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  • Shotgun proteomics provides an insight into pathogenesis related proteins using anamorphic stage of the biotroph, Erysiphe pisi pathogen of garden pea
    Microbiol. Res. (IF 2.777) Pub Date : 2019-02-12
    Malathi Bheri, Sheetal M. Bhosle, Ragiba Makandar

    E. pisi is an ascomycete member causing powdery mildew disease of garden pea. It is a biotrophic pathogen requiring a living host for its survival. Our understanding of molecular mechanisms underlying its pathogenesis is limited. The identification of proteins expressed in the pathogen is required to gain an insight into the functional mechanisms of an obligate biotrophic fungal pathogen. In this study, the proteome of the anamorphic stage of E. pisi pathogen has been elucidated through the nano LC-MS/MS approach. A total of 328 distinct proteins were detected from Erysiphe isolates infecting the susceptible pea cultivar, Arkel. The proteome is available via ProteomeXchange with identifier PXD010238. The functional classification of protein accessions based on Gene Ontology revealed proteins related to signal transduction, secondary metabolite formation and stress which might be involved in virulence and pathogenesis. The functional validation carried through differential expression of genes encoding G-protein beta subunit, a Cyclophilin (Peptidyl prolyl cis-transisomerase) and ABC transporter in a time course study confirmed their putative role in pathogenesis between resistant and susceptible genotypes, JI2480 and Arkel. The garden pea-powdery mildew pathosystem is largely unexplored, therefore, the identified proteome provides a first-hand information and will form a basis to analyse mechanisms involving pathogen survival, pathogenesis and virulence.

  • Enhanced Nitrogen and Phosphorus Activation with an Optimized Bacterial Community by Endophytic Fungus Phomopsis liquidambari in Paddy Soil
    Microbiol. Res. (IF 2.777) Pub Date : 2019-02-11
    Meng-Jun Tang, Qiang Zhu, Feng-Min Zhang, Wei Zhang, Jie Yuan, Kai Sun, Fang-Ji Xu, Chuan-Chao Dai
  • An alternative biocontrol agent of soil-borne phytopathogens: a new antifungal compound produced by a plant growth promoting bacterium isolated from North Algeria
    Microbiol. Res. (IF 2.777) Pub Date : 2019-02-10
    Bruna Agrillo, Sara Mirino, Rosarita Tatè, Lorena Gratino, Marta Gogliettino, Ennio Cocca, Nassira Tabli, Elhafid Nabti, Gianna Palmieri

    Bacteria isolated from different environments can be exploited for biocontrol purposes by the identification of the molecules involved in the antifungal activity. The present study was aimed at investigating antifungal protein compounds purified from a previously identified plant growth promoting bacterium, Pseudomonas protegens N isolated from agricultural land in northern Algeria. Therefore, a novel protein was purified by chromatographic and ultrafiltration steps and its antifungal activity together with growth-inhibition mechanism was evaluated against different fungi by plate-based assays. In addition, stereomicroscopy and transmission electron microscopy (TEM) was performed to explore the inhibition activity of the compound on spore germination processes. The protein, showing a molecular mass of about 100 kDa under native conditions, was revealed to be in the surface-membrane fraction and displayed an efficient activity against a variety of phytopathogenic fungi, being Alternaria the best target towards which it exhibited a marked fungicidal action and inhibition of spore germination. Moreover, the compound was able to significantly decrease fungal infection on tomato fruits producing also morphological aberrations on conidia. The obtained results suggested that the isolated compound could represent a promising agent for eco-friendly management of plant pathogens in agriculture.

  • Plant Beneficial Endophytic Bacteria: Mechanisms, Diversity, Host Range and Genetic Determinants
    Microbiol. Res. (IF 2.777) Pub Date : 2019-02-04
    Imran Afzal, Zabta Khan Shinwari, Shomaila Sikandar, Shaheen Shahzad

    Endophytic bacteria are the plant beneficial bacteria that thrive inside plants and can improve plant growth under normal and challenging conditions. They can benefit host plants directly by improving plant nutrient uptake and by modulating growth and stress related phytohormones. Indirectly, endophytic bacteria can improve plant health by targeting pests and pathogens with antibiotics, hydrolytic enzymes, nutrient limitation, and by priming plant defenses. To confer these benefits, the bacteria have to colonize the plant endosphere after colonizing the rhizosphere. The colonization is achieved using a battery of traits involving motility, attachment, plant-polymer degradation, and evasion of plant defenses. The diversity of endophytic colonizers depends on several bacteria, plant and environment specific factors. Some endophytic bacteria can have a broad host range and can be used as bioinoculants in developing a safe and sustainable agriculture system. This review elaborates the factors affecting diversity of bacterial endophytes, their host specificity and mechanisms of plant growth promotion. The review also accentuates various methods used to study endophytic communities, wild plants as a source of novel endophytic bacteria, and innovative approaches that may improve plant-endophyte association. Moreover, bacterial genes expressed in planta and challenges to study them are also discussed.

  • Muscodor brasiliensis sp. nov. produces volatile organic compounds with activity against Penicillium digitatum
    Microbiol. Res. (IF 2.777) Pub Date : 2019-01-18
    Lorena C. Pena, Gustavo H. Jungklaus, Daiani C. Savi, Lisandra Ferreira-Maba, André Servienski, Beatriz H.L.N.S. Maia, Vinicius Annies, Lygia V. Galli-Terasawa, Chirlei Glienke, Vanessa Kava
  • 更新日期:2019-01-23
  • Communication mechanisms in extremophiles: Exploring their existence and industrial applications
    Microbiol. Res. (IF 2.777) Pub Date : 2019-01-18
    Amandeep Kaur, Neena Capalash, Prince Sharma

    Quorum sensing plays important roles in the regulation of physiological and virulence processes in most bacteria, but its role in extremophiles is largely unknown. Comparative genomic, phylogenic, structural and signaling pathways analyses and deletion mutant studies have suggested the presence of three major quorum sensing systems (AI-1, Peptide based and AI-2) in extremophiles. Autoinducer-1(AI-1) system was found to be most prevalent (except in thermophiles where it is autoinducer-2) while peptide based system was least prevalent in extremophiles. Some unknown mechanisms of quorum sensing have also been reported which need further exploration. Quorum sensing is utilized by extremophiles for processes like cold adaptation, lowering the freezing point, biofilm formation, oxidative stress resistance and persister cell formation. Explication of quorum sensing in extreme environments may provide discernment regarding the role and functional strategies for survival of extremophiles. Here the role of quorum sensing in different classes of extremophiles and also in their survival strategies has been reviewed. Further, the applications and problems caused by quorum sensing regulated factors in extreme environments are discussed.

  • Comparative genomics of Lactobacillus sakei supports the development of starter strain combinations
    Microbiol. Res. (IF 2.777) Pub Date : 2019-01-14
    Lara Eisenbach, Andreas J. Geissler, Matthias A. Ehrmann, Rudi F. Vogel

    Strains of Lactobacillus sakei can be isolated from a variety of sources including meat, fermented sausages, sake, sourdough, sauerkraut or kimchi. Selected strains are widely used as starter cultures for sausage fermentation. Recently we have demonstrated that control about the lactic microbiota in fermenting sausages is achieved rather by pairs or strain sets than by single strains. In this work we characterized the pan genome of L. sakei to enable exploitation of the genomic diversity of L. sakei for the establishment of assertive starter strain sets. We have established the full genome sequences of nine L. sakei strains from different sources of isolation and included in the analysis the genome of L. sakei 23 K. Comparative genomics revealed an accessory genome comprising about 50% of the pan genome and different lineages of strains with no relation to their source of isolation. Group and strain specific differences could be found, which namely referred to agmatine and citrate metabolism. The presence of genes encoding metabolic pathways for fructose, sucrose and trehalose as well as gluconate in all strains suggests a general adaptation to plant/sugary environments and a life in communities with other genera. Analysis of the plasmidome did not reveal any specific mechanisms of adaptation to a habitat. The predicted differences of metabolic settings enable prediction of partner strains, which can occupy the meat environment to a large extent and establish competitive exclusion of autochthonous microbiota. This may assist the development of a new generation of meat starter cultures containing L. sakei strains.

  • Differential involvement of glycans in the binding of Staphylococcus epidermidis and Corynebacterium spp. to human sweat
    Microbiol. Res. (IF 2.777) Pub Date : 2018-12-30
    Chi-Hung Lin, Robyn A Peterson, Audrey Gueniche, Ségolène Adam de Beaumais, Virginie Hourblin, Lionel Breton, Maria Dalko, Nicolle H. Packer

    Sweat is a secretory fluid that can be a source of unpleasant body odour due to interaction of resident bacteria with sweat components. Identification of glycoproteins in sweat suggests that protein-conjugated glycans may act as binding epitopes for bacteria, as found in other secretory fluids such as human milk, tears and saliva which help to protect epithelial surfaces from infection. We conducted proteomic and glycomic analysis of sweat to reveal an abundance of glycoproteins, predominantly carrying bi-antennary sialylated N-glycans with or without fucose. A fluorescent plate assay was used to determine whether glycans on sweat proteins provide binding epitopes for odour-producing skin commensals Staphylococcus epidermidis and Corynebacterium. Sialic acid and fucose were found to be important binding epitopes for S. epidermidis 3-22-BD-6, a strain recently isolated from human sweat, whereas fucose (but not sialic acid) contributed to the binding of Type strain S. epidermidis ATCC 12228. In contrast, our results indicate that sweat N-glycans do not provide binding epitopes for Corynebacterium. Synthetic sugar mimics of Lewis blood group antigens were investigated as potential inhibitors of the binding of S. epidermidis 3-22-BD-6 to sweat. Pre-incubation of the bacterium with LeB, LeX, LeY and sLeX (pentaose) resulted in a significant reduction in sweat protein adhesion indicating that terminal fucose is a key binding epitope, particularly when linked to a Type 2 chain (Galβ1-4GlcNAc) configuration (LeY). Our results form an impetus for future studies seeking to elucidate the role of glycans in sweat associated malodour, with possible implications for cosmetic and medical fields.

  • Sestrin-like protein from Dictyostelium discoideum is involved in autophagy under starvation stress
    Microbiol. Res. (IF 2.777) Pub Date : 2018-12-28
    S Rafia, S Saran

    Sestrins are highly conserved; stress inducible proteins that help maintain metabolic homeostasis and protect cells under stress conditions. They are up-regulated during stress and influence AMPK and mTOR pathways. Our objective was to find the role of Sestrin protein from Dictyostelium discoideum (Dd), a lower eukaryote where starvation stress initiates multicellular development. The single DdSesn-like gene was expressed and its endogenous functions were characterized. Both, the knockout and constitutively expressing strains were made and their involvement in starvation-induced autophagy was analyzed. Autophagic fluxes and ROS levels were also monitored. Additionally, overexpression of DdSesn decreased cell growth and showed a longer lag phase. Upon starvation both DdSesn and ROS levels increased. SesnOE showed reduced ROS levels while sesn- showed increased ROS levels when compared to the wild type. Therefore, we suggest that increased sesn expression may be beneficial in reducing ROS levels during starvation. Deletion of sesn showed reduced autophagic flux and increased p4EBP1 levels. We show that DdSesn promotes autophagy in D. discoideum upon starvation.

