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Cynthia B Whitchurch - One of the best experts on this subject based on the ideXlab platform.
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a global genomic approach uncovers novel components for Twitching Motility mediated biofilm expansion in pseudomonas aeruginosa
Microbial genomics, 2018Co-Authors: Laura M Nolan, Cynthia B Whitchurch, Lars Barquist, Marilyn Katrib, Christine J Boinett, Matthew Mayho, David Goulding, Ian G Charles, Alain FillouxAbstract:Pseudomonas aeruginosa is an extremely successful pathogen able to cause both acute and chronic infections in a range of hosts, utilizing a diverse arsenal of cell-associated and secreted virulence factors. A major cell-associated virulence factor, the Type IV pilus (T4P), is required for epithelial cell adherence and mediates a form of surface translocation termed Twitching Motility, which is necessary to establish a mature biofilm and actively expand these biofilms. P. aeruginosa Twitching Motility-mediated biofilm expansion is a coordinated, multicellular behaviour, allowing cells to rapidly colonize surfaces, including implanted medical devices. Although at least 44 proteins are known to be involved in the biogenesis, assembly and regulation of the T4P, with additional regulatory components and pathways implicated, it is unclear how these components and pathways interact to control these processes. In the current study, we used a global genomics-based random-mutagenesis technique, transposon directed insertion-site sequencing (TraDIS), coupled with a physical segregation approach, to identify all genes implicated in Twitching Motility-mediated biofilm expansion in P. aeruginosa. Our approach allowed identification of both known and novel genes, providing new insight into the complex molecular network that regulates this process in P. aeruginosa. Additionally, our data suggest that the flagellum-associated gene products have a differential effect on Twitching Motility, based on whether components are intra- or extracellular. Overall the success of our TraDIS approach supports the use of this global genomic technique for investigating virulence genes in bacterial pathogens.
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a global genomic approach uncovers novel components for Twitching Motility mediated biofilm expansion in pseudomonas aeruginosa
bioRxiv, 2018Co-Authors: Laura M Nolan, Cynthia B Whitchurch, Lars Barquist, Marilyn Katrib, Christine J Boinett, Matthew Mayho, David Goulding, Ian G Charles, Alain Filloux, Julian ParkhillAbstract:Pseudomonas aeruginosa is an extremely successful pathogen able to cause both acute and chronic infections in a range of hosts, utilizing a diverse arsenal of cell-associated and secreted virulence factors. A major cell-associated virulence factor, the Type IV pilus (T4P), is required for epithelial cell adherence and mediates a form of surface translocation termed Twitching Motility, which is necessary to establish a mature biofilm and actively expand these biofilms. P. aeruginosa Twitching Motility-mediated biofilm expansion is a coordinated, multicellular behaviour, allowing cells to rapidly colonize surfaces, including implanted medical devices. Although at least 44 proteins are known to be involved in the biogenesis, assembly and regulation of the T4P, with additional regulatory components and pathways implicated, it is unclear how these components and pathways interact to control these processes. In the current study, we used a global genomics-based random-mutagenesis technique, transposon directed insertion-site sequencing (TraDIS), coupled with a physical segregation approach, to identify all genes implicated in Twitching Motility-mediated biofilm expansion in P. aeruginosa. Our approach allowed identification of both known and novel genes, providing new insight into the complex molecular network that regulates this process in P. aeruginosa. Additionally, our data suggests a differential effect on Twitching Motility by flagellum components based upon their cellular location. Overall the success of our TraDIS approach supports the use of this global genomic technique for investigating virulence genes in bacterial pathogens.
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a two component regulatory system modulates Twitching Motility in dichelobacter nodosus
Veterinary Microbiology, 2015Co-Authors: Lynne Turnbull, Ruth M Kennan, Carrie J Lovitt, Xiaoyan Han, Dane Parker, Cynthia B WhitchurchAbstract:Dichelobacter nodosus is the essential causative agent of footrot in sheep and type IV fimbriae-mediated Twitching Motility has been shown to be essential for virulence. We have identified a two-component signal transduction system (TwmSR) that shows similarity to chemosensory systems from other bacteria. Insertional inactivation of the gene encoding the response regulator, TwmR, led to a Twitching Motility defect, with the mutant having a reduced rate of Twitching Motility when compared to the wild-type and a mutant complemented with the wild-type twmR gene. The reduced rate of Twitching Motility was not a consequence of a reduced growth rate or decreased production of surface located fimbriae, but video microscopy indicated that it appeared to result from an overall loss of Twitching directionality. These results suggest that a chemotactic response to environmental factors may play an important role in the D. nodosus-mediated disease process.
