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  • Interaction Between BGLF2 and BBLF1 Is Required for the Efficient Production of Infectious Epstein–Barr Virus Particles
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-17
    Chien-Hui Hung; Ya-Fang Chiu; Wen-Hung Wang; Lee-Wen Chen; Pey-Jium Chang; Tsung-Yu Huang; Ying-Ju Lin; Wan-Ju Tsai; Chia-Ching Yang

    BGLF2 is a tegument protein of the Epstein–Barr virus (EBV). This study finds that BGLF2 is expressed in the late stage of the EBV lytic cycle. Microscopic investigations reveal that BGLF2 is present in both the nucleus and the cytoplasm and colocalized with BBLF1 and gp350 at juxtanuclear regions in the cytoplasm. This study also finds that the basic KKK69 motif of BGLF2 and acidic DYEE31 motif of BBLF1 are crucial for the interaction between BGLF2 and BBLF1, which is required for the recruitment of BGLF2 to the BBLF1 that is anchored on the trans-Golgi-network (TGN). In addition, BGLF2 in a density gradient is co-sedimented with un-enveloped capsids, revealing that BGLF2 associates with the EBV capsid before the final envelopment. The knockout of BGLF2 expression is demonstrated to reduce the numbers of infectious virions that are released into the culture medium, but they do not affect the expression of lytic proteins and viral DNA replication. The production of infectious viral particles by a BGLF2-knockout mutant can be rescued by exogenously expressed BGLF2 but only partially rescued by BGLF2-3KA, which is a mutant with reduced ability to interact with BBLF1 but does not affect its ability to activate the MAPK pathway and the expression of the EBV lytic proteins, suggesting that the interaction of BGLF2 with BBLF1 is important to the efficient production of infectious viral particles during the maturation. The results of this study improve our understanding of how BGLF2 promotes EBV viral production.

    更新日期:2020-01-26
  • Regulation of the ER Stress Response by the Ion Channel Activity of the Infectious Bronchitis Coronavirus Envelope Protein Modulates Virion Release, Apoptosis, Viral Fitness, and Pathogenesis
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-17
    Shumin Li; Lixia Yuan; Guo Dai; Rui Ai Chen; Ding Xiang Liu; To Sing Fung

    Coronavirus (CoV) envelope (E) protein is a small structural protein critical for virion morphogenesis and release. The recently characterized E protein ion channel activity (EIC) has also been implicated in modulating viral pathogenesis. In this study, we used infectious bronchitis coronavirus (IBV) as a model to study EIC. Two recombinant IBVs (rIBVs) harboring EIC-inactivating mutations – rT16A and rA26F – were serially passaged, and several compensatory mutations were identified in the transmembrane domain (TMD). Two rIBVs harboring these putative EIC-reverting mutations – rT16A/A26V and rA26F/F14N – were recovered. Compared with the parental rIBV-p65 control, all four EIC mutants exhibited comparable levels of intracellular RNA synthesis, structural protein production, and virion assembly. Our results showed that the IBV EIC contributed to the induction of ER stress response, as up-regulation of ER stress-related genes was markedly reduced in cells infected with the EIC-defective mutants. EIC-defective mutants also formed smaller plaques, released significantly less infectious virions into the culture supernatant, and had lower levels of viral fitness in cell culture. Significantly, all these defective phenotypes were restored in cells infected with the putative EIC revertants. EIC mutations were also implicated in regulating IBV-induced apoptosis, induction of pro-inflammatory cytokines, and viral pathogenicity in vivo. Taken together, this study highlights the importance of CoV EIC in modulating virion release and various aspects of CoV – host interaction.

    更新日期:2020-01-26
  • Development of a Sensitive Escherichia coli Bioreporter Without Antibiotic Markers for Detecting Bioavailable Copper in Water Environments
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-17
    Yilin Pang; Xiaojun Ren; Jianghui Li; Feng Liang; Xiaoyu Rao; Yang Gao; Wenhe Wu; Dong Li; Juanjuan Wang; Jianguo Zhao; Xufen Hong; Fengying Jiang; Wu Wang; Huaibin Zhou; Jianxin Lyu; Guoqiang Tan

    The whole-cell bioreporters based on the cop-operon sensing elements have been proven specifically useful in the assessment of bioavailable copper ions in water environments. In this study, a series of experiments was conducted to further improve the sensitivity and robustness of bioreporters. First, an Escherichia coli △copA△cueO△cusA mutant with three copper transport genes knocked out was constructed. Then, the copAp::gfpmut2 sensing element was inserted into the chromosome of E. coli △copA△cueO△cusA by gene knock-in method to obtain the bioreporter strain E. coli WMC-007. In optimized assay conditions, the linear detection range of Cu2+ was 0.025–5 mg/L (0.39–78.68 μM) after incubating E. coli WMC-007 in Luria–Bertani medium for 5 h. The limit of detection of Cu2+ was 0.0157 mg/L (0.25 μM). Moreover, fluorescence spectrometry and flow cytometry experiments showed more environmental robustness and lower background fluorescence signal than those of the sensor element based on plasmids. In addition, we found that the expression of GFPmut2 in E. coli WMC-007 was induced by free copper ions, rather than complex-bound copper, in a dose-dependent manner. Particularly, the addition of 40 mM 3-(N-Morpholino)propanesulfonic acid buffer to E. coli WMC-007 culture enabled accurate quantification of bioavailable copper content in aqueous solution samples within a pH range from 0.87 to 12.84. The copper recovery rate was about 95.88–113.40%. These results demonstrate potential applications of E. coli WMC-007 as a bioreporter to monitor copper contamination in acidic mine drainage, industrial wastewater, and drinking water. Since whole-cell bioreporters are relatively inexpensive and easy to operate, the combination of this method with other physicochemical techniques will in turn provide more specific information on the degree of toxicity in water environments.

    更新日期:2020-01-26
  • The Impact of Antiretroviral Therapy on Malaria Parasite Transmission
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-18
    Raquel Azevedo; António M. Mendes; Miguel Prudêncio

    Coendemicity between the human immunodeficiency virus (HIV) and Plasmodium parasites, the causative agents of acquired immunodeficiency syndrome (AIDS) and malaria, respectively, occurs in several regions around the world. Although the impact of the interaction between these two organisms is not well understood, it is thought that the outcome of either disease may be negatively influenced by coinfection. Therefore, it is important to understand how current first-line antiretroviral therapies (ART) might impact Plasmodium infection in these regions. Here, we describe the effect of 18 antiretroviral compounds and of first-line ART on the blood and sporogonic stages of Plasmodium berghei in vitro and in vivo. We show that the combination zidovudine + lamivudine + lopinavir/ritonavir (LPV/r), employed as first-line HIV treatment in the field, has a strong inhibitory activity on the sporogonic stages of P. berghei and that several non-nucleoside reverse transcriptase inhibitors (NNRTI) have a moderate effect on this stage of the parasite’s life cycle. Our results expose the effect of current first-line ART on Plasmodium infection and identify potential alternative therapies for HIV/AIDS that might impact malaria transmission.

    更新日期:2020-01-26
  • Current Status of Latency Reversing Agents Facing the Heterogeneity of HIV-1 Cellular and Tissue Reservoirs
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-18
    Amina Ait-Ammar; Anna Kula; Gilles Darcis; Roxane Verdikt; Stephane De Wit; Virginie Gautier; Patrick W. G. Mallon; Alessandro Marcello; Olivier Rohr; Carine Van Lint

    One of the most explored therapeutic approaches aimed at eradicating HIV-1 reservoirs is the “shock and kill” strategy which is based on HIV-1 reactivation in latently-infected cells (“shock” phase) while maintaining antiretroviral therapy (ART) in order to prevent spreading of the infection by the neosynthesized virus. This kind of strategy allows for the “kill” phase, during which latently-infected cells die from viral cytopathic effects or from host cytolytic effector mechanisms following viral reactivation. Several latency reversing agents (LRAs) with distinct mechanistic classes have been characterized to reactivate HIV-1 viral gene expression. Some LRAs have been tested in terms of their potential to purge latent HIV-1 in vivo in clinical trials, showing that reversing HIV-1 latency is possible. However, LRAs alone have failed to reduce the size of the viral reservoirs. Together with the inability of the immune system to clear the LRA-activated reservoirs and the lack of specificity of these LRAs, the heterogeneity of the reservoirs largely contributes to the limited success of clinical trials using LRAs. Indeed, HIV-1 latency is established in numerous cell types that are characterized by distinct phenotypes and metabolic properties, and these are influenced by patient history. Hence, the silencing mechanisms of HIV-1 gene expression in these cellular and tissue reservoirs need to be better understood to rationally improve this cure strategy and hopefully reach clinical success.

    更新日期:2020-01-26
  • Characterization of a Novel Moderately Thermophilic Solvent-Tolerant Esterase Isolated From a Compost Metagenome Library
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-19
    Ji-Min Park; Chul-Hyung Kang; Sung-Min Won; Ki-Hoon Oh; Jung-Hoon Yoon

    A novel esterase, EstCS1, was isolated from a compost metagenomics library. The EstCS1 protein, which consists of 309 amino acid residues with an anticipated molecular mass of 34 kDa, showed high amino acid sequence identities to predicted esterases and alpha/beta hydrolases (59%) from some cultured bacteria and to predicted lipases/esterases from uncultured bacteria. The phylogenetic analysis suggested that the EstCS1 belongs to the hormone-sensitive lipase family of lipolytic enzyme classification and contains a catalytic triad including Ser155–Asp255–His285. The Ser155 residue of the catalytic triad in the EstCS1 was located in the consensus active-site motif, GXSXG. Besides, a conserved HGGG motif placed in an oxyanion hole of the hormone-sensitive lipase family was discovered, too. The EstCS1 demonstrated the highest activity toward p-nitrophenyl propionate (C3) and caproate (C6) and was normally stable up to 60°C with optimal activity at 50°C. In addition, an optimal activity was observed at pH 8, and the EstCS1 possessed its stability within the pH range between 5 and 10. Interestingly, EstCS1 had an outstanding stability in up to 30% (v/v) organic solvents and activity over 50% in the presence of 50% (v/v) acetone, ethanol, dimethyl sulfoxide (DMSO), and N,N-dimethylformamide. The EstCS1 hydrolyzed sterically hindered tertiary alcohol esters of t-butyl acetate and linalyl acetate. Considering the properties, such as the moderate thermostability, stability against organic solvents, and activity toward esters of tertiary alcohols, the EstCS1 will be worthwhile to be used for organic synthesis and related industrial applications.

    更新日期:2020-01-26
  • Molecular and Biological Characterization of the First Hypovirus Identified in Fusarium oxysporum
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-24
    Almudena Torres-Trenas; M. Carmen Cañizares; M. Dolores García-Pedrajas; Encarnación Pérez-Artés

    A novel mycovirus named Fusarium oxysporum f. sp. dianthi hypovirus 2 (FodHV2) has been identified infecting isolates Fod 408 and Fod 409 of Fusarium oxysporum f. sp. dianthi from Morocco. The genome of FodHV2 is 9,444 nucleotides long excluding the poly(A) tail, and has a single open reading frame encoding a polyprotein. The polyprotein contains three highly conserved domains of UDP glucose/sterol glucosyltransferase, RNA-dependent RNA polymerase, and viral RNA helicase. In addition, particular residues of Cys, Hys, and Gly detected in the N-terminal region suggest the presence of the catalytic site of a highly diverged papain-like protease. Genomic organization, presence of particular conserved motifs, and phylogenetic analyses based on multiple alignments clearly grouped FodHV2 with the members of the family Hypoviridae. FodHV2 was transferred by hyphal anastomosis to a recipient HygR-tagged virus-free strain. The comparison of the infected and non-infected isogenic strains showed that FodHV2 did not alter the vegetative growth, neither the conidiation nor the virulence of its fungal host. Efficiency of FodHV2 transmission through the conidia was 100% in both the original and the recipient infected-isolates. To the best of our knowledge, this is the first report of a hypovirus infecting the plant pathogen F. oxysporum, and also the first one of a hypovirus detected in a fungal strain from the African continent.

    更新日期:2020-01-26
  • Salmonella enterica Serovar Typhimurium 14028s Genomic Regions Required for Colonization of Lettuce Leaves
    Front. Microbiol. (IF 4.259) Pub Date : 2020-01-03
    Jeanine Montano; Gabrielle Rossidivito; Joseph Torreano; Steffen Porwollik; Shlomo Sela Saldinger; Michael McClelland; Maeli Melotto

    Contamination of edible produce leaves with human bacterial pathogens has been associated with serious disease outbreaks and has become a major public health concern affecting all aspects of the market, from farmers to consumers. While pathogen populations residing on the surface of ready-to-eat produce can be potentially removed through thorough washing, there is no disinfection technology available that effectively eliminates internal bacterial populations. By screening 303 multi-gene deletion (MGD) mutants of Salmonella enterica serovar Typhimurium (STm) 14028s, we were able to identify ten genomic regions that play a role in opening the stomatal pore of lettuce leaves. The major metabolic functions of the deleted regions are associated with sensing the environment, bacterium movement, transport through the bacterial membrane, and biosynthesis of surface appendages. Interestingly, at 21 days post inoculation, seven of these mutants showed increased population titers inside the leaf, two mutants showed similar titers as the wild type bacterium, whereas one mutant with a large deletion that includes the Salmonella pathogenicity island 2 (SPI-2) showed significantly impaired persistence in the leaf apoplast. These findings suggest that not all the genomic regions required for initiation of leaf colonization (i.e., epiphytic behavior and tissue penetration) are essential for continuing bacterial survival as an endophyte. We also observed that mutants lacking either SPI-1 (Mut3) or SPI-2 (Mut9) induce callose deposition levels comparable to those of the wild type STm 14028s; therefore, these islands do not seem to affect this lettuce defense mechanism. However, the growth of Mut9, but not Mut3, was significantly impaired in the leaf apoplastic wash fluid (AWF) suggesting that the STm persistence in the apoplast may be linked to nutrient acquisition capabilities or overall bacterial fitness in this niche, which are dependent on the gene(s) deleted in the Mut9 strain. The genetic basis of STm colonization of leaves investigated in this study provides a foundation from which to develop mitigation tactics to enhance food safety.

