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  • American Society for Microbiology  (22)
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  • 1
    Online-Ressource
    Online-Ressource
    Structural and Mechanistic Insights into the Improvement of the Halotolerance of a Marine Microbial Esterase by Increasing Intra- and Interdomain Hydrophobic Interactions
    American Society for Microbiology ; 2017
    In:  Applied and Environmental Microbiology Vol. 83, No. 18 ( 2017-09-15)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 83, No. 18 ( 2017-09-15)
    Kurzfassung: Halotolerant enzymes are beneficial for industrial processes requiring high salt concentrations and low water activity. Most halophilic proteins are evolved to have reduced hydrophobic interactions on the surface and in the hydrophobic cores for their haloadaptation. However, in this study, we improved the halotolerance of a thermolabile esterase, E40, by increasing intraprotein hydrophobic interactions. E40 was quite unstable in buffers containing more than 0.3 M NaCl, and its k cat and substrate affinity were both significantly reduced in 0.5 M NaCl. By introducing hydrophobic residues in loop 1 of the CAP domain and/or α7 of the catalytic domain in E40, we obtained several mutants with improved halotolerance, and the M3 S202W I203F mutant was the most halotolerant. (“M3” represents a mutation in loop 1 of the CAP domain in which residues R22-K23-T24 of E40 are replaced by residues Y22-K23-H24-L25-S26 of Est2.) Then we solved the crystal structures of the S202W I203F and M3 S202W I203F mutants to reveal the structural basis for their improved halotolerance. Structural analysis revealed that the introduction of hydrophobic residues W202 and F203 in α7 significantly improved E40 halotolerance by strengthening intradomain hydrophobic interactions of F203 with W202 and other residues in the catalytic domain. By further introducing hydrophobic residues in loop 1, the M3 S202W I203F mutant became more rigid and halotolerant due to the formation of additional interdomain hydrophobic interactions between the introduced Y22 in loop 1 and W204 in α7. These results indicate that increasing intraprotein hydrophobic interactions is also a way to improve the halotolerance of enzymes with industrial potential under high-salt conditions. IMPORTANCE Esterases and lipases for industrial application are often subjected to harsh conditions such as high salt concentrations, low water activity, and the presence of organic solvents. However, reports on halotolerant esterases and lipases are limited, and the underlying mechanism for their halotolerance is still unclear due to the lack of structures. In this study, we focused on the improvement of the halotolerance of a salt-sensitive esterase, E40, and the underlying mechanism. The halotolerance of E40 was significantly improved by introducing hydrophobic residues. Comparative structural analysis of E40 and its halotolerant mutants revealed that increased intraprotein hydrophobic interactions make these mutants more rigid and more stable than the wild type against high concentrations of salts. This study shows a new way to improve enzyme halotolerance, which is helpful for protein engineering of salt-sensitive enzymes.
    Materialart: Online-Ressource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.01286-17
    RVK:
    WA 15000
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2017
    ZDB Id: 223011-2
    ZDB Id: 1478346-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 2
    Online-Ressource
    Online-Ressource
    Mechanistic Insight into the Fragmentation of Type I Collagen Fibers into Peptides and Amino Acids by a Vibrio Collagenase
    American Society for Microbiology ; 2022
    In:  Applied and Environmental Microbiology Vol. 88, No. 7 ( 2022-04-12)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 88, No. 7 ( 2022-04-12)
    Kurzfassung: Vibrio collagenases of the M9A subfamily are closely related to Vibrio pathogenesis for their role in collagen degradation during host invasion. Although some Vibrio collagenases have been characterized, the collagen degradation mechanism of Vibrio collagenase is still largely unknown. Here, an M9A collagenase, VP397, from marine Vibrio pomeroyi strain 12613 was characterized, and its fragmentation pattern on insoluble type I collagen fibers was studied. VP397 is a typical Vibrio collagenase composed of a catalytic module featuring a peptidase M9N domain and a peptidase M9 domain and two accessory bacterial prepeptidase C-terminal domains (PPC domains). It can hydrolyze various collagenous substrates, including fish collagen, mammalian collagens of types I to V, triple-helical peptide [(POG) 10 ] 3 , gelatin, and 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-o-Arg (Pz-peptide). Atomic force