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extracellular atp inhibits Twitching Motility mediated biofilm expansion by pseudomonas aeruginosa
BMC Microbiology, 2015Co-Authors: Laura M Nolan, Lynne Turnbull, Rosalia Cavaliere, Cynthia B WhitchurchAbstract:Background Pseudomonas aeruginosa is an opportunistic pathogen that exploits damaged epithelia to cause infection. Type IV pili (tfp) are polarly located filamentous structures which are the major adhesins for attachment of P. aeruginosa to epithelial cells. The extension and retraction of tfp powers a mode of surface translocation termed Twitching Motility that is involved in biofilm development and also mediates the active expansion of biofilms across surfaces. Extracellular adenosine triphosphate (eATP) is a key “danger” signalling molecule that is released by damaged epithelial cells to alert the immune system to the potential presence of pathogens. As P. aeruginosa has a propensity for infecting damaged epithelial tissues we have explored the influence of eATP on tfp biogenesis and Twitching Motility-mediated biofilm expansion by P. aeruginosa.
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Motility assay Twitching Motility
Methods of Molecular Biology, 2014Co-Authors: Lynne Turnbull, Cynthia B WhitchurchAbstract:Twitching Motility is a mode of solid surface translocation that occurs under humid conditions on semisolid or solid surfaces, is dependent on the presence of retractile type IV pili and is independent of the presence of a flagellum. Surface translocation via Twitching Motility is powered by the extension and retraction of type IV pili and can manifest as a complex multicellular collective behavior that mediates the active expansion of colonies cultured on the surface of solidified nutrient media, and of interstitial colonies that are cultured at the interface between solidified nutrient media and an abiotic material such as the base of a petri dish or a glass coverslip. Here we describe two methods for assaying Twitching Motility mediated interstitial colony expansion in P. aeruginosa. The first method, the "Macroscopic Twitching Assay," can be used to determine if a strain is capable of Twitching Motility mediated interstitial colony expansion and can also be used to quantitatively assess the influence of mutation or environmental signals on this process. The second method, the "Microscopic Twitching Assay," can be used for detailed interrogation of the movements of individual cells or small groups of bacteria during Twitching Motility mediated colony expansion.
Guoliang Qian - One of the best experts on this subject based on the ideXlab platform.
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a non flagellated biocontrol bacterium employs a pilz pilb complex to provoke Twitching Motility associated with its predation behavior
Phytopathology Research, 2020Co-Authors: Long Lin, Shanho Chou, Danyu Shen, Mimi Zhou, Sen Han, Alex M Fulano, Guoliang QianAbstract:Lysobacter enzymogenes OH11 is a non-flagellated, ubiquitous soil bacterium with broad-spectrum antifungal activities. Although lacking flagella, it employs another type of motile behavior, known as Twitching Motility that is powered by type IV pilus (T4P) to move towards neighboring crop fungal pathogens to kill them as food. At present, little is known about how this non-flagellated bacterium controls Twitching Motility that is crucial for its predatory lifestyle. Herein, we present a report on how a non-canonical PilZ domain, PilZLe3639, controls such Motility in the non-flagellated L. enzymogenes; it failed to bind with c-di-GMP but seemed to be required for Twitching Motility. Using bacterial two-hybrid and pull-down approaches, we identified PilBLe0708, one of the PilZLe3639-binding proteins that are essential for the bacterial Twitching Motility, could serve as an ATPase to supply energy for T4P extension. Through site-mutagenesis approaches, we identified one essential residue of PilZLe3639 that is required for its binding affinity with PilBLe0708 and its regulatory function. Besides, two critical residues within the ATPase catalytic domains of PilBLe0708 were detected to be essential for regulating Twitching behavior but not involved in binding with PilZLe3639. Overall, we illustrated that the PilZ-PilB complex formation is indispensable for Twitching Motility in a non-flagellated bacterium.