    更新日期:2020-01-26
  • Stimulated Biosynthesis of an C10-Deoxy Heptaene NPP B2 via Regulatory Genes Overexpression in Pseudonocardia autotrophica
    Front. Microbiol. (IF 4.259) Pub Date : 2020-01-07
    Heung-Soon Park; Hye-Jin Kim; Chi-Young Han; Hee-Ju Nah; Si-Sun Choi; Eung-Soo Kim

    Polyene macrolides, such as nystatin A1, amphotericin B, and NPP A1, belong to a large family of valuable antifungal polyketide compounds that are typically produced by soil actinomycetes. Previously, NPP B1, a novel NPP A1 derivative harboring a heptaene core structure, was generated by introducing two amino acid substitutions in the putative NADPH-binding motif of the enoyl reductase domain in module 5 of the NPP A1 polyketide synthase in Pseudonocardia autotrophica. This derivative showed superior antifungal activity to NPP A1. In this study, another novel derivative called NPP B2 was developed, which lacks a hydroxyl group at the C10 position by site-specific gene disruption of the P450 hydroxylase NppL. To stimulate the extremely low expression of the NPP B2 biosynthetic pathway genes, the 32-kb NPP-specific regulatory gene cluster was overexpressed via site-specific chromosomal integration. The extra copy of the six NPP-specific regulatory genes led to a significant increase in the NPP B2 yield from 0.19 to 7.67 mg/L, which is the highest level of NPP B2 production ever achieved by the P. autotrophica strain. Subsequent in vitro antifungal activity and toxicity studies indicated that NPP B2 exhibited similar antifungal activity but significantly lower hemolytic toxicity than NPP B1. These results suggest that an NPP biosynthetic pathway refactoring and overexpression of its pathway-specific regulatory genes is an efficient approach to stimulating the production of an extremely low-level metabolite, such as NPP B2 in a pathway-engineered rare actinomycete strain.

    更新日期:2020-01-26
  • A Pilot Study of the Humoral Response Against the AntiSense Protein (ASP) in HIV-1-Infected Patients
    Front. Microbiol. (IF 4.259) Pub Date : 2020-01-07
    Juliette Savoret; Nathalie Chazal; Jean-Pierre Moles; Edouard Tuaillon; Faroudy Boufassa; Laurence Meyer; Camille Lecuroux; Olivier Lambotte; Philippe Van De Perre; Jean-Michel Mesnard; Antoine Gross

    The existence of an antisense Open Reading Frame (ORF) that encodes a putative AntiSense Protein (ASP) on the proviral genome of Human Immunodeficiency Virus type 1 (HIV-1) was a source of debate for 30 years. During the last years, some progresses have been made to characterize the cellular immune response against ASP in HIV-1 seropositive patients. However, no tools were available for the detection of antibodies to ASP in the plasma of HIV-1-infected patients during the natural course of the infection. The aim of our study was to develop a Luciferase Immuno-Precipitation System (LIPS) to monitor the quantitative detection of ASP-specific antibodies in the plasma of HIV-1-infected patients [antiretroviral therapy (ART) naive-patients, patients under ART and HIV-1 controllers], patients who discontinued antiretroviral drugs (ARV). We further used this approach to delineate the epitopes of ASP targeted by antibodies. Antibodies directed against ASP were detected in 3 out of 19 patients who discontinued ARV (15%) and in 1 out of 10 ART-naive patients (10%), but were neither detected in HIV-1 infected patients under ART nor in HIV-1 controllers. Individual variations in levels of ASP-specific antibodies were detected overtime. Both the conserved prolin-rich motif and the core 60–189 region of ASP were found to be essential for antibody recognition in the four patients tested positive for anti-ASP antibodies, who were all untreated at the time of sampling. Moreover, for two of these patients, increased levels of ASP-specific antibodies were observed concomitantly to viremia declines. Overall, our method may represent a useful tool to detect a humoral response to ASP in HIV-1-infected patients, which allowed us to confirm the expression of ASP during the course of HIV-1 infection. Further studies will be needed to fully characterize the humoral response to ASP in HIV-1-infected patients.

    更新日期:2020-01-26
  • Triple HIV-1 Infection Is Associated With Faster CD4+ T-Cell Decline
    Front. Microbiol. (IF 4.259) Pub Date : 2020-01-07
    Yu Zhang; Bin Su; Hanping Li; Jingwan Han; Tong Zhang; Tianyi Li; Hao Wu; Xiaolin Wang; Jingyun Li; Yongjian Liu; Lin Li

    HIV-1 dual infection occurs when an individual is simultaneously or sequentially infected with two or more genetically distinct HIV-1 strains. According to the number of infected strains, HIV-1 dual infection can be divided in double infection and triple infection and so on. Currently, the majority of dual infection cases have been reported to be double infections which can result in detrimental clinical outcomes. The high incidence of double infection among specific high-risk populations increases the likelihood of triple infection, which has been sporadically described. There is no doubt that we are concerned about the association between triple infection and disease progression. However, this relationship is still unclear on the population level. In this study, 70 individuals from the Beijing PRIMO cohort were longitudinally followed up with a median time of 15.75 months for the purpose of investigating the incidence of dual infection. Phylogenetic analyses using bulk and single-genome sequences showed that nine individuals acquired double infection, with the incidence of 9.21 per 100 person-years, and three individuals with triple infection were identified, with the incidence of 3.07 per 100 person-years. The further survival analysis demonstrated that the triple infection group exhibited faster CD4+ T-cell decline. In summary, these results demonstrate for the first time that the triple HIV-1 infection might reduce CD4+ T-cell counts, which would predict a more rapid disease progression.

    更新日期:2020-01-26
  • Diversity of Sinorhizobium (Ensifer) meliloti Bacteriophages in the Rhizosphere of Medicago marina: Myoviruses, Filamentous and N4-Like Podovirus
    Front. Microbiol. (IF 4.259) Pub Date : 2020-01-07
    María Teresa Cubo; Cynthia Alías-Villegas; Eduardo Balsanelli; Dany Mesa; Emanuel de Souza; María Rosario Espuny

    Using different Sinorhizobium meliloti strains as hosts, we isolated eight new virulent phages from the rhizosphere of the coastal legume Medicago marina. Half of the isolated phages showed a very narrow host range while the other half exhibited a wider host range within the strains tested. Electron microscopy studies showed that phages M_ort18, M_sf1.2, and M_sf3.33 belonged to the Myoviridae family with feature long, contractile tails and icosaedral head. Phages I_sf3.21 and I_sf3.10T appeared to have filamentous shape and produced turbid plaques, which is a characteristic of phages from the Inoviridae family. Phage P_ort11 is a member of the Podoviridae, with an icosahedral head and a short tail and was selected for further characterization and genome sequencing. P_ort11 contained linear, double-stranded DNA with a length of 75239 bp and 103 putative open reading frames. BLASTP analysis revealed strong similarities to Escherichia phage N4 and other N4-like phages. This is the first report of filamentous and N4-like phages that infect S. meliloti.

    更新日期:2020-01-26
  • Role of the LytSR Two-Component Regulatory System in Staphylococcus lugdunensis Biofilm Formation and Pathogenesis
    Front. Microbiol. (IF 4.259) Pub Date : 2020-01-09
    Sandrine Dahyot; Virginie Oxaran; Maïté Niepceron; Eddy Dupart; Stéphanie Legris; Laurie Destruel; Jennifer Didi; Thomas Clamens; Olivier Lesouhaitier; Yasmine Zerdoumi; Jean-Michel Flaman; Martine Pestel-Caron

    Staphylococcus lugdunensis is a coagulase negative Staphylococcus recognized as a virulent pathogen. It is responsible for a wide variety of infections, some of which are associated with biofilm production, such as implanted medical device infections or endocarditis. However, little is known about S. lugdunensis regulation of virulence factor expression. Two-component regulatory systems (TCS) play a critical role in bacterial adaptation, survival, and virulence. Among them, LytSR is widely conserved but has variable roles in different organisms, all connected to metabolism or cell death and lysis occurring during biofilm development. Therefore, we investigated here the functions of LytSR in S. lugdunensis pathogenesis. Deletion of lytSR in S. lugdunensis DSM 4804 strain did not alter either susceptibility to Triton X-100 induced autolysis or death induced by antibiotics targeting cell wall synthesis. Interestingly, ΔlytSR biofilm was characterized by a lower biomass, a lack of tower structures, and a higher rate of dead cells compared to the wild-type strain. Virulence toward Caenorhabditis elegans using a slow-killing assay was significantly reduced for the mutant compared to the wild-type strain. By contrast, the deletion of lytSR had no effect on the cytotoxicity of S. lugdunensis toward the human keratinocyte cell line HaCaT. Transcriptional analyses conducted at mid- and late-exponential phases showed that lytSR deletion affected the expression of 286 genes. Most of them were involved in basic functions such as the metabolism of amino acids, carbohydrates, and nucleotides. Furthermore, LytSR appeared to be involved in the regulation of genes encoding known or putative virulence and colonization factors, including the fibrinogen-binding protein Fbl, the major autolysin AtlL, and the type VII secretion system. Overall, our data suggest that the LytSR TCS is implicated in S. lugdunensis pathogenesis, through its involvement in biofilm formation and potentially by the control of genes encoding putative virulence factors.

    更新日期:2020-01-26
  • Bacteriophages as an Up-and-Coming Alternative to the Use of Sulfur Dioxide in Winemaking
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-05
    Gustavo Cordero-Bueso; Javier Moraga; María Ríos-Carrasco; Marina Ruiz-Muñoz; Jesús Manuel Cantoral

    Certain acetic and lactic acid bacteria are major causes of quality defects in musts and wines, giving rise to defects such as a “vinegary,” “sharp, like nail polish-remover” taste or preventing alcoholic and/or malolactic fermentation. Sulfur dioxide is the major tool currently used in the control of these bacteria in wine. The aim of this work was to isolate bacteriophages from musts and wine of different grape varieties that were able to eliminate lactic and acetic acid bacteria spoilages at the laboratory scale. Musts obtained from grape-berries of Vitis vinifera cv. Chardonnay and Moscatel and a red wine made with V. vinifera cv. Tintilla de Rota were used to isolate bacteriophages. Bacteriophages were obtained from each of the musts and the wine and belonged to the order Caudovirals and the family Tectivirals. They were isolated by classical virology methods and identified by electron microscopy. The host bacteria used in the study were lactic acid bacteria of the species Lactobacillus hilgardii, Lactobacillus plantarum, and Oenococcus oeni and the acetic bacteria Acetobacter aceti. A comparative study was performed by adding phage titrations and SO2 to musts and wines, which had been previously inoculated with bacteria, to study the effectiveness of bacteriophages against bacteria. The comparative study showed that some bacteriophages were as effective as sulfur dioxide at low concentrations.

    更新日期:2020-01-26
  • Small Molecular Contaminant and Microorganism Can Be Simultaneously Detected Based on Nanobody-Phage: Using Carcinogen Aflatoxin and Its Main Fungal Aspergillus Section Flavi spp. in Stored Maize for Demonstration
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-17
    Xianfeng Ren; Xiaofeng Yue; Silivano Edson Mwakinyali; Wen Zhang; Qi Zhang; Peiwu Li

    Simultaneous detection technology has become a hot topic in analytical chemistry; however, very few reports on how to simultaneously detect small molecular contaminants and microorganisms have been in place. Aflatoxins are a group of highly toxic and carcinogenic compounds, which are produced mainly by Aspergillus flavus and Aspergillus parasiticus from section Flavi responsible for aflatoxin accumulation in stored cereals. Both aflatoxins and Aspergillus section Flavi were used to demonstrate the duplex real-time RCR method of simultaneously detecting small molecular contaminants and microorganisms. The detection of aflatoxins and Aspergillus section Flavi was carried out depending on the anti-idiotypic nanobody-phage V2–5 and aflatoxin-synthesis related gene nor-1 (=aflD), respectively. The quantitative standard curves for simultaneous detection of aflatoxins and Aspergillus section Flavi were constructed, with detection limits of 0.02 ng/ml and 8 × 102 spores/g, respectively. Naturally contaminated maize samples (n = 25) were analyzed for a further validation. The results were in good agreement between the new developed method and the referential methods (high-performance liquid chromatography and the conventional plating counts).

    更新日期:2020-01-26
  • The Hippo Pathway and Viral Infections
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-17
    Zhilong Wang; Wanhang Lu; Yiling Zhang; Feng Zou; Zhigang Jin; Tiejun Zhao

    The Hippo signaling pathway is a novel tumor suppressor pathway, initially found in Drosophila. Recent studies have discovered that the Hippo signaling pathway plays a critical role in a wide range of biological processes, including organ size control, cell proliferation, cancer development, and virus-induced diseases. In this review, we summarize the current understanding of the biological feature and pathological role of the Hippo pathway, focusing particularly on current findings in the function of the Hippo pathway in virus infection and pathogenesis.