microscopy (AFM) observation and biochemical analyses revealed that VP397 first assaults the C-telopeptide region to dismantle the compact structure of collagen and dissociate tropocollagen fragments, which are further digested into peptides and amino acids by VP397 mainly at the Y-Gly bonds in the repeating Gly-X-Y triplets. In addition, domain deletion mutagenesis showed that the catalytic module of VP397 alone is capable of hydrolyzing type I collagen fibers and that its C-terminal PPC2 domain functions as a collagen-binding domain during collagenolysis. Based on our results, a model for the collagenolytic mechanism of VP397 is proposed. This study sheds light on the mechanism of collagen degradation by Vibrio collagenase, offering a better understanding of the pathogenesis of Vibrio and helping in developing the potential applications of Vibrio collagenase in industrial and medical areas. IMPORTANCE Many Vibrio species are pathogens and cause serious diseases in humans and aquatic animals. The collagenases produced by pathogenic Vibrio species have been regarded as important virulence factors, which occasionally exhibit direct pathogenicity to the infected host or facilitate other toxins’ diffusion through the digestion of host collagen. However, our knowledge concerning the collagen degradation mechanism of Vibrio collagenase is still limited. This study reveals the degradation strategy of Vibrio collagenase VP397 on type I collagen. VP397 binds on collagen fibrils via its C-terminal PPC2 domain, and its catalytic module first assaults the C-telopeptide region and then attacks the Y-Gly bonds in the dissociated tropocollagen fragments to release peptides and amino acids. This study offers new knowledge regarding the collagenolytic mechanism of Vibrio collagenase, which is helpful for better understanding the role of collagenase in Vibrio pathogenesis and for developing its industrial and medical applications.
    Materialart: Online-Ressource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/aem.01677-21
    RVK:
    WA 15000
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2022
    ZDB Id: 223011-2
    ZDB Id: 1478346-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 3
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    Divergent Co-occurrence Patterns and Assembly Processes Structure the Abundant and Rare Bacterial Communities in a Salt Marsh Ecosystem
    American Society for Microbiology ; 2020
    In:  Applied and Environmental Microbiology Vol. 86, No. 13 ( 2020-06-17)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 86, No. 13 ( 2020-06-17)
    Kurzfassung: Understanding how species interaction and assembly processes structure the abundant and rare bacterial biospheres in soils is crucial for predicting how biodiversity influences ecosystem functioning. Here, we profiled the bacterial communities across a salt marsh ecosystem gradient to investigate the co-occurrence patterns across taxa and the relative influence of ecological processes mediating the assembly of the abundant and rare biospheres in soil. Our results revealed abundant taxa to be ubiquitous across all sites, whereas the distributions of the rare taxa were relatively more site specific. The α-diversity indices and β-diversity of rare subcommunities were significantly higher than those of the abundant subcommunities. Besides, both the taxonomic and functional composition of soil bacterial communities differed significantly between the two biospheres. Furthermore, the influence of stochasticity differed in each subcommunity. In particular, stochastic processes were relatively more important in constraining the assembly of rare taxa. Co-occurrence network analysis revealed that a few abundant taxa occupy central nodes within the networks, possibly indicating crucial roles as keystone taxa. Collectively, these findings suggest that abundant and rare bacterial biospheres have distinct distributions underpinned by a dynamic interplay of ecological processes and taxon co-occurrence patterns. IMPORTANCE Estuarine salt marshes are highly productive ecosystems subjected to regular disturbances by hydrodynamic exchange. However, little is known about how distinct assembly processes and co-occurrence of taxa influence the structure of the abundant and rare bacterial biospheres in these soil systems. This study aims at unravelling these intricacies by studying a typical estuarine salt marsh located in Hangzhou Bay, China. Our study provides important pieces of evidence on the diverse distribution of rare and abundant bacterial biospheres. We show that a few abundant taxa are central nodes in species co-occurrence, potentially playing important roles as keystone species in the system. In addition, we highlight a dynamic interplay of assembly processes structuring these two subcommunities.
