus aureus under Cold Atmospheric Plasma Treatment
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Yang, Y., Wang, H., Zhou, H., Hu, Z., Shang, W., Rao, Y., Peng, H., Zheng, Y., Hu, Q., Zhang, R., Luo, H., Rao, X.

    Staphylococcus aureus infection poses a serious threat to public health, and antibiotic resistance has complicated the clinical treatment and limited the solutions available to solve this problem. Cold atmospheric plasma (CAP) is a promising strategy for microorganism inactivation. However, the mechanisms of microbial inactivation or resistance remain unclear. In this study, we treated S. aureus strains with a self-assembled CAP device and found that CAP can kill S. aureus in an exposure time-dependent manner. In addition, the liquid environment can influence the survival rate of S. aureus post-CAP treatment. The S. aureus cells can be completely inactivated in normal saline and phosphate-buffered saline but not in tryptic soy broth culture medium. Scanning and transmission electron microscopy revealed that the CAP-treated S. aureus cells maintained integrated morphological structures, similar to the wild-type strain. Importantly, the CAP-treated S. aureus cells exhibited a reduced pigment phenotype. Deletion of the staphyloxanthin biosynthetic genes crtM and crtN deprived the pigmentation ability of S. aureus Newman. Both the Newman-crtM and Newman-crtN mutants presented high sensitivity to CAP treatment, whereas Newman-crtO exhibited a survival rate comparable to wild-type Newman after CAP treatment. Our data demonstrated that the yellow pigment intermediates of the staphyloxanthin biosynthetic pathway are responsible for the protection of S. aureus from CAP inactivation. The key enzymes, such as CrtM and CrtN, of the golden staphyloxanthin biosynthetic pathway could be important targets for the design of novel sterilization strategies against S. aureus infections. IMPORTANCE Staphylococcus aureus is an important pathogen that can be widely distributed in the community and clinical settings. The emergence of S. aureus with multiple-antibiotic resistance has complicated staphylococcal infection control. The development of alternative strategies with powerful bactericidal effects is urgently needed. Cold atmospheric plasma (CAP) is a promising strategy for microorganism inactivation. Nevertheless, the underlying mechanisms of microbial inactivation or resistance are not completely illustrated. In this study, we validated the bactericidal effects of CAP on S. aureus, including antibiotic-resistant strains. We also found that the golden staphyloxanthin, as well as its yellow pigment intermediates, protected S. aureus against CAP, and blocking the staphyloxanthin synthesis pathway at the early steps could strengthen the sensitivity of S. aureus to CAP treatment. These data provide insights into the germicidal mechanism of CAP from the aspect of bacteria and suggest new targets against S. aureus infections.

    更新日期:2020-01-22
  • Prevalence of Potentially Pathogenic Antibiotic-Resistant Aeromonas spp. in Treated Urban Wastewater Effluents versus Recipient Riverine Populations: a 3-Year Comparative Study
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Skwor, T., Stringer, S., Haggerty, J., Johnson, J., Duhr, S., Johnson, M., Seckinger, M., Stemme, M.

    Antibiotic resistance continues to be an emerging threat both in clinical and environmental settings. Among the many causes, the impact of postchlorinated human wastewater on antibiotic resistance has not been well studied. Our study compared antibiotic susceptibility among Aeromonas spp. in postchlorinated effluents to that of the recipient riverine populations for three consecutive years against 12 antibiotics. Aeromonas veronii and Aeromonas hydrophila predominated among both aquatic environments, although greater species diversity was evident in treated wastewater. Overall, treated wastewater contained a higher prevalence of nalidixic acid-, trimethoprim-sulfamethoxazole (SXT)-, and tetracycline-resistant isolates, as well as multidrug-resistant (MDR) isolates compared to upstream surface water. After selecting for tetracycline-resistant strains, 34.8% of wastewater isolates compared to 8.3% of surface water isolates were multidrug resistant, with nalidixic acid, streptomycin, and SXT being the most common. Among tetracycline-resistant isolates, efflux pump genes tetE and tetA were the most prevalent, though stronger resistance correlated with tetA. Over 50% of river and treated wastewater isolates exhibited cytotoxicity that was significantly correlated with serine protease activity, suggesting many MDR strains from effluent have the potential to be pathogenic. These findings highlight that conventionally treated wastewater remains a reservoir of resistant, potentially pathogenic bacterial populations being introduced into aquatic systems that could pose a threat to both the environment and public health. IMPORTANCE Aeromonads are Gram-negative, asporogenous rod-shaped bacteria that are autochthonous in fresh and brackish waters. Their pathogenic nature in poikilotherms and mammals, including humans, pose serious environmental and public health concerns especially with rising levels of antibiotic resistance. Wastewater treatment facilities serve as major reservoirs for the dissemination of antibiotic resistance genes (ARGs) and resistant bacterial populations and are, thus, a potential major contributor to resistant populations in aquatic ecosystems. However, few longitudinal studies exist analyzing resistance among human wastewater effluents and their recipient aquatic environments. In this study, considering their ubiquitous nature in aquatic environments, we used Aeromonas spp. as bacterial indicators of environmental antimicrobial resistance, comparing it to that in postchlorinated wastewater effluents over 3 years. Furthermore, we assessed the potential of these resistant populations to be pathogenic, thus elaborating on their potential public health threat.

    更新日期:2020-01-22
  • Comparative Genomics Guides Elucidation of Vitamin B12 Biosynthesis in Novel Human-Associated Akkermansia Strains
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Kirmiz, N., Galindo, K., Cross, K. L., Luna, E., Rhoades, N., Podar, M., Flores, G. E.

    Akkermansia muciniphila is a mucin-degrading bacterium found in the gut of most humans and is considered a "next-generation probiotic." However, knowledge of the genomic and physiological diversity of human-associated Akkermansia sp. strains is limited. Here, we reconstructed 35 metagenome-assembled genomes and combined them with 40 publicly available genomes for comparative genomic analysis. We identified at least four species-level phylogroups (AmI to AmIV), with distinct functional potentials. Most notably, we identified genes for cobalamin (vitamin B12) biosynthesis within the AmII and AmIII phylogroups. To verify these predictions, 10 Akkermansia strains were isolated from adults and screened for vitamin B12 biosynthesis genes via PCR. Two AmII strains were positive for the presence of cobalamin biosynthesis genes, while all 9 AmI strains tested were negative. To demonstrate vitamin B12 biosynthesis, we measured the production of acetate, succinate, and propionate in the presence and absence of vitamin supplementation in representative strains of the AmI and AmII phylogroups, since cobalamin is an essential cofactor in propionate metabolism. Results showed that the AmII strain produced acetate and propionate in the absence of supplementation, which is indicative of vitamin B12 biosynthesis. In contrast, acetate and succinate were the main fermentation products for the AmI strains when vitamin B12 was not supplied in the culture medium. Lastly, two bioassays were used to confirm vitamin B12 production by the AmII phylogroup. This novel physiological trait of human-associated Akkermansia strains may affect how these bacteria interact with the human host and other members of the human gut microbiome. IMPORTANCE There is significant interest in the therapeutic and probiotic potential of the common gut bacterium Akkermansia muciniphila. However, knowledge of both the genomic and physiological diversity of this bacterial lineage is limited. Using a combination of genomic, molecular biological, and traditional microbiological approaches, we identified at least four species-level phylogroups with differing functional potentials that affect how these bacteria interact with both their human host and other members of the human gut microbiome. Specifically, we identified and isolated Akkermansia strains that were able to synthesize vitamin B12. The ability to synthesize this important cofactor broadens the physiological capabilities of human-associated Akkermansia strains, fundamentally altering our understanding of how this important bacterial lineage may affect human health.

    更新日期:2020-01-22
  • Electronic Cigarette (E-Cigarette) Vapor Exposure Alters the Streptococcus pneumoniae Transcriptome in a Nicotine-Dependent Manner without Affecting Pneumococcal Virulence
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Bagale, K., Paudel, S., Cagle, H., Sigel, E., Kulkarni, R.

    The effects of electronic cigarette (e-cigarette) vapor (EV) exposure on the physiology of respiratory microflora are not fully defined. We analyzed the effects of exposure to vapor from nicotine-containing and nicotine-free e-liquid formulations on the virulence and transcriptome of Streptococcus pneumoniae strain TIGR4, a pathogen that asymptomatically colonizes the human nasopharyngeal mucosa. TIGR4 was preexposed for 2 h to nicotine-containing EV extract (EVE+NIC), nicotine-free EV extract (EVE–NIC), cigarette smoke extract (CSE), or nutrient-rich tryptic soy (TS) broth (control). The differences between the treatment and control strains were explored using transcriptome sequencing (RNA sequencing [RNA-Seq]), in vitro virulence assays, and an in vivo mouse model of acute pneumonia. The analysis of RNA-Seq profiles revealed modest changes in the expression of 14 genes involved in sugar transport and metabolism in EVE–NIC-preexposed TIGR4 compared to the control, while EVE+NIC or CSE exposure altered expression of 264 and 982 genes, respectively, most of which were involved in metabolism and stress response. Infection in a mouse model of acute pneumonia with control TIGR4 or with TIGR4 preexposed to EVE+NIC, EVE–NIC, or CSE did not show significant differences in disease parameters, such as bacterial organ burden and respiratory cytokine response. Interestingly, TIGR4 exposed to CSE or EVE+NIC (but not EVE–NIC) exhibited moderate induction of biofilm formation. However, none of the treatment groups showed significant alterations in pneumococcal hydrophobicity or epithelial cell adherence. In summary, our study reports that exposure to EV significantly alters the S. pneumoniae transcriptome in a nicotine-dependent manner without affecting pneumococcal virulence. IMPORTANCE With the increasing popularity of e-cigarettes among cigarette smoking and nonsmoking adults and children and the recent reports of vaping-related lung illness and deaths, further analysis of the adverse health effects of e-cigarette vapor (EV) exposure is warranted. Since pathogenic bacteria such as Streptococcus pneumoniae can colonize the human nasopharynx as commensals, they may be affected by exposure to bioactive chemicals in EV. Hence, in this study we examined the effects of EV exposure on the physiology of S. pneumoniae strain TIGR4. In order to differentiate between the effects of nicotine and nonnicotine components, we specifically compared the RNA-Seq profiles and virulence of TIGR4 exposed to vapor from nicotine-containing and nicotine-free e-liquid formulations. We observed that nicotine-containing EV augmented TIGR4 biofilms and altered expression of TIGR4 genes predominantly involved in metabolism and stress response. However, neither nicotine-containing nor nicotine-free EV affected TIGR4 virulence in a mouse model.

    更新日期:2020-01-22
  • Medium-Chain Fatty Acid Synthesis by "Candidatus Weimeria bifida" gen. nov., sp. nov., and "Candidatus Pseudoramibacter fermentans" sp. nov.
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Scarborough, M. J., Myers, K. S., Donohue, T. J., Noguera, D. R.

    Chain elongation is emerging as a bioprocess to produce valuable medium-chain fatty acids (MCFA; 6 to 8 carbons in length) from organic waste streams by harnessing the metabolism of anaerobic microbiomes. Although our understanding of chain elongation physiology is still evolving, the reverse β-oxidation pathway has been identified as a key metabolic function to elongate the intermediate products of fermentation to MCFA. Here, we describe two uncultured chain-elongating microorganisms that were enriched in an anaerobic microbiome transforming the residues from a lignocellulosic biorefining process. Based on a multi-omic analysis, we describe "Candidatus Weimeria bifida" gen. nov., sp. nov., and "Candidatus Pseudoramibacter fermentans" sp. nov., both predicted to produce MCFA but using different substrates. The analysis of a time series metatranscriptomic data set suggests that "Ca. Weimeria bifida" is an effective xylose utilizer since both the pentose phosphate pathway and the bifid shunt are active. Furthermore, the metatranscriptomic data suggest that energy conservation during MCFA production in this organism is essential and occurs via the creation of an ion motive force using both the RNF complex and an energy-conserving hydrogenase. For "Ca. Pseudoramibacter fermentans," predicted to produce MCFA from lactate, the metatranscriptomic analysis reveals the activity of an electron-confurcating lactate dehydrogenase, energy conservation via the RNF complex, H2 production for redox balance, and glycerol utilization. A thermodynamic analysis also suggests the possibility of glycerol being a substrate for MCFA production by "Ca. Pseudoramibacter fermentans." In total, this work reveals unknown characteristics of MCFA production in two novel organisms. IMPORTANCE Chain elongation by medium-chain fatty acid (MCFA)-producing microbiomes offers an opportunity to produce valuable chemicals from organic streams that would otherwise be considered waste. However, the physiology and energetics of chain elongation are only beginning to be studied, and many of these organisms remain uncultured. We analyzed MCFA production by two uncultured organisms that were identified as the main MCFA producers in a microbial community enriched from an anaerobic digester; this characterization, which is based on meta-multi-omic analysis, complements the knowledge that has been acquired from pure-culture studies. The analysis revealed previously unreported features of the metabolism of MCFA-producing organisms.

    更新日期:2020-01-22
  • A Plasmid-Encoded Putative Glycosyltransferase Is Involved in Hop Tolerance and Beer Spoilage in Lactobacillus brevis
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Feyereisen, M., Mahony, J., OSullivan, T., Boer, V., van Sinderen, D.