  • Differential gene expression in two grapevine cultivars recovered from “flavescence dorée”
    Microbiol. Res. (IF 2.777) Pub Date : 2018-12-28
    Davide Pacifico, Paolo Margaria, Luciana Galetto, Mauro Legovich, Simona Abbà, Flavio Veratti, Cristina Marzachì, Sabrina Palmano

    The biological bases of recovery of two grapevine cultivars, Nebbiolo and Barbera, showing different susceptibility and recovery ability to “flavescence dorée” (FD) phytoplasma infection were investigated. The expression over one vegetative season, in FD-recovered and healthy grapevines, of 18 genes involved in defence, hydrogen peroxide and hormone production was verified at two time points. Difference (Δ) between the relative expressions of August and July were calculated for each target gene of both cultivars. The significance of differences among groups assessed by univariate and multivariate statistical methods, and sPLS-DA analyses of the Δ gene expression values, showed that control and recovered grapevines of both cultivars were clearly separated. The Barbera-specific deregulation of defence genes supports a stronger response of this variety, within a general frame of interactions among H2O2, jasmonate and ethylene metabolisms, common to both varieties. This may strengthen the hypothesis that FD-recovered Barbera grapevines modulate transcription of their genes to cope with potential damages associated to the alteration of their oxidative status. Nebbiolo variety would fit into this picture, although with a less intense response, in line with its lower degree of susceptibility and recovery incidence to FD, compared to Barbera. The results evidenced a scenario where plant response to phytoplasma infection is highly affected by climatic and edaphic conditions. Nevertheless, even after several years from the original FD infection, it was still possible to distinguish, at molecular level, control and recovered grapevines of both cultivars by analyzing their overall-season response, rather than that of a single time point.

  • Reactive green dye remediation by Alternanthera philoxeroides in association with plant growth promoting Klebsiella sp. VITAJ23; A pot culture study
    Microbiol. Res. (IF 2.777) Pub Date : 2018-12-21
    Astha Sinha, Sajitha Lulu S, Vino S, W.Jabez Osborne

    Contamination of soil by textile effluent is a major threat found worldwide. These pollutants have a diverse negative effects on the ecosystem, therefore restoration through a cost biological strategy is the need of the hour. The aim of the current study was to enhance the decolourization of reactive green dye (RGD) by phytoremediation coupled with augmentation of effective bacteria to the rhizosphere. The isolate Klebsiella sp. VITAJ23 was isolated from textile polluted soil and was assessed for plant growth promoting traits (PGP) and the PGP functional genes were amplified. The soil was artificially polluted with RGD concentration ranging from 1000-3000 mg kg-1 and Alternanthera philoxeroides plantlets were planted in phyto and rhizoremediation treatments, setup was maintained upto 60 d. The isolate VITAJ23 was augmented in the rhizoremediation setup and the morphological were assessed at regular interval. There was a significant increase in the chlorophyll content as well as root and shoot length of the plant treated with bacterial suspension. Decolourization study revealed 79% removal of reactive green dye with an enhanced oxido-reductase enzyme activity in the plants bioaugmented with bacteria. The biodegraded metabolites were identified as 2-allylnapthalene, l-alanine, n-acetyl-and propenoic acid by GC-MS analysis and a plant-bacteria degradation pathway was predicted. Inoculation of PGP- Klebsiella sp. VITAJ23 enhanced the rate of plant growth and dye degradation.

  • Specific regions of the SulA protein recognized and degraded by the ATP-dependent ClpYQ (HslUV) protease in Escherichia coli
    Microbiol. Res. (IF 2.777) Pub Date : 2018-12-17
    Chun-Yang Chang, Yu-Ting Weng, Lin-Yi Hwang, Hui-Ting Hu, Po-Shu Shih, Jung-En Kuan, Ke-Feng Wu, Whei-Fen Wu

    In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease, in which ClpQ is the peptidase subunit and ClpY is the ATPase and unfoldase. ClpY functions to recognize protein substrates, and denature and translocate the unfolded polypeptides into the proteolytic site of ClpQ for degradation. However, it is not clear how the substrates are recognized by the ClpYQ protease and the mechanism by which the substrates are selected, unfolded and translocated by ClpY into the interior site of ClpQ hexamers. Both Lon and ClpYQ proteases can degrade SulA, a cell division inhibitor, in bacterial cells. In this study, using yeast two-hybrid and in vivo degradation analyses, we first demonstrated that the C-terminal internal hydrophobic region (139th~ 149th aa) of SulA is necessary for binding and degradation by ClpYQ. A conserved region, GFIMRP, between 142th and 147th residues of SulA, were identified among various Gram-negative bacteria. By using MBP-SulA(F143Y) (phenylalanine substituted with tyrosine) as a substrate, our results showed that this conserved residue of SulA is necessary for recognition and degradation by ClpYQ. Supporting these data, MBP-SulA(F143Y), MBP-SulA(F143 N) (phenylalanine substituted with asparagine) led to a longer half-life with ClpYQ protease in vivo. In contrast, MBP-SulA(F143D) and MBP-SulA(F143S) both have shorter half-lives. Therefore, in the E. coli ClpYQ protease complex, ClpY recognizes the C-terminal region of SulA, and F143 of SulA plays an important role for the recognition and degradation by ClpYQ protease.

  • Control of Fusarium wilt of lisianthus by reassembling the microbial community in infested soil through reductive soil disinfestation
    Microbiol. Res. (IF 2.777) Pub Date : 2018-12-06
    Xing Zhou, Chunyu Li, Liangliang Liu, Jun Zhao, Jinbo Zhang, Zucong Cai, Xinqi Huang

    Continuous monocropping often influences negatively the soil microbial community and leads to the occurrence of soil-borne diseases. In this study, a pre-cultivation soil management strategy, reductive soil disinfestation (RSD), involving amendment by the use of labile organic substrates, such as reed straw, bagasse, and rice straw, and creating anaerobic soil conditions, was used to regulate the microbial community in a soil infested by Fusarium wilt of lisianthus and make it suitable for plant cultivation. The results showed that RSD significantly decreased F. oxysporum population by 97.1%–99.1% and the incidence of lisianthus wilt disease to 3.0%–14.3% compared with that of the untreated soil. The lowest disease incidence was found in the soil treated with RSD where bagasse was incorporated. The replantation of the host plant differently stimulated the pathogen proliferations across the different soils. MiSeq sequencing and culture-dependent investigation showed that the RSD treatments established distinct microbial communities compared to that of the untreated soil. Furthermore, the relative abundances of representatives of the families Cytophagaceae, Chitinophagaceae, Chaetomiaceae, and an unclassified family within Sordariomycetes, as well as soil microbial activity and the proportions of antagonists were significantly and negatively correlated with the pathogen population increase. Overall, the RSD treatment contributed to the reassembly of the soil microbiome which contained more beneficial agents that successfully controlled the pathogen inoculum level and lisianthus Fusarium wilt disease.

  • The stringent response factor, RelA, positively regulates T6SS4 expression through the RovM/RovA pathway in Yersinia pseudotuberculosis
    Microbiol. Res. (IF 2.777) Pub Date : 2018-12-06
    Xiaobing Yang, Yunhong Song, Qingyun Dai, Hongyun Zhang, Li Song, Zhuo Wang, Junfeng Pan, Yao Wang

    The type VI secretion system (T6SS) is a versatile molecular machinery widely distributed in Gram-negative bacteria. The activity of the T6SS is tightly regulated by various mechanisms, including quorum sensing (QS), iron concentration, and transcriptional regulators. Here we demonstrated that the stringent response regulator, RelA, contributes to bacterial resistance to multiple environmental stresses in Yersinia pseudotuberculosis. We also revealed that the stress resistance function of stringent response (SR) was partially mediated by the general stress response T6SS4 system. RelA positively regulates the expression of T6SS4 to combat various stresses in response to nutrition starvation collectively mediated by the RovM and RovA regulators. These findings revealed not only the important role of T6SS4 in SR induced stress resistance, but also a new pathway to regulate T6SS4 expression in response to starvation stress.

  • Avocado rhizobacteria emit volatile organic compounds with antifungal activity against Fusarium solani, Fusarium sp. associated with Kuroshio shot hole borer, and Colletotrichum gloeosporioides
    Microbiol. Res. (IF 2.777) Pub Date : 2018-11-24
    Edgar Guevara-Avendaño, Alix Adriana Bejarano-Bolívar, Ana-Luisa Kiel-Martínez, Mónica Ramírez-Vázquez, Alfonso Méndez-Bravo, Eneas Aguirre von Wobeser, Diana Sánchez-Rangel, José A. Guerrero-Analco, Akif Eskalen, Frédérique Reverchon

    Recent studies showed that bacterial volatile organic compounds (VOCs) play an important role in the suppression of phytopathogens. The ability of VOCs produced by avocado (Persea americana Mill.) rhizobacteria to suppress the growth of common avocado pathogens was therefore investigated. We evaluated the antifungal activity of VOCs emitted by avocado rhizobacteria in a first screening against Fusarium solani, and in subsequent antagonism assays against Fusarium sp. associated with Kuroshio shot hole borer, Colletotrichum gloeosporioides and Phytophthora cinnamomi, responsible for Fusarium dieback, anthracnosis and Phytophthora root rot in avocado, respectively. We also analyzed the composition of the bacterial volatile profiles by solid phase microextraction (SPME) gas chromatography coupled to mass spectrometry (GC-MS). Seven isolates, belonging to the bacterial genera Bacillus and Pseudomonas, reduced the mycelial growth of F. solani with inhibition percentages higher than 20%. Isolate HA, related to Bacillus amyloliquefaciens, significantly reduced the mycelial growth of Fusarium sp. and C. gloeosporioides and the mycelium density of P. cinnamomi. Isolates SO and SJJ, also members of the genus Bacillus, reduced Fusarium sp. mycelial growth and induced morphological alterations of fungal hyphae whilst isolate HB, close to B. mycoides, inhibited C. gloeosporioides. The analysis of the volatile profiles revealed the presence of ketones, pyrazines and sulfur-containing compounds, previously reported with antifungal activity. Altogether, our results support the potential of avocado rhizobacteria to act as biocontrol agents of avocado fungal pathogens and emphasize the importance of Bacillus spp. for the control of emerging avocado diseases such as Fusarium dieback.