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the homologous components of flagellar type iii protein apparatus have acquired a novel function to control Twitching Motility in a non flagellated biocontrol bacterium
Biomolecules, 2020Co-Authors: Alex M Fulano, Shanho Chou, Danyu Shen, Miki Kinoshita, Guoliang QianAbstract:The bacterial flagellum is one of the best-studied surface-attached appendages in bacteria. Flagellarassembly in vivo is promoted by its own protein export apparatus, a type III secretion system (T3SS) in pathogenic bacteria. Lysobacter enzymogenes OH11 is a non-flagellated soil bacterium that utilizes type IV pilus (T4P)-driven Twitching Motility to prey upon nearby fungi for food. Interestingly, the strain OH11 encodes components homologous to the flagellar type III protein apparatus (FT3SS) on its genome, but it remains unknown whether this FT3SS-like system is functional. Here, we report that, despite the absence of flagella, the FT3SS homologous genes are responsible not only for the export of the heterologous flagellin in strain OH11 but also for Twitching Motility. Blocking the FT3SS-like system by in-frame deletion mutations in either flhB or fliI abolished the secretion of heterologous flagellin moleculesinto the culture medium, indicating that the FT3SS is functional in strain OH11. A deletion of flhA, flhB, fliI, or fliR inhibited T4P-driven Twitching Motility, whereas neither that of fliP nor fliQ did, suggesting that FlhA, FlhB, FliI, and FliR may obtain a novel function to modulate the Twitching Motility. The flagellar FliI ATPase was required for the secretion of the major pilus subunit, PilA, suggesting that FliI would have evolved to act as a PilB-like pilus ATPase. These observations lead to a plausible hypothesis that the non-flagellated L. enzymogenes OH11 could preserve FT3SS-like genes for acquiring a distinct function to regulate Twitching Motility associated with its predatory behavior.
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two forms of phosphomannomutase in gammaproteobacteria the overlooked membrane bound form of algc is required for Twitching Motility of lysobacter enzymogenes
Environmental Microbiology, 2019Co-Authors: Guoliang Qian, Shifang Fei, Michael Y GalperinAbstract:Lysobacter enzymogenes, a member of Xanthomonadaceae, is a promising tool to control crop-destroying fungal pathogens. One of its key antifungal virulence factors is the type IV pili that are required for Twitching Motility. Transposon mutagenesis of L. enzymogenes revealed that the production of type IV pili required the presence of the Le2152 gene, which encodes an AlgC-type phosphomannomutase/phosphoglucomutase (PMM). However, in addition to the cytoplasmic PMM domain, the Le2152 gene product contains a ~200-aa N-terminal periplasmic domain that is anchored in the membrane by two transmembrane segments and belongs to the dCache superfamily of periplasmic sensor domains. Sequence analysis identified similar membrane-anchored PMMs, encoded in conserved coaBC-dut-algC gene clusters, in a variety of gammaproteobacteria, either as the sole PMM gene in the entire genome or in addition to the gene encoding the stand-alone enzymatic domain. Previously overlooked N-terminal periplasmic sensor domains were detected in the well-characterized PMMs of Pseudomonas aeruginosa and Xanthomonas campestris, albeit not in the enzymes from Pseudomonas fluorescens, Pseudomonas putida or Azotobacter vinelandii. It appears that after the initial cloning of the enzymatically active soluble part of P. aeruginosa AlgC in 1991, all subsequent studies utilized N-terminally truncated open reading frames. The N-terminal dCache sensor domain of AlgC is predicted to modulate the PMM activity of the cytoplasmic domain in response to as yet unidentified environmental signal(s). AlgC-like membrane-bound PMMs appear to comprise yet another environmental signalling system that regulates the production of type IV pili and potentially other systems in certain gammaproteobacteria.
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two direct gene targets contribute to clp dependent regulation of type iv pilus mediated Twitching Motility in lysobacter enzymogenes oh11
Applied Microbiology and Biotechnology, 2018Co-Authors: Jiaojiao Chen, Shanho Chou, Danyu Shen, Sen Han, Benard Omondi Odhiambo, Guoliang QianAbstract:Lysobacter enzymogenes is an agriculturally important Gram-negative bacterium that employs a multitude of antifungal mechanisms to inhibit and infect filamentous fungal pathogens, through secretion of antifungal antibiotic HSAF (heat-stable antifungal factor), formation of T4P (type IV pilus)-mediated Twitching Motility, and production of extracellular chitinase. Interestingly, all such key antifungal factors seem to be controlled by Clp, a master regulator in L. enzymogenes; however, the underlying mechanisms are poorly understood. Here, employing strain OH11 as a working model, we show that Clp plays a dual role in controlling OH11 Twitching Motility. It controls transcription of pilA, a major T4P structure pilin gene, via directly binding to its promoter region, as well as regulates the gene transcription of pilMONOPQ operon, whose products were essential for T4P assembly, by directly binding to a similar promoter sequence. We also truncated the Clp-binding region of the pilA promoter fragment down to 41 bp to identify the potential Clp-binding sequence. In addition, the Clp-recognized pilM promoter motif of the L. enzymogenes strains is similarly conserved as the pilA promoter, both with a conserved 5′-GTG and a conserved CAC-3′, spaced by ten highly variable nucleotides. Thus, this study identified two direct and previously uncharacterized gene targets of Clp contributing to its regulation in the L. enzymogenes Twitching Motility. Overall, our findings further elucidate the molecular genetics of Clp-dependent Twitching Motility in Lysobacter.