    更新日期:2020-01-26
  • Isolate Specific Cold Response of Yersinia enterocolitica in Transcriptional, Proteomic, and Membrane Physiological Changes
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-17
    Chenyang Li; Jayaseelan Murugaiyan; Christian Thomas; Thomas Alter; Carolin Riedel

    Yersinia enterocolitica, a zoonotic foodborne pathogen, is able to withstand low temperatures. This psychrotrophic ability allows it to multiply in food stored in refrigerators. However, little is known about the Y. enterocolitica cold response. In this study, isolate-specific behavior at 4°C was demonstrated and the cold response was investigated by examining changes in phenotype, gene expression, and the proteome. Altered expression of cold-responsive genes showed that the ability to survive at low temperature depends on the capacity to acclimate and adapt to cold stress. This cold acclimation at the transcriptional level involves the transient induction and effective repression of cold-shock protein (Csp) genes. Moreover, the resumption of expression of genes encoding other non-Csp is essential during prolonged adaptation. Based on proteomic analyses, the predominant functional categories of cold-responsive proteins are associated with protein synthesis, cell membrane structure, and cell motility. In addition, changes in membrane fluidity and motility were shown to be important in the cold response of Y. enterocolitica. Isolate-specific differences in the transcription of membrane fluidity- and motility-related genes provided evidence to classify strains within a spectrum of cold response. The combination of different approaches has permitted the systematic description of the Y. enterocolitica cold response and gives a better understanding of the physiological processes underlying this phenomenon.

    更新日期:2020-01-26
  • Effect of Selected Environmental Factors on the Microbicidal Effectiveness of Radiant Catalytic Ionization
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-18
    Krzysztof Skowron; Ewa Wałecka-Zacharska; Katarzyna Grudlewska; Joanna Kwiecińska-Piróg; Natalia Wiktorczyk; Maria Kowalska; Zbigniew Paluszak; Katarzyna Kosek-Paszkowska; Klaudia Brożek; Jakub Korkus; Eugenia Gospodarek-Komkowska

    The aim of this study was the assessment of the effect of time exposure, temperature, distance, and organic contaminants on radiant catalytic ionization (RCI) microbicidal effectiveness. The number of all examined bacteria decreased together with time exposure of RCI. The lowest recovery was obtained, both from the rubber surface (6.36 log CFU × cm–2) and steel (6.04 log CFU × cm–2) in the case of Escherichia coli O157:H7. On the other hand, Staphylococcus aureus was isolated in the largest number (rubber: 7.88 log CFU × cm–2, steel: 7.79 log CFU × cm–2). Among the tested environmental conditions, the greatest bacterial population was re-isolated at 4°C (distance: 0.5 m, time: 24 h), whereas the lowest population was found at a distance of 0.5 m (temperature: 20°C, time: 24 h) and on surfaces without contamination. In the samples treated with RCI, the bacterial population was the lowest on non-contaminated surfaces, ranging from 3.76 log CFU × cm–2 (E. coli O157:H7) to 5.58 log CFU × cm–2 (S. aureus) for the rubber, and from 3.26 log CFU × cm–2 (E. coli O157:H7) to 5.20 log CFU × cm–2 (S. aureus) for the stainless steel. The highest bacteria number was isolated from surfaces contaminated with meat and fish pulp. The lowest bacterial reduction caused by RCI was found in the case of rubber contaminated with meat-fish pulp (24 h, 0.5 m, 20°C). The reduction rate was equal to 0.89 log CFU × cm–2 for S. aureus, 1.17 log CFU × cm–2 for Listeria monocytogenes, 1.43 log CFU × cm–2 for Salmonella Enteritidis and 1.61 log CFU × cm–2 for E. coli O157:H7. In turn, the greatest bacterial reduction was found in the case of non-contaminated steel (24 h, 0.5 m, 37°C). The reduction rate was equal to 4.52 log CFU × cm–2 for L. monocytogenes, 3.61 log CFU × cm–2 for S. Enteritidis, 2.98 log CFU × cm–2 for E. coli O157:H7 and 2.77 log CFU × cm–2 for S. aureus. RCI allows the inactivation of pathogens from stainless steel and rubber surfaces. Its efficacy is species-dependent and affected by environmental factors.

    更新日期:2020-01-26
  • The Uptake and Release of Amino Acids by Staphylococcus aureus at Mid-Exponential and Stationary Phases and Their Corresponding Responses to Changes in Temperature, pH and Osmolality
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-18
    Mousa M. Alreshidi; R. Hugh Dunstan; Margaret M. Macdonald; Johan Gottfries; Tim K. Roberts

    Staphylococcus aureus is an important pathogen that is associated with nosocomial infections, as well as food poisoning. This bacterium is resistant to antimicrobial agents and can survive in a wide range of environmental conditions. The aim of this study was to measure the uptake and release of amino acids by S. aureus at mid-exponential and stationary phases of growth following exposure to a combination of conditions including variations in temperature, pH and NaCl. Bacterial cells were grown up to mid-exponential and stationary phases in tryptic soy broth (TSB), where the supernatants were collected for analyses of amino acids to determine the uptake and release characteristics. The uptake/release of amino acids was estimated by subtracting the initial levels of the free amino acids in the media from those measured at mid-exponential and stationary phases of growth. When cells were grown at ideal conditions, the analyses revealed that significant uptake of amino acids had occurred by stationary phase compared with the mid-exponential phase. A substantial release of valine and tyrosine into the external media was observed by cells at stationary phase. At both phases, the uptake and release patterns were significantly different between cells grown under ideal control conditions, when compared with those grown under various combinations of sub-optimal environmental conditions. The analyses of the supernatants harvested from controls and treatment groups at exponential phase indicated that the total uptake of amino acids was reduced approximately five times by cells grown with addition of 2.5% NaCl or with pH6 at 35°C, and 2-fold by cells grown at pH8 at 35°C. However, the final quantities of amino acids taken up by cells grown to stationary phase did not significantly alter between control and treated samples. Valine was found to be the most abundant amino acid that was significantly released into the media at stationary phase by both control and treated samples. It was evident that diverse environmental conditions resulted in differential patterns of amino acid uptake and release during adaptation to designated conditions.

    更新日期:2020-01-26
  • The Preliminary Development of an in vitro Poultry Cecal Culture Model to Evaluate the Effects of Original XPCTM for the Reduction of Campylobacter jejuni and Its Potential Effects on the Microbiota
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-18
    Kristina M. Feye; Peter M. Rubinelli; William Evan Chaney; Hilary O. Pavlidis; Michael H. Kogut; Steven C. Ricke

    Poultry is a major reservoir for the pathogen Campylobacter jejuni. C. jejuni inhabits the poultry gastrointestinal tract as a part of the gut microbiota. The objective of this study was to evaluate both the survival of C. jejuni and the changes in the population dynamics of the cecal microbiome during an in vitro C. jejuni inoculation in the presence or absence of the functional metabolites of Diamond V Original XPCTM (XPC). Two independent trials were conducted. Broiler chickens (n = 6 per Trial 1 and n = 3 per Trial 2) were raised according to standard industry guidelines and euthanized on Day 41. The ceca were collected aseptically, their contents removed independently and then used in an in vitro microaerobic model with 0.1% cecal contents + Campylobacter with or without 1% XPC (w/v). Before the inoculation with a chloramphenicol resistant marker strain of C. jejuni, the cecal contents were pre-incubated with XPC at 42°C for 24 h, in a shaking incubator (200 rpm) under microaerobic conditions, then experimentally inoculated with 108/ml of C. jejuni into the appropriate treatment groups. At 0 and 24 h for Trial 1, and 48 h for Trial 2, sub-samples of the culture (n = 3 ceca, two technical replicates per ceca, XPC alone or ceca culture alone) were enumerated using a Petroff–Hausser counter, and the DNA was extracted for microbiome analysis. DNA was isolated using the Qiagen QIAamp Fast Stool DNA Mini Kit and sequenced using the Illumina MiSeq platform. The reads were filtered, normalized, and assigned taxonomical identities using the QIIME2 pipeline. The relative microbiota populations were identified via ANCOM. Altogether, evidence suggests that XPC alters the microbiome, and in turn reduces Campylobacter survival.

    更新日期:2020-01-23
  • In situ Treatment With Novel Microbiocide Inhibits Methicillin Resistant Staphylococcus aureus in a Murine Wound Infection Model
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-23
    Joseph P. Hoffmann; Jessica K. Friedman; Yihui Wang; James B. McLachlan; Mimi C. Sammarco; Lisa A. Morici; Chad J. Roy

    Increased prevalence of antibiotic resistance in skin and soft tissue infections is a concerning public health challenge currently facing medical science. A combinatory, broad spectrum biocidal antiseptic has been developed (“ASP”) as a topically applied solution to potential resistant and polymicrobial infected wounds that may be encountered in this context. The ASP-105 designate was evaluated in vitro by determining the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), against different strains of methicillin-resistant Staphylococcus aureus (MRSA), resulting estimates of which approximated the positive control (bacitracin). To evaluate in vivo microbicide efficacy, we utilized a murine full thickness wound model to study bacterial infection and wound healing kinetics. Mice were experimentally wounded dorsally and infected with bioluminescent MRSA. The infected wound was splinted, dressed and treated topically with either ASP-105, vehicle (-control), or bacitracin. Bacterial burden and wound healing was monitored using an in vivo imaging system and evaluation of biofilm formation using scanning electron microscopy of wound dressing. Treatment with ASP-105 significantly reduced bacterial burdens in the first 3 days of infection and inhibited MRSA biofilm formation on the surgical dressing. Notably, treatment with ASP-105 resulted in a sterilizing effect of any detectable MRSA in nearly all (80%; 4/5) of treatment group. All mice receiving vehicle control developed highly MRSA-luminescent and purulent wound beds as a result of experimental infection. The ASP-105 therapy facilitated natural healing in the absence of MRSA infection. Results of this study suggests that that the novel “ASP” combinatory topical antiseptic can be used directly in wounds as a potent, broad-spectrum microbicide against drug resistant S. aureus without injury to the wound bed and impediment of natural restorative processes associated with wound healing. Further studies are warranted to test the effectiveness of this biocidal formulation against other recalcitrant bacterial and fungal pathogens in the context of serious wound infections, and to assess utility of use in both clinical and self-treat scenarios.

    更新日期:2020-01-23
  • Beyond Taxonomic Analysis of Microbiomes: A Functional Approach for Revisiting Microbiome Changes in Colorectal Cancer
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-24
    Mohammad Hossein Norouzi-Beirami; Sayed-Amir Marashi; Ali Mohammad Banaei-Moghaddam; Kaveh Kavousi

    Colorectal cancer (CRC) is one of the most prevalent cancers in the world, especially in developed countries. In different studies, the association between CRC and dysbiosis of gut microbiome has been reported. However, most of these works focus on the taxonomic variation of the microbiome, which presents little, if any, functional insight about the reason behind and/or consequences of microbiome dysbiosis. In this study, we used a previously reported metagenome dataset which is obtained by sequencing 156 microbiome samples of healthy individuals as the control group (Co), as well as microbiome samples of patients with advanced colorectal adenoma (Ad) and colorectal carcinoma (Ca). Features of the microbiome samples have been analyzed at the level of species, as well as four functional levels, i.e., gene, KEGG orthology (KO) group, Enzyme Commission (EC) number, and reaction. It was shown that, at each of these levels, certain features exist which show significant changing trends during cancer progression. In the next step, a list of these features were extracted, which were shown to be able to predict the category of Co, Ad, and Ca samples with an accuracy of >85%. When only one group of features (species, gene, KO group, EC number, reaction) was used, KO-related features were found to be the most successful features for classifying the three categories of samples. Notably, species-related features showed the least success in sample classification. Furthermore, by applying an independent test set, we showed that these performance trends are not limited to our original dataset. We determined the most important classification features at each of the four functional levels. We propose that these features can be considered as biomarkers of CRC progression. Finally, we show that the intra-diversity of each sample at the levels of bacterial species and genes is much more than those of the KO groups, EC numbers, and reactions of that sample. Therefore, we conclude that the microbiome diversity at the species level, or gene level, is not necessarily associated with the diversity at the functional level, which again indicates the importance of KO-, EC-, and reaction-based features in metagenome analysis. The source code of proposed method is freely available from https://www.bioinformatics.org/mamed.

    更新日期:2020-01-23
  • sRNA scr5239 Involved in Feedback Loop Regulation of Streptomyces coelicolor Central Metabolism
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-24
    Franziska Engel; Elena Ossipova; Per-Johan Jakobsson; Michael-Paul Vockenhuber; Beatrix Suess

    In contrast to transcriptional regulation, post-transcriptional regulation and the role of small non-coding RNAs (sRNAs) in streptomycetes are not well studied. Here, we focus on the highly conserved sRNA scr5239 in Streptomyces coelicolor. A proteomics approach revealed that the sRNA regulates several metabolic enzymes, among them phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of the central carbon metabolism. The sRNA scr5239 represses pepck at the post-transcriptional level and thus modulates the intracellular level of phosphoenolpyruvate (PEP). The expression of scr5239 in turn is dependent on the global transcriptional regulator DasR, thus creating a feedback loop regulation of the central carbon metabolism. By post-transcriptional regulation of PEPCK and in all likelihood other targets, scr5239 adds an additional layer to the DasR regulatory network and provides a tool to control the metabolism dependent on the available carbon source.

    更新日期:2020-01-23
  • Genomics Evolutionary History and Diagnostics of the Alternaria alternata Species Group Including Apple and Asian Pear Pathotypes
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-24
    Andrew D. Armitage; Helen M. Cockerton; Surapareddy Sreenivasaprasad; James Woodhall; Charles R. Lane; Richard J. Harrison; John P. Clarkson

    The Alternaria section alternaria (Alternaria alternata species group) represents a diverse group of saprotroph, human allergens, and plant pathogens. Alternaria taxonomy has benefited from recent phylogenetic revision but the basis of differentiation between major phylogenetic clades within the group is not yet understood. Furthermore, genomic resources have been limited for the study of host-specific pathotypes. We report near complete genomes of the apple and Asian pear pathotypes as well as draft assemblies for a further 10 isolates representing Alternaria tenuissima and Alternaria arborescens lineages. These assemblies provide the first insights into differentiation of these taxa as well as allowing the description of effector and non-effector profiles of apple and pear conditionally dispensable chromosomes (CDCs). We define the phylogenetic relationship between the isolates sequenced in this study and a further 23 Alternaria spp. based on available genomes. We determine which of these genomes represent MAT1-1-1 or MAT1-2-1 idiomorphs and designate host-specific pathotypes. We show for the first time that the apple pathotype is polyphyletic, present in both the A. arborescens and A. tenuissima lineages. Furthermore, we profile a wider set of 89 isolates for both mating type idiomorphs and toxin gene markers. Mating-type distribution indicated that gene flow has occurred since the formation of A. tenuissima and A. arborescens lineages. We also developed primers designed to AMT14, a gene from the apple pathotype toxin gene cluster with homologs in all tested pathotypes. These primers allow identification and differentiation of apple, pear, and strawberry pathotypes, providing new tools for pathogen diagnostics.