  • Application of rice (Oryza sativa L.) root endophytic diazotrophic Azotobacter sp. strain Avi2 (MCC 3432) can increase rice yield under green house and field condition
    Microbiol. Res. (IF 2.777) Pub Date : 2018-11-23
    Avishek Banik, Gautam Kumar Dash, Padmini Swain, Upendra Kumar, Subhra Kanti Mukhopadhyay, Tushar Kanti Dangar

    Use of plant-associated beneficial microbes, especially endophytes are getting popular day by day as they occupy a relatively privileged niche inside different plant tissues with lesser competition for food and shelter than rhizosphere. The effects of different physical factors like temperature, rainfall, and seasonal variation and UV radiation on plant growth promoting endophytic communities are less pronounced than those on the rhizospheric and phylloplane microbes. This present work has been compromised with further utilization of an indigenous rice (Oryza sativa L.) root endophytic Azotobacter sp. strain Avi2 (MCC 3432) (AzA) as a bio-formulation for sustainable rice production based on several physiological parameters (plant height, root length/weight, leaf area, yield, chlorophyll contain), in-vitro comparative plant growth promoting assays, greenhouse and field experiments (dry and wet season). Treatments with AzA exhibited higher yield as well as maximal chlorophyll fluorescence (Fm) of flag leaves in flowering and grain filling stages indicating higher photosynthetic rates. Scanning electron microscopic image of rice roots demonstrated accumulation of bacterial biofilm at the junction of primary and lateral root confirming the root-colonizing ability of the bacterium. The results of the study were quite encouraging as AzA exhibited better vegetative and reproductive growth of rice in pot and field experiment compared to formulated rhizospheric Azotobacter sp. (commercial product). Apart from that plants treated with AzA (supplemented 50% nitrogenous fertilizer of recommended dose) exhibited similar yield parameters when it was compared with the recommended dose of fertilizer (RDF; 120:60:60 mg N:P:K kg-1 soil/ without any bacterial). Therefore, it can be concluded that application of this plant growth promoting endophyte can reduce a substantial amount of N-fertilizer for field application.

  • The methionine biosynthesis regulator AaMetR contributes to oxidative stress tolerance and virulence in Alternaria alternata
    Microbiol. Res. (IF 2.777) Pub Date : 2018-11-23
    Yunpeng Gai, Bing Liu, Haijie Ma, Lei Li, Xinglong Chen, Susan Moenga, Brendan Riely, Amna Fayyaz, Mingshuang Wang, Hongye Li

    The tangerine pathotype of A. alternata, which produces a unique host-selective ACT toxin causes brown spots on citrus leaves and fruits. In this study, we report a methionine biosynthesis regulator (MetR), which belong to bZIP transcription factor, is required for methionine metabolism, oxidative stress tolerance and pathogenicity. We generated two ΔAaMetR mutants in the tangerine pathotype of Alternaria alternata and investigated the resulting mutant phenotypes. The ΔAaMetR disruption mutant grew poorly in the absence of methionine and unable to produce conidia. Furthermore, pathogenicity tests have shown that ΔAaMetR mutant on their tangerine host can neither penetrate nor cause disease. These ΔAaMetR mutants exhibit an increased sensitivity to exogenous H2O2 and many ROS generating oxidants. To elucidate the transcription network of AaMetR, we performed RNA-Seq experiments on wild-type and ΔAaMetR mutant and identified genes that were differentially expressed between the two genotypes. Transcriptome data demonstrated that AaMetR contributes in many other biological processes including ROS detoxification, sulfur transfer, and amino acid metabolism. Comparative transcriptome analysis indicated that the ΔAaMetR mutant up-regulated several genes involved in cysteine and methionine metabolism. In conclusion, our results highlight the global regulatory role of AaMetR in cysteine and methionine metabolism and provide new insights into the crucial role of ROS detoxification, sporulation and pathogenicity in the tangerine pathotype of A. alternata.

  • Interactions between Pseudomonas spp. and their role in improving the red pepper plant growth under salinity stress
    Microbiol. Res. (IF 2.777) Pub Date : 2018-11-23
    Sandipan Samaddar, Poulami Chatterjee, Aritra Roy Choudhury, Shamim Ahmed, Tongmin Sa

    Solitary inoculation of bacteria has been studied widely for plant growth development and amelioration of salinity stress but co-inoculation of bacteria for salt stress amelioration in red pepper plants has been less studied till date. Here, we investigated the co-inoculation effect of Pseudomonas frederiksbergensis OB139 and Pseudomonas vancouverensis OB155 in red pepper plant growth characteristics, plant photosynthesis pigments, ethylene emission, and antioxidant properties under 0, 50, 100 and 150 mM salt stress and compared them with non-inoculated control and single inoculation of each isolate. Results showed increasing concentrations of salinity stress arrested the normal plant growth, increased the stress ethylene levels, disrupted the photosynthetic parameters and also influenced the antioxidant enzymatic activities in non-inoculated control plants. Co-inoculation of 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing Pseudomonas spp. significantly reduced the stress ethylene emission and contributed to a significant increase in plant growth compared to single inoculation and non-inoculated control. Catalase activity which was significantly increased in co-inoculated red pepper plants compared to other treatments imply its ability to efficiently neutralize the hydrogen peroxide ions formed as a result of oxidative stress in plants under salinity stress. Besides, significant reduction in malondialdehyde (MDA) content can be correlated to the increased salt tolerance in co-inoculated red pepper plants. Lastly, the increased content of photosynthetic pigments suggest the importance of co-inoculation in improving photosynthesis of red pepper plants. Together, the data demonstrated the functional compatibility of the ACC deaminase producing bacterial isolates and their role in improving the plant physical and biochemical characteristics under salinity stress.

  • Pseudomonas protegens MP12: A plant growth-promoting endophytic bacterium with broad-spectrum antifungal activity against grapevine phytopathogens
    Microbiol. Res. (IF 2.777) Pub Date : 2018-11-23
    Marco Andreolli, Giacomo Zapparoli, Elisa Angelini, Gianluca Lucchetta, Silvia Lampis, Giovanni Vallini

    Pseudomonas sp. MP12 was isolated from a soil sample collected in a typical warm-temperate deciduous forest near Brescia, northern Italy. Phylogenetic analysis identified the species as Pseudomonas protegens. We detected the genes phlD, pltB and prnC, which are required to synthesize the antifungal compounds 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin and pyrrolnitrin, respectively. P. protegens MP12 was also shown to produce siderophores and ammonia, yielded positive results in the indole-3-acetic acid test, and was capable of phosphate solubilization. P. protegens MP12 was able to inhibit the in vitro mycelial growth of prominent grapevine (Vitis vinifera) phytopathogens such as Botrytis cinerea, Alternaria alternata, Aspergillus niger, Penicillium expansum and Neofusicoccum parvum. The strain also showed activity against Phaeomoniella chlamydospora and Phaeoacremonium aleophilum, which cause the devastating tracheomycosis/esca disease of grapevine trunks for which there are no efficacious control methods. Moreover, MP12 strain showed in vivo antifungal activity against B. cinerea on grapevine leaves. Culture-dependent and culture-independent analysis revealed the ability of P. protegens MP12 to efficiently and permanently colonize inner grapevine tissues. These results suggest that P. protegens MP12 could be developed as an antifungal biocontrol agent with applications in viticulture.

  • MarTrack: A versatile toolbox of mariner transposon derivatives used for functional genetic analysis of bacterial genomes
    Microbiol. Res. (IF 2.777) Pub Date : 2018-11-23
    Lifan Wei, Haoxian Qiao, Bing Liu, Kaiyu Yin, Qin Liu, Yuanxing Zhang, Yue Ma, Qiyao Wang

    The mariner transposon family of Himar1 has been widely used for the random mutagenesis of bacteria to generate single insertions into the chromosome. Here, a versatile toolbox of mariner transposon derivatives was generated and applied to the functional genomics investigation of fish pathogen Edwardsiella piscicida. In this study, we combined the merits of the random mutagenesis of mariner transposon and common efficient reporter marker genes or regulatory elements, mcherry, gfp, luxAB, lacZ, sacBR, and PBAD and antibiotic resistance cassettes to construct a series of derivative transposon vectors, pMmch, pMKGR, pMCGR, pMXKGR, pMLKGR, pMSGR, and pMPR, based on the initial transposon pMar2xT7. The function and effectiveness of the modified transposons were verified by introducing them into E. piscicida EIB202. Based on the toolbox, a transposon insertion mutant library containing approximately 3.0 × 105 separated mutants was constructed to explore the upstream regulators of esrB, the master regulator of the type III and type VI secretion systems (T3/T6SS) in E. piscicida. Following analysis by Con-ARTIST, ETAE_2184 (renamed as EsrR) was screened out and identified as a novel regulator mediating T3SS expression. In addition, the esrR mutants displayed critical virulence attenuation. Due to the broad-range host compatibility of mariner transposons, the newly built transposon toolbox can be broadly applied for functional genomics studies in various bacteria.

  • Screening, plant growth promotion and root colonization pattern of two rhizobacteria (Pseudomonas fluorescens Ps006 and Bacillus amyloliquefaciens Bs006) on banana c.v Williams (Musa acuminata)
    Microbiol. Res. (IF 2.777) Pub Date : 2018-11-23
    R. Gamez, M. Cardinale, M. Montes, S. Ramirez, S. Schnell, F. Rodriguez

    Banana is the second largest export crop in Colombia. To meet the demand of international markets, high amounts of chemical fertilizers are required, which represent high costs and can be hazardous to the environment. Plan growth promoting rhizobacteria (PGPR) can at least partially replace chemical fertilizers. In this paper, we evaluated the effect of nine PGPR of the genera Bacillus and Pseudomonas on Banana growth. Banana seedlings were produced through tissue culture and acclimatized in the greenhouse core. Plants were inoculated with the rhizobacteria and plant growth features (plant height, leaf number, leaf area, pseudostem thickness, root and shoot fresh weight, root and shoot dry weight) were assessed after 55 days of growth. The two best performing PGPR, Bs006 and Ps006 previously identified as Bacillus amyloliquefaciens and Pseudomonas fluorescens, respectively, promoted Banana growth similarly or slightly superior to 100% chemical fertilization, and were selected for further characterization of root colonization by both eletron microscopy and confocal microscopy of fluorescence in situ hybridization (FISH)–stained root tissues. Both bacteria P. fluorescens Ps006 and B. amyloquifaciens Bs006 showed ability to colonize Banana roots, but Bs006 appeared faster than Ps006 in the colonization dynamics. This work demonstrated that inoculation of rhizobacteria Bacillus amyloliquefaciens Bs006 and Pseudomonas fluorescens Ps006 could partially replace the chemical fertilization of tissue cultured Banana plants, and therefore could be used for the formulation of a new biofertilizer.

  • Bacterial chemotaxis coupling protein: Structure, function and diversity
    Microbiol. Res. (IF 2.777) Pub Date : 2018-11-06
    Zhiwei Huang, Xiaoyue Pan, Nan Xu, Minliang Guo

    In most signal transduction systems, coupling or scaffold proteins establish crucial connections between receptors and histidine kinases. These connections are important for signal transduction. The bacterial chemotaxis system is a canonical signal transduction system that relies on coupling proteins. The coupling proteins in the chemotaxis system have two architectures: CheW or CheV. In a typical chemotaxis signal transduction system, two CheW coupling protein molecules bridge a histidine kinase CheA dimer and two chemoreceptor (also called as methyl-accepting chemotaxis protein, MCP) trimers of dimers to form a core signaling complex and couple CheA activity to chemoreceptor control. Although CheW is a small cytoplasmic protein, it plays multiple functions in chemotaxis. CheW also builds connections between core signaling complexes, which leads to the formation of large chemosensory arrays that are responsible for collecting and amplifying signals from various chemoreceptors. Another coupling protein, CheV, shares a largely redundant ability with CheW; however, the function of CheV is not identical to that of CheW in chemotaxis. In this article, we summarize the molecular mechanism of chemotaxis in Escherichia coli and review the recent advances in the structural details and functions of CheW and CheV. Furthermore, we focus on the diversity of coupling proteins and discuss the relationship among multiple coupling proteins in one organism.