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sigma factor rpon employs a dual transcriptional regulation for controlling Twitching Motility and biofilm formation in lysobacter enzymogenes oh11
Current Genetics, 2018Co-Authors: Danyu Shen, Shanho Chou, Dan Xu, Yun Zhao, Guoliang QianAbstract:Lysobacter is a Gram-negative genus comprising a group of environmental bacteria with abilities to produce abundant novel antibiotics, as well as adopting a unique type IV pilus (T4P)-mediated Twitching Motility (TM) that remains poorly understood. Here, we employ L. enzymogenes OH11 exhibiting significant antifungal activity as a working model to address this issue. Via mutating the 28 potential sigma factors in strain OH11, we have identified one protein RpoNOH11 (sigma 54) that is indispensable for T4P formation and TM. We further showed that RpoNOH11 not only regulates the transcription of pilA, but also another crucial gene chpA that encodes a hybrid two-component transduction system. The L. enzymogenes RpoNOH11 was found to directly bind to the promoter of chpA to control its transcription, which is found to be essential for the T4P-mediated TM. To our knowledge, such a transcriptional regulation performed by RpoN in control of bacterial TM has never been reported. Finally, we showed that L. enzymogenes OH11 could also produce biofilm that is likely employed by this strain to infect fungal pathogens. Mutation of rpoN OH11, pilA and chpA all led to a significant decrease in biofilm formation, suggesting that the dual transcriptional regulation of pilA and chpA by RpoNOH11 plays a key role for RpoNOH11 to modulate the biofilm formation in L. enzymogenes. Overall, this study identified chpA as a new target of RpoN for controlling the T4P-mediated Twitching Motility and biofilm formation in L. enzymogenes OH11.
John S Mattick - One of the best experts on this subject based on the ideXlab platform.
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extragenic suppressor mutations that restore Twitching Motility to fiml mutants of pseudomonas aeruginosa are associated with elevated intracellular cyclic amp levels
MicrobiologyOpen, 2012Co-Authors: Laura M Nolan, John S Mattick, Lynne Turnbull, Scott A Beatson, Larry Croft, P Jones, Anthony M George, Cynthia B WhitchurchAbstract:Cyclic AMP (cAMP) is a signaling molecule that is involved in the regulation of multiple virulence systems of the opportunistic pathogen Pseudomonas aeruginosa. The intracellular concentration of cAMP in P. aeruginosa cells is tightly controlled at the levels of cAMP synthesis and degradation through regulation of the activity and/or expression of the adenylate cyclases CyaA and CyaB or the cAMP phosphodiesterase CpdA. Interestingly, mutants of fimL, which usually demonstrate defective Twitching Motility, frequently revert to a wild-type Twitching-Motility phenotype presumably via the acquisition of an extragenic suppressor mutation(s). In this study, we have characterized five independent fimL Twitching-Motility revertants and have determined that all have increased intracellular cAMP levels compared with the parent fimL mutant. Whole-genome sequencing revealed that only one of these fimL revertants has acquired a loss-of-function mutation in cpdA that accounts for the elevated levels of intracellular cAMP. As mutation of cpdA did not account for the restoration of Twitching Motility observed in the other four fimL revertants, these observations suggest that there is at least another, as yet unidentified, site of extragenic suppressor mutation that can cause phenotypic reversion in fimL mutants and modulation of intracellular cAMP levels of P. aeruginosa.
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tonb3 is required for normal Twitching Motility and extracellular assembly of type iv pili
Journal of Bacteriology, 2004Co-Authors: Bixing Huang, Cynthia B Whitchurch, Zheng Yuan, John S MattickAbstract:Three mutants with Tn5-B21 insertion in tonB3 (PA0406) of Pseudomonas aeruginosa exhibited defective Twitching Motility and reduced assembly of extracellular pili. These defects could be complemented with wild-type tonB3.