    Lactobacillus brevis beer-spoiling strains harbor plasmids that contain genes such as horA, horC, and hitA which are known to confer hop tolerance. The L. brevis beer-spoiling strain UCCLBBS124, which possesses four plasmids, was treated with novobiocin, resulting in the isolation of UCCLBBS124 derivatives exhibiting hop sensitivity and an inability to grow in beer. One selected derivative was shown to have lost a single plasmid, here designated UCCLBBS124_D, which harbors the UCCLBBS124_pD0015 gene, predicted to encode a glycosyltransferase. Hop tolerance and growth in beer were restored when UCCLBBS124_pD0015 was introduced in one of these hop-sensitive derivatives on a plasmid. We hypothesize that this gene modifies the surface composition of the polysaccharide cell wall, conferring protection against hop compounds. Furthermore, the introduction of this gene in trans in L. brevis UCCLB521, a strain that cannot grow in and spoil beer, was shown to furnish the resulting strain with the ability to grow in beer, while its expression also conferred phage resistance. This study underscores how the acquisition of certain mobile genetic elements plays a role in hop tolerance and beer spoilage for strains of this bacterial species. IMPORTANCE Lactobacillus brevis is a member of the lactic acid bacteria and is often reported as the causative agent of food or beverage spoilage, in particular, that of beer. Bacterial spoilage of beer may result in product withdrawal or recall, with concomitant economic losses for the brewing industry. A very limited number of genes involved in beer spoilage have been identified and primarily include those involved in hop resistance, such as horA, hitA, and horC. However, since none of these genes are universal, it is clear that there are likely (many) other molecular players involved in beer spoilage. Here, we report on the importance of a plasmid-encoded glycosyltransferase associated with beer spoilage by L. brevis that is involved in hop tolerance. The study highlights the complexity of the genetic requirements to facilitate beer spoilage and the role of multiple key players in this process.

    更新日期:2020-01-22
  • Dynamics of Solitary Predation by Myxococcus xanthus on Escherichia coli Observed at the Single-Cell Level
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Zhang, W., Wang, Y., Lu, H., Liu, Q., Wang, C., Hu, W., Zhao, K.

    The predatory behavior of Myxococcus xanthus has attracted extensive attention due to its unique social traits and inherent biological activities. In addition to group hunting, individual M. xanthus cells are able to kill and lyse prey cells; however, there is little understanding of the dynamics of solitary predation. In this study, by employing a bacterial tracking technique, we investigated M. xanthus predatory dynamics on Escherichia coli at the single-cell level. The killing and lysis of E. coli by a single M. xanthus cell was monitored in real time by microscopic observation, and the plasmolysis of prey cells was identified at a relatively early stage of solitary predation. After quantitative characterization of their solitary predatory behavior, M. xanthus cells were found to respond more dramatically to direct contact with live E. coli cells than heat-killed or UV-killed cells, showing slower predator motion and faster lysing of prey. Among the three contact-dependent killing modes classified according to the major subareas of M. xanthus cells in contact with prey, leading pole contact was observed most. After killing the prey, approximately 72% of M. xanthus cells were found to leave without thorough degradation of the lysed prey, and this postresidence behavior is described as a lysis-leave pattern, indicating that solitary predation has low efficiency in terms of prey-cell consumption. Our results provide a detailed description of the single-cell level dynamics of M. xanthus solitary predation from both prey and predator perspectives. IMPORTANCE Bacterial predation plays multiple essential roles in bacterial selection and mortality within microbial ecosystems. In addition to its ecological and evolutionary importance, many potential applications of bacterial predation have been proposed. The myxobacterium Myxococcus xanthus is a well-known predatory member of the soil microbial community. Its predation is commonly considered a collective behavior comparable to a wolf pack attack; however, individual M. xanthus cells are also able to competently lead to the lysis of a prey cell. Using a bacterial tracking technique, we are able to observe and analyze solitary predation by M. xanthus on Escherichia coli at the single-cell level and reveal the dynamics of both predator and prey during the process. The present study will not only provide a comprehensive understanding of M. xanthus solitary predation but also help to explain why M. xanthus often displays multicellular characteristic predatory behaviors in nature, while a single cell is capable of predation.

    更新日期:2020-01-22
  • Understanding and Eliminating the Detrimental Effect of Thiamine Deficiency on the Oleaginous Yeast Yarrowia lipolytica
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Walker, C., Ryu, S., Giannone, R. J., Garcia, S., Trinh, C. T.

    Thiamine is a vitamin that functions as a cofactor for key enzymes in carbon and energy metabolism in all living cells. While most plants, fungi, and bacteria can synthesize thiamine de novo, the oleaginous yeast Yarrowia lipolytica cannot. In this study, we used proteomics together with physiological characterization to elucidate key metabolic processes influenced and regulated by thiamine availability and to identify the genetic basis of thiamine auxotrophy in Y. lipolytica. Specifically, we found that thiamine depletion results in decreased protein abundance for the lipid biosynthesis pathway and energy metabolism (i.e., ATP synthase), leading to the negligible growth and poor sugar assimilation observed in our study. Using comparative genomics, we identified the missing 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (THI13) gene for the de novo thiamine biosynthesis in Y. lipolytica and discovered an exceptional promoter, P3, that exhibits strong activation and tight repression by low and high thiamine concentrations, respectively. Capitalizing on the strength of our thiamine-regulated promoter (P3) to express the missing gene from Saccharomyces cerevisiae (scTHI13), we engineered a thiamine-prototrophic Y. lipolytica strain. By comparing this engineered strain to the wild-type strain, we revealed the tight relationship between thiamine availability and lipid biosynthesis and demonstrated enhanced lipid production with thiamine supplementation in the engineered thiamine-prototrophic Y. lipolytica strain. IMPORTANCE Thiamine plays a crucial role as an essential cofactor for enzymes involved in carbon and energy metabolism in all living cells. Thiamine deficiency has detrimental consequences for cellular health. Yarrowia lipolytica, a nonconventional oleaginous yeast with broad biotechnological applications, is a native thiamine auxotroph whose affected cellular metabolism is not well understood. Therefore, Y. lipolytica is an ideal eukaryotic host for the study of thiamine metabolism, especially because mammalian cells are also thiamine auxotrophic and thiamine deficiency is implicated in several human diseases. This study elucidates the fundamental effects of thiamine deficiency on cellular metabolism in Y. lipolytica and identifies genes and novel thiamine-regulated elements that eliminate thiamine auxotrophy in Y. lipolytica. Furthermore, the discovery of thiamine-regulated elements enables the development of thiamine biosensors with useful applications in synthetic biology and metabolic engineering.

    更新日期:2020-01-22
  • Influences of a Prolific Gut Fungus (Zancudomyces culisetae) on Larval and Adult Mosquito (Aedes aegypti)-Associated Microbiota
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Frankel-Bricker, J., Buerki, S., Feris, K. P., White, M. M.

    Adult mosquitoes inherit a bacterial community from larvae via transstadial transmission, an understudied process that may influence host-microbe interactions. Microbes contribute to important host life history traits, and analyzing transmitted microbial communities, the interrelationship between larval and adult-associated microbiota, and factors influencing host-microbe relationships provides targets for research. During its larval stage, the yellow fever mosquito (Aedes aegypti) hosts the trichomycete gut fungus Zancudomyces culisetae, and fungal colonization coincides with environmental perturbations in the digestive tract microecosystem. Natural populations are differentially exposed to fungi, thereby potentially harboring distinct microbiota and experiencing disparate host-microbe interactions. This study’s objectives were to characterize larval and initial adult microbiomes, investigate variation in diversity and distribution of microbial communities across individuals, and assess whether larval fungal colonization impacted microbiomes at these developmental stages. Laboratory-based fungal infestation assays, sequencing of 16S rRNA gene amplicons, and bacterial load quantification protocols revealed that initial adult microbiomes varied in diversity and distribution. Larval fungal colonization had downstream effects on initial adult microbiomes, significantly reducing microbial community variation, shifting relative abundances of certain bacterial families, and influencing transstadial transmission outcomes of particular genera. Further, abundances of several families consistently decreased in adults relative to levels in larvae, possibly reflecting impacts of host development on specific bacterial taxa. These findings demonstrated that a prolific gut fungus impacted mosquito-associated microbiota at two developmental stages in an insect connected with global human health. IMPORTANCE Mosquitoes are widespread vectors of numerous human pathogens and harbor microbiota known to affect host phenotypic traits. However, little research has directly investigated how bacterial communities associated with larvae and adults are connected. We characterized whole-body bacterial communities in mosquito larvae preceding pupation and in newly emerged adults, and investigated whether a significant biotic factor, fungal colonization of the larval hindgut, impacted these microbiomes. Results showed that fungal colonization reduced microbial community variation across individuals and differentially impacted the outcomes of transstadial transmission for certain bacterial genera, revealing downstream effects of the fungus on initial adult microbiomes. The importance of our research is in providing a thorough comparative analysis of whole-body microbiota harbored in larvae and adults of the yellow fever mosquito (Aedes aegypti) and in demonstrating the important role a widespread gut fungus played in a host-associated microbiome.

    更新日期:2020-01-22
  • Chemical Targeting and Manipulation of Type III Secretion in the Phytopathogen Xanthomonas campestris for Control of Disease
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Zhou, L., Wang, C., Wang, G.-H., Wei, Z.-W., Fu, Q.-X., Hang, X.-H., Yang, M., Jiang, B.-L., Tang, J.-L.

    Xanthomonas campestris pv. campestris is the causative agent of black rot disease in crucifer plants. This Gram-negative bacterium utilizes the type III secretion system (T3SS), encoded by the hrp gene cluster, to aid in its resistance to host defenses and the ability to cause disease. The T3SS injects a set of proteins known as effectors into host cells that come into contact with the bacterium. The T3SS is essential for the virulence and hypersensitive response (HR) of X. campestris pv. campestris, making it a potential target for disease control strategies. Using a unique and straightforward high-throughput screening method, we examined a large collection of diverse small molecules for their potential to modulate the T3SS without affecting the growth of X. campestris pv. campestris. Screening of 13,129 different compounds identified 10 small molecules that had a significant inhibitory influence on T3SS. Moreover, reverse transcription-quantitative PCR (qRT-PCR) assays demonstrated that all 10 compounds repress the expression of the hrp genes. Interestingly, the effect of these small molecules on hrp genes may be through the HpaS and ColS sensor kinase proteins that are key to the regulation of the T3SS in planta. Five of the compounds were also capable of inhibiting X. campestris pv. campestris virulence in a Chinese radish leaf-clipping assay. Furthermore, seven of the small molecules significantly weakened the HR in nonhost pepper plants challenged with X. campestris pv. campestris. Taken together, these small molecules may provide potential tool compounds for the further development of antivirulence agents that could be used in disease control of the plant pathogen X. campestris pv. campestris. IMPORTANCE The bacterium Xanthomonas campestris pv. campestris is known to cause black rot disease in many socioeconomically important vegetable crops worldwide. The management and control of black rot disease have been tackled with chemical and host resistance methods with variable success. This has motivated the development of alternative methods for preventing this disease. Here, we identify a set of novel small molecules capable of inhibiting X. campestris pv. campestris virulence, which may represent leading compounds for the further development of antivirulence agents that could be used in the control of black rot disease.

    更新日期:2020-01-22
  • New Sipanmycin Analogues Generated by Combinatorial Biosynthesis and Mutasynthesis Approaches Relying on the Substrate Flexibility of Key Enzymes in the Biosynthetic Pathway
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Malmierca, M. G., Perez-Victoria, I., Martin, J., Reyes, F., Mendez, C., Salas, J. A., Olano, C.

    The appearance of new infectious diseases, the increase in multidrug-resistant bacteria, and the need for more effective chemotherapeutic agents have oriented the interests of researchers toward the search for metabolites with novel or improved bioactivities. Sipanmycins are disaccharyl glycosylated macrolactams that exert antibiotic and cytotoxic activities. By applying combinatorial biosynthesis and mutasynthesis approaches, we have generated eight new members of the sipanmycin family. The introduction of plasmids harboring genes responsible for the biosynthesis of several deoxysugars into sipanmycin-producing Streptomyces sp. strain CS149 led to the production of six derivatives with altered glycosylation patterns. After structural elucidation of these new metabolites, we conclude that some of these sugars are the result of the combination of the enzymatic machinery encoded by the introduced plasmids and the native enzymes of the d-sipanose biosynthetic pathway of the wild-type CS149 strain. In addition, two analogues of the parental compounds with a modified polyketide backbone were generated by a mutasynthesis approach, feeding cultures of a mutant strain defective in sipanmycin biosynthesis with 3-aminopentanoic acid. The generation of new sipanmycin analogues shown in this work relied on the substrate flexibility of key enzymes involved in sipanmycin biosynthesis, particularly the glycosyltransferase pair SipS9/SipS14 and enzymes SipL3, SipL1, SipL7, and SipL2, which are involved in the incorporation of the polyketide synthase starting unit. IMPORTANCE Combinatorial biosynthesis has proved its usefulness in generating derivatives of already known compounds with novel or improved pharmacological properties. Sipanmycins are a family of glycosylated macrolactams produced by Streptomyces sp. strain CS149, whose antiproliferative activity is dependent on the sugar moieties attached to the aglycone. In this work, we report the generation of several sipanmycin analogues with different deoxysugars, showing the high degree of flexibility exerted by the glycosyltransferase machinery with respect to the recognition of diverse nucleotide-activated sugars. In addition, modifications in the macrolactam ring were introduced by mutasynthesis approaches, indicating that the enzymes involved in incorporating the starter unit have a moderate ability to introduce different types of β-amino acids. In conclusion, we have proved the substrate flexibility of key enzymes involved in sipanmycin biosynthesis, especially the glycosyltransferases, which can be exploited in future experiments.

    更新日期:2020-01-22
  • Responses of Marine Diatom Skeletonema marinoi to Nutrient Deficiency: Programmed Cell Death
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-21
    Wang, H., Chen, F., Mi, T., Liu, Q., Yu, Z., Zhen, Y.