  • Physiological response of Lactuca sativa exposed to 2-nonanone emitted by Bacillus sp. BCT9
    Microbiol. Res. (IF 2.777) Pub Date : 2018-11-03
    Paola Fincheira, Andrés Quiroz

    Volatile organic compounds (VOCs) released from bacterial species have been reported as plant growth inducers. In this sense, Lactuca sativa was used as model vegetable to prospect the effects of 2-nonanone released by Bacillus sp. BCT9 at cellular and organ structure level, so we present preliminary results about the physiological effects. In this study, 2-day-old L. sativa were exposed to 2-nonanone for 10 days under two delivery systems: 1) 2-nonanone (abrupt delivery) and 2) 2-nonanone + lanolin (controlled delivery). The X-ray elemental microanalysis, scanning electron and confocal laser microscopies techniques were used to evaluate physiological changes “in vivo” conditions. The results indicated that 2-nonanone increased root and shoot length independently of 2-nonanone delivery system after 7 days of exposition. Additionally, 2-nonanone elicited the increase of anthocyanin and not affects chlorophyll content and electrolyte leakage percentage. The abrupt delivery elicited the increase of both length and density of root hair without causing changes in size of cell epidermis, while controlled delivery induced stomatal opening. Besides, 2-nonanone exposition did not modify the composition and distribution of carbon, nitrogen, phosphorus, potassium, and chlorine in the surface of plant tissue. The results suggested that 2-nonanone acts as a bacterial signal molecule to elicit changes related to root development without damaging the external morphology while epidermal cells at leaf level are not affected, suggesting that 2-nonanone can be an important tool to apply to vegetables.

  • The Potential of Bacilli Rhizobacteria for Sustainable Crop Production and Environmental Sustainability
    Microbiol. Res. (IF 2.777) Pub Date : 2018-11-02
    B.N. Aloo, B.A. Makumba, E.R. Mbega

    Conventional agricultural practices often rely on synthetic fertilizers and pesticides which have immense and adverse effects on humans, animals and environments. To minimize these effects, scientists world over are now deeply engaged in finding alternative approached for crop production which are less dependent on chemical inputs. One such approach is the use of rhizospheric bacteria as vital components of soil fertility and plant growth promotion (PGP) through their direct and indirect processes in plant rhizospheres. Among the most studied rhizobacteria are the Bacilli, particularly for production of antibiotics, enzymes and siderophores all of which are important aspects of PGP. Despite this, little information is available especially on their potentiality in crop production and their direct application only involves a few species, leaving a majority of these important rhizobacteria exploited. This paper gives an overview of the unique properties of Bacilli rhizobacteria as well as their different PGP mechanisms that if mined can lead to their successful application and agricultural sustainability. It further points out the missing aspects with regards to these important rhizobacteria that should be considered for future research. This information will be useful in analyzing the PGP abilities of Bacilli rhizobacteria with an aim of fully mining their potential for crop production and environmental sustainability.

  • Current situation of biofuel production and its enhancement by CRISPR/Cas9-mediated genome engineering of microbial cells
    Microbiol. Res. (IF 2.777) Pub Date : 2018-11-02
    Muhammad Rizwan Javed, Muhammad Noman, Muhammad Shahid, Temoor Ahmed, Mohsin Khurshid, Muhammad Hamid Rashid, Muhammad Ismail, Maria Sadaf, Fahad Khan

    Geopolitical and economic factors have motivated the scientific community to utilize renewable energy resources. In addition to the modifications in major steps and processes of biofuel production, manipulation of microbial genome engineering tools is essential in order to find sustainable solution of continuous depletion of fossil-fuels. Recently, the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated nuclease 9), a prokaryotic molecular immunity system, has emerged as a novel technology for targeted genomic engineering. This genetic machinery seems to be a groundbreaking discovery to engineer the microbial genomes for desired traits such as enhancing the biofuel tolerance, inhibitor tolerance and thermotolerance as well as modifying the cellulases and hemicelluloses enzymes. In this review, a summary of different generations of biofuels, integrated processes of bioconversion of raw materials into biofuels and role of microbes in biofuel production has been presented. However, the ultimate focus of the review is on major discoveries of CRISPR/Cas9-mediated genome editing in microorganisms and exploitation of these discoveries for enhanced biofuel production.

  • Development of low-cost formulations of plant growth-promoting bacteria to be used as inoculants in beneficial agricultural technologies
    Microbiol. Res. (IF 2.777) Pub Date : 2018-11-02
    Constanza Belén Lobo, María Silvina Juárez Tomás, Emilce Viruel, Marcela Alejandra Ferrero, María Ester Lucca
  • Far-reaching cellular consequences of tat deletion in Escherichia coli revealed by comprehensive proteome analyses
    Microbiol. Res. (IF 2.777) Pub Date : 2018-11-01
    Katarzyna M. Dolata, Isabel Guerrero Montero, Wayne Miller, Susanne Sievers, Thomas Sura, Christian Wolff, Rabea Schlüter, Katharina Riedel, Colin Robinson
  • Transcriptome analysis of hopanoid deficient mutant of Rhodopseuodomonas palustris TIE-1
    Microbiol. Res. (IF 2.777) Pub Date : 2018-10-29
    Tushar D. Lodha, Indu B., Sasikala Ch., Ramana Ch.V.

    All three domains of life have an ordered plasma membrane which is pivotal in the selective fitness of primitive life. Like cholesterol in eukaryotes, hopanoids are important in bacteria to modulate membrane order. Hopanoids are pentacyclic triterpenoid lipids biosynthesised in many eubacteria, few ferns and lichens. Hopanoid modulates outer membrane order and hopanoid deficiency results in the weakened structural integrity of the membrane which may intern affect the other structures within or spanning the cell envelope and contributing to various membrane functions. Hence, to decipher the role of hopanoid, genome-wide transcriptome of wild-type and Δshc mutant of Rhodopseudomonas palustris TIE-1 was studied which indicated 299 genes were upregulated and 306 genes were downregulated in hopanoid deficient mutant, representing ~11.5% of the genome. Thirty-eight genes involved in chemotaxis, response to stimuli and signal transduction were differentially regulated and impaired motility in hopanoid deficient mutant showed that hopanoid plays a crucial role in chemotaxis. The docking study demonstrated that diguanylate cyclase which catalyses the synthesis of secondary messenger exhibited the capability to interact with hopanoids and might be confederating in chemotaxis and signal transduction. Seventy-four genes involved in membrane transport were differentially expressed and cell assays have explicit that the multidrug transport is compromised in Δshc mutant. Membrane transport is reliant on hopanoids which may explain the basis for previous observations linking hopanoids to antibiotic resistance. Disturbing the membrane order by targeting lipid synthesis can be a possible novel approach in developing new antimicrobials and hopanoid biosynthesis could be a potential target.

  • The histidine kinase slnCl1 of Colletotrichum lindemuthianum as a pathogenicity factor against Phaseolus vulgaris L
    Microbiol. Res. (IF 2.777) Pub Date : 2018-10-28
    Guilherme Bicalho Nogueira, Leandro Vieira dos Santos, Casley Borges de Queiroz, Thamy Lívia Ribeiro Corrêa, Renato Pedrozo Menicucci, Denise Mara Soares Bazzolli, Elza Fernandes de Araújo, Marisa Vieira de Queiroz

    Colletotrichum lindemuthianum, the causal agent of anthracnose, is responsible for significant damage in the common bean (Phaseolus vulgaris L.). Unraveling the genetic mechanisms involved in the plant/pathogen interaction is a powerful approach for devising efficient methods to control this disease. In the present study, we employed the Restriction Enzyme-Mediated Integration (REMI) methodology to identify the gene slnCl1, encoding a histidine kinase protein, as involved in pathogenicity. The mutant strain, MutCl1, generated by REMI, showed an insertion in the slnCl1 gene, deficiency of the production and melanization of appressoria, as well as the absence of pathogenicity on bean leaves when compared with the wild-type strain. The slnCl1 gene encodes a histidine kinase class IV called SlnCl1 showing identity of 97% and 83% with histidine kinases from Colletotrichum orbiculare and Colletotrichum gloesporioides, respectively. RNA interference was used for silencing the histidine kinase gene and confirm slnCl1 as a pathogenicity factor. Furthermore, we identified four major genes involved in the RNA interference-mediated gene silencing in Colletotrichum spp. and demonstrated the functionality of this process in C. lindemuthianum. Silencing of the EGFP reporter gene and slnCl1 were demonstrated using qPCR. This work reports for the first time the isolation and characterization of a HK in C. lindemuthianum and the occurrence of gene silencing mediated by RNA interference in this organism, demonstrating its potential use in the functional characterization of pathogenicity genes.

  • Plant-microbe interactions endorse growth by uplifting microbial community structure of Bacopa monnieri rhizosphere under nematode stress
    Microbiol. Res. (IF 2.777) Pub Date : 2018-10-26
    Rupali Gupta, Akanksha Singh, Madhumita Srivastava, Karuna Shanker, Rakesh Pandey

    The modification of rhizosphere microbial diversity and ecological processes are of rising interest as shifting in microbial community structure impacts the mutual role of host–microbe interactions. Nevertheless, the connection between host-microbial community diversity, their function under biotic stress in addition to their impact on plant performances is poorly understood. The study was designed with the aim to analyze the tripartite interactions among Chitiniphilus sp., Streptomyces sp. and their combination with indigenous rhizospheric microbial population of Bacopa monnieri for enhancing the plant growth and bacoside A content under Meloidogyne incognita stress. Overall, plants treated with the microbial combination recorded enhanced growth as illustrated by significantly higher biomass (2.0 fold), nitrogen uptake (1.8 fold) and bacoside A content (1.3 fold) along with biocontrol efficacy (58.5 %) under nematode infected field. The denaturing gradient gel electrophoresis (DGGE) fingerprints of 16S-rDNA revealed that microbial inoculations are major initiators of bacterial community structure in the plant rhizosphere. Additionally, the plants treated with microbial combination showed maximum diversity viz., Shannon’s (3.29), Margalef’s (4.21), and Simpson’s (0.96) indices. Likewise the metabolic profiling data also showed a significant variation among the diversity and evenness indices upon microbial application on the native microflora. We surmise that the application of beneficial microbes in combinational mode not only helped in improving the microbial community structure but also successfully enhanced plant and soil health under biotic stress.