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fimx a multidomain protein connecting environmental signals to Twitching Motility in pseudomonas aeruginosa
Journal of Bacteriology, 2003Co-Authors: Bixing Huang, Cynthia B Whitchurch, John S MattickAbstract:Twitching Motility is a form of surface translocation mediated by the extension, tethering, and retraction of type IV pili. Three independent Tn5-B21 mutations of Pseudomonas aeruginosa with reduced Twitching Motility were identified in a new locus which encodes a predicted protein of unknown function annotated PA4959 in the P. aeruginosa genome sequence. Complementation of these mutants with the wild-type PA4959 gene, which we designated fimX, restored normal Twitching Motility. fimX mutants were found to express normal levels of pilin and remained sensitive to pilus-specific bacteriophages, but they exhibited very low levels of surface pili, suggesting that normal pilus function was impaired. The fimX gene product has a molecular weight of 76,000 and contains four predicted domains that are commonly found in signal transduction proteins: a putative response regulator (CheY-like) domain, a PAS-PAC domain (commonly involved in environmental sensing), and DUF1 (or GGDEF) and DUF2 (or EAL) domains, which are thought to be involved in cyclic di-GMP metabolism. Red fluorescent protein fusion experiments showed that FimX is located at one pole of the cell via sequences adjacent to its CheY-like domain. Twitching Motility in fimX mutants was found to respond relatively normally to a range of environmental factors but could not be stimulated by tryptone and mucin. These data suggest that fimX is involved in the regulation of Twitching Motility in response to environmental cues.
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phosphorylation of the pseudomonas aeruginosa response regulator algr is essential for type iv fimbria mediated Twitching Motility
Journal of Bacteriology, 2002Co-Authors: Cynthia B Whitchurch, Tatiana E Erova, Jacqui A Emery, Jennifer L Sargent, Jonathon M Harris, Analese B T Semmler, Michael D Young, John S Mattick, Daniel J WozniakAbstract:The response regulator AlgR is required for both alginate biosynthesis and type IV fimbria-mediated Twitching Motility in Pseudomonas aeruginosa. In this study, the roles of AlgR signal transduction and phosphorylation in Twitching Motility and biofilm formation were examined. The predicted phosphorylation site of AlgR (aspartate 54) and a second aspartate (aspartate 85) in the receiver domain of AlgR were mutated to asparagine, and mutant algR alleles were introduced into the chromosome of P. aeruginosa strains PAK and PAO1. Assays of these mutants demonstrated that aspartate 54 but not aspartate 85 of AlgR is required for Twitching Motility and biofilm initiation. However, strains expressing AlgR D85N were found to be hyperfimbriate, indicating that both aspartate 54 and aspartate 85 are involved in fimbrial biogenesis and function. algD mutants were observed to have wild-type Twitching Motility, indicating that AlgR control of Twitching Motility is not mediated via its role in the control of alginate biosynthesis. In vitro phosphorylation assays showed that AlgR D54N is not phosphorylated by the enteric histidine kinase CheA. These findings indicate that phosphorylation of AlgR most likely occurs at aspartate 54 and that aspartate 54 and aspartate 85 of AlgR are required for the control of the molecular events governing fimbrial biogenesis, Twitching Motility, and biofilm formation in P. aeruginosa.
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differential regulation of Twitching Motility and elastase production by vfr in pseudomonas aeruginosa
Journal of Bacteriology, 2002Co-Authors: Scott A Beatson, Cynthia B Whitchurch, Jennifer L Sargent, Roger C Levesque, John S MattickAbstract:Vfr, a homolog of Escherichia coli cyclic AMP (cAMP) receptor protein, has been shown to regulate quorum sensing, exotoxin A production, and regA transcription in Pseudomonas aeruginosa. We identified a Twitching Motility-defective mutant that carries a transposon insertion in vfr and confirmed that vfr is required for Twitching Motility by construction of an independent allelic deletion-replacement mutant of vfr that exhibited the same phenotype, as well as by the restoration of normal Twitching Motility by complementation of these mutants with wild-type vfr. Vfr-null mutants exhibited severely reduced Twitching Motility with barely detectable levels of type IV pili, as well as loss of elastase production and altered pyocyanin production. We also identified reduced-Twitching variants of quorum-sensing mutants (PAK lasI::Tc) with a spontaneous deletion in vfr (S. A. Beatson, C. B. Whitchurch, A. B. T. Semmler, and J. S. Mattick, J. Bacteriol., 184:3598-3604, 2002), the net result of which was the loss of five residues (EQERS) from the putative cAMP-binding pocket of Vfr. This allele (VfrΔEQERS) was capable of restoring elastase and pyocyanin production to wild-type levels in vfr-null mutants but not their defects in Twitching Motility. Furthermore, structural analysis of Vfr and VfrΔEQERS in relation to E. coli CRP suggests that Vfr is capable of binding both cAMP and cyclic GMP whereas VfrΔEQERS is only capable of responding to cAMP. We suggest that Vfr controls Twitching Motility and quorum sensing via independent pathways in response to these different signals, bound by the same cyclic nucleotide monophosphate-binding pocket.
Laura M Nolan - One of the best experts on this subject based on the ideXlab platform.