    Materialart: Online-Ressource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.00322-20
    RVK:
    WA 15000
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2020
    ZDB Id: 223011-2
    ZDB Id: 1478346-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 4
    Online-Ressource
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    Novel Insights into Dimethylsulfoniopropionate Catabolism by Cultivable Bacteria in the Arctic Kongsfjorden
    American Society for Microbiology ; 2022
    In:  Applied and Environmental Microbiology Vol. 88, No. 2 ( 2022-01-25)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 88, No. 2 ( 2022-01-25)
    Kurzfassung: Dimethylsulfoniopropionate (DMSP) is one of the most abundant organic sulfur compounds in the oceans, which is mainly degraded by bacteria through two pathways, a cleavage pathway and a demethylation pathway. Its volatile catabolites dimethyl sulfide (DMS) and methanethiol (MT) in these pathways play important roles in the global sulfur cycle and have potential influences on the global climate. Intense DMS/DMSP cycling occurs in the Arctic. However, little is known about the diversity of cultivable DMSP-catabolizing bacteria in the Arctic and how they catabolize DMSP. Here, we screened DMSP-catabolizing bacteria from Arctic samples and found that bacteria of four genera ( Psychrobacter , Pseudoalteromonas , Alteromonas, and Vibrio ) could grow with DMSP as the sole carbon source, among which Psychrobacter and Pseudoalteromonas are predominant. Four representative strains ( Psychrobacter sp. K31L, Pseudoalteromonas sp. K222D, Alteromonas sp. K632G, and Vibrio sp. G41H) from different genera were selected to probe their DMSP catabolic pathways. All these strains produce DMS and MT simultaneously during their growth on DMSP, indicating that all strains likely possess the two DMSP catabolic pathways. On the basis of genomic and biochemical analyses, the DMSP catabolic pathways in these strains were proposed. Bioinformatic analysis indicated that most Psychrobacter and Vibrio bacteria have the potential to catabolize DMSP via the demethylation pathway and that only a small portion of Psychrobacter strains may catabolize DMSP via the cleavage pathway. This study provides novel insights into DMSP catabolism in marine bacteria. IMPORTANCE Dimethylsulfoniopropionate (DMSP) is abundant in the oceans. The catabolism of DMSP is an important step of the global sulfur cycle. Although Gammaproteobacteria are widespread in the oceans, the contribution of Gammaproteobacteria in global DMSP catabolism is not fully understood. Here, we found that bacteria of four genera belonging to Gammaproteobacteria ( Psychrobacter , Pseudoalteromonas , Alteromonas and Vibrio ), which were isolated from Arctic samples, were able to grow on DMSP. The DMSP catabolic pathways of representative strains were proposed. Bioinformatic analysis indicates that most Psychrobacter and Vibrio bacteria have the potential to catabolize DMSP via the demethylation pathway and that only a small portion of Psychrobacter strains may catabolize DMSP via the cleavage pathway. Our results suggest that novel DMSP dethiomethylases/demethylases may exist in Pseudoalteromonas , Alteromonas, and Vibrio and that Gammaproteobacteria may be important participants in the marine environment, especially in polar DMSP cycling.
    Materialart: Online-Ressource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.01806-21
    RVK:
    WA 15000
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2022
    ZDB Id: 223011-2
    ZDB Id: 1478346-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 5
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    Enhanced Control of Cucumber Wilt Disease by Bacillus amyloliquefaciens SQR9 by Altering the Regulation of Its DegU Phosphorylation
    American Society for Microbiology ; 2014
    In:  Applied and Environmental Microbiology Vol. 80, No. 9 ( 2014-05), p. 2941-2950
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 80, No. 9 ( 2014-05), p. 2941-2950
    Kurzfassung: Bacillus amyloliquefaciens strain SQR9, isolated from the cucumber rhizosphere, suppresses the growth of Fusarium oxysporum in the cucumber rhizosphere and protects the host plant from pathogen invasion through efficient root colonization. In the Gram-positive bacterium Bacillus , the response regulator DegU regulates genetic competence, swarming motility, biofilm formation, complex colony architecture, and protease production. In this study, we report that stepwise phosphorylation of DegU in B. amyloliquefaciens SQR9 can influence biocontrol activity by coordinating multicellular behavior and regulating the synthesis of antibiotics. Results from in vitro and in situ experiments and quantitative PCR (qPCR) studies demonstrate the following: (i) that the lowest level of phosphorylated DegU (DegU∼P) (the degQ mutation) impairs complex colony architecture, biofilm formation, colonization activities, and biocontrol efficiency of Fusarium wilt disease but increases the production of macrolactin and bacillaene, and (ii) that increasing the level of DegU∼P by degQ and degSU overexpression significantly improves complex colony architecture, biofilm formation, colonization activities, production of the antibiotics bacillomycin D and difficidin, and efficiency of biocontrol of Fusarium wilt disease. The results offer a new strategy to enhance the biocontrol efficacy of Bacillus amyloliquefaciens SQR9.