    Diatoms are important phytoplankton and contribute greatly to the primary productivity of marine ecosystems. Despite the ecological significance of diatoms and the importance of programmed cell death (PCD) in the fluctuation of diatom populations, little is known about the molecular mechanisms of PCD triggered by different nutrient stresses. Here we describe the physiological, morphological, biochemical, and molecular changes in response to low levels of nutrients in the ubiquitous diatom Skeletonema marinoi. The levels of gene expression involved in oxidation resistance and PCD strongly increased upon nitrogen (N) or phosphorus (P) starvation. The enzymatic activity of caspase 3-like protein also increased. Differences in mRNA levels and protein activities were observed between the low-N and low-P treatments, suggesting that PCD could have a differential response to different nutrient stresses. When cultures were replete with N or P, the growth inhibition stopped. Meanwhile, the enzymatic activity of caspase 3-like protein and the number of cells with damaged membranes decreased. These results suggest that PCD is an important cell fate decision mechanism in the marine diatom S. marinoi. Our results provide important insight into how diatoms adjust phenotypic and genotypic features of their cell-regulated death programs when stressed by nutrient limitations. Overall, this study could allow us to better understand the molecular mechanism behind the formation and termination of diatom blooms in the marine environment. IMPORTANCE Our study showed how the ubiquitous diatom S. marinoi responded to different nutrient limitations with PCD in terms of physiological, morphological, biochemical, and molecular characteristics. Some PCD-related genes (PDCD4, GOX, and HSP90) induced by N deficiency were relatively upregulated compared to those induced by P deficiency. In contrast, the expression of the TSG101 gene in S. marinoi showed a clear and constant increase during P limitation compared to N limitation. These findings suggest that PCD is a complex mechanism involving several different proteins. The systematic mRNA level investigations provide new insight into understanding the oxidative stress- and cell death-related functional genes of diatoms involved in the response to nutrient fluctuations (N or P stress) in the marine environment.

    更新日期:2020-01-22
  • Akkermansia muciniphila Prevents Fatty Liver, Decreases Serum Triglycerides, and Maintains Gut Homeostasis
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-17
    Kim, S., Lee, Y., Kim, Y., Seo, Y., Lee, H., Ha, J., Lee, J., Choi, Y., Oh, H., Yoon, Y.

    The objective of this study was to elucidate the effect of intestinal Akkermansia muciniphila on fatty liver disease. Five-week-old C57BL/6N mice were administered either phosphate buffer saline (PBS, control) or A. muciniphila at 108-109 CFU/mL, and were fed either a 45% fat diet (HFD, high-fat diet) or a 10% fat diet (ND) for 10 weeks. After 10 weeks, the mice were euthanized, and the blood and tissue samples, including adipose tissue, caecum, liver, and brain, were immediately collected. Biochemical and histological analyses were conducted and the expression levels of related factors were compared to determine the anti-obesity effects of Akkermansia muciniphila. The gut microbiome was analyzed in fecal samples. Oral administration of A. muciniphila significantly (p < 0.05) lowered serum triglyceride (TG), and alanine aminotransferase (ALT) levels in obese mice. Compared to the non-A. muciniphila-treated group, the expression of SREBP (regulator of TG synthesis in liver tissue) was decreased in the A. muciniphila-treated group. The expression of IL-6 in the liver of obese mice was decreased following the administration of A. muciniphila. Furthermore, alterations in the Firmicutes to Bacteroidetes ratio and decrease in bacterial diversity caused by the HFD were restored upon the administration of A. muciniphila. These results indicate that A. muciniphila prevents fatty liver disease in obese mice by regulating TG synthesis in the liver and maintaining gut homeostasis. Importance This study investigated the effect of Akkermansia muciniphila on fatty liver disease. Though some researches about effects of A. muciniphila on host health were published, the study on relation between A. muciniphila administration and fatty liver as well as changes of gut microbiota have not been conducted. In this study we specifically focused on composition of gut microbiota, and also demonstrate gene expression levels, regulating fat synthesis and inflammation in the liver.

    更新日期:2020-01-21
  • Freshwater cyanobacterium Synechococcus elongatus PCC 7942 adapts to salt stress environment via ion-induced enzymatic balance of compatible solute
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-17
    Liang, Y., zhang, M., Wang, M., Zhang, W., Qiao, C., Luo, Q., Lu, X.

    Salinity is one of the most important abiotic factors in various natural habitats of microbes. Cyanobacteria are the most widely distributed family of photosynthetic microorganisms in environments with fluctuating salinity. In response to salt stress, many cyanobacteria de novo synthesize compatible solutes to maintain osmotic balance in the cell. However, the regulation of intracellular accumulation of these compounds is still not well understood. The freshwater cyanobacterium Synechococcus elongatus PCC 7942 (Syn7942) exclusively accumulates sucrose as compatible solute upon salt stress, and is thereby an ideal model microorganism to study the metabolism of compatible solute dynamics. Here, we focused on elucidating the regulatory mechanisms involved in salt-induced sucrose accumulation of Syn7942. Using a series of physiological and biochemical experiments, we showed that the ionic effect of salt stress plays an important role in inducing sucrose synthesis, whereby elevated ion concentration directly activates the sucrose synthesizing enzyme sucrose-phosphate synthase and simultaneously inhibits the sucrose degrading enzyme invertase, resulting in a rapid sucrose accumulation. Thus, we propose a novel mechanism for cyanobacterial adaption to salt stress and fluctuating salinity, i.e., the ion-induced synergistic modulation of the enzymes synthesizing and degrading compatible solutes. These findings greatly develop our current understandings on microbial adaptation to salt. IMPORTANCE Most of microbes de novo synthesize compatible solutes for the adaption to salt stress or fluctuating salinity environments. However, to date, one of the core questions involved in these physiological processes, i.e. the regulation of salt-induced compatible solute biosynthesis is still not well understood. Here, this issue was systematically investigated employing a model freshwater cyanobacterium Synechococcus elongatus PCC 7942. A novel mechanism for cyanobacterial adaption to salt stress and fluctuating salinity, i.e. the ion-induced synergistic modulation of key synthesizing and degrading enzymes of compatible solutes, is proposed. Because the ion-induced activation/inhibition of enzymes is a fast and efficient progress, it may represent a common strategy of microbes for the adaption to the fluctuating salinity environments.

    更新日期:2020-01-21
  • A pathway for degradation of uracil to acetyl coenzyme A in Bacillus megaterium
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-17
    Zhu, D., Wei, Y., Yin, J., Liu, D., Ang, E. L., Zhao, H., Zhang, Y.

    Bacteria utilize diverse biochemical pathways for the degradation of the pyrimidine ring. The function of the pathways studied to date has been the release of nitrogen for assimilation. The most widespread of these pathways is the reductive pyrimidine catabolic pathway, which converts uracil into ammonia, carbon dioxide and β-alanine. Here we report the characterization of a β-alanine:pyruvate aminotransferase (PydD2), and a NAD+-dependent malonic semialdehyde dehydrogenase (MSDH), from a reductive pyrimidine catabolism gene cluster in Bacillus megaterium. Together, these enzymes convert β-alanine into acetyl-CoA, a key intermediate in carbon and energy metabolism. We demonstrate the growth of B. megaterium in defined medium with uracil as its sole carbon and energy source. Homologs of PydD2 and MSDH are found in association with reductive pyrimidine pathway genes in many Gram positive bacteria in the order Bacillales. Our study provides a basis for further investigations of the utilization of pyrimidines as a carbon and energy source by bacteria. Importance Pyrimidine has wide occurrence in natural environments, where bacteria use it as nitrogen and carbon source for growth. Detailed biochemical pathways have been investigated with focus mainly on nitrogen assimilation in the past decades. Here we report the discovery and characterization of two important enzymes, PydD2 and MSDH, which constitute an extension for the reductive pyrimidine catabolic pathway. These two enzymes, prevalent in Bacillales based on our bioinformatics studies, allow stepwise conversion of β-alanine, a previous "end product" of the reductive pyrimidine degradation pathway to acetyl-CoA, as carbon and energy source.

    更新日期:2020-01-21
  • Site-specific profiling of the dental mycobiome reveals strong taxonomic shifts during progression of early childhood caries
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-17
    O'Connell, L. M., Santos, R., Springer, G., Burne, R. A., Nascimento, M. M., Richards, V. P.

    Dental caries is one of the most common diseases worldwide. Bacteria and fungi are both commensals in the oral cavity; however, most research regarding caries has focused on bacterial impacts. The oral fungal mycobiome associated with caries is not well characterized and its role in disease is unclear. ITS1 amplicon sequencing was used to generate taxonomic profiles from site-specific supragingival plaque samples (n=82) obtained from 33 children with different caries status. Children were either caries-free (CF), caries-active with enamel lesions (CAE), or caries-active with dentin lesions (CA). Plaque samples were collected from caries-free surfaces (PF), and from enamel (PE) and dentin (PD) lesions. Taxonomic profiles representing the different categorizations (CF-PF, CAE-PF, CAE-PE, CA-PF, CA-PE and CA-PD) were utilized to characterize the mycobiome and its change through disease progression. A total of 139 fungal species were identified. Candida albicans was the most abundant species followed by Candida dubliniensis. We found that severely progressed plaque communities (CA-PD) were significantly different from healthy plaque communities (CF-PF). A total of 32 taxa were differentially abundant across the plaque categories. C. albicans, C. dubliniensis, Nigrospora oryzae, and an unclassified Microdichium sp. were correlated with caries, whereas 12 other taxa were correlated with health. C. dubliniensis increased steadily as caries progressed, suggesting that C. dubliniensis may play an important role in caries pathogenicity. In contrast, four health-associated fungal taxa have the potential to antagonize the cariogen S. mutans via xylitol production, suggesting a possible fungal mechanism that could contribute to maintenance of dental health. IMPORTANCE Early childhood caries is one of the most prevalent diseases in children worldwide, and while preventable, remains a global public health concern. Untreated cavities are painful, expensive, and can lead to tooth loss and a lower quality of life. Caries are driven by acid production via microbial fermentation of dietary carbohydrates resulting in enamel erosion. While caries is a well-studied disease, most research has focused on bacterial impacts even though fungi are commensal organisms living within the plaque biofilm. There is very little known about how fungi impact caries pathogenicity. The elucidation of fungal taxa involved in caries disease progression is necessary for a more holistic view of the human oral microbiome. Data from this study will improve our understanding of how the fungal community changes as disease progresses and provides insight into the complex etiology of dental caries which is necessary for development of treatment plans and preventative measures.

    更新日期:2020-01-21
  • Metagenomic shotgun analyses reveal complex patterns of intra- and interspecific variation in the intestinal microbiomes of codfishes
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-17
    Riiser, E. S., Haverkamp, T. H. A., Varadharajan, S., Borgan, O., Jakobsen, K. S., Jentoft, S., Star, B.

    The relative importance of host-specific selection or environmental factors in determining the composition of the intestinal microbiome in wild vertebrates remains poorly understood. Here, we use metagenomic shotgun sequencing of individual specimens to compare the intra- and interspecific variation of intestinal microbiome communities in two ecotypes (NEAC and NCC) of Atlantic cod (Gadus morhua) –that have distinct behavior and habitats– and three Gadidae species that occupy a range of ecological niches. Interestingly, we find significantly diverged microbiomes amongst the two Atlantic cod ecotypes. Interspecific patterns of variation are more variable, with significantly diverged communities for most species' comparisons, apart from the comparison between coastal cod (NCC) and Norway pout (Trisopterus esmarkii), whose community compositions are not significantly diverged. The absence of consistent species-specific microbiomes suggests that external environmental factors, such as temperature, diet or a combination there-off comprise major drivers of the intestinal community composition of codfishes. Importance The composition of the intestinal microbial community associated with teleost fish is influenced by a diversity of factors, ranging from internal factors (such as host-specific selection) to external factors (such as niche occupation). These factors are often difficult to separate, as differences in niche occupation (e.g. diet, temperature or salinity) may correlate with distinct evolutionary trajectories. Here, we investigate four gadoid species with contrasting levels of evolutionary separation and niche occupation. Using metagenomic shotgun sequencing, we observe distinct microbiomes amongst two Atlantic cod (Gadus morhua) ecotypes (NEAC and NCC) with distinct behavior and habitats. In contrast, interspecific patterns of variation are more variable. For instance, we do not observe interspecific differentiation between the microbiomes of coastal cod (NCC) and Norway pout (Trisopterus esmarkii) whose lineages have evolutionary separated over 20 million years ago. The observed pattern of microbiome variation in these gadoid species is therefore most parsimoniously explained by differences in niche occupation.

    更新日期:2020-01-21
  • Respiratory physiology of Lactococcus lactis in chemostat cultures and its effect on cellular robustness in frozen and freeze-dried starter cultures
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-17
    Johanson, A., Goel, A., Olsson, L., Franzen, C. J.

    In this study, we used chemostat cultures to analyze the quantitative effects of the specific growth rate and respiration on the metabolism in L. lactis CHCC2862, and on the downstream robustness of cells after freezing or freeze-drying. Under anaerobic conditions, metabolism remained homofermentative, although biomass yields varied with the dilution rate (D). In contrast, metabolism shifted with the dilution rate under respiration-permissive conditions. At D = 0.1 h-1, no lactate was produced, while lactate formation increased with higher dilution rates. Thus, a clear metabolic shift was observed, from flavor-forming respiratory metabolism at low specific growth rate, to mixed-acid respiro-fermentative metabolism at higher specific growth rates. Quantitative analysis of the respiratory activity, lactose uptake rate and metabolite production rates showed that aerobic acetoin formation provided most of the NADH consumed in respiration. Moreover, the maintenance-associated lactose consumption under respiration-permissive conditions was only 10 % of the anaerobic value, either due to higher respiratory yield of ATP on consumed lactose or due to lower maintenance-related ATP demand. The cultivation conditions also affected the quality of the starter cultures produced. Cells harvested under respiration-permissive conditions at D = 0.1 h-1 were less robust after freeze-drying and had a lower acidification activity for subsequent milk acidification, whereas respiration-permissive conditions at the higher dilution rates led to robust cells that performed equally well or better than anaerobic cells. Importance Lactococcus lactis is used in large quantities by the food and biotechnological industries. L. lactis can use oxygen for respiration if heme is supplied in the growth medium. This has been extensively studied in batch cultures using various mutants, but quantitative studies of how the cell growth affects respiratory metabolism, energetics and cell quality are surprisingly scarce. Our results demonstrate that the respiratory metabolism of L. lactis is remarkably flexible and can be modulated by controlling the specific growth rate. We also link the physiological state of cells during cultivation to the quality of frozen or freeze-dried cells, which is relevant to the industry that may lack understanding of such relationships. This study extends our knowledge on respiratory metabolism in L. lactis and its impact on frozen and freeze-dried starter culture products, and it illustrates the influence of cultivation conditions and microbial physiology on the quality of starter cultures.