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chpc controls Twitching Motility mediated expansion of pseudomonas aeruginosa biofilms in response to serum albumin mucin and oligopeptides
bioRxiv, 2019Co-Authors: Cb Whitchurch, Laura M Nolan, Laura C Mccaughey, Jessica Merjane, Lynne TurnbullAbstract:Abstract Twitching Motility-mediated biofilm expansion occurs via coordinated, multi-cellular collective behaviour to allow bacteria to actively expand across surfaces. Type-IV pili (T4P) are cell-associated virulence factors which mediate this expansion via rounds of extension, surface attachment and retraction. The Chp chemosensory system is thought to respond to environmental signals to regulate the biogenesis, assembly and Twitching Motility function of T4P. In other well characterised chemosensory systems, methyl-accepting chemotaxis proteins (MCPs) feed environmental signals through a CheW adapter protein to the histidine kinase CheA to modulate Motility. The Pseudomonas aeruginosa Chp system has two CheW adapter proteins, PilI and ChpC, and an MCP PilJ that likely interacts via PilI with the histidine kinase ChpA. It is thought that ChpC associates with other MCPs to feed environmental signals into the system, however no such signals have been identified. In the current study we show that ChpC is involved in the response to host-derived signals serum albumin, mucin and oligopeptides. We demonstrate that these signals stimulate an increase in Twitching Motility, as well as in levels of 3’-5’-cyclic adenosine monophosphate (cAMP) and surface-assembled T4P. Interestingly, our data shows that changes in cAMP and surface piliation levels are independent of ChpC but that the Twitching Motility response to these environmental signals requires ChpC. Based upon our data we propose a model whereby ChpC associates with an MCP other than PilJ to feed these environmental signals through the Chp system to control Twitching Motility. The MCP PilJ and the CheW adapter PilI then modulate T4P surface levels to allow the cell to continue to undergo Twitching Motility. Our study is the first to link environmental signals to the Chp chemosensory system and refines our understanding of how this system controls Twitching Motility-mediated biofilm expansion in P. aeruginosa.
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a global genomic approach uncovers novel components for Twitching Motility mediated biofilm expansion in pseudomonas aeruginosa
Microbial genomics, 2018Co-Authors: Laura M Nolan, Cynthia B Whitchurch, Lars Barquist, Marilyn Katrib, Christine J Boinett, Matthew Mayho, David Goulding, Ian G Charles, Alain FillouxAbstract:Pseudomonas aeruginosa is an extremely successful pathogen able to cause both acute and chronic infections in a range of hosts, utilizing a diverse arsenal of cell-associated and secreted virulence factors. A major cell-associated virulence factor, the Type IV pilus (T4P), is required for epithelial cell adherence and mediates a form of surface translocation termed Twitching Motility, which is necessary to establish a mature biofilm and actively expand these biofilms. P. aeruginosa Twitching Motility-mediated biofilm expansion is a coordinated, multicellular behaviour, allowing cells to rapidly colonize surfaces, including implanted medical devices. Although at least 44 proteins are known to be involved in the biogenesis, assembly and regulation of the T4P, with additional regulatory components and pathways implicated, it is unclear how these components and pathways interact to control these processes. In the current study, we used a global genomics-based random-mutagenesis technique, transposon directed insertion-site sequencing (TraDIS), coupled with a physical segregation approach, to identify all genes implicated in Twitching Motility-mediated biofilm expansion in P. aeruginosa. Our approach allowed identification of both known and novel genes, providing new insight into the complex molecular network that regulates this process in P. aeruginosa. Additionally, our data suggest that the flagellum-associated gene products have a differential effect on Twitching Motility, based on whether components are intra- or extracellular. Overall the success of our TraDIS approach supports the use of this global genomic technique for investigating virulence genes in bacterial pathogens.
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a global genomic approach uncovers novel components for Twitching Motility mediated biofilm expansion in pseudomonas aeruginosa
bioRxiv, 2018Co-Authors: Laura M Nolan, Cynthia B Whitchurch, Lars Barquist, Marilyn Katrib, Christine J Boinett, Matthew Mayho, David Goulding, Ian G Charles, Alain Filloux, Julian ParkhillAbstract:Pseudomonas aeruginosa is an extremely successful pathogen able to cause both acute and chronic infections in a range of hosts, utilizing a diverse arsenal of cell-associated and secreted virulence factors. A major cell-associated virulence factor, the Type IV pilus (T4P), is required for epithelial cell adherence and mediates a form of surface translocation termed Twitching Motility, which is necessary to establish a mature biofilm and actively expand these biofilms. P. aeruginosa Twitching Motility-mediated biofilm expansion is a coordinated, multicellular behaviour, allowing cells to rapidly colonize surfaces, including implanted medical devices. Although at least 44 proteins are known to be involved in the biogenesis, assembly and regulation of the T4P, with additional regulatory components and pathways implicated, it is unclear how these components and pathways interact to control these processes. In the current study, we used a global genomics-based random-mutagenesis technique, transposon directed insertion-site sequencing (TraDIS), coupled with a physical segregation approach, to identify all genes implicated in Twitching Motility-mediated biofilm expansion in P. aeruginosa. Our approach allowed identification of both known and novel genes, providing new insight into the complex molecular network that regulates this process in P. aeruginosa. Additionally, our data suggests a differential effect on Twitching Motility by flagellum components based upon their cellular location. Overall the success of our TraDIS approach supports the use of this global genomic technique for investigating virulence genes in bacterial pathogens.