    Materialart: Online-Ressource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.03943-13
    RVK:
    WA 15000
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2014
    ZDB Id: 223011-2
    ZDB Id: 1478346-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 6
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    Online-Ressource
    Lack of N-Terminal Segment of the Flagellin Protein Results in the Production of a Shortened Polar Flagellum in the Deep-Sea Sedimentary Bacterium Pseudoalteromonas sp. Strain SM9913
    American Society for Microbiology ; 2021
    In:  Applied and Environmental Microbiology Vol. 87, No. 21 ( 2021-10-14)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 87, No. 21 ( 2021-10-14)
    Kurzfassung: Bacterial polar flagella, comprised of flagellin, are essential for bacterial motility. Pseudoalteromonas sp. strain SM9913 is a bacterium isolated from deep-sea sediments. Unlike other Pseudoalteromonas strains that have a long polar flagellum, strain SM9913 has an abnormally short polar flagellum. Here, we investigated the underlying reason for the short flagellum and found that a single-base mutation was responsible for the altered flagellar assembly. This mutation leads to the fragmentation of the flagellin gene into two genes, PSM_A2281 , encoding the core segment and the C-terminal segment, and PSM_A2282 , encoding the N-terminal segment, and only gene PSM_A2281 is involved in the production of the short polar flagellum. When a chimeric gene of PSM_A2281 and PSM_A2282 encoding an intact flagellin, A2281::82, was expressed, a long polar flagellum was produced, indicating that the N-terminal segment of flagellin contributes to the production of a polar flagellum of a normal length. Analyses of the simulated structures of A2281 and A2281::82 and that of the flagellar filament assembled with A2281::82 indicate that due to the lack of two α-helices, the core of the flagellar filament assembled with A2281 is incomplete and is likely too weak to support the stability and movement of a long flagellum. This mutation in strain SM9913 had little effect on its growth and only a small effect on its swimming motility, implying that strain SM9913 can live well with this mutation in natural sedimentary environments. This study provides a better understanding of the assembly and production of bacterial flagella. IMPORTANCE Polar flagella, which are essential organelles for bacterial motility, are comprised of multiple flagellin subunits. A flagellin molecule contains an N-terminal segment, a core segment, and a C-terminal segment. The results of this investigation of the deep-sea sedimentary bacterium Pseudoalteromonas sp. strain SM9913 demonstrate that a single-base mutation in the flagellin gene leads to the production of an incomplete flagellin without the N-terminal segment and that the loss of the N-terminal segment of the flagellin protein results in the production of a shortened polar flagellar filament. Our results shed light on the important function of the N-terminal segment of flagellin in the assembly and stability of bacterial flagellar filament.
    Materialart: Online-Ressource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.01527-21
    RVK:
    WA 15000
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2021
    ZDB Id: 223011-2
    ZDB Id: 1478346-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 7
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    Gene Deletion Strategy To Examine the Involvement of the Two Chondroitin Lyases in Flavobacterium columnare Virulence
    American Society for Microbiology ; 2015
    In:  Applied and Environmental Microbiology Vol. 81, No. 21 ( 2015-11), p. 7394-7402
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 81, No. 21 ( 2015-11), p. 7394-7402
    Kurzfassung: Flavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (P ompA ) was fused to sacB to construct a counterselectable marker for F. columnare . F. columnare carrying P ompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying P ompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB , were deleted. The Δ cslA and Δ cslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (Δ cslA Δ cslB ) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G 4 and of the chondroitin lyase-deficient Δ cslA Δ cslB mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of F. columnare but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum Bacteroidetes .