    更新日期:2020-01-21
  • Dissemination of quinolone resistant Escherichia coli in the Norwegian broiler and pig production chain, and possible persistence in the broiler production environment
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-17
    Kaspersen, H., Sekse, C., Fiskebeck, E. Z., Slettemeas, J. S., Simm, R., Norström, M., Urdahl, A. M., Lagesen, K.

    In Norway, the use of quinolones in livestock populations is very low, and prophylactic use is prohibited. Despite this, quinolone resistant E. coli (QREC) are present at low levels in several animal species. The source of these QREC is unknown. The aim of this study was to characterize and compare QREC from different animal species to identify putative factors that may promote the occurrence of QREC. A total of 280 QREC isolates, from broilers, pigs, red foxes and wild birds, were whole genome sequenced and analysed. Well-known chromosomal and plasmid-mediated resistance mechanisms were identified. In addition, mutations in marR, marA and rpoB causing novel amino acid substitutions in their respective proteins were detected. Phylogenetic analyses were used to determine the relationships between the isolates. Quinolone resistance mechanism patterns appeared to follow sequence type groups. Similar QREC isolates with similar resistance mechanism patterns were detected from the samples, and further phylogenetic analysis indicated close evolutionary relationships between specific isolates from different sources. This suggests dissemination of highly similar QREC isolates between animal species, and also persistence of QREC strains within the broiler production chain. This highlights the importance of both control measures at the top of the production chain, as well as biosecurity measures to avoid further dissemination and persistence of QREC in these environments. Significance of study Since antimicrobial usage is low in Norwegian animal husbandry, Norway is an ideal country to study antimicrobial resistance in the absence of selective pressure from antimicrobial usage. In particular, the usage of quinolones is very low, which makes it possible to investigate the spread and development of quinolone resistance in natural environments. Comparing quinolone resistant E. coli (QREC) from livestock and wild animals in light of this low quinolone usage provides new insights into the development and dissemination of QREC in both natural- and production environments. With this information, preventive measures may be taken to prevent further dissemination within Norwegian livestock and between other animals, thus maintaining the favourable situation in Norway.

    更新日期:2020-01-21
  • GlnR negatively regulates glutamate-dependent acid resistance in Lactobacillus brevis
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-17
    Gong, L., Ren, C., Xu, Y.

    Lactic acid bacteria often encounter a variety of multiple stresses in their natural and industrial fermentation environments. Glutamate decarboxylase (GAD) system is one of the most important acid resistance systems in lactic acid bacteria. In this study, we demonstrated that GlnR, a nitrogen regulator in Gram-positive bacteria, directly modulated -aminobutyric acid (GABA) conversion from glutamate and was involved in glutamate-dependent acid resistance in Lactobacillus brevis. The glnR-deletion strain (glnR) achieved a titer of 284.7 g/L GABA, a 9.8-fold higher than that of the wild-type strain. The cell survival of the glnR-deletion strain was significantly higher than that of the wild-type strain under the condition of acid challenge and was positively correlated with initial glutamate concentration and GABA production. Quantitative reverse transcription PCR assays demonstrated that GlnR inhibited the transcription of glutamate decarboxylase encoding gene (gadB), glutamate/GABA antiporter encoding gene (gadC), glutamine synthetase encoding gene (glnA) as well as the specific transcriptional regulator encoding gene (gadR) involved in gadCB operon regulation. Moreover, GABA production and glutamate-dependent acid resistance were absolutely abolished in the gadR-glnR-deletion strain. Electrophoretic mobility shift and DNase I footprinting assays revealed that GlnR directly bound to the 5'-untranslated regions of the gadR gene and gadCB operon, thus inhibited their transcription. These results revealed a novel regulatory mechanism of GlnR on glutamate-dependent acid resistance in Lactobacillus. IMPORTANCE Free-living lactic acid bacteria often encounter acid stresses because of their organic acids producing features. Several acid resistant mechanisms, such as glutamate decarboxylase system, F1F0-ATPase proton pump and alkali production, are usually employed to relieve growth inhibition caused by acids. Glutamate decarboxylase system is vital for GAD-containing lactic acid bacteria to protect cells from DNA damage, enzyme inactivation and product yield loss in acidic habitats. In this study, we found that a MerR-type regulator GlnR was involved in glutamate-dependent acid resistance by directly regulating the transcription of the gadR gene and gadCB operon, resulting in an inhibition of GABA conversion from glutamate in L. brevis. This study represents a novel regulatory mechanism of GlnR to glutamate-dependent acid resistance and also provides a simple and novel strategy to engineer Lactobacillus strains to elevate their acid resistance as well as GABA conversion from glutamate.

    更新日期:2020-01-21
  • Molecular Characterization of the Interactions Between Biotite and the Mineral-Weathering Bacterium Pseudomonas azotoformans F77
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-17
    Wang, Y.-L., Sun, L.-J., Xian, C.-M., Kou, F.-L., Zhu, Y., He, L.-Y., Sheng, X.-F.

    In this study, the mineral-weathering bacterium Pseudomonas azotoformans F77, isolated from the soil of a debris flow area, was evaluated for its weathering activity under direct contact with biotite or without contact. Then, biotite-weathering behaviors of strain F77, its mutants that were created by deleting the gcd and adh genes, which are involved in gluconic acid metabolism and pilus formation, respectively and the double mutant F77gcdadh, were compared. The relative gene expression levels of F77 and its mutants F77gcd and F77adh were also analyzed in the presence of biotite. Direct contact with biotite increased Fe and Al release from the mineral in the presence of F77. All strains had a similar ability to release Fe and Al from the mineral except for F77gcd and F77adh. Mobilized Fe and Al concentrations were decreased by up to 72, 26, and 87% in the presence of F77gcd, F77adh, and F77gcdadh, respectively, compared to those observed in the presence of F77 during the mineral-weathering process. Gluconic acid production was decreased for F77gcd and F77gcdadh, while decreased cell attachment on the mineral surface was observed for F77adh compared to that observed for F77. The F77 genes involved in pilus formation and gluconic acid metabolism showed increased expression levels in the presence of biotite. The results of this study showed the important role of the genes involved in gluconic acid metabolism and pilus formation in mineral weathering by F77 and demonstrated the distinctive effect of these genes on mineral weathering by F77. IMPORTANCE Bacteria play an important role in mineral weathering and soil formation; although the molecular mechanisms underlying the interactions between bacteria and silicate minerals are poorly understood. In this study, the interactions between biotite and the highly effective mineral-weathering bacterium P. azotoformans F77 were characterized. Our results showed that the genes involved in gluconic acid metabolism and pilus formation play important roles in mineral weathering by F77. The presence of biotite could promote the expression of these genes in F77, and a distinctive effect of these genes on mineral weathering by F77 was observed in this study. Our results provide new knowledge and promote better understanding regarding the interaction between silicate minerals and mineral-weathering bacteria and of the molecular mechanisms involved in these processes.

    更新日期:2020-01-21
  • Potential control of potato soft rot disease by the obligate predators Bdellovibrio and like organisms
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-17
    Youdkes, D., Helman, Y., Burdman, S., Matan, O., Jurkevitch, E.

    Bacterial soft rot diseases caused by Pectobacterium spp. and Dickeya spp. affect a wide range of crops including potato, a major food crop. As of today, farmers mostly rely on sanitary practices, water management and plant nutrition for control. We tested the bacterial predators Bdellovibrio and like organisms (BALOs) to control potato soft rot. BALOs are small, motile predatory bacteria found in terrestrial and aquatic environments. They predate on a wide range of Gram-negative bacteria, including animal and plant pathogens. To this end, BALO strains HD100, 109J and merRNA were shown to efficiently prey on various rot-causing strains of Pectobacterium and Dickeya solani. BALO control of maceration caused by a highly virulent strain of P. carotovorum subsp. brasilense (Pcb), was then tested in situ using a potato slice assay. All BALO strains were highly effective at reducing disease up to complete prevention. Effectivity was concentration-dependent, and BALOs applied before Pcb inoculation performed significantly better than when applied after the disease-causing agent, maybe due to in situ consumption of glucose by the prey, as glucose metabolism by live prey bacteria was shown to prevent predation. Dead predators and the supernatant of BALO cultures did not significantly prevent maceration, indicating that predation was the major mechanism for the prevention of the disease. Finally, plastic resistance to predation was affected by prey and predator population parameters, suggesting that population dynamics affects prey response to predation. Importance Bacterial soft rot diseases caused by Pectobacterium spp. and Dickeya spp. are among the most important disease-causing bacteria in plants. Among other crops, they inflict large scale damage to potatoes. As of today, farmers have little options to control them. The bacteria Bdellovibrio and like organisms (BALOs) are obligate predators of bacteria. We tested their potential to prey on Pectobacterium spp. and Dickeya spp. and to protect potato. We show that different BALOs can prey on soft rot-causing bacteria, and prevent their growth in-situ, precluding tissue maceration. Dead predators and the supernatant of BALO cultures did not significantly prevent maceration showing the effect is due to predation. Soft-rot control by the predators was concentration-dependent, and higher when the predator was inoculated ahead of the prey. As residual prey remained, we investigated what determines their level, finding that initial prey and predator population parameters affects prey response to predation.

    更新日期:2020-01-21
  • Phylosymbiosis across deeply diverging lineages in omnivorous cockroaches
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-17
    Tinker, K. A., Ottesen, E. A.

    The gut microbiome is shaped by both host diet and by host phylogeny. However, separating the relative influence of these two factors over long periods of evolutionary time is often difficult. We conducted a 16S rRNA gene amplicon-based survey of the gut microbiome from 237 individuals and 19 species of omnivorous cockroaches from the order Blattodea. The order Blattodea represents an ancient lineage of insects that emerged over 300 million years ago and share a diverse gut microbiota and a typically gregarious lifestyle. All cockroaches share a broadly similar gut microbiota, with 66 microbial families present across all species and 13 present in every individual examined. Although our network analysis of the cockroach gut microbiome showed a high amount of connectivity, we demonstrated that gut microbiota cluster strongly by host species. We conducted follow-up tests to determine if cockroaches exhibit phylosymbiosis, or the tendency of host-associated microbial communities to parallel the phylogeny of related host species. Across the full dataset, gut microbial community similarity was not found to correlate with host phylogenetic distance. However, a weak but significant phylosymbiotic signature was observed using the matching cluster metric, which allows for localized changes within a phylogenetic tree that are more likely to occur over long evolutionary distances. This suggests that host phylogeny plays a large role in structuring the cockroach gut microbiome over shorter evolutionary distances, and a weak but significant role in shaping the gut microbiome over extended periods of evolutionary time. IMPORTANCE The gut microbiome plays a key role in host health. Therefore, it is important to understand the evolution of the gut microbiota and how it impacts, and is impacted by, host evolution. In this study, we explore the relationship between host phylogeny and gut microbiome composition in omnivorous, gregarious cockroaches within Blattodea order, an ancient lineage that spans 300 million years of evolutionary divergence. We demonstrate a strong relationship between host species identity and gut microbiome composition, and a found weaker but significant role for host phylogeny in determining microbiome similarity over extended periods of evolutionary time. This study advances our understanding of the role of host phylogeny in shaping the gut microbiome over different evolutionary distances.

    更新日期:2020-01-21
  • Faster diffusive dynamics of histone-like nucleoid structuring proteins in live bacteria caused by silver ions
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-17
    Sadoon, A. A., Khadka, P., Freeland, J., Gundampati, R. K., Manso, R., Ruiz, M., Krishnamurthi, V. R., Thallapuranam, S. K., Chen, J., Wang, Y.

    The antimicrobial activity and mechanism of silver ions (Ag+) have gained broad attention in recent years. However, dynamic studies are rare in this field. Here, we report our measurement of the effects of Ag+ ions on the dynamics of histone-like nucleoid structuring (H-NS) proteins in live bacteria using single-particle tracking photoactivated localization microscopy (sptPALM). It was found that treating the bacteria with Ag+ ions led to faster diffusive dynamics of H-NS proteins. Several techniques were used to understand the mechanism of the observed faster dynamics. Electrophoretic mobility shift assay on purified H-NS proteins indicated that Ag+ ions weaken the binding between H-NS proteins and DNA. Isothermal titration calorimetry confirmed that DNA and Ag+ ions interact directly. Our recently developed sensing method based on bent DNA suggested that Ag+ ions caused dehybridization of double-stranded DNA (i.e., dissociation into single strands). These evidences led us to a plausible mechanism for the observed faster dynamics of H-NS proteins in live bacteria when subjected to Ag+ ions: Ag+-induced DNA dehybridization weakens the binding between H-NS proteins and DNA. This work highlighted the importance of dynamic study of single proteins in the live cells for understanding the functions of antimicrobial agents to the bacteria. Importance As "superbug" bacteria resistant to commonly prescribed antibiotics become a global threat to the public threat in recent years, noble metals, such as silver, in various forms have been attracting broad attention due to their antimicrobial activities. However, most of the existing literature relied on the traditional bioassays for studying the antimicrobial mechanism of silver; in addition, temporal resolution is largely missing for understanding the effects of silver the molecular dynamics inside the bacteria. Here, we report our study on the antimicrobial effect of silver ions at the nanoscale on the diffusive dynamics of histone-like nucleoid structuring (H-NS) proteins in live bacteria using single-particle tracking photoactivated localization microscopy. This work highlights the importance of dynamic study of single proteins in the live cells for understanding the functions of antimicrobial agents to the bacteria.