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extracellular atp inhibits Twitching Motility mediated biofilm expansion by pseudomonas aeruginosa
BMC Microbiology, 2015Co-Authors: Laura M Nolan, Lynne Turnbull, Rosalia Cavaliere, Cynthia B WhitchurchAbstract:Background Pseudomonas aeruginosa is an opportunistic pathogen that exploits damaged epithelia to cause infection. Type IV pili (tfp) are polarly located filamentous structures which are the major adhesins for attachment of P. aeruginosa to epithelial cells. The extension and retraction of tfp powers a mode of surface translocation termed Twitching Motility that is involved in biofilm development and also mediates the active expansion of biofilms across surfaces. Extracellular adenosine triphosphate (eATP) is a key “danger” signalling molecule that is released by damaged epithelial cells to alert the immune system to the potential presence of pathogens. As P. aeruginosa has a propensity for infecting damaged epithelial tissues we have explored the influence of eATP on tfp biogenesis and Twitching Motility-mediated biofilm expansion by P. aeruginosa.
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extragenic suppressor mutations that restore Twitching Motility to fiml mutants of pseudomonas aeruginosa are associated with elevated intracellular cyclic amp levels
MicrobiologyOpen, 2012Co-Authors: Laura M Nolan, John S Mattick, Lynne Turnbull, Scott A Beatson, Larry Croft, P Jones, Anthony M George, Cynthia B WhitchurchAbstract:Cyclic AMP (cAMP) is a signaling molecule that is involved in the regulation of multiple virulence systems of the opportunistic pathogen Pseudomonas aeruginosa. The intracellular concentration of cAMP in P. aeruginosa cells is tightly controlled at the levels of cAMP synthesis and degradation through regulation of the activity and/or expression of the adenylate cyclases CyaA and CyaB or the cAMP phosphodiesterase CpdA. Interestingly, mutants of fimL, which usually demonstrate defective Twitching Motility, frequently revert to a wild-type Twitching-Motility phenotype presumably via the acquisition of an extragenic suppressor mutation(s). In this study, we have characterized five independent fimL Twitching-Motility revertants and have determined that all have increased intracellular cAMP levels compared with the parent fimL mutant. Whole-genome sequencing revealed that only one of these fimL revertants has acquired a loss-of-function mutation in cpdA that accounts for the elevated levels of intracellular cAMP. As mutation of cpdA did not account for the restoration of Twitching Motility observed in the other four fimL revertants, these observations suggest that there is at least another, as yet unidentified, site of extragenic suppressor mutation that can cause phenotypic reversion in fimL mutants and modulation of intracellular cAMP levels of P. aeruginosa.
Danyu Shen - One of the best experts on this subject based on the ideXlab platform.
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a non flagellated biocontrol bacterium employs a pilz pilb complex to provoke Twitching Motility associated with its predation behavior
Phytopathology Research, 2020Co-Authors: Long Lin, Shanho Chou, Danyu Shen, Mimi Zhou, Sen Han, Alex M Fulano, Guoliang QianAbstract:Lysobacter enzymogenes OH11 is a non-flagellated, ubiquitous soil bacterium with broad-spectrum antifungal activities. Although lacking flagella, it employs another type of motile behavior, known as Twitching Motility that is powered by type IV pilus (T4P) to move towards neighboring crop fungal pathogens to kill them as food. At present, little is known about how this non-flagellated bacterium controls Twitching Motility that is crucial for its predatory lifestyle. Herein, we present a report on how a non-canonical PilZ domain, PilZLe3639, controls such Motility in the non-flagellated L. enzymogenes; it failed to bind with c-di-GMP but seemed to be required for Twitching Motility. Using bacterial two-hybrid and pull-down approaches, we identified PilBLe0708, one of the PilZLe3639-binding proteins that are essential for the bacterial Twitching Motility, could serve as an ATPase to supply energy for T4P extension. Through site-mutagenesis approaches, we identified one essential residue of PilZLe3639 that is required for its binding affinity with PilBLe0708 and its regulatory function. Besides, two critical residues within the ATPase catalytic domains of PilBLe0708 were detected to be essential for regulating Twitching behavior but not involved in binding with PilZLe3639. Overall, we illustrated that the PilZ-PilB complex formation is indispensable for Twitching Motility in a non-flagellated bacterium.