  • Deletion of the epigenetic regulator GcnE in Aspergillus niger FGSC A1279 activates the production of multiple polyketide metabolites
    Microbiol. Res. (IF 2.777) Pub Date : 2018-10-15
    Bin Wang, Xuejie Li, Dou Yu, Xiaoyi Chen, Jioji Tabudravu, Hai Deng, Pan Li

    Epigenetic modification is an important regulatory mechanism in the biosynthesis of secondary metabolites in Aspergillus species, which have been considered to be the treasure trove of new bioactive secondary metabolites. In this study, we reported that deletion of the epigenetic regulator gcnE, a histone acetyltransferase in the SAGA/ADA complex, resulted in the production of 12 polyketide secondary metabolites in A. niger FGSC A1279, which was previously not known to produce toxins or secondary metabolites. Chemical workup and structural elucidation by 1D/2D NMR and high resolution electrospray ionization mass (HR-ESIMS) yielded the novel compound nigerpyrone (1) and five known compounds: carbonarone A (2), pestalamide A (3), funalenone (4), aurasperone E (5), and aurasperone A (6). Based on chemical information and the literature, the biosynthetic gene clusters of funalenone (4), aurasperone E (5), and aurasperone A (6) were located on chromosomes of A. niger FGSC A1279. This study found that inactivation of GcnE activated the production of secondary metabolites in A. niger. The biosynthetic pathway for nigerpyrone and its derivatives was identified and characterized via gene knockout and complementation experiments. A biosynthetic model of this group of pyran-based fungal metabolites was proposed.

  • Partner-triggered proteome changes in the cell wall of Bacillus sonorensis and roots of groundnut benefit each other
    Microbiol. Res. (IF 2.777) Pub Date : 2018-10-09
    Sravani Ankati, T. Swaroopa Rani, Appa Rao Podile

    Plant growth promoting rhizobacteria (PGPR) promote plant growth and activate defense response against phytopathogens. At the subcellular level plant-PGPR interaction is less understood, which would be essential for future improvement(s) of PGPR formulations. In a rigorous screening process, that also involved efficient PGPR strains, Bacillus sonorensis RS4 was selected to study partner-triggered interactions. The potential of B. sonorensis RS4 to improve growth of groundnut, efficiency to colonize roots, and influence on root topology was assessed. Twenty four cell wall proteins of B. sonorensis RS4 [in presence of groundnut root exudates (REs)], and 22 groundnut root proteins (in RS4-bacterized plants) were differentially expressed. The alterations in cell wall proteins of B. sonorensis RS4 were primarily related to the amino acids synthesis, chemotaxis, antioxidant-metabolism, carbohydrate metabolism, transporters, and antibiosis-related secondary metabolites. Root proteins that were differentially expressed during the interaction may be involved in plant growth, defense responses, and in transportation. The changes in B. sonorensis RS4 cell wall proteome and groundnut root proteome, suggest that at least a part of the proteome changes triggered by each of the partners appear to play a significant role in helping each other akin to symbiosis.

  • Antimicrobial, plant growth-promoting and genomic properties of the peanut endophyte Bacillus velezensis LDO2
    Microbiol. Res. (IF 2.777) Pub Date : 2018-10-09
    Liang Chen, Hao Shi, Junying Heng, Dianxuan Wang, Ke Bian

    Peanut suffer from a number of fungal and bacterial pathogens, while plant endophytes were considered excellent candidates as biocontrol agents. In this study, the peanut endophytic bacterium LDO2 was evaluated for the potential of peanut pathogens inhibition and growth-promotion, and the genetic mechanisms were explored by genome mining. Strain LDO2 significantly inhibited the growth of peanut pathogenic fungi and pathogenic bacteria, and specifically, it showed pronounced inhibition on mycelia growth of Aspergillus flavus mycelia and caused mycelial deformity. Gene clusters responsible for antifungal metabolites (fengycin, surfactin, bacilysin) and antibacterial metabolites (butirosin, bacillaene, difficidin, macrolactin, surfactin, bacilysin) were identified. Strain LDO2 also exhibited several growth-promoting related features including phosphate solubilization, siderophore production and growth promotion of peanut root. Genes associated with plant growth promotion were also identified and analyzed, as well as genes related to secreted proteins. These findings suggested that this peanut endophyte could be a potential biocontrol agent in peanut production and a source of antimicrobial compounds for further exploitation.

  • Recombineering Pseudomonas protegens CHA0: An innovative approach that improves nitrogen fixation with impressive bactericidal potency
    Microbiol. Res. (IF 2.777) Pub Date : 2018-10-06
    Fangnan Yu, Xiaoshu Jing, Xiaochen Li, Hailong Wang, Hanna Chen, Lin Zhong, Jia Yin, Deng Pan, Yulong Yin, Jun Fu, Liqiu Xia, Xiaoying Bian, Qiang Tu, Youming Zhang
  • Environmental conditions shape the biofilm of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125
    Microbiol. Res. (IF 2.777) Pub Date : 2018-10-06
    Annarita Ricciardelli, Angela Casillo, Alessandro Vergara, Nicole Balasco, Maria Michela Corsaro, Maria Luisa Tutino, Ermenegilda Parrilli

    Biofilms are the most widely distributed and successful microbial modes of life. The capacity of bacteria to colonize surfaces provides stability in the growth environment, allows the capturing of nutrients and affords protection from a range of environmental challenges and stress. Bacteria living in cold environments, like Antarctica, can be found as biofilms, even though the mechanisms of how this lifestyle is related to their environmental adaptation have been poorly investigated. In this paper, the biofilm of Pseudoalteromonas haloplanktis TAC125, one of the model organisms of cold-adapted bacteria, has been characterized in terms of biofilm typology and matrix composition. The characterization was performed on biofilms produced by the bacterium in response to different nutrient abundance and temperatures; in particular, this is the first report describing the structure of a biofilm formed at 0 °C. The results reported demonstrate that PhTAC125 produces biofilms in different amount and endowed with different physico-chemical properties, like hydrophobicity and roughness, by modulating the relative amount of the different macromolecules present in the biofilm matrix. The capability of PhTAC125 to adopt different biofilm structures in response to environment changes appears to be an interesting adaptation strategy and gives the first hints about the biofilm formation in cold environments.

  • Isolation and characterization of endophytes from nodules of Mimosa pudica with biotechnological potential
    Microbiol. Res. (IF 2.777) Pub Date : 2018-10-06
    Ricardo Sánchez-Cruz, Irán Tapia Vázquez, Ramón Alberto Batista-García, Erick Williams Méndez Santiago, María del Rayo Sánchez Carbente, Alfonso Leija, Verónica Lira-Ruan, Gerogina Hernández, Arnoldo Wong-Villarreal, Jorge Luis Folch-Mallol

    Legumes establish symbiotic relationships with different microorganisms, which could function as plant growth promotion microorganisms (PGPM). The finding of new PGPM strains is important to increase plant production avoiding or diminishing the use of industrial fertilizers. Thus, in this work we evaluated the plant growth promotion traits of ten strains isolated from Mimosa pudica root nodules. According to the 16S rDNA sequence, the microorganisms were identified as Enterobacter sp. and Serratia sp.. To the best of our knowledge this is the first report describing and endophytic interaction between Mimosa pudica and Enterobacter sp. These strains have some plant growth promoting traits such as phosphate solubilization, auxin production and cellulase and chitinase activity. Strains identified as Serratia sp. inhibited the growth of the phytopathogenic fungi Fusarium sp., and Alternaria solani and the oomycete Phytophthora capsici. According to their biochemical characteristics, three strains were selected to test their plant growth promoting activity in a medium with an insoluble phosphate source. These bacteria show low specificity for their hosts as endophytes, since they were able to colonize two very different legumes: Phaseolus vulgaris and M. pudica. Seedlings of P. vulgaris were inoculated and grown for fifteen days. Enterobacter sp. NOD1 and NOD10, promoted growth as reflected by an increase in shoot height as well as an increase in the size and emergence of the first two trifolia. We could localize NOD5 as an endophyte in roots in P. vulgaris by transforming the strain with a Green Fluorescent Protein carrying plasmid. Experiments of co-inoculation with different Rhizobium etli strains allowed us to discard that NOD5 can fix nitrogen in the nodules formed by a R. etli Fix- strain. The isolates described in this work show biotechnological potential for plant growth promoting activity and production of indoleacetic acid and siderophores.

  • Helicobacter pylori evasion strategies of the host innate and adaptive immune responses to survive and develop gastrointestinal disease
    Microbiol. Res. (IF 2.777) Pub Date : 2018-10-06
    Ahmad Karkhah, Soheil Ebrahimpour, Maryam Rostamtabar, Veerendra Koppolu, Sorena Darvish, Veneela Krishna Rekha Vasigala, Majid Validi, Hamid Reza Nouri

    Helicobacter pylori (H. pylori) is a bacterial pathogen that resides in more than half of the human population and has co-evolved with humans for more than 58000 years. This bacterium is orally transmitted during childhood and is a key cause of chronic gastritis, peptic ulcers and two malignant cancers including MALT (mucosa-associated lymphoid tissue) lymphoma and adenocarcinoma. Despite the strong innate and adaptive immune responses, H. pylori has a long-term survival in the gastric mucosa. In addition to the virulence factors, survival of H. pylori is strongly influenced by the ability of bacteria to escape, disrupt and manipulate the host immune system. This bacterium can escape from recognition by innate immune receptors via altering its surface molecules. Moreover, H. pylori subverts adaptive immune response by modulation of effector T cell. In this review, we discuss the immune-pathogenicity of H. pylori by focusing on its ability to manipulate the innate and acquired immune responses to increase its survival in the gastric mucosa, leading up to gastrointestinal disorders. We also highlight the mechanisms that resulted to the persistence of H. pylori in gastric mucosa.

  • Organization of multi-binding to host proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Mycoplasma pneumoniae
    Microbiol. Res. (IF 2.777) Pub Date : 2018-09-26
    Juliane Grimmer, Roger Dumke

    Mycoplasma pneumoniae is a frequent cause of community-acquired infections of the human respiratory tract. During the evolutionary adaptation of the bacteria to the host, the genome of the pathogen is strongly reduced resulting in the loss of cell wall, limited metabolic pathways and a relatively small number of virulence factors. As interacting with host proteins, surface-exposed proteins with a primary function in cytosol-located processes of metabolism and regulation such as glycolytic enzymes, heat-shock proteins and chaperones have been considered as contributing to pathogenesis. Among these moonlighting proteins, some members are confirmed as binding to several host components. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of M. pneumoniae is a typical example of such multi-binding proteins. To investigate the organization of these interactions, GAPDH was divided into four parts. Recombinant proteins were successfully expressed in Escherichia coli and polyclonal antisera were produced. Binding of full length and parts of GAPDH to human A549 cells was proven. Furthermore, interactions with human plasminogen, vitronectin, fibronectin and fibrinogen were demonstrated for nearly all recombinant GAPDH proteins. In the presence of these proteins, plasminogen can be activated to the protease plasmin. In contrast, the localization on the surface of bacterial cell was confirmed for the C-terminal part of GAPDH only. By using overlapping peptides covering this region, binding of the investigated host components to the sequence 326QLVRVVNYCAKL337 was found. The results of the study suggest a prominent role of the surface-localized C-terminal part of GAPDH in associations with different human proteins indicating its importance for host-pathogen-interactions.