    更新日期:2020-01-21
  • A negative regulator of carotenogenesis in Blakeslea trispora
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-17
    Luo, W., Gong, Z., Li, N., Zhao, Y., Zhang, H., Yang, X., Liu, Y., Rao, Z., Yu, X.

    As an ideal carotenoids producer, Blakeslea trispora has gained lots of attentions due to its large biomass and high production of β-carotene and lycopene. However, carotenogenesis regulation in B. trispora still needs to be clarified since few investigations have been conducted at molecular level in B. trispora. In this study, the homologous gene of carotenogenesis regulatory gene (crgA) was cloned from the mating type (-) of B. trispora and the deduced CrgA protein was analyzed for its primary structure and domains. In order to clarify the crgA-mediated regulation in B. trispora, we used the strategies of gene knockout and complementation to investigate the effect of crgA expression on the phenotype of B. trispora. In contrast to the wild-type strain, the crgA null mutant was defective in sporulation, but accumulated much more β-carotene (31.2% improvement at the end) accompanied by the enhanced transcription levels of three structural genes (hmgR, carB and carRA) for carotenoids over whole culture time. When the wild-type copy of crgA was complemented into the crgA null mutant, sporulation, transcription level of structural gene and carotenoids production were restored as present in wild-type strain. Gas chromatography-mass spectrometry (GC-MS)-based metabolomic approach and multivariate statistical analyses were performed to investigate the intracellular metabolite profiles. The reduced levels of tricarboxylic acid (TCA) cycle components and some amino acids and enhanced levels of glycolysis intermediates and fatty acids indicate that more metabolic flux was driven into the mevalonate (MVA) pathway, thus the increase of precursors and fat content contributes to the accumulation of carotenoids. IMPORTANCE The zygomycete Blakeslea trispora is an important strain for the production of carotenoids in large scale. However, the regulation mechanism of carotenoid biosynthesis is still not well understood in this filamentous fungus. In the current study, we sought to investigate how crgA influences the expression of structural genes for carotenoids, carotenoid biosynthesis and other anabolic phenotypes. This will lead to a better understanding of global regulation mechanism of carotenoid biosynthesis and facilitate engineering this strain in the future for enhanced production of carotenoids.

    更新日期:2020-01-21
  • DNA stable-isotope probing delineates carbon flows from rice residues into soil microbial communities depending on fertilization
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-17
    Kong, Y., Kuzyakov, Y., Ruan, Y., Zhang, J., Wang, T., Wang, M., Guo, S., Shen, Q., Ling, N.

    Decomposition of crop residues in soil is mediated by microorganisms whose activities varied with fertilization. The complexity of active microorganisms and their interactions by utilizing residues is impossible to disentangle without isotope applications. Thus, 13C-labeled rice residues were employed and DNA stable-isotope probing (DNA-SIP) combined with high-throughput sequencing was applied to identify microbes active in assimilating residue carbon (C). Manure addition strongly modified microbial community compositions involved in the C flow from rice-residues. Relative abundances of bacterial genus Lysobacter and fungal genus Syncephalis were increased, but that of bacterial genus Streptomyces and fungal genus Trichoderma were decreased in soils receiving mineral fertilizers plus manure (NPKM) compared to soils receiving only mineral fertilizers (NPK). Microbes involved in the flow of residue-C formed more complex network in NPKM than that in NPK soils, because of necessity to decompose more diverse organic compounds. The fungal species (Jugulospora rotula and Emericellopsis terricola in NPK and NPKM soils, respectively) were identified as keystone species in network and may significantly contribute to residue-C decomposition. Most of fungal genera in NPKM soils, especially Chaetomium, Staphylotrichum, Penicillium and Aspergillus, responded faster to residue addition than those in NPK soils. This is connected with the changes in the composition of the rice residue during the degradation and with fungal adaptation (abundance and activity) to continuous manure input. Our findings provide fundamental information about the roles of key microbial groups in residue decomposition and offer important cue on manipulating the soil microbiome for residue utilization and C sequestration in soil. Importance Identifying and understanding the active microbial communities and interactions involved in plant-residue utilization is a key question to elucidate the transformation of soil organic matter (SOM) in agricultural ecosystems. Microbial community composition responds strongly to management, but little is known about specific microbial groups involved in plant-residue utilization and consequently microbial functions under distinct fertilization. We combined DNA stable-isotope (13C) probing and high-throughput sequencing to identify active fungal and bacterial groups degrading residues in soils after 3-year mineral fertilization with and without manure. Manuring changed the active microbial composition and complexified microbial interactions involved in residue-C flow. Most fungal genera, especially Chaetomium, Staphylotrichum, Penicillium and Aspergillus, responded to residue addition faster in soils historically received manure. We generated a valuable "library of microorganisms" involved in plant residue utilization for future targeted research to exploit specific functions of microbial groups in organic matter utilization and C sequestration.

    更新日期:2020-01-21
  • Short-term stable isotope probing of proteins reveals taxa incorporating inorganic carbon in a hot spring microbial mat
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-17
    Steinke, L., Slysz, G. W., Lipton, M. S., Klatt, C., Moran, J. J., Romine, M. F., Wood, J. .M., Anderson, G., Bryant, D. A., Ward, D. M.

    The upper green layer of the chlorophototrophic microbial mats associated with the alkaline siliceous hot springs of Yellowstone National Park consist of oxygenic cyanobacteria (Synechococcus spp.), anoxygenic Roseiflexus spp., and several other anoxygenic chlorophototrophs. Synechococcus spp. are believed to be the main fixers of inorganic carbon (Ci), but some evidence suggests that Roseiflexus spp. also contribute to inorganic carbon fixation during low-light, anoxic morning periods. Contributions of other phototrophic taxa have not been investigated. In order to follow the pathway of Ci incorporation into different taxa, mat samples were incubated with 13C-bicarbonate for 3 h during the early-morning, low-light anoxic period. Extracted proteins were treated with trypsin and analyzed by mass spectrometry, leading to peptide identifications and peptide isotopic profile signatures containing evidence of 13C-label incorporation. 25,483 peptides, corresponding to 7,221 proteins, were identified from spectral features and associated with mat taxa by comparison to metagenomic assembly sequences. 1,417 peptides, derived from 720 proteins, were detectably labeled with 13C. Most 13C-labeled peptides were derived from proteins of Synechococcus spp. and Roseiflexus spp. Chaperones and proteins of carbohydrate metabolism were most abundantly labeled. Proteins involved in photosynthesis, Ci fixation, and N2 fixation were also labeled in Synechococcus spp. Importantly, most proteins of the 3-hydroxypropionate bi-cycle for Ci fixation in Roseiflexus spp. were labeled, establishing that members of this taxocene contribute to Ci fixation. Other taxa showed much lower 13C-bicarbonate incorporation. Importance Yellowstone hot spring mats have been studied as natural models for understanding microbial community ecology and as modern analogs of stromatolites, the earliest community fossils on Earth. Protein-SIP permitted short-term interrogation of the taxa that are involved in the important process of light-driven Ci fixation in this highly active community and will be useful in linking other metabolic processes to mat taxa. Here, evidence is presented that Roseiflexus spp., which use the 3-hydroxypropionate bi-cycle, are active in Ci fixation. Because this pathway imparts a lower degree of selection of isotopically heavy Ci than does the Calvin-Benson-Bassham Cycle, the results suggest a mechanism for why the natural abundance of 13C in mat biomass is greater than expected if only the latter pathway were involved. Understanding how mat community members influence the 13C/12C ratios of mat biomass will help geochemists interpret the 13C/12C ratios of organic carbon in the fossil record.

    更新日期:2020-01-21
  • Reconstruction of the "archaeal" mevalonate pathway from the methanogenic archaeon Methanosarcina mazei in Escherichia coli cells
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-10
    Yoshida, R., Yoshimura, T., Hemmi, H.

    The mevalonate pathway is a well-known metabolic route that provides biosynthetic precursors for myriad isoprenoids. An unexpected variety of the pathway has been discovered from recent studies on microorganisms, mainly on archaea. The most recently discovered example, called the "archaeal" mevalonate pathway, is a modified version of the canonical eukaryotic mevalonate pathway and was elucidated in our previous study using the hyperthermophilic archaeon Aeropyrum pernix. This pathway comprises four known enzymes that can produce mevalonate 5-phosphate from acetyl-CoA, two recently discovered enzymes designated as phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase, and two more known enzymes: isopentenyl phosphate kinase and isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase. To show its wide distribution in archaea and to confirm if its enzyme configuration is identical among species, the putative genes of a lower portion of the pathway – from mevalonate to isopentenyl pyrophosphate – were isolated from the methanogenic archaeon Methanosarcina mazei, which is taxonomically distant from A. pernix, and were introduced in the engineered Escherichia coli strain that produces lycopene, a red carotenoid pigment. Lycopene production, as a measure of isoprenoid productivity, was enhanced when the cells were grown semi-anaerobically with the supplementation of mevalonolactone, which demonstrates that the archaeal pathway can function in bacterial cells to convert mevalonate into isopentenyl pyrophosphate. Gene deletion and complementation analysis using the carotenogenic E. coli strain suggested that both phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase from M. mazei are required for the enhancement of lycopene production. Importance Two enzymes that have recently been identified from the hyperthermophilic archaeon A. pernix as components of the archaeal mevalonate pathway do not require ATP for their reactions. This pathway, therefore, might consume less energy than other mevalonate pathways to produce precursors for isoprenoids. Thus, the pathway might be applicable to metabolic engineering toward the production of valuable isoprenoids that have applications as pharmaceuticals. The archaeal mevalonate pathway was successfully reconstructed in E. coli cells by introducing several genes from the methanogenic or hyperthermophilic archaeon, which demonstrated that the pathway requires the same components even in distantly related archaeal species and can function in bacterial cells.

    更新日期:2020-01-13
  • Comparative Genomics and Phylogenetic Analyses Suggest Several Novel Species within Clavibacter sp. Including Non-Pathogenic Tomato-Associated Strains
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-10
    Osdaghi, E., Rahimi, T., Taghavi, S. M., Ansari, M., Zarei, S., Portier, P., Briand, M., Jacques, M.-A.

    Members of Clavibacter spp. are economically important bacterial plant pathogens infecting a set of diverse agricultural crops (e.g. alfalfa, corn, potato, tomato, and wheat). Tomato-associated Clavibacter spp. strains occupy a great portion of genetic diversity of the genus, and C. michiganensis sensu stricto (formerly C. michiganensis subsp. michiganensis) causing bacterial canker disease considered one of the destructive seed-borne agents of the crop worldwide. However, current taxonomic descriptions of the genus do not reflect the existing diversity of the strains, resulting in unsatisfactory consequences in quarantine surveys for the pathogens. In this study, we used all the available genome sequences of Clavibacter spp. strains - including the type strains of newly described subspecies - to provide a precise insight into the diversity of tomato-associated members of the genus, and further clarify taxonomic status of the strains using genotypic and phenotypic features. Results of phylogenetic analyses revealed the existence of nine hypothetical new species among the investigated strains. None of the three new subspecies (i.e. C. michiganensis subsp. californiensis, C. michiganensis subsp. chilensis and C. michiganensis subsp. phaseoli) is included within the tomato-pathogenic C. michiganensis sensu stricto lineage. Although comparative genomics revealed the lack of chp and tomA pathogenicity determinant gene clusters in the non-pathogenic strains, a number of pathogenicity related genes were noted to be present in all the strains regardless of their pathogenicity characteristics. Altogether, our results advocate a need for a formal taxonomic reconsideration of tomato-associated Clavibacter spp. strains to facilitate differentiation of the lineages in quarantine inspections. Importance Clavibacter spp. are economically important bacterial plant pathogens infecting a set of diverse agricultural crops such as alfalfa, corn, pepper, potato, tomato, and wheat. A number of plant pathogenic members of the genus (e.g. C. michiganensis sensu stricto and C. sepedonicus infecting tomato and potato plants, respectively) are included in the A2 (high risk) list of quarantine pathogens by the European and Mediterranean Plant Protection Organization (EPPO). Although tomato-associated members of Clavibacter spp. occupy a significant portion of the genetic diversity in the genus, only the strains belonging to C. michiganensis sensu stricto (formerly C. michiganensis subsp. michiganensis) cause bacterial canker disease of tomato and subjected to the quarantine inspections. Hence, discrimination of the pathogenic and non-pathogenic Clavibacter spp. strains associated with tomato seeds and transplants plays a pivotal role in the accurate detection and cost efficiently management of the disease. On the other hand, detailed information on the genetic contents of different lineages of the genus would lead to the development of genome-informed specific detection techniques. In this study, we have provided an overview on the phylogenetic and genomic differences between the pathogenic and non-pathogenic tomato-associated Clavibacter spp. strains. We have also noted that the taxonomic status of newly introduced subspecies of C. michiganensis (i.e. C. michiganensis subsp. californiensis, C. michiganensis subsp. chilensis and C. michiganensis subsp. phaseoli) should be reconsidered.

    更新日期:2020-01-13
  • Genomic and phenotypic stability of Lactobacillus rhamnosus GG in an industrial production process
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-10
    Stage, M., Gustafsson, A. W., Jorgensen, M., Vera-Jimenez, N. I., Wielje, M., Nielsen, D. S., Sandelin, A., Chen, Y., Baker, A.