    更新日期:2020-01-23
  • Necroptosis and Caspase-2-Mediated Apoptosis of Astrocytes and Neurons, but Not Microglia, of Rat Hippocampus and Parenchyma Caused by Angiostrongylus cantonensis Infection
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-24
    Hongli Zhou; Zhe Chen; Yanin Limpanont; Yue Hu; Yubin Ma; Ping Huang; Paron Dekumyoy; Minyu Zhou; Yixin Cheng; Zhiyue Lv

    Infection with the roundworm Angiostrongylus cantonensis is the main cause of eosinophilic meningitis worldwide. The underlying molecular basis of the various pathological outcomes in permissive and non-permissive hosts infected with A. cantonensis remains poorly defined. In the present study, the histology of neurological disorders in the central nervous system (CNS) of infected rats was assessed by using hematoxylin and eosin staining. Quantitative reverse transcription polymerase chain reaction (RT-qPCR), western blot and immunofluorescence (IF) were used in evolutions of the transcription and translation levels of the apoptosis-, necroptosis-, autophagy-, and pyroptosis-related genes. The distribution of apoptotic and necroptotic cells in the rat hippocampus and parenchyma was further detected using flow cytometry, and the features of the ultrastructure of the cells were examined by transmission electron microscopy (TEM). The inflammatory response upon CNS infection with A. cantonensis evolved, as characterized by the accumulation of a small number of inflammatory cells under the thickened meninges, which peaked at 21 days post-infection (dpi) and returned to normal by 35 dpi. The transcription levels and translation of caspase-2, caspase-8, RIP1 and RIP3 increased significantly at 21 and 28 dpi but decreased sharply at 35 dpi compared to those in the normal control group. However, the changes in the expression of caspase-1, caspase-3, caspase-11, Beclin-1 and LC3B were not obvious, suggesting that apoptosis and necroptosis but not autophagy or pyroptosis occurred in the brains of infected animals at 21 and 28 dpi. The results of RT-qPCR, western blot analysis, IF, flow cytometry and TEM further illustrated that necroptosis and caspase-2-mediated apoptosis occurred in astrocytes and neurons but not in microglia in the parenchyma and hippocampus of infected animals. This study provides the first evidence that neuronal and astrocytic necroptosis and caspase-2-mediated apoptosis are induced by A. cantonensis infection in the parenchymal and hippocampal regions of rats at 21 and 28 dpi but these processes are negligible at 35 dpi. These findings enhance our understanding of the pathogenesis of A. cantonensis infection and provide new insights into therapeutic approaches targeting the occurrence of cell death in astrocytes and neurons in infected patients.

    更新日期:2020-01-23
  • Comparative Transcriptomics Reveals Features and Possible Mechanisms of Glucose-Mediated Soil Fungistasis Relief in Arthrobotrys oligospora
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-27
    Tong Liu; Ying Huang; Xiang-Xiang Chen; Xi Long; Yun-He Yang; Ming-Liang Zhu; Ming-He Mo; Ke-Qin Zhang

    Soil-borne pest diseases result in large annual agricultural losses globally. Fungal bio-control agents are an alternative means of controlling pest diseases; however, soil fungistasis limits the effect of fungal agents. Nutrients can relieve soil fungistasis, but the mechanisms behind this process remain poorly understood. In this study, we determined and quantified the transcriptomes of Arthrobotrys oligospora, a nematode-trapping fungus, derived from samples of fresh conidia, germinated conidia, soil fungistatic conidia, and glucose-relieved conidia. The transcriptomes of fungistatic and glucose-relieved conidia were significantly different from those of the other two conidia samples. KEGG pathway analyses showed that those genes upregulated in fungistatic and glucose-relieved conidia were mainly involved in translation and substance metabolism, and the downregulated genes were mainly involved in MAPK pathway, autophagy, mitophagy, and endocytosis. As being different from the transcriptome of fungistatic conidia, upregulated genes in the transcriptome of glucose-relieved conidia are also related to replication and repair, spliceosome, oxidative phosphorylation, autophagy, and degradation pathway (lysosome, proteasome, and RNA degradation). And the upregulated genes resulted from comparison of glucose-relieved conidia and fungistatic conidia were enriched in metabolic pathways, cycle, DNA replication, and repair. The differentially splicing events in the transcriptome of glucose-relieved conidia are far more than that of other two transcriptomes, and genes regulated by differentially splicing were analyzed through KEGG pathway analysis. Furthermore, autophagy genes were proved to play important role in resisting soil fungistasis and glucose-mediated soil fungistasis relief. These data indicate that, in addition to being a carbon and energy source for conidia germination, glucose may also help to relieve soil fungistasis by activating many cellular processes, including autophagy, DNA replication and repair, RNA alternative splicing, and degradation pathways.

    更新日期:2020-01-23
  • Morphologically Different Pectobacterium brasiliense Bacteriophages PP99 and PP101: Deacetylation of O-Polysaccharide by the Tail Spike Protein of Phage PP99 Accompanies the Infection
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-29
    Anna A. Lukianova; Mikhail M. Shneider; Peter V. Evseev; Anna M. Shpirt; Eugenia N. Bugaeva; Anastasia P. Kabanova; Ekaterina A. Obraztsova; Kirill K. Miroshnikov; Sofiya N. Senchenkova; Alexander S. Shashkov; Stepan V. Toschakov; Yuriy A. Knirel; Alexander N. Ignatov; Konstantin A. Miroshnikov

    Soft rot caused by numerous species of Pectobacterium and Dickeya is a serious threat to the world production of potatoes. The application of bacteriophages to combat bacterial infections in medicine, agriculture, and the food industry requires the selection of comprehensively studied lytic phages and the knowledge of their infection mechanism for more rational composition of therapeutic cocktails. We present the study of two bacteriophages, infective for the Pectobacterium brasiliense strain F152. Podoviridae PP99 is a representative of the genus Zindervirus, and Myoviridae PP101 belongs to the still unclassified genomic group. The structure of O-polysaccharide of F152 was established by sugar analysis and 1D and 2D NMR spectroscopy: → 4)-α-D-Manp6Ac-(1→ 2)-α-D-Manp-(1→ 3)-β-D-Galp-(1→ 3↑1α-l-6dTalpAc0−2 The recombinant tail spike protein of phage PP99, gp55, was shown to deacetylate the side chain talose residue of bacterial O-polysaccharide, thus providing the selective attachment of the phage to the cell surface. Both phages demonstrate lytic behavior, thus being prospective for therapeutic purposes.

    更新日期:2020-01-23
  • Phage Lytic Protein LysRODI Prevents Staphylococcal Mastitis in Mice
    Front. Microbiol. (IF 4.259) Pub Date : 2020-01-06
    Diana Gutiérrez; Victoria Garrido; Lucía Fernández; Silvia Portilla; Ana Rodríguez; María Jesús Grilló; Pilar García

    Phage lytic proteins are promising antimicrobials that could complement conventional antibiotics and help to combat multi-drug resistant bacteria that cause important human and animal infections. Here, we report the characterization of endolysin LysRODI (encoded by staphylophage phiIPLA-RODI) and its application as a prophylactic mastitis treatment. The main properties of LysRODI were compared with those of endolysin LysA72 (encoded by staphylophage phiIPLA35) and the chimeric protein CHAPSH3b (derived from the virion-associated peptidoglycan hydrolase HydH5 and lysostaphin). Time-kill experiments performed with Staphylococcus aureus and Staphylococcus epidermidis demonstrated that the killing rate of LysRODI and CHAPSH3b is higher than that of LysA72 (0.1 μM protein removed 107 CFU/ml of S. aureus in 30 min). Of note, all proteins failed to select resistant mutants as bacterial exposure to sub-lethal concentrations of the proteins did not alter the MIC values. Additionally, LysRODI and CHAPSH3b were non-toxic in a zebrafish embryo model at concentrations near the MIC (0.5 and 0.7 μM, respectively). Moreover, these two proteins significantly reduced mortality in a zebrafish model of systemic infection. In contrast to LysRODI, the efficacy of CHAPSH3b was dose-dependent in zebrafish, requiring higher-dose treatments to achieve the maximum survival rate. For this reason, LysRODI was selected for further analysis in mice, demonstrating great efficacy to prevent mammary infections by S. aureus and S. epidermidis. Our findings strongly support the use of phage lytic proteins as a new strategy to prevent staphylococcal mastitis.

    更新日期:2020-01-23
  • Study on a Novel Cold-Active and Halotolerant Monoacylglycerol Lipase Widespread in Marine Bacteria Reveals a New Group of Bacterial Monoacylglycerol Lipases Containing Unusual C(A/S)HSMG Catalytic Motifs
    Front. Microbiol. (IF 4.259) Pub Date : 2020-01-06
    Ping-Yi Li; Yan-Qi Zhang; Yi Zhang; Wen-Xin Jiang; Yan-Jun Wang; Yi-Shuo Zhang; Zhong-Zhi Sun; Chun-Yang Li; Yu-Zhong Zhang; Mei Shi; Xiao-Yan Song; Long-Sheng Zhao; Xiu-Lan Chen

    Monoacylglycerol lipases (MGLs) are present in all domains of life. However, reports on bacterial MGLs are still limited. Until now, reported bacterial MGLs are all thermophilic/mesophilic enzymes from warm terrestrial environments or deep-sea hydrothermal vent, and none of them originates from marine environments vastly subject to low temperature, high salts, and oligotrophy. Here, we characterized a novel MGL, GnMgl, from the marine cold-adapted and halophilic bacterium Glaciecola nitratireducens FR1064T. GnMgl shares quite low sequence similarities with characterized MGLs (lower than 31%). GnMgl and most of its bacterial homologs harbor a catalytic Ser residue located in the conserved C(A/S)HSMG motif rather than in the typical GxSxG motif reported on other MGLs, suggesting that GnMgl-like enzymes might be different from reported MGLs in catalysis. Phylogenetic analysis suggested that GnMgl and its bacterial homologs are clustered as a separate group in the monoglyceridelipase_lysophospholipase family of the Hydrolase_4 superfamily. Recombinant GnMgl has no lysophospholipase activity but could hydrolyze saturated (C12:0-C16:0) and unsaturated (C18:1 and C18:2) MGs and short-chain triacylglycerols, displaying distinct substrate selectivity from those of reported bacterial MGLs. The substrate preference of GnMgl, predicted to be a membrane protein, correlates to the most abundant fatty acids within the strain FR1064T, suggesting the role of GnMgl in the lipid catabolism in this marine bacterium. In addition, different from known bacterial MGLs that are all thermostable enzymes, GnMgl is a cold-adapted enzyme, with the maximum activity at 30°C and retaining 30% activity at 0°C. GnMgl is also a halotolerant enzyme with full activity in 3.5M NaCl. The cold-adapted and salt-tolerant characteristics of GnMgl may help its source strain FR1064T adapt to the cold and saline marine environment. Moreover, homologs to GnMgl are found to be abundant in various marine bacteria, implying their important physiological role in these marine bacteria. Our results on GnMgl shed light on marine MGLs.

    更新日期:2020-01-23
  • Transcriptomic Profiles of Zymomonas mobilis 8b to Furfural Acute and Long-Term Stress in Both Glucose and Xylose Conditions
    Front. Microbiol. (IF 4.259) Pub Date : 2020-01-06
    Shihui Yang; Mary Ann Franden; Xia Wang; Yat-Chen Chou; Yun Hu; Steven D. Brown; Philip T. Pienkos; Min Zhang

    Zymomonas mobilis 8b is an ethanologenic bacterium engineered to utilize both glucose and xylose. The impacts of lignocellulosic hydrolyzate inhibitors on the growth of Zymomonas mobilis 8b have been investigated. However, the molecular responses of these inhibitors have not been completely elucidated yet. In this study, molecular responses to furfural were investigated using transcriptomic approaches of both chip-based microarray and a directional mRNA-Seq. Furfural acute shock time-course experiment with 3 g/L furfural supplemented when cells reached exponential phase and stress response experiment in the presence of 2 g/L furfural from the beginning of fermentation were carried out to study the physiological and transcriptional profiles of short-term and long-term effects of furfural on 8b. Furfural negatively affected 8b growth in terms of final biomass and the fermentation time. Transcriptomic studies indicated that the response of 8b to furfural was dynamic and complex, and differences existed between short-term shock and long-term stress responses. However, the gene function categories were similar with most down-regulated genes related to translation and biosynthesis, while the furfural up-regulated genes were mostly related to general stress responses. Several gene candidates have been identified and genetic studies indicated that expression of ZMO0465 and cysteine synthase operon ZMO0003-0006 driven by its native promoter in a shuttle vector enhanced the furfural tolerance of 8b. In addition, the relationship between microarray and mRNA-Seq was compared with good correlations. The directional mRNA-Seq data not only provided the gene expression profiling, but also can be applied for transcriptional architecture improvement to identify and confirm operons, novel transcripts, hypothetical gene functions, transcriptional start sites, and promoters with different strength.