    Materialart: Online-Ressource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.01586-15
    RVK:
    WA 15000
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2015
    ZDB Id: 223011-2
    ZDB Id: 1478346-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 8
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    ResDE Two-Component Regulatory System Mediates Oxygen Limitation-Induced Biofilm Formation by Bacillus amyloliquefaciens SQR9
    American Society for Microbiology ; 2018
    In:  Applied and Environmental Microbiology Vol. 84, No. 8 ( 2018-04-15)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 84, No. 8 ( 2018-04-15)
    Kurzfassung: Efficient biofilm formation and root colonization capabilities facilitate the ability of beneficial plant rhizobacteria to promote plant growth and antagonize soilborne pathogens. Biofilm formation by plant-beneficial Bacillus strains is triggered by environmental cues, including oxygen deficiency, but the pathways that sense these environmental signals and regulate biofilm formation have not been thoroughly elucidated. In this study, we showed that the ResDE two-component regulatory system in the plant growth-promoting rhizobacterium Bacillus amyloliquefaciens strain SQR9 senses the oxygen deficiency signal and regulates biofilm formation. ResE is activated by sensing the oxygen limitation-induced reduction of the NAD + /NADH pool through its PAS domain, stimulating its kinase activity, and resulting in the transfer of a phosphoryl group to ResD. The phosphorylated ResD directly binds to the promoter regions of the qoxABCD and ctaCDEF operons to improve the biosynthesis of terminal oxidases, which can interact with KinB to activate biofilm formation. These results not only revealed the novel regulatory function of the ResDE two-component system but also contributed to the understanding of the complicated regulatory network governing Bacillus biofilm formation. This research may help to enhance the root colonization and the plant-beneficial efficiency of SQR9 and other Bacillus rhizobacteria used in agriculture. IMPORTANCE Bacillus spp. are widely used as bioinoculants for plant growth promotion and disease suppression. The exertion of their plant-beneficial functions is largely dependent on their root colonization, which is closely related to their biofilm formation capabilities. On the other hand, Bacillus is the model bacterium for biofilm study, and the process and molecular network of biofilm formation are well characterized (B. Mielich-Süss and D. Lopez, Environ Microbiol 17:555–565, 2015, https://doi.org/10.1111/1462-2920.12527 ; L. S. Cairns, L. Hobley, and N. R. Stanley-Wall, Mol Microbiol 93:587–598, 2014, https://doi.org/10.1111/mmi.12697 ; H. Vlamakis, C. Aguilar, R. Losick, and R. Kolter, Genes Dev 22:945–953, 2008, https://doi.org/10.1101/gad.1645008 ; S. S. Branda, A. Vik, L. Friedman, and R. Kolter, Trends Microbiol 13:20–26, 2005, https://doi.org/10.1016/j.tim.2004.11.006 ; C. Aguilar, H. Vlamakis, R. Losick, and R. Kolter, Curr Opin Microbiol 10:638–643, 2007, https://doi.org/10.1016/j.mib.2007.09.006 ; S. S. Branda, J. E. González-Pastor, S. Ben-Yehuda, R. Losick, and R. Kolter, Proc Natl Acad Sci U S A 98:11621–11626, 2001, https://doi.org/10.1073/pnas.191384198 ). However, the identification and sensing of environmental signals triggering Bacillus biofilm formation need further research. Here, we report that the oxygen deficiency signal inducing Bacillus biofilm formation is sensed by the ResDE two-component regulatory system. Our results not only revealed the novel regulatory function of the ResDE two-component regulatory system but also identified the sensing system of a biofilm-triggering signal. This knowledge can help to enhance the biofilm formation and root colonization of plant-beneficial Bacillus strains and also provide new insights of bacterial biofilm formation regulation.
    Materialart: Online-Ressource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.02744-17
    RVK:
    WA 15000
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2018
    ZDB Id: 223011-2
    ZDB Id: 1478346-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 9
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    MtTRC-1, a Novel Transcription Factor, Regulates Cellulase Production via Directly Modulating the Genes Expression of the Mthac-1 and Mtcbh-1 in Myceliophthora thermophila
    American Society for Microbiology ; 2022
    In:  Applied and Environmental Microbiology Vol. 88, No. 19 ( 2022-10-11)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 88, No. 19 ( 2022-10-11)
    Kurzfassung: The thermophilic fungus Myceliophthora thermophila has been used to produce industrial enzymes and biobased chemicals. In saprotrophic fungi, the mechanisms regulating cellulase production have been studied, which revealed the involvement of multiple transcription factors. However, in M. thermophila , the transcription factors influencing cellulase gene expression and secretion remain largely unknown. In this study, we identified and characterized a novel cellulase regulator (MtTRC-1) in M. thermophila through a combination of functional genomics and genetic analyses. Deletion of Mttrc-1 resulted in significantly decreased cellulase production and activities. Transcriptome analysis revealed downregulation of not only the encoding genes of main cellulases but also the transcriptional regulator MtHAC-1 of UPR pathway after disruption of MtTRC-1 under cellulolytic induction conditions. Herein, we also characterized the ortholog of the yeast HAC1p in M. thermophila . We show that Mthac-1 mRNA undergoes an endoplasmic reticulum (ER) stress-induced splicing by removing a 23-nucleotide (nt) intron. Notably, the protein secretion on cellulose was dramatically impaired by the deletion of MtHAC-1. Moreover, the colonial growth on various carbon sources was defective in the absence of MtHAC-1. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays verified MtTRC-1 regulates the transcription of Mthac-1 and the major cellulase gene Mtcbh-1 by binding directly to the promoters in vitro and in vivo. Furthermore, DNase I footprinting assays identified the putative consensus binding site (5′-GNG/C-3′). These results revealed the importance of MtTRC-1 for positively regulating cellulase production. This finding has clarified the complex regulatory pathways involved in cellulolytic enzyme production. IMPORTANCE In the present study, we characterized a novel regulator MtTRC-1 in M. thermophila , which regulated cellulase production through direct transcriptional regulation of the Mthac-1 and Mtcbh-1 genes. Our data demonstrated that MtHAC-1 is a key factor for the cellulase secretion capacity of M. thermophila . Our data indicate that this thermophilic fungus regulates cellulase production through a multilevels network, in which the protein secretory pathway is modulated by MtHAC-1-dependent UPR pathway and the cellulase gene expression is directly regulated in parallel by transcription factors. The conservation of Mttrc1 in filamentous fungi suggests this mechanism may be exploited to engineer filamentous fungal cell factories capable of producing proteins on an industrial scale.