    Lactobacillus rhamnosus GG is one of the most widely marketed and studied probiotic strains. In L. rhamnosus GG the spaCBA-srtC1 gene cluster encodes pili, which are important for some of the probiotic properties of the strain. A previous study showed that the DNA sequence of the spaCBA-srtC1 gene cluster was not present in some L. rhamnosus GG variants isolated from liquid dairy products. To examine the stability of the L. rhamnosus GG genome in an industrial production process, we sequenced the genome of samples of L. rhamnosus GG (DSM 33156) collected at specific steps of the industrial production process, including the culture collection stock, intermediate fermentations, and final freeze-dried products. We found that the L. rhamnosus GG genome sequence was unchanged throughout the production process. Consequently, the spaCBA-srtC1 gene locus was intact and fully conserved in all 31 samples examined. In addition, different production batches of L. rhamnosus GG exhibited consistent phenotypes, including the presence of pili on final freeze-dried products and consistent characteristics on in vitro assays of probiotic properties. Our data show that L. rhamnosus GG is highly stable in this industrial production process. Importance Lactobacillus rhamnosus GG is one of the best studied probiotic strains. One of the well-characterized features of the strain are the pili encoded by the spaCBA-srtC1 gene cluster. These pili are involved in persistence in the gastrointestinal tract and are important for the probiotic properties of L. rhamnosus GG. Previous studies demonstrated that the L. rhamnosus GG genome can be unstable under certain conditions and lose the spaCBA-srtC1 gene cluster. Since in vitro studies have shown that loss of the spaCBA-srtC1 gene cluster decreases certain L. rhamnosus GG probiotic properties, we assessed both the genomic stability and phenotypic properties of L. rhamnosus GG throughout an industrial production process. We found that neither genomic nor phenotypic changes occurred in the samples. Therefore, we demonstrate that L. rhamnosus GG retains the spaCBA-srtC1 cluster and exhibits excellent genomic and phenotypic stability in the specific industrial process examined here.

    更新日期:2020-01-13
  • Molecular and enzymatic verification of the dual agarolytic pathways in a marine bacterium, Vibrio sp. strain EJY3
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-10
    Yu, S., Yun, E. J., Kim, D. H., Park, S. Y., Kim, K. H.

    Vibrio sp. EJY3 is an agarolytic marine bacterium that catabolizes 3,6-anhydro-l-galactose (AHG), a monomeric sugar unit of agarose. While the AHG catabolic pathway in EJY3 has been recently discovered, the complete agarolytic system of EJY3 remains unclear. We have identified five enzymes, namely the β-agarases VejGH50A, VejGH50B, VejGH50C, and VejGH50D and the α-neoagarooligosaccharide (NAOS) hydrolase VejGH117, involved in the agarolytic system of EJY3. Based on the characterization of recombinant enzymes and intracellular metabolite analysis, we found that EJY3 catabolizes agarose via two different agarolytic pathways. Among the four EJY3 β-agarases, VejGH50A, VejGH50B, and VejGH50C, were found to be extracellular agarases, producing mainly neoagarotetraose (NeoDP4) and neoagarobiose. By detecting intracellular NeoDP4 in EJY3 grown on agarose, NeoDP4 was observed being taken up by cells. Intriguingly, intracellular NeoDP4 acted as a branching point for the two different downstream agarolytic pathways. First, via the well-known agarolytic pathway, NeoDP4 was depolymerized into monomeric sugars by the exo-type β-agarase, VejGH50D and α-NAOS hydrolase, VejGH117. Second, via the newly found alternative agarolytic pathway, NeoDP4 was depolymerized into AHG and agarotriose (AgaDP3) by VejGH117, and AgaDP3 was then completely depolymerized into monomeric sugars by sequential enzymatic reactions of the agarolytic β-galactosidase (ABG), VejABG and VejGH117. Therefore, by experimentally verifying agarolytic enzymatic activity and transport of NeoDP4 into EJY3 cells, we revealed that EJY3 possesses both the known pathway and a newly discovered alternative pathway that involves α-NAOS hydrolase and ABG. IMPORTANCE Agarose is the main polysaccharide of red macroalgae and is composed of galactose and 3,6-anhydro-l-galactose. Many marine bacteria possess enzymes capable of depolymerizing agarose into oligomers and then depolymerizing the oligomers into monomers. Here, we experimentally verified that both a well-known and a novel agarolytic pathway exist in a marine bacterium, Vibrio sp. EJY3. In agarolytic pathways, agarose is depolymerized mainly into 4-sugar-unit oligomers by extracellular enzymes, which are then transported into cells. The imported oligomers are intracellularly depolymerized into galactose and 3,6-anhydro-l-galactose by two different agarolytic pathways, using different combinations of intracellular enzymes. These results elucidate the depolymerization routes of red macroalgal biomass in the ocean by marine bacteria and provide clues for developing industrial processes for efficiently producing sugars from red macroalgae.

    更新日期:2020-01-13
  • A highly prevalent and pervasive densovirus discovered among sea stars from the North American Atlantic Coast
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-10
    Jackson, E. W., Pepe-Ranney, C., Johnson, M. R., Distel, D. L., Hewson, I.

    The etiology of Sea Star Wasting Syndrome is hypothesized to be caused by a densovirus, SSaDV, that has previously been reported on the Pacific and Atlantic coasts of the United States. In this study, we reevaluated the presence of SSaDV among sea stars from the North American Atlantic Coast and in doing so discovered a novel densovirus we have named Asterias forbesi associated densovirus (AfaDV) that shares 78% nucleotide pairwise identity to SSaDV. In contrast to previous studies, SSaDV was not detected in sea stars from the North American Atlantic Coast. Using a variety of PCR-based techniques, we investigated the tissue tropism, host specificity, and prevalence of AfaDV among populations of sea stars at five locations along the Atlantic Coast. AfaDV was detected in three sea star species (Asterias forbesi, Asterias rubens, and Henricia sp.) found in this region and was highly prevalent (>80% of individuals tested, n=134), among sampled populations. AfaDV was detected in the body wall, gonads, and pyloric caeca (digestive gland) of specimens but was not detected in their coelomic fluid. A significant difference in viral load was found between tissue types with the pyloric caeca having the highest viral loads. Further investigation of Asterias forbesi gonad tissue found germline cells (oocytes) to be virus positive, suggesting a potential route of vertical transmission. Taken together, these observations show that the presence of AfaDV is not an indicator of Sea Star Wasting Syndrome because AfaDV is a common constituent of these animals' microbiome, regardless of health. Importance Sea Star Wasting Syndrome is a disease primarily observed on the Pacific and Atlantic coast of North America that has significantly impacted sea star populations. The etiology of this disease is unknown though hypothesized to be caused by a densovirus, SSaDV. However, previous studies have not found a correlation between SSaDV to Sea Star Wasting Syndrome on the North American Atlantic Coast. This study suggests that this observation may be explained by the presence of a genetically similar densovirus, AfaDV, that may have confounded previous studies. SSaDV was not present in sea stars screened in this study, and instead AfaDV was commonly found in sea star populations across the New England region with no apparent signs of disease. These results suggest that sea star densoviruses may be common constituents of the animal's microbiome, and the diversity and extent of these viruses among wild populations may be greater than previously recognized.

    更新日期:2020-01-13
  • Direct Electron Transfer between the frhAGB-Encoding Hydrogenase and Thioredoxin Reductase in the Non-Methanogenic Archaeon Thermococcus onnurineus NA1
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-10
    Jung, H.-C., Lim, J. K., Yang, T.-J., Kang, S. G., Lee, H. S.

    To date, NAD(P)H, ferredoxin, and coenzyme F420 have been identified as electron donors for thioredoxin reductase (TrxR). In this study, we present a novel electron source for TrxR. In the hyperthermophilic archaeon Thermococcus onnurineus NA1, the frhAGB-encoding hydrogenase, a homolog of the F420-reducing hydrogenase of methanogens, was demonstrated to interact with TrxR in coimmunoprecipitation experiments and in vitro pull-down assays. Electrons derived from H2 oxidation by the frhAGB-encoding hydrogenase were transferred to TrxR and reduced Pdo, a redox partner of TrxR. Interaction and electron transfer were observed between TrxR and the heterodimeric hydrogenase complex (FrhAG) as well as the heterotrimeric complex (FrhAGB). Hydrogen-dependent reduction of TrxR was seven-fold less efficient than when NADPH was electron donor. This study not only presents a different type of electron donor for TrxR but also reveals new functionality of the frhAGB-encoding hydrogenase utilizing a protein as an electron acceptor. IMPORTANCE This study has importance in that TrxR can use H2 as an electron donor with the aid of the frhAGB-encoding hydrogenase as well as NAD(P)H in T. onnurineus NA1. Further studies are needed to explore the physiological significance of this protein. This study also has importance as a significant step toward understanding the functionality of the frhAGB-encoding hydrogenase in a non-methanogen: the hydrogenase can transfer electrons derived from oxidation of H2 to a protein target by direct contact without the involvement of an electron carrier, which is distinct from the mechanism of its homologs, F420-reducing hydrogenases of methanogens.

    更新日期:2020-01-13
  • Distribution of microbial communities and core microbiome in successive wound grades of diabetic foot ulcer individuals
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-10
    Jnana, A., Muthuraman, V., Varghese, V. K., Chakrabarty, S., Murali, T. S., Ramachandra, L., Shenoy, K. R., Rodrigues, G. S., Prasad, S. S., Dendukuri, D., Morschhauser, A., Nestler, J., Peter, H., Bier, F. F., Satyamoorthy, K.

    Diabetic foot ulcer (DFU) is a major complication of diabetes with high morbidity and mortality rates. Pathogenesis of DFUs is governed by a complex milieu of environmental and host factors. The empirical treatment is initially based on wound severity since culturing and profiling the antibiotic sensitivity of wound-associated microbes is time consuming. Hence, a thorough and rapid analysis of the microbial landscape is a major requirement towards devising evidence-based interventions. Towards this, 122 wound (100 diabetic and 22 non-diabetic) samples were sampled for their bacterial community structure using both culture-based and next-generation 16S rRNA based metagenomics approach. Both the approaches showed that the Gram-negative microbes were more abundant in the wound microbiome. The core microbiome consisted of bacterial genera including Alcaligenes, Pseudomonas, Burkholderia, and Corynebacterium in decreasing order of average relative abundance. Despite the heterogenous nature and extensive sharing of microbes, an inherent community structure was apparent as revealed by a cluster analysis based on Euclidean distances. Facultative anaerobes (26.5%) were predominant in Wagner grade 5 while strict anaerobes were abundant in Wagner grade 1 (26%). A non-metric dimensional scaling analysis could not clearly discriminate samples based on HbA1c levels. Sequencing approach revealed the presence of major culturable species even in samples with no bacterial growth in culture-based approach. Our study indicates that a) composition of core microbial community varies with wound severity, b) polymicrobial species distribution is individual-specific, and c) antibiotic susceptibility varies with individuals. Our study suggests the need to evolve better-personalized care for better wound management therapies. IMPORTANCE: Chronic non-healing diabetic foot ulcers (DFU) is a serious complication of diabetes and is further exacerbated by bacterial colonization. Microbial burden in the wound of each individual displays diverse morphological and physiological characteristics with unique patterns of host-pathogen interactions, antibiotic resistance and virulence. Treatment involves empirical decisions until definitive results on the causative wound pathogens and their antibiotic susceptibility profiles are available. Hence, there is a need for rapid and accurate detection of these polymicrobial communities for effective wound management. Deciphering microbial communities will aid clinicians to tailor their treatment specifically to the microbes prevalent in the DFU at the time of assessment. This may reduce DFU associated morbidity and mortality while impeding the rise of multi drug resistant microbes.

    更新日期:2020-01-13
  • Potential and limitations of metagenomics as a public health risk assessment tool in a study of natural creek sediments influenced by agricultural and livestock runoff.
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-10
    Suttner, B. J., Johnston, E. R., Orellana, L. H., Rodriguez-R, L. M., Hatt, J. K., Carychao, D., Carter, M. Q., Cooley, M. B., Konstantinidis, K. T.

    Little is known about the public health risks associated with natural creek sediments that are affected by runoff and fecal pollution from agricultural and livestock practices. For instance, the persistence of foodborne pathogens originating from these practices such as Shiga Toxin-producing Escherichia coli (STEC) remains poorly quantified. Towards closing these knowledge gaps, the water-sediment interface of two creeks in the Salinas River Valley of California was sampled over a nine-month period using metagenomics and traditional culture-based tests for STEC. Our results revealed that these sediment communities are extremely diverse and comparable to the functional and taxonomic diversity observed in soils. With our sequencing effort (~4Gbp per library), we were unable to detect any pathogenic E. coli in the metagenomes of 11 samples that had tested positive using culture-based methods, apparently due to relatively low abundance. Further, there were no significant differences in the abundance of human- or cow-specific gut microbiome sequences in the downstream, impacted sites compared to upstream, more pristine (control) sites, indicating natural dilution of anthropogenic inputs. Notably, a high number of metagenomic reads carrying antibiotic resistance genes (ARGs) was found in all samples that was significantly higher compared to ARG reads in other available freshwater and soil metagenomes, suggesting that these communities may be natural reservoirs of ARGs. The work presented here should serve as guide for sampling volumes, amount of sequencing to apply, and what bioinformatics analyses to perform when using metagenomics for public health risk studies of environmental samples such as sediments. IMPORTANCE Current agricultural and livestock practices contribute to fecal contamination in the environment and the spread of food and water-borne disease and antibiotic resistance genes (ARGs). Traditionally, the level of pollution and risk to public health is assessed by culture-based tests for the intestinal bacterium, E. coli. However, the accuracy of these traditional methods (e.g., low accuracy in quantification, and false positive signal when PCR-based) and their suitability for sediments remains unclear. We collected sediments for a time series metagenomics study from one of the most highly productive agricultural regions in the U.S. in order to assess how agricultural runoff affects the native microbial communities and if the presence of STEC in sediment samples can be detected directly by sequencing. Our study provided important information on the potential for using metagenomics as a tool for assessment of public health risk in natural environments.