  • Genome sequence and comparative genomics of Rhizobium sp. Td3, a novel plant growth promoting phosphate solubilizing Cajanus cajan symbiont
    Microbiol. Res. (IF 2.777) Pub Date : 2018-09-24
    Bhagya Iyer, Shalini Rajkumar

    Rhizobium sp. Td3 is a Sesbania plant growth promoting, Cajanus cajan nodulating rhizobia. Studying its whole genome was important as it is a potent phosphate solubilizer with constitutive gluconic acid production ability through operation of the periplasmic glucose oxidation pathway even under conditions of catabolite repression. This is in contrast to the other explored phosphate solubilizers. Rhizobial isolates sequenced so far are known to lack components of the direct glucose oxidation pathway and cannot produce gluconic acid on its own. Here, we present the genome sequence of Rhizobium sp. Td3. Genome comprises of a single chromosome of size 5,606,547 bp (5.6 Mb) with no symbiotic plasmid. Rhizobium leguminosarum bv. viciae USDA2370 was the closest whole genome known. 109 genes responsible for diverse plant growth promoting activities like P solubilization, synthesis of acetoin, nitric oxide, indole-3 acetic acid, exopolysaccharide, siderophore and trehalose have been identified. Flagellar proteins, genes encoding antibiotic and metal resistance, enzymes required for combating oxidative stress as well as attachment and colonization in the plant rhizosphere are also present. Availability of genome sequence of such a versatile plant growth promoting agent will help in exploiting all the phyto-beneficial traits of Td3 for its use as a biofertilizer.

  • Cordycepin and Pentostatin Biosynthesis Gene Identified through Transcriptome and Proteomics Analysis of Cordyceps kyushuensis Kob
    Microbiol. Res. (IF 2.777) Pub Date : 2018-09-17
    Xuan Zhao, Guoying Zhang, Caiyi Li, Jianya Ling

    Cordyceps kyushuensis is the only species of cordyceps growing on the larvae of Clanis bilineata Walker, and has been demonstrated that there are lots of pharmacological components including cordycepin. Cordycepin shows lots of pharmacological action but it could be converted to 3’-deoxyinosine by adenosine deaminase in vivo, which weakens the efficiency of cordycepin. That pentostatin, which has been reported to inhibit adenosine deaminase, combining cordycepin could enhance the efficiency of cordycepin in vivo. During transcriptome and proteomics analysis of Cordyceps kyushuensis, a single gene cluster including four genes we named ck1-ck4 which can synthesis both cordycepin and pentostatin has been identified using BLAST. Meanwhile, KEGG, KOG, GO analysis and differentially expressed genes were analyzed in transcriptome and proteomics. This study first sequenced transcriptome and proteomics of C. kyushuensis, and demonstrated that there is a single gene cluster related to biosynthesis of cordycepin and pentostatin, which can be employed to improve the yield of cordycepin and find more functional proteins.

  • Enhanced molecular visualization of root colonization and growth promotion by Bacillus subtilis EA-CB0575 in different growth systems
    Microbiol. Res. (IF 2.777) Pub Date : 2018-09-11
    Luisa F. Posada, J.C. Álvarez, Magally Romero-Tabarez, Luz de-Bashan, Valeska Villegas-Escobar

    Bacillus subtilis EA-CB0575 is a plant growth-promoting bacterium (PGPB) associated with banana and tomato crops. Root colonization is an important trait for PGPB microorganisms and potentiates the bacterial effect related to the mechanisms of plant growth promotion. Therefore, detection of bacterial colonization of roots in different culture systems is important in the study of plant–microorganism interactions. In this study, fluorescent in situ hybridization (FISH) and catalyzed reporter deposition–FISH (CARD–FISH) were evaluated to determine the colonization ability of B. subtilis EA-CB0575 on banana and tomato roots planted on solid and liquid Murashige and Skoog medium (MS(S) and MS(L), respectively) and in soil for tomato plants. Results showed B. subtilis colonization 0–30 days post inoculation for banana and tomato plants in different culture systems with differential distribution of bacterial cells along tomato and banana roots. FISH and CARD–FISH methodologies were both successful in detecting B. subtilis colonies, but CARD–FISH proved to be superior due to its enhanced fluorescence signal. The presence of bacteria correlated with the promotion of plant growth in both plant species, providing clues to relate rhizospheric colonization with improvement in plant growth. FISH and CARD–FISH analysis results suggested the presence of native microbiota on the roots of in vitro banana plants, but not on those of tomato plants.

  • Application of laser microdissection to study phytoplasma site-specific gene expression in the model plant Arabidopsis thaliana
    Microbiol. Res. (IF 2.777) Pub Date : 2018-09-10
    Marika Rossi, Massimo Pesando, Marta Vallino, Luciana Galetto, Cristina Marzachì, Raffaella Balestrini

    Many aspects of plant diseases caused by phytoplasmas are still unknown, as these pathogens are phloem restricted, uncultivable wall-less bacteria and must be studied always in association with their host. Phytoplasma transcripts are strongly underrepresented within host tissues and this poses problems for gene expression analyses. In this study, a procedure was established to infect the model plant Arabidopsis thaliana with the phytoplasma Flavescence dorée, a serious threat to European viticulture. Rates of phytoplasma infective insects and transmission efficiency to A. thaliana as well as pathogen loads were measured in different tissues of infected A. thaliana plants, and modification of phloem cell ultrastructure was observed in infected plant tissues at microscopic level. Moreover, a protocol for the application of laser microdissection to analyze plant and phytoplasma gene expression profiles in the specific colonized tissue was designed. The procedure allowed a good preservation of the plant tissue anatomy. Results showed that the extracted RNA was suitable for qualitative and quantitative RT-PCR, since both plant and pathogen transcripts, either abundant or rare ones, could be detected without any pre-amplification step. The combined use of laser microdissection approach and A. thaliana to study phytoplasmas opens the way to exploit biological, molecular and bioinformatic tools available for the model plant and to elucidate key pathways of the infection mechanisms of these important plant pathogen.

  • The exopolysaccharide gene cluster pea is transcriptionally controlled by RpoS and repressed by AmrZ in Pseudomonas putida KT2440
    Microbiol. Res. (IF 2.777) Pub Date : 2018-09-10
    Huizhong Liu, Huaduo Yan, Yujie Xiao, Hailing Nie, Qiaoyun Huang, Wenli Chen

    In Pseudomonas putida KT2440, the exopolysaccharide Pea is associated with biofilm stability and pellicle formation; however, little is known about its regulatory pathway. In this study, we identified that the gene cluster pea was transcribed from 25 bp upstream of the operon and the stationary phase alternative sigma factor RpoS regulated the transcription of pea. When RpoS was absent, another sigma factor, likely the housekeeping sigma factor RpoD, could also mediate pea transcription but at a low level. The function of Pea polysaccharide was further confirmed to be necessary for full production of biofilm, formation of pellicle and c-di-GMP-dependent wrinkly colony morphology. Additionally, evidences were provided to demonstrate that the transcriptional regulator AmrZ was a negative regulator for pea expression. DNase I footprinting studies verified that AmrZ bound directly to the site overlapping the pea promoter, which might interfere with the binding of RNA polymerase to the promoter and resulted in inhibition of transcription initiation.

  • Blue light exposure and nutrient conditions influence the expression of genes involved in simultaneous hyphal knot formation in Coprinopsis cinerea
    Microbiol. Res. (IF 2.777) Pub Date : 2018-09-08
    Yuichi Sakamoto, Shiho Sato, Miyuki Ito, Yuki Ando, Kiyoshi Nakahori, Hajime Muraguchi
  • Isolation, characterization, and selection of heavy metal-resistant and plant growth-promoting endophytic bacteria from root nodules of Robinia pseudoacacia in a Pb/Zn mining area
    Microbiol. Res. (IF 2.777) Pub Date : 2018-09-07
    Miaochun Fan, Zhenshan Liu, Lijun Nan, Entao Wang, Weimin Chen, Yanbing Lin, Gehong Wei

    Multiple heavy metals (HMs) commonly coexist in mining areas, which highlights the necessity to select multiple HM-resistant plant growth-promoting bacteria for improving phytoremediation efficiency. In this study, we isolated and characterized 82 endophytic bacteria from the root nodules of black locust (Robinia pseudoacacia) grown in a Pb-Zn mining area. There were 80 isolates showing resistance to four HMs, 0.01–18.0 mM/L for Cd, 0.2–40.0 mM/L for Zn, 0.3–2.2 mM/L for Pb, and 0.2–1.4 mM/L for Cu. Indole-3-acetic acid production, siderophore production, and 1-aminocyclopropane-1-carboxylate deaminase activity were detected in 43, 50, and 17 isolates, respectively. Two symbiotic isolates selected with the highest potential for HM resistance and PGP traits, designated Mesorhizobium loti HZ76 and Agrobacterium radiobacter HZ6, were evaluated for promotion of plant growth and metal uptake by R. pseudoacacia seedlings grown in pots containing different levels of Cd, Zn, Pb, or Cu. HZ76 significantly increased plant shoot biomass, while HZ6 did not, compared with non-inoculated controls. The results indicate that inoculation with HZ76 or HZ6 relieved HM stress in the plants, depending on the type and concentration of HM in the treatment. Mesorhizobium loti HZ76 may be a better candidate for application in phytoremediation than A. radiobacter HZ6. The microsymbiosis between HM-resistant rhizobia and R. pseudoacacia is an interesting mutualistic system for phytoremediation in mining areas contaminated with multiple HMs.

  • Biological control of the soft rot bacterium Pectobacterium carotovorum by Bacillus amyloliquefaciens strain Ar10 producing glycolipid-like compounds
    Microbiol. Res. (IF 2.777) Pub Date : 2018-08-31
    Sana Azaiez, Imen Ben Slimene, Ines Karkouch, Rym Essid, Selim Jallouli, Naceur Djebali, Salem Elkahoui, Ferid Limam, Olfa Tabbene

    Four hundred and fifty bacteria were evaluated for antagonistic activity against bacterial soft rot of potato caused by Pectobacterium carotovorum sp strain II16. A strain Ar10 exhibiting potent antagonist activity has been identified as Bacillus amyloliquefaciens on the basis of biochemical and molecular characterization. Cell free supernatant showed a broad spectrum of antibacterial activity against human and phytopathogenic bacteria in the range of 10-60 AU / mL. Incubation of P. carotovorum cells with increasing concentrations of the antibacterial compound showed a killing rate of 94.8 and 96% at MIC and 2xMIC respectively. In addition, the antibacterial agent did not exert haemolytic activity at the active concentration and has been preliminary characterized by TLC and GC-MS as a glycolipid compound. Treatment of potato tubers with strain Ar10 for 72 h significantly reduced the severity of disease symptoms (100 and 85.05% reduction of necrosis deep / area and weight loss respectively). The same levels in disease symptoms severity was also recorded following treatment of potato tubers with cell free supernatant for 1 h. Data suggest that protection from potato soft rot disease may be related to glycolipid production by strain Ar10. The present study affords new alternatives for anti-Pectobacterium carotovorum bioactive compounds against the soft rot disease of potato.