    更新日期:2020-01-23
  • Short-Term Adaptation Modulates Anaerobic Metabolic Flux to Succinate by Activating ExuT, a Novel D-Glucose Transporter in Escherichia coli
    Front. Microbiol. (IF 4.259) Pub Date : 2020-01-08
    Hyun Ju Kim; Haeyoung Jeong; Sang Jun Lee

    The sugar phosphotransferase system (PTS) is an essential energy-saving mechanism, particularly under anaerobic conditions. Since the PTS consumes equimolar phosphoenolpyruvate to phosphorylate each molecule of internalized glucose in the process of pyruvate generation, its absence can adversely affect the mixed acid fermentation profile and cell growth under anaerobic conditions. In this study, we report that the ΔptsG mutant cells of Escherichia coli K-12 strain exhibited inefficient glucose utilization, produced a significant amount of succinate, and exhibited a low growth rate. However, cells adapted soon after and started to grow rapidly in the same batch culture. As a result, the adapted ΔptsG cells showed the same mixed acid fermentation profiles as the wild-type cells, which was attributed to the mutation of the mlc gene, a repressor of the D-mannose PTS, another transporter for D-glucose. Similar adaptations were observed in the cells with ΔptsGΔmanX and the cells with ΔptsI that resulted in the production of a substantial amount of succinate and fast growth rate. The genome sequencing showed the presence of null mutations in the exuR gene, which encodes a modulator of exuT-encoded non-PTS sugar transporter, in adapted ΔptsGΔmanX and ΔptsI strains. Results from the RT-qPCR analysis and genetic test confirmed that the enhanced expression of ExuT, a non-PTS sugar transporter, was responsible for the uptake of D-glucose, increased succinate production, and fast growth of adapted cells. In conclusion, our study showed that the regulatory network of sugar transporters can be modulated by short-term adaptation and that downstream metabolic flux could be significantly determined by the choice of sugar transporters.

    更新日期:2020-01-23
  • Role of the Dihydrodipicolinate Synthase DapA1 on Iron Homeostasis During Cyanide Assimilation by the Alkaliphilic Bacterium Pseudomonas pseudoalcaligenes CECT5344
    Front. Microbiol. (IF 4.259) Pub Date : 2020-01-08
    Alfonso Olaya-Abril; María Dolores Pérez; Purificación Cabello; Diego Martignetti; Lara Paloma Sáez; Víctor Manuel Luque-Almagro; Conrado Moreno-Vivián; María Dolores Roldán

    Cyanide is a toxic compound widely used in mining and jewelry industries, as well as in the synthesis of many different chemicals. Cyanide toxicity derives from its high affinity for metals, which causes inhibition of relevant metalloenzymes. However, some cyanide-degrading microorganisms like the alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 may detoxify hazardous industrial wastewaters that contain elevated cyanide and metal concentrations. Considering that iron availability is strongly reduced in the presence of cyanide, mechanisms for iron homeostasis should be required for cyanide biodegradation. Previous omic studies revealed that in the presence of a cyanide-containing jewelry residue the strain CECT5344 overproduced the dihydrodipicolinate synthase DapA1, a protein involved in lysine metabolism that also participates in the synthesis of dipicolinates, which are excellent metal chelators. In this work, a dapA1– mutant of P. pseudoalcaligenes CECT5344 has been generated and characterized. This mutant showed reduced growth and cyanide consumption in media with the cyanide-containing wastewater. Intracellular levels of metals like iron, copper and zinc were increased in the dapA1– mutant, especially in cells grown with the jewelry residue. In addition, a differential quantitative proteomic analysis by LC-MS/MS was carried out between the wild-type and the dapA1– mutant strains in media with jewelry residue. The mutation in the dapA1 gene altered the expression of several proteins related to urea cycle and metabolism of arginine and other amino acids. Additionally, the dapA1– mutant showed increased levels of the global nitrogen regulator PII and the glutamine synthetase. This proteomic study has also highlighted that the DapA1 protein is relevant for cyanide resistance, oxidative stress and iron homeostasis response, which is mediated by the ferric uptake regulator Fur. DapA1 is required to produce dipicolinates that could act as iron chelators, conferring protection against oxidative stress and allowing the regeneration of Fe-S centers to reactivate cyanide-damaged metalloproteins.

    更新日期:2020-01-23
  • Degradation of Recalcitrant Polyurethane and Xenobiotic Additives by a Selected Landfill Microbial Community and Its Biodegradative Potential Revealed by Proximity Ligation-Based Metagenomic Analysis
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-10
    Itzel Gaytán; Ayixon Sánchez-Reyes; Manuel Burelo; Martín Vargas-Suárez; Ivan Liachko; Maximilian Press; Shawn Sullivan; M. Javier Cruz-Gómez; Herminia Loza-Tavera

    Polyurethanes (PU) are the sixth most produced plastics with around 18-million tons in 2016, but since they are not recyclable, they are burned or landfilled, generating damage to human health and ecosystems. To elucidate the mechanisms that landfill microbial communities perform to attack recalcitrant PU plastics, we studied the degradative activity of a mixed microbial culture, selected from a municipal landfill by its capability to grow in a water PU dispersion (WPUD) as the only carbon source, as a model for the BP8 landfill microbial community. The WPUD contains a polyether-polyurethane-acrylate (PE-PU-A) copolymer and xenobiotic additives (N-methylpyrrolidone, isopropanol and glycol ethers). To identify the changes that the BP8 microbial community culture generates to the WPUD additives and copolymer, we performed chemical and physical analyses of the biodegradation process during 25 days of cultivation. These analyses included Nuclear magnetic resonance, Fourier transform infrared spectroscopy, Thermogravimetry, Differential scanning calorimetry, Gel permeation chromatography, and Gas chromatography coupled to mass spectrometry techniques. Moreover, for revealing the BP8 community structure and its genetically encoded potential biodegradative capability we also performed a proximity ligation-based metagenomic analysis. The additives present in the WPUD were consumed early whereas the copolymer was cleaved throughout the 25-days of incubation. The analysis of the biodegradation process and the identified biodegradation products showed that BP8 cleaves esters, C-C, and the recalcitrant aromatic urethanes and ether groups by hydrolytic and oxidative mechanisms, both in the soft and the hard segments of the copolymer. The proximity ligation-based metagenomic analysis allowed the reconstruction of five genomes, three of them from novel species. In the metagenome, genes encoding known enzymes, and putative enzymes and metabolic pathways accounting for the biodegradative activity of the BP8 community over the additives and PE-PU-A copolymer were identified. This is the first study revealing the genetically encoded potential biodegradative capability of a microbial community selected from a landfill, that thrives within a WPUD system and shows potential for bioremediation of polyurethane- and xenobiotic additives-contamitated sites.

    更新日期:2020-01-23
  • Computational Identification of the Proteins Associated With Quorum Sensing and Biofilm Formation in Mycobacterium tuberculosis
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-16
    Shubhada R. Hegde

    With prolonged therapy and increased instances of drug resistance, tuberculosis is viewed as a serious infectious disease causing high mortality. Emerging concepts in Mycobacterium tuberculosis pathogenicity include biofilm formation, which endows bacterial survival in the host for a long time. To tackle chronic tuberculosis infection, a detailed understanding of the bacterial survival mechanisms is crucial. Using comparative genomics and literature mining, 115 M. tuberculosis proteins were shortlisted for their likely association with biofilm formation or quorum sensing. These include essential genes such as secA2, lpqY-sugABC, Rv1176c, and Rv0195, many of which are also known virulence factors. Furthermore, the functional relationship among these proteins was established by considering known protein-protein interactions, regulatory interactions, and gene expression correlation data/information. Graph centrality and motif analyses predicted the importance of proteins, such as Rv0081, DevR, RegX3, Rv0097, and Rv1996 in M. tuberculosis biofilm formation. Analysis of conservation across other biofilm-forming bacteria suggests that most of these genes are conserved in mycobacteria. As the processes, such as quorum sensing, leading to biofilm formation involve diverse pathways and interactions between proteins, these system-wide studies provide a novel perspective toward understanding mycobacterial persistence.

    更新日期:2020-01-23
  • Droplet Digital PCR Is an Improved Alternative Method for High-Quality Enumeration of Viable Probiotic Strains
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-17
    Sarah J. Z. Hansen; Peipei Tang; Anthony Kiefer; Kevin Galles; Connie Wong; Wesley Morovic

    Traditional microbiological enumeration methods have long been employed as the standard evaluation procedure for probiotic microorganisms. These methods are labor intensive, have long-time to results and inherently have a high degree of variability – up to 35%. As clinical probiotic and microbiome science continues to grow and develop, it is increasingly important that researchers thoroughly define and deliver the targeted probiotic dose. Furthermore, to establish high quality commercial products, the same dosage level must be administered to consumers. An ISO method for the use of flow cytometry has been established which does speed up the time to results and reduce variability, but the method has not yet gained widespread adoption across the probiotic industry. This is possibly due to expertise needed to implement and maintain a new testing platform in an established quality system. In this study we compare enumeration using plate counts and flow cytometry to the use of droplet digital PCR (ddPCR), which in addition to giving faster time to results than plate count and less variability than both plate count and flow cytometry, has additional benefits such as strain-specific counts. Use of ddPCR gives the ability to design primers to target deletions and single base pair differences which will allow for strain profiling in microbiome analyses. We demonstrate that ddPCR probiotic enumeration results are positively correlated to both plate count and flow cytometry results and should be considered a viable, next generation enumeration method for the evaluation of probiotics.

    更新日期:2020-01-23
  • Mechanism of Action of Surface Immobilized Antimicrobial Peptides Against Pseudomonas aeruginosa
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-18
    Muhammad Yasir; Debarun Dutta; Khondker R. Hossain; Renxun Chen; Kitty K. K. Ho; Rajesh Kuppusamy; Ronald J. Clarke; Naresh Kumar; Mark D. P. Willcox

    Bacterial colonization and biofilm development on medical devices can lead to infection. Antimicrobial peptide-coated surfaces may prevent such infections. Melimine and Mel4 are chimeric cationic peptides showing broad-spectrum antimicrobial activity once attached to biomaterials and are highly biocompatible in animal models and have been tested in Phase I and II/III human clinical trials. These peptides were covalently attached to glass using an azidobenzoic acid linker. Peptide attachment was confirmed using X-ray photoelectron spectroscopy and amino acid analysis. Mel4 when bound to glass was able to adopt a more ordered structure in the presence of bacterial membrane mimetic lipids. The ability of surface bound peptides to neutralize endotoxin was measured along with their interactions with the bacterial cytoplasmic membrane which were analyzed using DiSC(3)-5 and Sytox green, Syto-9, and PI dyes with fluorescence microscopy. Leakage of ATP and nucleic acids from cells were determined by analyzing the surrounding fluid. Attachment of the peptides resulted in increases in the percentage of nitrogen by 3.0% and 2.4%, and amino acid concentrations to 0.237 nmole and 0.298 nmole per coverslip on melimine and Mel4 coated surfaces, respectively. The immobilized peptides bound lipopolysaccharide and disrupted the cytoplasmic membrane potential of Pseudomonas aeruginosa within 15 min. Membrane depolarization was associated with a reduction in bacterial viability by 82% and 63% for coatings melimine and Mel4, respectively (p < 0.001). Disruption of membrane potential was followed by leakage of ATP from melimine (1.5 ± 0.4 nM) or Mel4 (1.3 ± 0.2 nM) coated surfaces compared to uncoated glass after 2 h (p < 0.001). Sytox green influx started after 3 h incubation with either peptide. Melimine coatings yielded 59% and Mel4 gave 36% PI stained cells after 4 h. Release of the larger molecules (DNA/RNA) commenced after 4 h for melimine (1.8 ± 0.9 times more than control; p = 0.008) and after 6 h with Mel4 (2.1 ± 0.2 times more than control; p < 0.001). The mechanism of action of surface bound melimine and Mel4 was similar to that of the peptides in solution, however, their immobilization resulted in much slower (approximately 30 times) kinetics.

    更新日期:2020-01-23
  • CRISPR-Cas Systems and the Paradox of Self-Targeting Spacers
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-19
    Franziska Wimmer; Chase L. Beisel

    CRISPR-Cas immune systems in bacteria and archaea record prior infections as spacers within each system’s CRISPR arrays. Spacers are normally derived from invasive genetic material and direct the immune system to complementary targets as part of future infections. However, not all spacers appear to be derived from foreign genetic material and instead can originate from the host genome. Their presence poses a paradox, as self-targeting spacers would be expected to induce an autoimmune response and cell death. In this review, we discuss the known frequency of self-targeting spacers in natural CRISPR-Cas systems, how these spacers can be incorporated into CRISPR arrays, and how the host can evade lethal attack. We also discuss how self-targeting spacers can become the basis for alternative functions performed by CRISPR-Cas systems that extend beyond adaptive immunity. Overall, the acquisition of genome-targeting spacers poses a substantial risk but can aid in the host’s evolution and potentially lead to or support new functionalities.

    更新日期:2020-01-23
  • Computer-Aided Design of Antimicrobial Peptides: Are We Generating Effective Drug Candidates?
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-20
    Marlon H. Cardoso; Raquel Q. Orozco; Samilla B. Rezende; Gisele Rodrigues; Karen G. N. Oshiro; Elizabete S. Cândido; Octávio L. Franco

    Antimicrobial peptides (AMPs), especially antibacterial peptides, have been widely investigated as potential alternatives to antibiotic-based therapies. Indeed, naturally occurring and synthetic AMPs have shown promising results against a series of clinically relevant bacteria. Even so, this class of antimicrobials has continuously failed clinical trials at some point, highlighting the importance of AMP optimization. In this context, the computer-aided design of AMPs has put together crucial information on chemical parameters and bioactivities in AMP sequences, thus providing modes of prediction to evaluate the antibacterial potential of a candidate sequence before synthesis. Quantitative structure-activity relationship (QSAR) computational models, for instance, have greatly contributed to AMP sequence optimization aimed at improved biological activities. In addition to machine-learning methods, the de novo design, linguistic model, pattern insertion methods, and genetic algorithms, have shown the potential to boost the automated design of AMPs. However, how successful have these approaches been in generating effective antibacterial drug candidates? Bearing this in mind, this review will focus on the main computational strategies that have generated AMPs with promising activities against pathogenic bacteria, as well as anti-infective potential in different animal models, including sepsis and cutaneous infections. Moreover, we will point out recent studies on the computer-aided design of antibiofilm peptides. As expected from automated design strategies, diverse candidate sequences with different structural arrangements have been generated and deposited in databases. We will, therefore, also discuss the structural diversity that has been engendered.