    更新日期:2020-01-13
  • Vanillin production in Pseudomonas: whole-genome sequencing of Pseudomonas sp. 9.1 and re-annotation of Pseudomonas putida calA as a vanillin reductase
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-10
    Garcia-Hidalgo, J., Brink, D. P., Ravi, K., Paul, C. J., Liden, G., Gorwa-Grauslund, M. F.

    Microbial degradation of lignin and its related aromatic compounds has great potential for sustainable production of chemicals and bioremediation of contaminated soils. We previously isolated Pseudomonas sp. 9.1 from historical waste deposits (forming so-called fiber banks) released from pulp and paper mills along the Baltic Sea coast. The strain accumulated vanillyl alcohol during growth on vanillin, and while reported in other microbes, this phenotype is less common in wild-type pseudomonads. As reduction of vanillin to vanillyl alcohol is an undesired trait in Pseudomonas strains engineered to accumulate vanillin, connecting the strain 9.1 phenotype with a genotype would increase the fundamental understanding and genetic engineering potential of microbial vanillin metabolism. The genome of Pseudomonas sp. 9.1 was sequenced and assembled. Annotation identified oxidoreductases with homology to a Saccharomyces cerevisiae alcohol dehydrogenase (ScADH6p) known to reduce vanillin to vanillyl alcohol, in both the 9.1 genome and the model strain Pseudomonas putida KT2440. Recombinant expression of Pseudomonas sp. 9.1 FEZ21_09870 and P. putida KT2440 PP_2426 (calA) genes in Escherichia coli revealed that these open reading frames encode aldehyde reductases that convert vanillin to vanillyl alcohol, and that P. putida KT2440 PP_3839 encodes a coniferyl alcohol dehydrogenase that oxidizes coniferyl alcohol to coniferyl aldehyde (i.e. the function previously assigned to calA). Deletion of PP_2426 in P. putida GN442 engineered to accumulate vanillin, resulted in a decrease in by-product (vanillyl alcohol) yield from 17% to ~1%. Based on these results, we propose the reannotation of PP_2426 and FEZ21_09870 as areA and PP_3839 as calA-II. Importance Valorization of lignocellulose (non-edible plant matter) is of key interest for the sustainable production of chemicals from renewable resources. Lignin, one of the main constituents of lignocellulose, is a heterogeneous aromatic biopolymer that can be chemically depolymerized into a heterogeneous mixture of aromatic building blocks; those can be further converted by certain microbes into value-added aromatic chemicals, e.g. the flavoring agent vanillin. We previously isolated a Pseudomonas sp. strain with the - for the genus - unusual trait of vanillyl alcohol production during growth on vanillin. Whole-genome sequencing of the isolate led to the identification of a vanillin reductase candidate gene whose deletion in a recombinant vanillin-accumulating P. putida strain, almost completely alleviated the undesired vanillyl alcohol by-product yield. These results represent an important step towards biotechnological production of vanillin from lignin using bacterial cell factories.

    更新日期:2020-01-13
  • Pigmentiphaga sp. strain D-2 uses a novel amidase to initiate the catabolism of the neonicotinoid insecticide acetamiprid
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-10
    Yang, H., Hu, S., Wang, X., Chuang, S., Jia, W., Jiang, J.

    Acetamiprid, a chloronicotinyl neonicotinoid insecticide, is among the most commonly used insecticides worldwide, and its environmental fate has caused considerable concern. The compound 1-(6-chloropyridin-3-yl)-N-methylmethanamine (IM 1-4) has been reported as the main intermediate during acetamiprid catabolism in microorganisms, honeybees and spinach. However, the molecular mechanism underlying the hydrolysis of acetamiprid to IM 1-4 has not yet been elucidated. In this study, a novel amidase (AceAB) that initially hydrolyzes the C-N bond of acetamiprid to generate IM 1-4 was purified and characterized from the acetamiprid-degrading strain Pigmentiphaga sp. D-2. Based on peptide profiling of the purified AceAB and the draft genome sequence of strain D-2, aceA (372 bp) and aceB (2,295 bp) encoding the α and β subunits of AceAB, respectively, were cloned and found to be necessary for acetamiprid hydrolysis in strain D-2. The characteristics of AceAB were also systematically investigated. Though AceA and AceB showed 35%-56% identities to the α and β subunits of the N,N-dimethylformamidase from Paracoccus aminophilus, AceAB was specific for the hydrolysis of acetamiprid and showed no activities to N,N-dimethylformamide or its structural analogs. IMPORTANCE Acetamiprid, belonging to the top world-widely used neonicotinoid insecticides, is one of the most important commercial insecticides. Due to its extensive use, the environmental fate of acetamiprid, especially the microbial degradation of acetamiprid, has caused considerable concern. Although the catabolic pathways of acetamiprid in microorganisms have been extensively studied, the molecular mechanisms underlying acetamiprid biodegradation (except for a nitrile hydratase) remain largely unknown, and the enzyme responsible for the biotransformation of acetamiprid to its main intermediate IM 1-4 has not yet been elucidated. The amidase AceAB and its encoding genes aceA and aceB, characterized in this study, was found to be necessary and specific for the initial hydrolysis of C-N bond of acetamiprid to generate IM 1-4 in Pigmentiphaga sp. strain D-2. The finding of the novel amidase AceAB will greatly enhance our understanding of the microbial catabolism of the widely used insecticide acetamiprid on the molecular level.

    更新日期:2020-01-13
  • Geographic Patterns of Bacterioplankton among Lakes of the Middle and Lower Reaches of the Yangtze River Basin, China
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-10
    Bai, C., Cai, J., zhou, L., Jiang, X., Hu, Y., Dai, J., Shao, K., Tang, X., Yang, X., Gao, G.

    The revolution of molecular techniques has revealed that the composition of natural bacterial communities normally includes a few abundant taxa and many rare taxa. Unraveling the mechanisms underlying the spatial assembly process of both abundant and rare bacterial taxa had become a central goal in microbial ecology. Here we used high-throughput sequencing to explore geographic patterns and the relative importance of ecological processes in the assembly of abundant and rare bacterial subcommunities from 25 lakes across the middle and lower reaches of Yangtze River basin (MLYB, located in southeast China), where most of the lakes are interconnected by river networks. We found a similar biogeographic pattern of abundant and rare subcommunities which could significantly separate between the two lake groups that were far from each other, while could not separate among the nearby lakes. Both abundant and rare bacteria followed a strong distance-decay relationship. These findings suggest that the interconnectivity between lakes homogenizes the bacterial communities in local areas, and the abundant and rare taxa therein may be affected by the same ecological process. In addition, based on the measured environmental variables, the deterministic processes explain a small fraction of variation within both abundant and rare subcommunities. While both neutral and null model revealed a high stochasticity ratio for the spatial distribution patterns of both abundant and rare taxa. These findings indicate that the stochastic processes exhibited a greater influence on both abundant and rare bacterial subcommunities assembly among interconnected lakes. Importance The Middle and Lower Yangtze Plain is a typical floodplain, in which many lakes will connect with each other, especially in the wet season. More importantly, with the frequent change of regional water level in the wet season, there is a mutual hydrodynamic exchange among these lakes. The microbial biogeography among these interconnected lakes is still poorly understood. This study aims to unravel the mechanisms underlying the assembly process of abundant and rare bacteria among the interconnected lakes in the Middle and Lower Yangtze Plain. Our findings will provide a deeper understanding of the biogeographic patterns of rare and abundant bacterial taxa and their determined processes among interconnected aquatic habitats.

    更新日期:2020-01-13
  • Exploiting the natural diversity of the acyltransferase RhlA for the synthesis of the rhamnolipid precursor 3-(3-hydroxyalkanoyloxy)alkanoic acid
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-10
    Germer, A., Tiso, T., Müller, C., Behrens, B., Vosse, C., Scholz, K., Froning, M., Hayen, H., Blank, L. M.

    While rhamnolipids of Pseudomonas aeruginosa type are commercially available, the natural diversity of rhamnolipids and its origin are barely investigated. Here we collected known and identified new rhlA genes encoding the acyltransferase responsible for the synthesis of the lipophilic rhamnolipid precursor 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA). Generally, all homologs were found in β- and -Proteobacteria. A likely horizontal gene transfer event into Actinobacteria is the only identified exception. The phylogeny of the RhlA homologs from Pseudomonas and Burkholderia species is consistent with the organism phylogeny and genes involved in rhamnolipid synthesis are located in operons. In contrast, RhlA homologs from the Enterobacterales do not follow the organisms' phylogeny but form an own branch. Furthermore, in many Enterobacterales and Halomonas from the Oceanospirillales, an isolated rhlA homolog can be found in the genome. The RhlAs from Pseudomonas aeruginosa PA01, Pseudomonas fluorescens LMG 05825, Pantoea ananatis LMG 20103, Burkholderia plantarii PG1, Burkholderia ambifaria LMG 19182, Halomonas sp. R57-5, Dickeya dadantii Ech586, and Serratia plymuthica PRI-2C were expressed in Escherichia coli and tested for HAA production. Indeed, except for the Serratia RhlA, HAAs were produced with the engineered strains. A detailed analysis of the produced HAA congeners by HPLC-MS/MS highlights the congener specificity of the RhlA proteins. The congener length varies between four and 18 carbon atoms with main congeners consisting of different combinations of saturated or monounsaturated C10, C12, and C14 fatty acids. The results are discussed in the context of the phylogeny of this unusual enzymatic activity. Importance: The RhlA specificity explains the observed differences in HAA congeners. Whole-cell catalysts can now be designed for the synthesis of different congener mixtures of HAAs and rhamnolipids, thereby contributing to the envisaged synthesis of designer HAAs.

    更新日期:2020-01-13
  • Wolbachia endosymbiont of the horn fly Haematobia irritans irritans: a supergroup A strain with multiple horizontally acquired cytoplasmic incompatibility genes
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-03
    Madhav, M., Parry, R., Morgan, J. A. T., James, P., Asgari, S.

    The horn fly, Haematobia irritans irritans, is a hematophagous parasite of livestock distributed throughout Europe, Africa, Asia, and the Americas. Welfare losses on livestock due to horn fly infestation are estimated to cost between USD 1-2.5 billion annually in North America and Brazil. The endosymbiotic bacterium Wolbachia pipientis is a maternally inherited manipulator of reproductive biology in arthropods and naturally infects laboratory colonies of horn flies from Kerrville, USA and Alberta, Canada, but has also been identified in wild-caught samples from Canada, USA, Mexico and Hungary. Re-assembly of PacBio long-read and Illumina genomic DNA libraries from the Kerrville H. i. irritans genome project allowed for a complete and circularised 1.3 Mb Wolbachia genome (wIrr). Annotation of wIrr yielded 1249 coding genes, 34 tRNAs, three rRNAs, and five prophage regions. Comparative genomics and whole genome Bayesian evolutionary analysis of wIrr compared to published Wolbachia genomes suggests that wIrr is most closely related to and diverged from Wolbachia supergroup A strains known to infect Drosophila spp. Whole-genome synteny analyses between wIrr and closely related genomes indicates that wIrr has undergone significant genome rearrangements while maintaining high nucleotide identity. Comparative analysis of the cytoplasmic incompatibility (CI) genes of wIrr suggests two phylogenetically distinct CI loci and acquisition of another CifB homolog from phylogenetically distant supergroup A Wolbachia strains suggesting horizontal acquisition of these loci. The wIrr genome provides a resource for future examination of the impact Wolbachia may have in both biocontrol and potential insecticide resistance of horn flies. Importance Horn flies, Haematobia irritans irritans, are obligate hematophagous parasites of cattle having significant effects on production and animal welfare. Control of horn flies mainly relies on the use of insecticides, but issues with resistance have increased interest in development of alternative means of control. Wolbachia pipientis is an endosymbiont bacterium known to have a range of effects on host reproduction such as induction of cytoplasmic incompatibility, feminization, male killing, and also impacts on vector transmission. These characteristics of Wolbachia have been exploited in biological control approaches for a range of insect pests. Here we report the assembly and annotation of the circular genome of the Wolbachia strain of the Kerrville, USA horn fly (wIrr). Annotation of wIrr suggests its unique features including the horizontal acquisition of additional transcriptionally active cytoplasmic incompatibility loci. This study will provide the foundation for future Wolbachia-induced biological effect studies for control of horn flies.

    更新日期:2020-01-04
  • Evolution of Listeria monocytogenes in a food-processing plant involves limited single nucleotide substitutions, but considerable diversification by gain and loss of prophages
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-03
    Harrand, A. S., Jagadeesan, B., Baert, L., Wiedmann, M., Orsi, R. H.

    Whole genome sequencing (WGS) is becoming the standard method for subtyping Listeria monocytogenes. Interpretation of WGS data for isolates from foods and associated environments is however challenging due to a lack of detailed data on Listeria evolution in processing facilities. Here, we used previously collected WGS data for 40 L. monocytogenes obtained from a cold-smoked salmon processing facility between 1998 and 2015 to probe the L. monocytogenes molecular evolution in this facility, combined with phenotypic assessment of selected isolates. Isolates represented three clusters (1, 2, and 3); cluster 3 isolates (n=32) were obtained over 18 years. The average mutation rate for cluster 3 was estimated as 1.15x10-7 changes per nucleotide per year (~0.35 changes per genome per year); the most recent common ancestors (MRCA) of sub-clusters 3a and 3b were estimated to have occurred around 1958 and 1974, respectively, within the age of the facility, suggesting long term persistence in this facility. Extensive prophage diversity was observed within sub-clusters 3a and 3b, which have one shared and six unique prophage profiles for each sub-cluster (with 16 prophage profiles found among all 40 isolates). The plasmid-borne sanitizer tolerance operon bcrABC was found in all cluster 2 and 3 isolates, while the transposon-borne sanitizer tolerance gene qacH was found in one cluster 1 isolate; presence of these genes was correlated with the ability to survive increased concentrations of sanitizers. Selected isolates showed a significant variation in the ability to attach to surface with persistent isolates attaching better than transient isolates at 21°C. IMPORTANCE Knowledge about the genetic evolution of L. monocytogenes in food processing facilities over multiple years is generally lacking. This information is critical to interpret WGS findings involving food or food-associated isolates. This study suggests that L. monocytogenes that persist in processing facilities may evolve with a slow single nucleotide mutation rate mostly driven by negative (i.e., purifying) selection, but with rapid diversification of prophages. Hence, isolation of L. monocytogenes with few SNP differences in different locations (e.g., supplier plants and receiving plants) is possible, highlighting the importance of epidemiological and detailed isolate metadata for interpreting WGS data in traceback investigation. Our study also shows how advanced WGS data analyses can be used to support root cause analysis efforts and may, for example, pinpoint the time when a persistence event started (which then potentially could be linked to facility changes, introduction of new equipment etc.).