  • Genome-wide transcriptome response of Streptomyces tsukubaensis to N-acetylglucosamine: effect on tacrolimus biosynthesis
    Microbiol. Res. (IF 2.777) Pub Date : 2018-08-31
    María Ordóñez-Robles, Antonio Rodríguez-García, Juan F. Martín

    Chitin is the second most abundant carbohydrate biopolymer present in soils and is utilized by antibiotic–producing Streptomyces species. Its monomer, N-acetylglucosamine (GlcNAc), regulates the developmental program of the model organism Streptomyces coelicolor. GlcNAc blocks differentiation when growing on rich medium whilst it promotes development on poor culture media. However, it is unclear if the same GlcNAc regulatory profile observed in S. coelicolor applies also to other industrially important Streptomyces species. We report here the negative effect of GlcNAc on differentiation and tacrolimus (FK506) production by Streptomyces tsukubaensis NRRL 18488. Using microarrays technology, we found that GlcNAc represses the transcription of fkbN, encoding the main transcriptional activator of the tacrolimus biosynthetic cluster, and of ppt1, encoding a phosphopantheteinyltransferase involved in tacrolimus biosynthesis. On the contrary, GlcNAc stimulated transcription of genes related to amino acid and nucleotide biosynthesis, DNA replication, RNA translation, glycolysis and pyruvate metabolism. The results obtained support those previously reported for S. coelicolor, but some important differences were observed; for example genes involved in GlcNAc transport and metabolism and genes encoding transcriptional regulators such as crr, ptsI, nagE1, nagE2, nagB, chiA, chiJ, ngcE, dasR or atrA are not significantly induced in S. tsukubaensis by GlcNAc addition. Differences in the GlcNAc transport systems in the physiology of S. tsukubaensis and S. coelicolor and/or the different composition of the culture media used are likely to be responsible for the discrepancies observed between these species.

  • Prospects of endophytic fungal entomopathogens as biocontrol and plant growth promoting agents: an insight on how artificial inoculation methods affect endophytic colonization of host plants
    Microbiol. Res. (IF 2.777) Pub Date : 2018-08-31
    Bamisope Steve Bamisile, Chandra Kanta Dash, Komivi Senyo Akutse, Ravindran Keppanan, Oluwatoyin Grace Afolabi, Mubasher Hussain, Muhammad Qasim, Liande Wang

    Entomopathogenic fungi (EPF) can be established as endophytes in the host plants to offer a long-term preventive measure for pests and diseases. This practice serves as a better alternative to the common practice of periodic direct application of EPF on plants or the target pests as a short-term defense strategy against pests and diseases. These fungal endophytes, aside from their role in pests and diseases prevention, also act as plant growth promoters. Several fungal endophytes have been associated with improvement in plant height, dry and wet weight and other growth parameters. However, many limiting factors have been identified as mitigating the successful colonization of the host plants by EPF. The inoculation methods used have been identified as one, but sadly, this has received little or less attention. Some previous studies carried out comparison between various artificial inoculation methods; foliar application, seedling dipping, soil drenching, seed inoculation, direct injection and others. In separate studies, some authors had suggested different application methods that are best suitable for certain fungal entomopathogens. For instance, leaf inoculation with conidial suspensions was suggested to be the best inoculation method for Beauveria bassiana in sorghum, stem injection was suggested as the most suitable for coffee, while, root dipping method proved the most successful for B. bassiana colonization of tomato plants for the management of Helicoverpa armigera. Here, we discussed entomopathogenic fungal endophytes as bio-control agents, plant growth promoters and highlighted the effect of various artificial inoculation methods on their endophytic colonization of the host plants.

  • Profiles of quorum sensing (QS)-related sequences in phycospheric microorganisms during a marine dinoflagellate bloom, as determined by a metagenomic approach
    Microbiol. Res. (IF 2.777) Pub Date : 2018-08-30
    Xinqing Huang, Jianming Zhu, Zhonghua Cai, Yongmin Lao, Hui Jin, Ke Yu, Boya Zhang, Jin Zhou

    The complicated relationships among environmental microorganisms are regulated by quorum sensing (QS). Understanding QS-based signals could shed light on the interactions between microbial communities in certain environments. Although QS characteristics have been widely discussed, few studies have been conducted on the role of QS in phycospheric microorganisms. Here, we used metagenomics to examine the profile of AI-1 (AinS, HdtS, LuxI) and AI-2 (LuxS) autoinducers from a deeply sequenced microbial database, obtained from a complete dinoflagellate bloom. A total of 3001 putative AI-1 homologs and 130 AI-2 homologs were identified. The predominant member among the AI groups was HdtS. The abundance of HdtS, AinS, and LuxS increased as the bloom developed, whereas the abundance of LuxI showed the opposite trend. Phylogenetic analysis suggested that HdtS and LuxI synthase originated mainly from alpha-, beta-, and gamma-Proteobacteria, whereas AinS synthase originated solely from Vibrionales. In comparison to AI-1, the sequences related to AI-2 (LuxS) demonstrated a much wider taxonomic coverage. Some significant correlations were found between dominant species and QS signals. In addition to the QS, we also performed parallel analysis of the quorum quenching (QQ) sequences. In comparison to QS, the relative abundance of QQ signals was lower; however, an obvious frequency correlation was observed. These results suggested that QS and QQ signals co-participate in regulating microbial communities during an algal bloom. These data helped to reveal the characteristic behavior of algal symbiotic bacteria, and facilitated a better understanding of microbial dynamics during an algal bloom event from a chemical ecological perspective.

  • Antifungal activity of spider venom-derived peptide lycosin-I against Candida tropicalis
    Microbiol. Res. (IF 2.777) Pub Date : 2018-08-27
    Li Tan, Le Bai, Ling Wang, Lagu He, Guangdi Li, Wenhan Du, Ting Shen, Zheyi Xiang, Jiali Wu, Zhonghua Liu, Min Hu

    Candida species are a major cause of human mucosal and deep tissue fungal infections, but few antifungal treatments are available. Here, we showed that lycosin-I, a peptide isolated from venom of the spider Lycosa singoriensis, acted as a potent antifungal inhibitor against Candida species. The MIC50 values of lycosin-I reached 8 µg/mL to treat fluconazole-susceptible and fluconazole-resistant C. tropicalis isolates. Time-kill kinetics assays revealed that after a 2-hour exposure, lycosin-I reduced colony-forming units/mL in fluconazole-susceptible and fluconazole-resistant C. tropicalis isolates approximately 70%. Furthermore, salinity tolerance assays suggested that even in the presence of Mg2+, lycosin-I maintained its potent antifungal ability at a high concentration. When the concentration of lycosin-I was increased from 1 × MIC to 8 × MIC, a significant decrease of the biofilm metabolic activity was observed in both fluconazole-susceptible and fluconazole-resistant C. tropicalis isolates. Moreover, the biofilm inhibitory concentration 50 (BIC50) and the biofilm eradicating concentration 50 (BEC50) were approximately 32 µg/mL and 128 µg/mL, respectively. Confocal laser scanning microscopy showed the localization of CY5-labeled lycosin-I mainly in the cell cytoplasm, and lycosin-I was likely to be localized in the cytoplasm after its transportation across the cell wall and membrane. Overall, our work shows that lycosin-I is a potent antifungal agent with a high efficacy, a high salinity tolerance, and potent anti-biofilm properties.

  • Transcriptional regulation of galF by RcsAB affects capsular polysaccharide formation in Klebsiella pneumoniae NTUH-K2044
    Microbiol. Res. (IF 2.777) Pub Date : 2018-08-25
    Dan Peng, Xuan Li, Pin Liu, Xipeng Zhou, Mei Luo, Kewen Su, Shuai Chen, Zhongshuang Zhang, Qiang He, Jingfu Qiu, Yingli Li

    RcsAB is an atypical two-component regulatory system that can regulate exopolysaccharide biosynthesis and is involved in the virulence of K. pneumoniae. The gene galF is well known as a gene involved in the biosynthesis of capsular polysaccharide (CPS). The specific DNA identification sequence for transcriptional regulation of RcsAB was found to be present in the promoter region of galF. This study aimed to detect the function of RcsAB in virulence and in biofilm and CPS formation. In addition, the transcriptional regulation of the galF gene in K. pneumoniae was studied. To determine the function of rcsAB gene, the wild-type K. pneumoniae strain NTUH-K2044 and the rcsAB knockout and complemented strains were used. The results showed decreased virulence, biofilm formation, and CPS levels in the rcsAB knockout strain. Complementation of the knockout by introducing an rcsAB fragment on an expression plasmid partially restored the virulence, biofilm, and CPS functions of the knockout strain. It indicated that the rcsAB genes might affect CPS formation and virulence of K. pneumonia. RT-qPCR, EMSA and DNase I footprinting assays were conducted to identify the transcriptional regulation of galF by RcsAB. RcsAB was seen to bind to the galF promoter-proximal region, and the binding site was further identified to be located from -177 bp to -152 bp upstream of the galF promoter. In conclusion, RcsAB could regulate the transcription of the galF gene positively by binding to the galF promoter DNA directly, and then affects the CPS formation of K. pneumonia.

  • Complete genome sequence of the marine fish pathogen Vibrio anguillarum and genome-wide transposon mutagenesis analysis of genes essential for in vivo infection
    Microbiol. Res. (IF 2.777) Pub Date : 2018-08-24
    Yang Guanhua, Cheng Wang, Xuetong Wang, Ruiqing Ma, Huajun Zheng, Qin Liu, Yuanxing Zhang, Yue Ma, Qiyao Wang

    Vibrio anguillarum is a notorious bacterial pathogen that causes vibriosis in various marine farmed fish species. The highly pathogenic V. anguillarum strain MVM425 has been isolated from moribund turbot in the China Yellow Sea, but the genetic basis for its pathogenesis is undefined. Complete genome sequencing uncovered that the bacterium encodes 3985 protein-coding sequences (CDSs) on two chromosomes and 62 CDSs on the virulence plasmid pEIB1. To investigate the genes that are essential for its in vitro and in vivo growth, we applied transposon insertion sequencing technology (Tn-seq) to screen a highly saturated transposon insertion mutant library grown in Luria-Bertani broth with 2% NaCl (LB20) and in its natural host turbot. Tn-seq identified 473 and ~173 putative genes as essential for its growth in rich medium and the in vivo infection process, respectively. Additionally, our analysis revealed that the genes are associated to various fitness levels in the liver, spleen and kidney, suggesting different genetic requirements for colonization in these organs. Furthermore, we validated our Tn-seq data using gene knockout mutants and in vivo infection experiments. Comprehensive functional genomics analysis highlighted the conditionally essential genes for important pathways involved in energy metabolism, nitrogen metabolism, nucleotide synthesis, amino acid synthesis, cofactors, siderophore synthesis, secretion (T1SS, T2SS, and T6SS), quorum sensing, and flagellum biosynthesis. This study provides a wealth of information about V. anguillarum genes related to infection in fish and will facilitate the understanding of its pathogenesis as well as the development of diagnostics and vaccines against the pathogen.