    更新日期:2020-01-23
  • Poplar Rows in Temperate Agroforestry Croplands Promote Bacteria, Fungi, and Denitrification Genes in Soils
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-23
    Lukas Beule; Ena Lehtsaar; Marife D. Corre; Marcus Schmidt; Edzo Veldkamp; Petr Karlovsky

    Agroforestry, which is the integration of trees into monoculture cropland, can alter soil properties and nutrient cycling. Temperate agroforestry practices have been shown to affect soil microbial communities as indicated by changes in enzyme activities, substrate-induced respiration, and microbial biomass. Research exploring soil microbial communities in temperate agroforestry with the help of molecular tools which allow for the quantification of microbial taxa and selected genes is scarce. Here, we quantified 13 taxonomic groups of microorganisms and nine genes involved in N cycling (N2 fixation, nitrification, and denitrification) in soils of three paired temperate agroforestry and conventional monoculture croplands using real-time PCR. The agroforestry croplands were poplar-based alley-cropping systems in which samples were collected in the tree rows as well as within the crop rows at three distances from the tree rows. The abundance of Acidobacteria, Actinobacteria, Alpha- and Gammaproteobacteria, Firmicutes, and Verrucomicrobia increased in the vicinity of poplar trees, which may be accounted for by the presence of persistent poplar roots as well as by the input of tree litter. The strongest population increase was observed for Basidiomycota, which was likely related to high soil moisture, the accumulation of tree litter, and the absence of tillage in the tree rows. Soil microorganisms carrying denitrification genes were more abundant in the tree rows than in the crop rows and monoculture systems, suggesting a greater potential for nitrate removal through denitrification, which may reduce nitrate leaching. Since microbial communities are involved in critical soil processes, we expect that the combination of real-time PCR with soil process measurements will greatly enhance insights into the microbial control of important soil functions in agroforestry systems.

    更新日期:2020-01-23
  • Shifts Between and Among Populations of Wheat Rhizosphere Pseudomonas, Streptomyces and Phyllobacterium Suggest Consistent Phosphate Mobilization at Different Wheat Growth Stages Under Abiotic Stress
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-23
    Claudia Breitkreuz; François Buscot; Mika Tarkka; Thomas Reitz

    Climate change models predict more frequent and prolonged drought events in Central Europe, which will exert extraordinary pressure on agroecosystems. One of the consequences is drought-related nutrient limitations for crops negatively affecting agricultural productivity. These effects can be mitigated by beneficial plant growth promoting rhizobacteria. In this study, we investigated the potential of cultivable bacterial species for phosphate solubilization in the rhizosphere of winter wheat at two relevant growth stages - stem elongation and grain filling stages. Rhizosphere samples were collected in the Global Change Experimental Facility in Central Germany, which comprises plots with conventional and organic farming systems under ambient and future climate. Phosphate-solubilizing bacteria were selectively isolated on Pikovskaya medium, phylogenetically classified by 16S rRNA sequencing, and tested for in vitro mineral phosphate solubilization and drought tolerance using plate assays. The culture isolates were dominated by members of the genera Phyllobacterium, Pseudomonas and Streptomyces. Cultivation-derived species richness and abundance of dominant taxa, especially within the genera Phyllobacterium and Pseudomonas, as well as composition of Pseudomonas species were affected by wheat growth stage. Pseudomonas was found to be more abundant at stem elongation than at grain filling, while for Phyllobacterium the opposite pattern was observed. The abundance of Streptomyces isolates remained stable throughout the studied growth stages. The temporal shifts in the cultivable fraction of the community along with considerable P solubilization potentials of Phyllobacterium and Pseudomonas species suggest functional redundancy between and among genera at different wheat growth stages. Phosphate-solubilizing Phyllobacterium species were assigned to Phyllobacterium ifriqiyense and Phyllobacterium sophorae. It is the first time that phosphate solubilization potential is described for these species. Since Phyllobacterium species showed the highest drought tolerance along all isolates, they may play an increasingly important role in phosphate solubilization in a future dryer climate.

    更新日期:2020-01-23
  • The Endophyte Pantoea alhagi NX-11 Alleviates Salt Stress Damage to Rice Seedlings by Secreting Exopolysaccharides
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-23
    Liang Sun; Peng Lei; Qian Wang; Junjie Ma; Yijing Zhan; Kang Jiang; Zongqi Xu; Hong Xu

    Endophytes have the potential to enhance the ability of plants to resist salt stress, improving crop development and yield. Therefore, in this study, we isolated an endophyte that produced large amounts of exopolysaccharides (EPSs) from the roots of sea rice and examined its effects on the physiological responses of rice (Oryza sativa L. ssp. japonica “Nipponbare”) seedlings to salt stress using hydroponic experiments. The endophyte was named Pantoea alhagi NX-11 based on its morphological characteristics and 16S ribosomal DNA (rDNA) sequence alignment. Rice seedlings that had been inoculated with P. alhagi NX-11 exhibited a 30.3% increase in fresh weight, a 28.6% increase in root length, a 51.6% increase in shoot length, and a 26.3% increase in chlorophyll content compared with control seedlings under normal conditions. In addition, inoculated rice seedlings had a 37.5% lower malondialdehyde content, a 133% higher K+/Na+ ratio, and a 52.8% higher proline content after 7 days under salt stress, as well as up-regulated expression of proline synthase, down-regulated expression of proline dehydrogenase, and enhanced antioxidant enzyme activities. Interestingly, rice seedlings that were inoculated with an EPS-deficient strain named NX-11eps– that was obtained by atmospheric and room temperature plasma (ARTP) mutagenesis were damaged by salt stress and had similar physiological and biochemical indicators to the control group. Therefore, we speculate that the ability of P. alhagi NX-11 to enhance the salt tolerance of rice seedlings is related to the EPSs it produces.

    更新日期:2020-01-23
  • The Balance Metabolism Safety Net: Integration of Stress Signals by Interacting Transcriptional Factors in Streptomyces and Related Actinobacteria
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-24
    Juan F. Martín; Paloma Liras

    Soil dwelling Streptomyces species are faced with large variations in carbon or nitrogen sources, phosphate, oxygen, iron, sulfur, and other nutrients. These drastic changes in key nutrients result in an unbalanced metabolism that have undesirable consequences for growth, cell differentiation, reproduction, and secondary metabolites biosynthesis. In the last decades evidence has accumulated indicating that mechanisms to correct metabolic unbalances in Streptomyces species take place at the transcriptional level, mediated by different transcriptional factors. For example, the master regulator PhoP and the large SARP-type regulator AfsR bind to overlapping sequences in the afsS promoter and, therefore, compete in the integration of signals of phosphate starvation and S-adenosylmethionine (SAM) concentrations. The cross-talk between phosphate control of metabolism, mediated by the PhoR–PhoP system, and the pleiotropic orphan nitrogen regulator GlnR, is very interesting; PhoP represses GlnR and other nitrogen metabolism genes. The mechanisms of control by GlnR of several promoters of ATP binding cassettes (ABC) sugar transporters and carbon metabolism are highly elaborated. Another important cross-talk that governs nitrogen metabolism involves the competition between GlnR and the transcriptional factor MtrA. GlnR and MtrA exert opposite effects on expression of nitrogen metabolism genes. MtrA, under nitrogen rich conditions, represses expression of nitrogen assimilation and regulatory genes, including GlnR, and competes with GlnR for the GlnR binding sites. Strikingly, these sites also bind to PhoP. Novel examples of interacting transcriptional factors, discovered recently, are discussed to provide a broad view of this interactions. Altogether, these findings indicate that cross-talks between the major transcriptional factors protect the cell metabolic balance. A detailed analysis of the transcriptional factors binding sequences suggests that the transcriptional factors interact with specific regions, either by overlapping the recognition sequence of other factors or by binding to adjacent sites in those regions. Additional interactions on the regulatory backbone are provided by sigma factors, highly phosphorylated nucleotides, cyclic dinucleotides, and small ligands that interact with cognate receptor proteins and with TetR-type transcriptional regulators. We propose to define the signal integration DNA regions (so called integrator sites) that assemble responses to different stress, nutritional or environmental signals. These integrator sites constitute nodes recognized by two, three, or more transcriptional factors to compensate the unbalances produced by metabolic stresses. This interplay mechanism acts as a safety net to prevent major damage to the metabolism under extreme nutritional and environmental conditions.

    更新日期:2020-01-23
  • Initial Transcriptomic Response and Adaption of Listeria monocytogenes to Desiccation on Food Grade Stainless Steel
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-26
    Martin Laage Kragh; Lisbeth Truelstrup Hansen

    The foodborne pathogen Listeria monocytogenes survives exposure to a variety of stresses including desiccation in the food industry. Strand-specific RNA sequencing was applied to analyze changes in the transcriptomes of two strains of L. monocytogenes (Lm 568 and Lm 08-5578) during desiccation [15°C, 43% relative humidity (RH)] on food grade stainless steel surfaces over 48 h to simulate a weekend with no food production. Both strains showed similar survival during desiccation with a 1.8–2 Log CFU/cm2 reduction after 48 h. Analysis of differentially expressed (DE) genes (>twofold, adjusted p-value <0.05) revealed that the initial response to desiccation was established after 6 h and remained constant with few new genes being DE after 12, 24, and 48 h. A core of 81 up- and 73 down-regulated DE genes were identified as a shared, strain independent response to desiccation. Among common upregulated genes were energy and oxidative stress related genes e.g., qoxABCD (cytochrome aa3) pdhABC (pyruvate dehydrogenase complex) and mntABCH (manganese transporter). Common downregulated genes related to anaerobic growth, proteolysis and the two component systems lmo1172/lmo1173 and cheA/cheY, which are involved in cold growth and flagellin production, respectively. Both strains upregulated additional genes involved in combatting oxidative stress and reactive oxygen species (ROS), including sod (superoxide dismutase), kat (catalase), tpx (thiol peroxidase) and several thioredoxins including trxAB, lmo2390 and lmo2830. Osmotic stress related genes were also upregulated in both strains, including gbuABC (glycine betaine transporter) and several chaperones clpC, cspA, and groE. Significant strain differences were also detected with the food outbreak strain Lm 08-5578 differentially expressing 1.9 × more genes (726) compared to Lm 568 (410). Unique to Lm 08-5578 was a significant upregulation of the expression of the alternative transcription factor σB and its regulon. A number of long antisense transcripts (lasRNA) were upregulated during desiccation including anti0605, anti0936, anti1846, and anti0777, with the latter controlling flagellum biosynthesis and possibly the downregulation of motility genes observed in both strains. This exploration of the transcriptomes of desiccated L. monocytogenes provides further understanding of how this bacterium encounters and survives the stress faced when exposed to dry conditions in the food industry.

    更新日期:2020-01-23
  • Designing Probiotic Therapies With Broad-Spectrum Activity Against a Wildlife Pathogen
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-26
    Xavier A. Harrison; Thomas Sewell; Matthew Fisher; Rachael E. Antwis

    Host-associated microbes form an important component of immunity that protect against infection by pathogens. Treating wild individuals with these protective microbes, known as probiotics, can reduce rates of infection and disease in both wild and captive settings. However, the utility of probiotics for tackling wildlife disease requires that they offer consistent protection across the broad genomic variation of the pathogen that hosts can encounter in natural settings. Here we develop multi-isolate probiotic consortia with the aim of effecting broad-spectrum inhibition of growth of the lethal amphibian pathogen Batrachochytrium dendrobatidis (Bd) when tested against nine Bd isolates from two distinct lineages. Though we achieved strong growth inhibition between 70 and 100% for seven Bd isolates, two isolates appeared consistently resistant to inhibition, irrespective of probiotic strategy employed. We found no evidence that genomic relatedness of the chytrid predicted similarity of inhibition scores, nor that increasing the genetic diversity of the bacterial consortia could offer stronger inhibition of pathogen growth, even for the two resistant isolates. Our findings have important consequences for the application of probiotics to mitigate wildlife diseases in the face of extensive pathogen genomic variation.

    更新日期:2020-01-23
  • A Novel Cold-Adaptive Endo-1,4-β-Glucanase From Burkholderia pyrrocinia JK-SH007: Gene Expression and Characterization of the Enzyme and Mode of Action
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-26
    Feifei Chen; Jianren Ye; Ayyappa Kumar Sista Kameshwar; Xuelian Wu; Jiahong Ren; Wensheng Qin; De-Wei Li

    The efficient industrial conversion of plant-derived cellulose to simple sugars and other value-added chemicals requires various highly stable and reactive enzymes. Industrial processes especially synchronous saccharification and fermentation (SSF)-based production of cellulosic bio-ethanol require enzymes that are active at lower temperatures. In this study, we have identified, characterized, and expressed the cold-adaptive endo-1,4-β-glucanase (BpEG) isolated from the Burkholderia pyrrocinia JK-SH007. The analysis of the predicted amino acid sequence indicated that BpEG belongs to GH family 8. The BpEG without the signal peptide was cloned into the expression vector pET32a and significantly expressed in Escherichia coli BL21 (DE3) competent cells. The SDS-PAGE and Western blot analysis of BpEG revealed that the recombinant BpEG was approximately 60 kDa. Purified recombinant BpEG exhibited hydrolytic activity against carboxymethyl cellulose (CMC) and phosphoric acid swollen cellulose (PASC), but not crystalline cellulose and xylan substrates. High performance, anion exchange, chromatography-pulsed amperometric detector (HPAEC-PAD) analysis of the enzymatic products obtained from depolymerization of 1,4-β-linked biopolymers of different lengths revealed an interesting cutting mechanism employed by endoglucanases. The recombinant BpEG exhibited 6.0 of optimum pH and 35°C of optimum temperature, when cultured with CMC substrate. The BpEG enzyme exhibited stable activity between pH 5.0 and 9.0 at 35°C. Interestingly, BpEG retained about 42% of its enzymatic activity at 10°C compared to its optimal temperature. This new cold-adaptive cellulase could potentially achieve synchronous saccharification and fermentation (SSF) making BpEG a promising candidate in the fields of biofuel, biorefining, food and pharmaceutical industries.