    更新日期:2020-01-04
  • Biodegradation of ephedrine isomers by Arthrobacter sp. TS-15: The discovery of novel ephedrine and pseudoephedrine dehydrogenases
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-03
    Shanati, T., Ansorge-Schumacher, M. B.

    The gram-positive soil bacterium Arthrobacter sp. TS-15 (DSM 32400), which is capable of metabolizing ephedrine as a sole source of carbon and energy, was isolated. According to 16S rRNA gene sequences and comparative genomic analysis, Arthrobacter sp. TS-15 is closely related to Arthrobacter aurescens. Distinct from all known physiological paths, ephedrine metabolism by Arthrobacter sp. TS-15 is initiated by the selective oxidation of the hydroxyl function at the α-C-atom, yielding methcathinone as the primary degradation product. Rational genome mining revealed a gene cluster potentially encoding the novel pathway. Two genes from the cluster, which encoded putative short-chain dehydrogenases, were cloned and expressed in Escherichia coli. The obtained enzymes were strictly NAD+-dependent and catalyzed the oxidation of ephedrine to methcathinone. Pseudoephedrine dehydrogenase (PseDH) selectively converted (S,S)-(+)-pseudoephedrine and (S,R)-(+)-ephedrine to (S- and R-)-methcathinone, respectively. Ephedrine dehydrogenase (EDH) exhibited strict selectivity for the oxidation of the diastereomers (R,S)-(–)-ephedrine and (R,R)-(–)-pseudoephedrine. Importance Arthrobacter sp. TS-15 is a newly isolated bacterium with the unique ability to degrade ephedrine isomers. The initiating steps of the novel metabolic pathway are described. Arthrobacter sp. TS-15 and its isolated ephedrine-oxidizing enzymes have potential for use in decontamination and synthetic applications.

    更新日期:2020-01-04
  • A high-throughput microbial growth data assembly and analysis protocol to elucidate the strain-specific NaCl tolerance variation in Listeria monocytogenes
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-03
    Aalto-Araneda, M., Pöntinen, A., Pesonen, M., Corander, J., Markkula, A., Tasara, T., Stephan, R., Korkeala, H.

    Listeria monocytogenes causes the severe foodborne illness listeriosis and survives in food-associated environments due to its high stress tolerance. A data assembly and analysis protocol for microbial growth experiments was compiled to elucidate the strain variability of L. monocytogenes stress tolerance. The protocol includes 1) measurement of growth ability under stress; 2) selection of a suitable method for growth parameter calculation; 3) comparison of growth patterns between strains; and 4) biological interpretation of the discovered differences. In step 1, L. monocytogenes strains (n = 388) of various serovars and origins grown on media with 9.0% NaCl were measured using Bioscreen C Microbiology Reader. Technical variability of the growth measurements was assessed and eliminated. In step 2, the growth parameters determined by Gompertz, modified-Gompertz, logistic, and Richards models and model-free splines were compared, illustrating differences in the suitability of these methods to describe the experimental data. In step 3, hierarchical clustering was used to describe the NaCl tolerance of L. monocytogenes measured by strain-specific variation in growth ability; tolerant strains had higher growth rates and maximum optical densities and shorter lag phases than susceptible strains. The spline parameter area under the curve best classified "poor", "average", and "good" growers. In step 4, the tested L. monocytogenes lineage I strains (serovars 4b and 1/2b) proved to be significantly more tolerant towards NaCl 9.0% than lineage II strains (serovars 1/2a, 1/2c, and 3a). Our protocol provides systematic tools to gain comparable data for investigating strain-specific variation of bacterial growth under stress. IMPORTANCE The pathogen Listeria monocytogenes causes the foodborne disease listeriosis, which can be fatal in immunocompromised individuals. L. monocytogenes tolerates several environmental stressors and can persist in food-processing environments and grow in foodstuffs despite traditional control measures such as high salt content. Nonetheless, L. monocytogenes strains differ in their ability to withstand stressors. Elucidating the intra-species strain variability of L. monocytogenes stress tolerance is crucial for the identification of particularly tolerant strains. To enhance reliable identification of variability in bacterial stress tolerance phenotypes, we compiled a large-scale protocol for the entire data assembly and analysis of microbial growth experiments, providing a systematic approach and check-list for experiments on strain-specific growth ability. Our study illustrated the diversity and strain-specific variation of L. monocytogenes stress tolerance with an unprecedented scope and discovered biologically relevant serovar- and lineage-dependent phenotypes of NaCl tolerance.

    更新日期:2020-01-04
  • Suboptimal Bacillus licheniformis and Bacillus weihenstephanensis spore incubation conditions increase heterogeneity of spore outgrowth time
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2020-01-03
    Trunet, C., Mtimet, N., Mathot, A.-G., Postollec, F., Leguerinel, I., Couvert, O., Broussolle, V., Carlin, F., Coroller, L.

    Changes with time of a population of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 dormant spores into germinated spores and vegetative cells were followed by flow cytometry, at pH ranges from 4.7 to 7.4, and temperature from 10°C to 37°C for B. weihenstephanensis and from 18°C to 59°C for B. licheniformis. Incubation conditions lower than optimal temperatures or pH led to lower proportions of dormant spores able to germinate and extended time of germination, lower proportion of germinated spores able to outgrow, an extension of their times of outgrowth, and an increase of the heterogeneity of spore outgrowth time. A model based on the strain growth limits, was proposed to quantify the impact of incubation temperature and pH on the passage through each physiological stage. The heat-treatment temperature or time acted independently on spore recovery. Indeed, a treatment at 85°C during 12 min or at 95°C during 2 min did not have the same impact on spore germination and outgrowth kinetics of B. weihenstephanensis despite they both led to a tenfold reduction of the population. Moreover, acidic sporulation pH increased the time of outgrowth by 1.2 fold and lowered the proportion of spores able to germinate and outgrow by 1.4 fold. Interestingly, we showed by a proteomic analysis that some proteins involved in germination and outgrowth were detected at a lower abundance in spores produced at pH 5.5 compared to those produced at pH 7.0, maybe at the origin of germination and outgrowth behavior of spores produced at suboptimal pH. Importance Sporulation and incubation conditions have an impact on the numbers of spores able to recover after exposure to sub-lethal heat-treatment. Using flow cytometry we were able to follow at a single cell level the changes in the physiological states of heat-stressed spores of Bacillus sp. and to discriminate between dormant spores, germinated spores and outgrowing vegetative cells. We developed original mathematical models that describe (i) the changes with time of the proportion of cells in their different states during germination and outgrowth, and (ii) the influence of temperature and pH on the kinetics of spore recovery using the growth limits of the tested strains as model parameters. We think that these models better predict spore recovery after a sub-lethal heat-treatment, a common situation in food processing and a concern for food preservation and safety.

    更新日期:2020-01-04
  • Characterization of a novel D-Allulose 3-epimerase gene from the metagenome of a thermal aquatic habitat and D-Allulose production by Bacillus subtilis whole-cell catalysis
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2019-12-20
    Patel, S. N., Kaushal, G., Singh, S. P.

    A novel D-Allulose 3-epimerase gene (daeM) has been identified from the metagenomic resource of the hot-water reservoir. The enzyme epimerizes D-Fructose into D-Allulose, a functional sugar of rare abundance in nature. The metagenomic DNA fragment was cloned and expressed in Escherichia coli. The purified recombinant protein (DaeM) was found to be metal-dependent (Co++ or Mn++). It displayed the maximal levels of catalytic activity in a pH and temperature range of 6 to 11, and 75 °C to 80 °C, respectively. The enzyme exhibited remarkably high thermal stability at 60 °C and 70 °C, with a half-life of 9,900 and 3,240 min, respectively. To the best of our knowledge, this is the highest thermal stability demonstrated by a D-Allulose 3-epimerase, characterized to date. The enzymatic treatment of 700 mg.mL-1 D-Fructose yielded about 217 mg.mL-1 D-Allulose, under optimal condition. The catalytic product was purified, and its NMR spectra were found indistinguishable from that of standard D-Allulose. For biomolecule production, the whole-cell catalysis procedure avoids the tedious process of extraction and purification of enzyme and also offers more excellent biocatalyst stability. Further, it is desirable to involve safe-grade microorganisms for the biosynthesis of a product. The daeM gene was expressed intracellularly in Bacillus subtilis. Whole-cell catalysis reaction performed in a reaction volume of 1 L at 60 °C yielded approximately 196 g.L-1 D-Allulose from 700 g.L-1 D-Fructose. Further, the whole recombinant cells were able to biosynthesize D-Allulose in apple juice, mixed fruit juice, and honey. IMPORTANCE D-Allulose is a non-caloric sugar substitute with anti-diabetic and anti-obesity potential. With several physiological significance, D-Allulose has wide applications in the food and pharma industries. The development of thermostable biocatalyst is mainstream research for the industrial acceptability of the enzyme. Aquatic habitats of extreme temperatures are considered as a metagenomic resource with thermal tolerant biocatalysts of industrial importance. The present study explored the thermal-spring metagenome of Tattapani geothermal region, Chhattisgarh, India, for the discovery of a novel D-Allulose 3-epimerase gene (daeM), encoding the enzyme of excessive heat-stability. The daeM was expressed in the microbial cells of non-pathogenic and safe-grade species, B. subtilis, which was capable of performing D-Fructose to D-Allulose interconversion via whole-cell catalysis reaction. The results propose DaeM as a potential biocatalyst for commercial production of the rare sugar, D-Allulose. The study established that extreme environmental niches are the genomic resource of functional sugar-related biocatalysts.

    更新日期:2019-12-21
  • PA0335, a Novel Gene Encoding Histidinol Phosphate Phosphatase Mediates Histidine Auxotrophy in Pseudomonas aeruginosa
    Appl. Environ. Microb. (IF 4.077) Pub Date : 2019-12-20
    Wang, Y., Wang, L., Zhang, J., Duan, X., Feng, Y., Wang, S., Shen, L.

    The biosynthesis of histidine, a proteinogenic amino acid, has been extensively studied due to its importance in bacterial growth and survival. Histidinol-phosphate phosphatase (Hol-Pase), which is responsible for the penultimate step of histidine biosynthesis, is generally the last enzyme to be characterized in many bacteria because its origin and evolution are more complex compared to other enzymes in histidine biosynthesis. However, none of the enzymes in histidine biosynthesis, including Hol-Pase, have been characterized in Pseudomonas aeruginosa, which is an important opportunistic gram-negative pathogen that can cause serious human infections. In our previous work, a transposon mutant of P. aeruginosa was found to display a growth defect on glucose-containing minimal solid medium. In this study, we found that the growth defect was due to incomplete histidine auxotrophy caused by PA0335 inactivation. Subsequently, PA0335 was shown to encode Hol-Pase, and its function and enzymatic activity were investigated using genetic and biochemical methods. In addition to PA0335, the roles of 12 other predicted genes involved in histidine biosynthesis in P. aeruginosa were examined. Among them, hisC2(PA3165), hisH2(PA3152), and hisF2(PA3151) were found to be dispensable for histidine synthesis, whereas hisG(PA4449), hisE(PA5067), hisF1(PA5140), hisB(PA5143), hisI(PA5066), hisC1(PA4447), and hisA(PA5141) were essential because deletion of each resulted in complete histidine auxotrophy; similar to PA0335, hisH1(PA5142) or hisD(PA4448) deletion caused incomplete histidine auxotrophy. Taken together, our results outline the histidine synthesis pathway of P. aeruginosa. IMPORTANCE Histidine is a common amino acid in proteins. Because it plays critical roles in bacterial metabolism, its biosynthetic pathway in many bacteria has been elucidated. However, the pathway remains unclear in P. aeruginosa, an important opportunistic pathogen in clinical settings; in particular, there is scant knowledge about histidinol-phosphate phosphatase (Hol-Pase), which has a complex origin and evolution. In this study, P. aeruginosa Hol-Pase was identified and characterized. Furthermore, the roles of all other predicted genes involved in histidine biosynthesis were examined. Our results illustrate the histidine synthesis pathway of P. aeruginosa. The knowledge obtained from this study may help in developing strategies to control P. aeruginosa-related infections. In addition, some enzymes of the histidine synthesis pathway from P. aeruginosa might be used as elements of histidine synthetic biology in other industrial microorganisms.

    更新日期:2019-12-21
Contents have been reproduced by permission of the publishers.
导出
全部期刊列表>>
2020新春特辑
限时免费阅读临床医学内容
ACS材料视界
科学报告最新纳米科学与技术研究
清华大学化学系段昊泓
自然科研论文编辑服务
加州大学洛杉矶分校
上海纽约大学William Glover
南开大学化学院周其林
课题组网站
X-MOL
北京大学分子工程苏南研究院
华东师范大学分子机器及功能材料
中山大学化学工程与技术学院
试剂库存
天合科研
down
wechat
bug