  • Dimethyl adenosine transferase (KsgA) contributes to cell-envelope fitness in Salmonella Enteritidis
    Microbiol. Res. (IF 2.777) Pub Date : 2018-08-23
    Kim Lam Chiok, Narayan C. Paul, Ezekiel O. Adekanmbi, Soumya K. Srivastava, Devendra H. Shah

    We previously reported that inactivation of a universally conserved dimethyl adenosine transferase (KsgA) attenuates virulence and increases sensitivity to oxidative and osmotic stress in Salmonella Enteritidis. Here, we show a role of KsgA in cell-envelope fitness as a potential mechanism underlying these phenotypes in Salmonella. We assessed structural integrity of the cell-envelope by transmission electron microscopy, permeability barrier function by determining intracellular accumulation of ethidium bromide and electrophysical properties by dielectrophoresis, an electrokinetic tool, in wild-type and ksgA knock-out mutants of S. Enteritidis. Deletion of ksgA resulted in disruption of the structural integrity, permeability barrier and distorted electrophysical properties of the cell-envelope. The cell-envelope fitness defects were alleviated by expression of wild-type KsgA (WT-ksgA) but not by its catalytically inactive form (ksgAE66A), suggesting that the dimethyl transferase activity of KsgA is important for cell-envelope fitness in S. Enteritidis. Upon expression of WT-ksgA and ksgAE66A in inherently permeable E. coli cells, the former strengthened and the latter weakened the permeability barrier, suggesting that KsgA also contributes to the cell-envelope fitness in E. coli. Lastly, expression of ksgAE66A exacerbated the cell-envelope fitness defects, resulting in impaired S. Enteritidis interactions with human intestinal epithelial cells, and human and avian phagocytes. This study shows that KsgA contributes to cell-envelope fitness and opens new avenues to modulate cell-envelopes via use of KsgA-antagonists.

  • The exoproteome profiles of three Staphylococcus saprophyticus strains reveal diversity in protein secretion contents
    Microbiol. Res. (IF 2.777) Pub Date : 2018-08-23
    Andrea Santana de Oliveira, Isabella Inês Rodrigues Rosa, Evandro Novaes, Lucas Silva de Oliveira, Lilian Cristiane Baeza, Clayton Luiz Borges, Lennart Marlinghaus, Célia Maria de Almeida Soares, Marcia Giambiagi-deMarval, Juliana Alves Parente-Rocha

    Staphylococcus saprophyticus is a gram-positive microorganism responsible for urinary tract infections (UTIs). Although some virulence factors are characterized, such as urease, autolysins, adhesins and hemagglutinins, large-scale proteomic studies have not been performed within this species. We performed the characterization of the exoproteome from three S. saprophyticus strains: the reference strain ATCC 15305, a non-capsular strain 7108 and the 9325 strain containing a thick capsule which were cultured in BHI medium and culture supernatants were analysed by using mass spectrometry approach. We observed a core of 72 secreted proteins. In addition, it was possible to detect diversity in the protein profiles of the exoproteomes. Interestingly, strain 7108 presented no secretion of three antigenic proteins, including the classical SsaA antigen. In addition, the level of antigenic proteins secreted by strain 9325 was higher than in ATCC 15305. This result was confirmed by Western blot analysis using anti-SsaA polyclonal antibodies, and no production/ secretion of SsaA was detected in strain 7108. Transcriptional data shows that 7108 strain produces transcripts encoding SsaA, suggesting post-transcriptional regulation occurs in this strain. Moreover, when compared with the other strains that were analyzed, it was possible to detect higher levels of proteases secreted by strain 7108 and higher levels of antigenic proteins and transglycosylases secreted by 9325 strain. The results reveal diversity in protein secretion among strains. This research is an important first step towards understanding the variability in S. saprophyticus exoproteome profile and could be significant in explaining differences among strains.

  • Isolation and characterization of actinomycetes from Mural paintings of Snu- Sert-Ankh tomb, their antimicrobial activity, and their biodeterioration
    Microbiol. Res. (IF 2.777) Pub Date : 2018-08-15
    Abeer F. Elhagrassy

    A total of 35 actinomycetes were isolated from the surface of mural paintings of snu- sort- ankh in El Leasht, Egypt during four seasons all over 2016-2017. In 2009 this tomb was deteriorated by "Aspergillus niger, A. flavus, Fusarium moniliforme, Alternaria alternate, Rhizopus stolonifera, Bacillus subtilis, Bacillus cereus, and micrococcus iuteus." In 2017 the isolation of swabs presents only Aspergillus niger and about 35 actinomycetes classified to three different groups "Streptomyces, Nocardia, and Micromonospora" only five species belong to Streptomyces group showed antimicrobial activity against the previous microorganisms. These actinomycetes were identified according to their sequences in the GenBank to "Streptomyces spectabilis, S. alborgriseolus, S. globsus, S. corchorstt, S. ambofactens." In the other hand, the pigments of the wall paintings of the tomb (Egyptian blue, Egyptian green, Goethite) that analyzed by SEM-EDX, FTIR were measured by the spectrophotometer in both 2009 and 2017. The results showed that the actinomycetes could produce extracellular pigments causing a color change of archaeological pigments otherwise it helps in inhibition the growth of the previous microorganisms found in 2009. The optimization factors for increasing the antibiotic production of the Streptomyces were 3% NaCl and temperature between 30:35°c in Alkin pH (pH = 7.5).

  • Pseudomonas sp. AF-54 containing multiple plant beneficial traits acts as growth enhancer of Helianthus annuus L.under reduced fertilizer input
    Microbiol. Res. (IF 2.777) Pub Date : 2018-08-13
    Afshan Majeed, M. Kaleem Abbasi, Sohail Hameed, Sumera Yasmin, Muhammad Kashif Hanif, Tahir Naqqash, Asma Imran

    Plant growth promoting rhizobacteria (PGPR) are capable to increase the growth and yield of crops in eco-friendly and sustainable manner. To evaluate the response of sunflower towards inoculation with PGPR, a sunflower root associated bacterium AF-54 isolated from Diyar Gali Himalayan Mountain region, Azad Jammu and Kashmir (AJK), identified as Pseudomonas sp. by 16S rRNA sequence analysis and was characterized using polyphasic approach. The bacterium produced 23.9 µgmL-1 indole-3-acetic acid in tryptophan-supplemented medium, showed 44.28 nmoles mg-1 protein h-1 nitrogenase activity through acetylene reduction assay and released 48.80 μg mL−1 insoluble phosphorus in Pikovskaya’s broth. During P-solubilization, the pH of the Pikovskaya’s medium decreased from 7 to 3.04 due to the production of acetic acid, malic acid and gluconic acid. Pseudomonas sp. AF-54 showed metabolic versatility by utilizing79 carbon sources from BIOLOG GN2 plates and resistance to many antibiotics. Furthermore, it inhibited the growth of Fusarium oxysporum in dual culture assay. To evaluate the plant-inoculation response, series of experiments conducted in hydroponic, sterilized soil and fields at AJK, and Faisalabad where inoculated plants with reduced fertilizer showed a significant increase in growth, yield, oil contents and achene NP uptake as compared to non-inoculated control. AF-54 showed extensive root colonization in sterilized and non-sterile conditions documented through yfp-labeling and fluorescent in situ hybridization coupled with confocal laser scanning microscopy. This study concludes that the Pseudomonas sp. strain AF-54 containing multiple plant growth promoting traits can be a potential candidate for biofertilizer production to enhance sunflower crop yield with reduced application of chemical (NP) fertilizers.

  • Emergence of boscalid-resistant strains of Erysiphe necator in French vineyards
    Microbiol. Res. (IF 2.777) Pub Date : 2018-08-11
    Semcheddine Cherrad, Aline Charnay, Catalina Hernandez, Herve Steva, Lassaad Belbahri, Sébastien Vacher

    The grapevine powdery mildew Erysiphe necator (E. necator) is an obligate pathogen. Powdery mildew-diseased vines show an important reduction in plant size, winter hardiness and grape yield. Even a low-level infection with powdery mildew was shown to taint wine and ultimately reduce wine quality. For many years, succinate dehydrogenase inhibitor (SDHI) fungicides, mainly the new generation active ingredients (AIs) boscalid, penthiopyrad and fluopyram, have been widely used to control powdery mildew in grapevines. The repeated use of fungicides (mainly boscalid) has resulted in the emergence of resistant microorganisms such as Botrytis cinerea (B. cinerea). However, boscalid resistance was never observed in E. necator. In this study, a large-scale survey of French grapevine field populations of E. necator revealed many field populations with low sensitivity to boscalid. Single spore strains originating from collected resistant populations showed Half maximal effective concentration (EC50) values greater than 100 mg L-1, and strains originating from boscalid sensitive populations showed EC50 values lower than 1 mg L-1. The complete nucleotide sequences of the EnSdhB succinate dehydrogenase of sensitive and resistant single spore strains revealed that H242R and H242Y substitutions in the EnSdhB succinate dehydrogenase subunit conferred E. necator resistance to boscalid. No cross-resistance of E. necator strains bearing H242R and H242Y substitutions in EnSdhB succinate dehydrogenase to fluxapyroxad and fluopyram was noticed. Therefore, our results highlight the emergence of resistance to boscalid activity in French vineyards and warrant the need of the implementation of risk assessment strategies to maintain effective grapevine protection against powdery mildew.

  • Biological roles of indole-3-acetic acid in Acinetobacter baumannii
    Microbiol. Res. (IF 2.777) Pub Date : 2018-08-07
    Huei-Ru Lin, Hung-Yu Shu, Guang-Huey Lin

    Indole-3-acetic acid (IAA) is an important plant hormone, and many types of bacteria interact with plants by producing or degrading IAA in the rhizosphere. The iac (indole-3-acetic acid catabolism) gene locus in Acinetobacter baumannii ATCC19606 was previously associated with IAA degradative capability, and in this study, transcriptome analysis results derived from A. baumannii cultured with IAA showed that the expression of catechol-degrading and phenylacetate-degrading genes was elevated, indicating that IAA is likely degraded through these pathways. This study further found that A. baumannii also has IAA productive capability, primarily involving the ipdC gene, and transcriptome and spent media analysis of wild-type and mutant cultures grown in minimal media revealed that A. baumannii likely produces IAA through the indole-3-pyruvic acid (IPyA) pathway. Exogenously applied IAA improved tolerance against oxidative stress in wild-type A. baumannii and iacA mutants unable to degrade IAA, but not in ipdC mutants incapable of producing IAA, suggesting that endogenous IAA is important for stress tolerance. Meanwhile, ipdC mutants also had reduced virulence against human A549 epithelial cells as compared to wild-type. Endogenously produced IAA was found to enhance root growth in A. baumannii and kidney bean plant co-cultures, indicating that A. baumannii can interact with plants through the production and degradation of IAA. Taken together, this study sheds light on the biosynthesis pathways and functional significance of IAA in A. baumannii, and may be useful in exploring other IAA-mediated plant-microbe interactions as well.

  • Phenotype responses to abiotic stresses, asexual reproduction and virulence among isolates of the entomopathogenic fungus Cordyceps javanica (Hypocreales: Cordycipitaceae)
    Microbiol. Res. (IF 2.777) Pub Date : 2018-08-04
    Gabriel Moura Mascarin, Ronaldo Alves Pereira-Junior, Éverton Kort Kamp Fernandes, Eliane Dias Quintela, Christopher A. Dunlap, Steven Paul Arthurs
Some contents have been Reproduced with permission of the American Chemical Society.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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