    Materialart: Online-Ressource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/aem.01263-22
    RVK:
    WA 15000
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2022
    ZDB Id: 223011-2
    ZDB Id: 1478346-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 10
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    Volatile Organic Compounds from Pythium oligandrum Play a Role in Its Parasitism on Plant-Pathogenic Pythium myriotylum
    American Society for Microbiology ; 2023
    In:  Applied and Environmental Microbiology Vol. 89, No. 2 ( 2023-02-28)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 89, No. 2 ( 2023-02-28)
    Kurzfassung: The oomycete Pythium oligandrum is a soil-inhabiting parasite and predator of both fungi and oomycetes, and uses hydrolytic enzymes extensively to penetrate and hydrolyze its host or prey. Other mechanisms have been studied less, and we investigated the contribution of P. oligandrum -produced volatile organic compounds (VOCs) to parasitism. The growth-inhibiting activity of P. oligandrum VOCs was tested on Pythium myriotylum —a host or prey of P. oligandrum —coupled with electron microscopy, and biochemical and transcriptomic analyses. The P. oligandrum -produced VOCs reduced P. myriotylum growth by 80% and zoospore levels by 60%. Gas chromatography–mass spectrometry (GC-MS) identified 23 VOCs, and methyl heptenone, d-limonene, 2-undecanone, and 1-octanal were potent inhibitors of P. myriotylum growth and led to increased production of reactive oxygen species at a concentration that did not inhibit P. oligandrum growth. Exposure to the P. oligandrum VOCs led to shrinkage of P. myriotylum hyphae and lysis of the cellular membranes and organelles. Transcriptomics of P. myriotylum exposed to the P. oligandrum VOCs at increasing levels of growth inhibition initially showed a strong upregulation of putative detoxification-related genes that was not maintained later. The inhibition of P. myriotylum growth continued immediately after the exposure to the VOCs was discontinued and led to the reduced infection of its plant hosts. The VOCs produced by P. oligandrum could be another factor alongside hydrolytic enzymes contributing to its ecological role as a microbial parasite in particular ecological niches such as in soil, and may also contribute to the biocontrol of diseases using P. oligandrum commercial preparations. IMPORTANCE Microbe–microbe interactions in nature are multifaceted, with multiple mechanisms of action, and are crucial to how plants interact with microbes. Volatile organic compounds (VOCs) have diverse functions, including contributing to parasitism in ecological interactions and potential applications in biocontrol. The microbial parasite P. oligandrum is well known for using hydrolytic enzymes as part of its parasitism. We found that P. oligandrum VOCs reduced the growth of, and caused major damage to, the hyphae of P. myriotylum (a host or prey of P. oligandrum ). Transcriptomic analyses of P. myriotylum exposed to the VOCs revealed the upregulation of genes potentially involved in an attempt to detoxify the VOCs. The inhibitory effects of the VOCs had a knock-on effect by reducing the virulence of P. myriotylum toward its plant hosts. The P. oligandrum VOCs could contribute to its ecological role as a microbial parasite. The VOCs analyzed here may also contribute to the biocontrol of diseases using P. oligandrum commercial preparations.
    Materialart: Online-Ressource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/aem.02036-22
    RVK:
    WA 15000
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2023
    ZDB Id: 223011-2
    ZDB Id: 1478346-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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