    更新日期:2020-01-23
  • A Critical Mutualism – Competition Interplay Underlies the Loss of Microbial Diversity in Sedentary Lifestyle
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-27
    Nazareth Castellanos; Gustavo G. Diez; Carmen Antúnez-Almagro; María Bailén; Carlo Bressa; Rocío González Soltero; Margarita Pérez; Mar Larrosa

    Physical exercise improves the overall health status by preventing the development of several diseases. In recent years, it has been observed that physical exercise impacts gut microbiota by increasing the presence of beneficial bacteria and microbial diversity. In contrast, a sedentary lifestyle increases the incidence of chronic diseases that often have an associated loss of microbial diversity. The gut microbiota is a vast ecosystem in which microorganisms interact with each other in different ways; however, microbial ecosystem interactions are scarcely studied. The goal of this study was to determine whether individuals with a sedentary lifestyle have lower diversity in their gut microbiota and how microbial diversity is associated with changes in bacterial network interactions. For that purpose, diet, body composition, physical activity, and sedentarism behavior were characterized for individuals who did or did not comply with the World Health Organization recommendations for physical activity. The composition of the gut microbiome was determined by 16S rRNA gene sequencing. Reorganization of microbial structure with lifestyle was approached from network analysis, where network complexity and the topology of positive and negative interdependences between bacteria were compared and correlated with microbial diversity. Sedentary lifestyle was significantly associated with a diet low in fiber and rich in sugars and processed meat, as well as with high visceral and total corporal fat composition. The diversity (phylogenic diversity, Chao, observed species, and Shannon’s index) and network complexity of the gut microbiota were significantly lower in sedentary compared to active individuals. Whereas mutualism or co-occurrence interactions were similar between groups, competitiveness was significantly higher in the active lifestyle group. The mutualism-competition ratio was moderate and positively associated with diversity in sedentary individuals, but not in active individuals. This finding indicates that there is a critical point in this ratio beyond which the stability of the microbial community is lost, inducing a loss of diversity.

    更新日期:2020-01-23
  • Comparative Analysis of Lactobacillus gasseri and Lactobacillus crispatus Isolated From Human Urogenital and Gastrointestinal Tracts
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-29
    Meichen Pan; Claudio Hidalgo-Cantabrana; Yong Jun Goh; Rosemary Sanozky-Dawes; Rodolphe Barrangou

    Lactobacillus crispatus and Lactobacillus gasseri are two of the main Lactobacillus species found in the healthy vaginal microbiome and have also previously been identified and isolated from the human gastrointestinal (GI) tract. These two ecological niches are fundamentally different, notably with regards to the epithelial cell type, nutrient availability, environmental conditions, pH, and microbiome composition. Given the dramatic differences between these two environments, we characterized strains within the same Lactobacillus species isolated from either the vaginal or intestinal tract to assess whether they are phenotypically and genetically different. We compared the genomes of the Lactobacillus strains selected in this study for genetic features of interest, and performed a series of comparative phenotypic assays including small intestinal juice and acid resistance, carbohydrate fermentation profiles, lactic acid production, and host interaction with intestinal Caco-2 and vaginal VK2 cell lines. We also developed a simulated vaginal fluid (SVF) to study bacterial growth in a proxy vaginal environment and conducted differential transcriptomic analysis between SVF and standard laboratory MRS medium. Overall, our results show that although strain-specific variation is observed, some phenotypic differences seem associated with the isolation source. We encourage future probiotic formulation to include isolation source and take into consideration genetic and phenotypic features for use at various body sites.

    更新日期:2020-01-23
  • The Complex Effect of Food Matrix Fat Content on Thermal Inactivation of Listeria monocytogenes: Case Study in Emulsion and Gelled Emulsion Model Systems
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-29
    Davy Verheyen; Marlies Govaert; Ti Kian Seow; Jonela Ruvina; Vivek Mukherjee; Maria Baka; Torstein Skåra; Jan F. M. Van Impe

    Previous studies on the influence of food matrix fat content on thermal inactivation kinetics of food pathogens have shown contradictory results due to the combined influence of fat content and other factors such as composition. Therefore, thermal inactivation of Listeria monocytogenes at 59, 64, and 69°C was systematically investigated in emulsion and gelled emulsion food model systems with various fat content (1, 5, 10, and 20%), such that the effect of fat content was isolated. Thermal conductivity and rheological properties of the model systems were quantified, as well as the effect of these properties on the thermal load of the model systems. Thermal conductivity was complexly related to fat content, the nature of the food matrix (i.e., viscous or gelled), and temperature. For the emulsions, the consistency index K increased with increasing fat content, while the flow behavior index n followed the opposite trend. For the gelled emulsions, the storage modulus G′ was always larger than the loss modulus G″ (i.e., measure of elastic and viscous properties, respectively). The phase angle δ [i.e., arctan (G″/G′)] was proportional with fat content, but this relation became more complex at higher temperatures. The thermal load of the model systems was not largely affected by food matrix fat content. Thermal inactivation of L. monocytogenes was investigated by means of the maximum specific inactivation rate kmax, log reductions, and sublethal injury (SI). Both for emulsions and gelled emulsions, kmax decreased with increasing fat content below approximately 60°C, while a more complex behavior was observed at higher temperatures. In the emulsions, log reductions were considerably lower (i.e., 2–3 log) at 1% fat than in systems with higher fat content. In the gelled emulsions, log reductions generally decreased with increasing fat content. SI decreased with increasing fat content, both in emulsions and gelled emulsions. In conclusion, the inactivation rate (i.e., kmax) of L. monocytogenes was affected by a complex relation between food matrix fat content, thermal conductivity, rheological properties, and inactivation temperature. Due to the small scale of the model systems, differences in kmax did not directly affect the final log reductions in a similar fashion.

    更新日期:2020-01-23
  • Spectral-Based Screening Approach Evaluating Two Specific Maize Lines With Divergent Resistance to Invasion by Aflatoxigenic Fungi
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-29
    Zuzana Hruska; Haibo Yao; Russell Kincaid; Feifei Tao; Robert L. Brown; Thomas E. Cleveland; Kanniah Rajasekaran; Deepak Bhatnagar

    In an effort to control aflatoxin contamination in food and/or feed grains, a segment of research has focused on host resistance to eliminate aflatoxin from susceptible crops, including maize. To this end, screening tools are key to identifying resistant maize genotypes. The traditional field screening techniques, the kernel screening laboratory assay (KSA), and analytical methods (e.g., ELISA) used for evaluating corn lines for resistance to fungal invasion, all ultimately require sample destruction. A technological advancement on the basic BGYF presumptive screening test, fluorescence hyperspectral imaging offers an option for non-destructive and rapid image-based screening. The present study aimed to differentiate fluorescence spectral signatures of representative resistant and susceptible corn hybrids infected by a toxigenic (SRRC-AF13) and an atoxigenic (SRRC-AF36) strain of Aspergillus flavus, at several time points (5, 7, 10, and 14 days), in order to evaluate fluorescence hyperspectral imaging as a viable technique for early, non-invasive aflatoxin screening in resistant and susceptible corn lines. The study utilized the KSA to promote fungal growth and aflatoxin production in corn kernels inoculated under laboratory conditions and to provide actual aflatoxin values to relate with the imaging data. Each time point consisted of 78 kernels divided into four groups (30-susceptible, 30-resistant, 9-susceptible control, and 9-resistant control), per inoculum. On specified days, kernels were removed from the incubator and dried at 60°C to terminate fungal growth. Dry kernels were imaged with a VNIR hyperspectral sensor (image spectral range of 400–1000 nm), under UV excitation centered at 365 nm. Following imaging, kernels were submitted for the chemical AflaTest assay (VICAM). Fluorescence emissions were compared for all samples over 14 days. Analysis of strain differences separating the fluorescence emission peaks of resistant from the susceptible strain indicated that the emission peaks of the resistant strain and the susceptible strains differed significantly (p < 0.01) from each other, and there was a significant difference in fluorescence intensity between the treated and control kernels of both strains. These results indicate a viable role of fluorescence hyperspectral imaging for non-invasive screening of maize lines with divergent resistance to invasion by aflatoxigenic fungi.

    更新日期:2020-01-23
  • Interacting Temperature, Nutrients and Zooplankton Grazing Control Phytoplankton Size-Abundance Relationships in Eight Swiss Lakes
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-30
    Francesco Pomati; Jonathan B. Shurin; Ken H. Andersen; Christoph Tellenbach; Andrew D. Barton

    Biomass distribution among size classes follows a power law where the Log-abundance of taxa scales to Log-size with a slope that responds to environmental abiotic and biotic conditions. The interactions between ecological mechanisms controlling the slope of locally realized size-abundance relationships (SAR) are however not well understood. Here we tested how warming, nutrient levels, and grazing affect the slope of phytoplankton community SARs in decadal time-series from eight Swiss lakes of the peri-alpine region, which underwent environmental forcing due to climate change and oligotrophication. We expected rising temperature to have a negative effect on slope (favoring small phytoplankton), and increasing nutrient levels and grazing pressure to have a positive effect (benefiting large phytoplankton). Using a random forest approach to extract robust patterns from the noisy data, we found that the effects of temperature (direct and indirect through water column stability), nutrient availability (phosphorus and total biomass), and large herbivore (copepods and daphnids) grazing and selectivity on slope were non-linear and interactive. Increasing water temperature or total grazing pressure, and decreasing phosphorus levels, had a positive effect on slope (favoring large phytoplankton, which are predominantly mixotrophic in the lake dataset). Our results therefore showed patterns that were opposite to the expected long-term effects of temperature and nutrient levels, and support a paradigm in which (i) small phototrophic phytoplankton appear to be favored under high nutrients levels, low temperature and low grazing, and (ii) large mixotrophic algae are favored under oligotrophic conditions when temperature and grazing pressure are high. The effects of temperature were stronger under nutrient limitation, and the effects of nutrients and grazing were stronger at high temperature. Our study shows that the phytoplankton local SARs in lakes respond to both the independent and the interactive effects of resources, grazing and water temperature in a complex, unexpected way, and observations from long-term studies can deviate significantly from general theoretical expectations.

    更新日期:2020-01-23
  • Structural Insights Into the Transcriptional Regulation of HigBA Toxin–Antitoxin System by Antitoxin HigA in Pseudomonas aeruginosa
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-30
    Ying Liu; Zengqiang Gao; Guangfeng Liu; Zhi Geng; Yuhui Dong; Heng Zhang

    HigB-HigA is a bacterial toxin–antitoxin (TA) system in which the antitoxin HigA can mask the endoribonuclease activity of toxin HigB and repress the transcription of the TA operon by binding to its own promoter region. The opportunistic pathogen Pseudomonas aeruginosa HigBA (PaHigBA) is closely associated with the pathogenicity by reducing the production of multiple virulence factors and biofilm formation. However, the molecular mechanism underlying HigBA TA operon transcription by PaHigA remains elusive. Here, we report the crystal structure of PaHigA binding to the promoter region of higBA operon containing two identical palindromic sequences at 3.14 Å resolution. The promoter DNA is bound by two cooperative dimers to essentially encircle the intact palindrome region. The helix-turn-helix (HTH) motifs from the two dimers insert into the major grooves of the DNA at the opposite sides. The DNA adopts a canonical B-DNA conformation and all the hydrogen bonds between protein and DNA are mediated by the DNA phosphate backbone. A higher resolution structure of PaHigA-DNA complex at 2.50 Å further revealed three water molecules bridged the DNA-binding interface and mediated the interactions between the bases of palindromic sequences and PaHigA (Thr40, Asp43, and Arg49). Structure-based mutagenesis confirmed these residues are essential for the specific DNA-binding ability of PaHigA. Our structure–function studies therefore elucidated the cooperative dimer–dimer transcription repression mechanism, and may help to understand the regulation of multiple virulence factors by PaHigA in P. aeruginosa.

    更新日期:2020-01-23
  • Microbiome Dynamics Associated With the Atacama Flowering Desert
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-30
    Juan Pablo Araya; Máximo González; Massimiliano Cardinale; Sylvia Schnell; Alexandra Stoll

    In a desert, plants as holobionts quickly respond to resource pulses like precipitation. However, little is known on how environment and plants modulate the rhizosphere-associated microbiome. As a model species to represent the Atacama Desert bloom, Cistanthe longiscapa (Montiaceae family) was selected to study the influence of abiotic and biotic environment on the diversity and structure of the microbiota associated to its rhizosphere. We analyzed the rhizosphere and soil microbiome along a North-South precipitation gradient and between a dry and rainy year by using Illumina high−throughput sequencing of 16S rRNA gene fragments and ITS2 regions for prokaryotes and fungi, respectively. In the rhizosphere of C. longiscapa the microbiota clearly differs in composition and structure from the surrounding bulk soil. The fungal and bacterial communities respond differently to environmental conditions. The diversity and richness of fungal OTUs were negatively correlated with aridity, as predicted. The community structure was predominantly influenced by other soil characteristics (pH, organic matter content) but not by aridity. In contrast, diversity, composition, and structure of the bacterial community were not influenced by aridity or any other evaluated soil parameter. These findings coincide with the identification of mainly site-specific microbial communities, not shared along the sites. These local communities contain a group of OTUs, which are exclusive to the rhizosphere of each site and presumably vertically inherited as seed endophytes. Their ecological functions and dispersal mechanisms remain unclear. The analysis of co-occurrence patterns highlights the strong effect of the desert habitat over the soil- and rhizosphere-microbiome. The site-independent enrichment of only a small bacterial cluster consistently associated with the rhizosphere of C. longiscapa further supports this conclusion. In a rainy year, the rhizosphere microbiota significantly differed from bulk and bare soil, whereas in a dry year, the community structure of the former rhizosphere approximates to the one found in the bulk. In the context of plant–microbe interactions in desert environments, our study contributes new insights into the importance of aridity in microbial community structure and composition, discovering the influence of other soil parameters in this complex dynamic network, which needs further to be investigated.

    更新日期:2020-01-23
  • Oral Supplementation of Lead-Intolerant Intestinal Microbes Protects Against Lead (Pb) Toxicity in Mice
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-31
    Qixiao Zhai; Dingwu Qu; Saisai Feng; Yaqi Yu; Leilei Yu; Fengwei Tian; Jianxin Zhao; Hao Zhang; Wei Chen

    Oral exposure to the heavy metal lead (Pb) causes various dysfunctions in animals. However, the influence of gut bacteria on Pb absorption, bioaccumulation, and excretion is largely unknown. In this study, we use a mouse model to investigate the relationship between gut microbiota, Pb-intolerant intestinal microbes and Pb toxicity. First, mice were treated with a broad-spectrum antibiotic cocktail to deplete their gut microbiota, and were then acutely and orally exposed to Pb at 1304 mg/kg for 3 days. Compared to the control mice, antibiotic-treated mice had increased Pb concentrations in the blood and primary organs and decreased Pb fecal concentrations, suggesting that gut microbiota limited the Pb burden that developed from acute oral Pb exposure. Next, three Pb-intolerant gut microbes, Akkermansia muciniphila, Faecalibacterium prausnitzii, and Oscillibacter ruminantium, were orally administered to mice, and their effects against Pb toxicity were evaluated. F. prausnitzii treatment significantly promoted the fecal Pb excretion and reduced Pb concentrations in blood (from 152.70 ± 25.62 μg/dL to 92.20 ± 24.33 μg/dL) and primary tissues. Supplementation with O. ruminantium significantly decreased Pb concentrations in blood (from 152.70 ± 25.62 μg/dL to 104.60 ± 29.85 μg/dL) and kidney (from 7.30 ± 1.08 μg/g to 5.64 ± 0.79 μg/g). Treatment with F. prausnitzii and O. ruminantium also upregulated tight junction (TJ) protein expression and the production of short-chain fatty acids by colonic microbiota, and showed protective effects against liver and kidney toxicity. These results indicate the potential for reducing Pb toxicity by the modulation of gut microbiota.

    更新日期:2020-01-22
  • WHI-2 Regulates Intercellular Communication via a MAP Kinase Signaling Complex
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-31
    A. Pedro Gonçalves; Karen M. Chow; Sara Cea-Sánchez; N. Louise Glass

    The formation of the fungal mycelial network is facilitated by somatic cell fusion of germinating asexual spores (or germlings). Neurospora crassa germlings in close proximity display chemotropic growth that is dependent upon an intracellular network of mitogen-activated protein kinase (MAPK) signaling cascades. Approximately 80 genes involved in intercellular communication and fusion have been identified, including three mutants with similar morphological phenotypes: Δwhi-2, Δcsp-6, and Δamph-1. Here we show that WHI-2 localizes to the cell periphery and regulates endocytosis, mitochondrial organization, sporulation, and cell fusion. WHI-2 was required to transduce signals through a conserved MAPK pathway (NRC-1/MEK-2/MAK-2) and target transcription factors (PP-1/ADV-1). The amph-1 locus encodes a Bin/Amphiphysin/Rvs domain-containing protein and mis-expression of whi-2 compensated for the cell fusion and endocytosis deficiencies of a Δamph-1 mutant. The csp-6 locus encodes a haloacid dehalogenase phosphatase whose activity was essential for cell fusion. Although fusion-deficient with themselves, cells that lacked whi-2, csp-6, or amph-1 showed a low frequency of chemotropic interactions with wild type cells. We hypothesize that WHI-2 could be important for signal perception during chemotropic interactions via a role in endocytosis.

    更新日期:2020-01-22
  • Identification of Small-Molecule Inhibitors of Brucella Diaminopimelate Decarboxylase by Using a High-Throughput Screening Assay
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-06
    Pengfei Bie; Xiaowen Yang; Cunrui Zhang; Qingmin Wu

    Brucellosis, caused by intracellular gram-negative pathogens of the genus Brucella, continues to be one of the most pandemic zoonotic diseases in most countries. At present, the therapeutic treatment of brucellosis relies on a combination of multiple antibiotics that involves a long course of treatment, easy relapse, and high side effects from the use of certain antibiotics (such as streptomycin). Thus, the need to identify novel drugs or targets to control this disease is urgent. Diaminopimelate decarboxylase (DAPDC), a key enzyme involved in the bacterial diaminopimelate (DAP) biosynthetic pathway, was suggested to be a promising anti-Brucella target in our previous study. In this work, the biological activity of Brucella melitensis DAPDC was characterized, and a library of 1,591 compounds was screened for inhibitors of DAPDC. The results of a high-throughput screening (HTS) assay showed that 24 compounds inhibited DAPDC activity. In a further in vitro bacterial inhibition experiment, five compounds exhibited anti-Brucella activity (SID3, SID4, SID14, SID15, and SID20). These results suggested that the identified compounds can be used as potent molecules against brucellosis and that the application ranges of these approved drugs can be expanded in the future.

    更新日期:2020-01-22
  • Characterization of Influenza A Virus Infection in Mouse Pulmonary Stem/Progenitor Cells
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-06
    Tai-Ling Chao; Sing-Yi Gu; Pi-Han Lin; Yu-Tien Chou; Thai-Yen Ling; Sui-Yuan Chang

    The pulmonary stem/progenitor cells, which could be differentiated into downstream cells to repair tissue damage caused by influenza A virus, have also been shown to be the target cells of influenza virus infection. In this study, mouse pulmonary stem/progenitor cells (mPSCs) with capability to differentiate into type I or type II alveolar cells were used as an in vitro cell model to characterize replication and pathogenic effects of influenza viruses in PSCs. First, mPSCs and its immortalized cell line mPSCsOct4+ were shown to be susceptible to PR8, seasonal H1N1, 2009 pandemic H1N1, and H7N9 influenza viruses and can generate infectious virus particles, although with a lower virus titer, which could be attributed by the reduced vRNA replication and nucleoprotein (NP) aggregation in the cytoplasm. Nevertheless, a significant increase of interleukin (IL)-6 and interferon (IFN)-γ at 12 h and IFN-β at 24 h post infection in mPSCs implicates that mPSCs might function as a sensor to modulate immune responses to influenza virus infection. In summary, our results demonstrated mPSCs, as one of the target cells for influenza A viruses, could modulate early proinflammatory responses to influenza virus infection.

    更新日期:2020-01-22
  • Cyclic AMP-CRP Modulates the Cell Morphology of Klebsiella pneumoniae in High-Glucose Environment
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-10
    Long Liu; Feiyu Li; Li Xu; Jingjie Wang; Moran Li; Jie Yuan; Hui Wang; Ruiping Yang; Bei Li

    Bacteria can modify their morphology in response to environmental stimuli for survival or host defense evasion. The rich glucose in vivo or in the Luria–Bertani (LB) medium shortened the cell length of Klebsiella pneumoniae. The environmental glucose decreased the levels of cyclic AMP (cAMP) and the transcription of crp, which declined the cAMP–cAMP receptor protein (cAMP-CRP) activity. The cell length of crp deletion mutant was significantly shorter than that of the wild type (0.981 ± 0.057 μm vs. 2.415 ± 0.075 μm, P < 0.001). These results indicated that the high environmental glucose alters the bacterial morphology to a round form through regulating the activity of cAMP-CRP complex. Comparative proteomics analysis showed increased expression of 10 proteins involved in cell division or cell wall biosynthesis in the crp deletion strain. Five of them (ompA, tolB, ybgC, ftsI, and rcsF) were selected to verify their expression in the high-glucose environment, and overexpression of tolB or rcsF shortened the bacterial length similar to that of the crp deletion strain. Electrophoretic mobility shift assay indicated that CRP directly negatively regulates the transcription of tolB and rcsF by binding to the promoter regions. This study first proved the role and partial regulation mechanism of CRP in altering cell morphology during infection and provided a theoretical basis for elucidating the mechanism in diabetes mellitus susceptible to K. pneumoniae.

    更新日期:2020-01-22
  • Evolutionary Histories of Type III Polyketide Synthases in Fungi
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-16
    Jorge Carlos Navarro-Muñoz; Jérôme Collemare

    Type III polyketide synthases (PKSs) produce secondary metabolites with diverse biological activities, including antimicrobials. While they have been extensively studied in plants and bacteria, only a handful of type III PKSs from fungi has been characterized in the last 15 years. The exploitation of fungal type III PKSs to produce novel bioactive compounds requires understanding the diversity of these enzymes, as well as of their biosynthetic pathways. Here, phylogenetic and reconciliation analyses of 522 type III PKSs from 1,193 fungal genomes revealed complex evolutionary histories with massive gene duplications and losses, explaining their discontinuous distribution in the fungal tree of life. In addition, horizontal gene transfer events from bacteria to fungi and, to a lower extent, between fungi, could be inferred. Ancestral gene duplication events have resulted in the divergence of eight phylogenetic clades. Especially, two clades show ancestral linkage and functional co-evolution between a type III PKS and a reducing PKS genes. Investigation of the occurrence of protein domains in fungal type III PKS predicted gene clusters highlighted the diversity of biosynthetic pathways, likely reflecting a large chemical landscape. Type III PKS genes are most often located next to genes encoding cytochrome P450s, MFS transporters and transcription factors, defining ancestral core gene clusters. This analysis also allowed predicting gene clusters for the characterized fungal type III PKSs and provides working hypotheses for the elucidation of the full biosynthetic pathways. Altogether, our analyses provide the fundamental knowledge to motivate further characterization and exploitation of fungal type III PKS biosynthetic pathways.

    更新日期:2020-01-21
  • Key Transitions in the Evolution of Rapid and Slow Growing Mycobacteria Identified by Comparative Genomics
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-16
    Nathan L. Bachmann; Rauf Salamzade; Abigail L. Manson; Richard Whittington; Vitali Sintchenko; Ashlee M. Earl; Ben J. Marais

    Mycobacteria have been classified into rapid and slow growing phenotypes, but the genetic factors that underlie these growth rate differences are not well understood. We compared the genomes of 157 mycobacterial species, representing all major branches of the mycobacterial phylogenetic tree to identify genes and operons enriched among rapid and slow growing mycobacteria. Overlaying growth phenotype on a phylogenetic tree based on 304 core genes suggested that ancestral mycobacteria had a rapid growth phenotype with a single major evolutionary separation into rapid and slow growing sub-genera. We identified 293 genes enriched among rapid growing sub-genera, including genes encoding for amino acid transport/metabolism (e.g., livFGMH operon) and transcription, as well as novel ABC transporters. Loss of the livFGMH and ABC transporter operons among slow growing species suggests that reduced cellular amino acid transport may be growth limiting. Comparative genomic analysis suggests that horizontal gene transfer, from non-mycobacterial genera, may have contributed to niche adaptation and pathogenicity, especially among slow growing species. Interestingly, the mammalian cell entry (mce) operon was found to be ubiquitous, irrespective of growth phenotype or pathogenicity, although protein sequence homology between rapid and slow growing species was low (<50%). This suggests that the mce operon was present in ancestral rapid growing species, but later adapted by slow growing species for use as a mechanism to establish an intra-cellular lifestyle.

    更新日期:2020-01-21
  • Prediction of Neighbor-Dependent Microbial Interactions From Limited Population Data
    Front. Microbiol. (IF 4.259) Pub Date : 2019-12-18
    Joon-Yong Lee; Shin Haruta; Souichiro Kato; Hans C. Bernstein; Stephen R. Lindemann; Dong-Yup Lee; Jim K. Fredrickson; Hyun-Seob Song

    Modulation of interspecies interactions by the presence of neighbor species is a key ecological factor that governs dynamics and function of microbial communities, yet the development of theoretical frameworks explicit for understanding context-dependent interactions are still nascent. In a recent study, we proposed a novel rule-based inference method termed the Minimal Interspecies Interaction Adjustment (MIIA) that predicts the reorganization of interaction networks in response to the addition of new species such that the modulation in interaction coefficients caused by additional members is minimal. While the theoretical basis of MIIA was established through the previous work by assuming the full availability of species abundance data in axenic, binary, and complex communities, its extension to actual microbial ecology can be highly constrained in cases that species have not been cultured axenically (e.g., due to their inability to grow in the absence of specific partnerships) because binary interaction coefficients – basic parameters required for implementing the MIIA – are inestimable without axenic and binary population data. Thus, here we present an alternative formulation based on the following two central ideas. First, in the case where only data from axenic cultures are unavailable, we remove axenic populations from governing equations through appropriate scaling. This allows us to predict neighbor-dependent interactions in a relative sense (i.e., fractional change of interactions between with versus without neighbors). Second, in the case where both axenic and binary populations are missing, we parameterize binary interaction coefficients to determine their values through a sensitivity analysis. Through the case study of two microbial communities with distinct characteristics and complexity (i.e., a three-member community where all members can grow independently, and a four-member community that contains member species whose growth is dependent on other species), we demonstrated that despite data limitation, the proposed new formulation was able to successfully predict interspecies interactions that are consistent with experimentally derived results. Therefore, this technical advancement enhances our ability to predict context-dependent interspecies interactions in a broad range of microbial systems without being limited to specific growth conditions as a pre-requisite.

    更新日期:2020-01-21
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