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the homologous components of flagellar type iii protein apparatus have acquired a novel function to control Twitching Motility in a non flagellated biocontrol bacterium
Biomolecules, 2020Co-Authors: Alex M Fulano, Shanho Chou, Danyu Shen, Miki Kinoshita, Guoliang QianAbstract:The bacterial flagellum is one of the best-studied surface-attached appendages in bacteria. Flagellarassembly in vivo is promoted by its own protein export apparatus, a type III secretion system (T3SS) in pathogenic bacteria. Lysobacter enzymogenes OH11 is a non-flagellated soil bacterium that utilizes type IV pilus (T4P)-driven Twitching Motility to prey upon nearby fungi for food. Interestingly, the strain OH11 encodes components homologous to the flagellar type III protein apparatus (FT3SS) on its genome, but it remains unknown whether this FT3SS-like system is functional. Here, we report that, despite the absence of flagella, the FT3SS homologous genes are responsible not only for the export of the heterologous flagellin in strain OH11 but also for Twitching Motility. Blocking the FT3SS-like system by in-frame deletion mutations in either flhB or fliI abolished the secretion of heterologous flagellin moleculesinto the culture medium, indicating that the FT3SS is functional in strain OH11. A deletion of flhA, flhB, fliI, or fliR inhibited T4P-driven Twitching Motility, whereas neither that of fliP nor fliQ did, suggesting that FlhA, FlhB, FliI, and FliR may obtain a novel function to modulate the Twitching Motility. The flagellar FliI ATPase was required for the secretion of the major pilus subunit, PilA, suggesting that FliI would have evolved to act as a PilB-like pilus ATPase. These observations lead to a plausible hypothesis that the non-flagellated L. enzymogenes OH11 could preserve FT3SS-like genes for acquiring a distinct function to regulate Twitching Motility associated with its predatory behavior.
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two direct gene targets contribute to clp dependent regulation of type iv pilus mediated Twitching Motility in lysobacter enzymogenes oh11
Applied Microbiology and Biotechnology, 2018Co-Authors: Jiaojiao Chen, Shanho Chou, Danyu Shen, Sen Han, Benard Omondi Odhiambo, Guoliang QianAbstract:Lysobacter enzymogenes is an agriculturally important Gram-negative bacterium that employs a multitude of antifungal mechanisms to inhibit and infect filamentous fungal pathogens, through secretion of antifungal antibiotic HSAF (heat-stable antifungal factor), formation of T4P (type IV pilus)-mediated Twitching Motility, and production of extracellular chitinase. Interestingly, all such key antifungal factors seem to be controlled by Clp, a master regulator in L. enzymogenes; however, the underlying mechanisms are poorly understood. Here, employing strain OH11 as a working model, we show that Clp plays a dual role in controlling OH11 Twitching Motility. It controls transcription of pilA, a major T4P structure pilin gene, via directly binding to its promoter region, as well as regulates the gene transcription of pilMONOPQ operon, whose products were essential for T4P assembly, by directly binding to a similar promoter sequence. We also truncated the Clp-binding region of the pilA promoter fragment down to 41 bp to identify the potential Clp-binding sequence. In addition, the Clp-recognized pilM promoter motif of the L. enzymogenes strains is similarly conserved as the pilA promoter, both with a conserved 5′-GTG and a conserved CAC-3′, spaced by ten highly variable nucleotides. Thus, this study identified two direct and previously uncharacterized gene targets of Clp contributing to its regulation in the L. enzymogenes Twitching Motility. Overall, our findings further elucidate the molecular genetics of Clp-dependent Twitching Motility in Lysobacter.
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sigma factor rpon employs a dual transcriptional regulation for controlling Twitching Motility and biofilm formation in lysobacter enzymogenes oh11
Current Genetics, 2018Co-Authors: Danyu Shen, Shanho Chou, Dan Xu, Yun Zhao, Guoliang QianAbstract:Lysobacter is a Gram-negative genus comprising a group of environmental bacteria with abilities to produce abundant novel antibiotics, as well as adopting a unique type IV pilus (T4P)-mediated Twitching Motility (TM) that remains poorly understood. Here, we employ L. enzymogenes OH11 exhibiting significant antifungal activity as a working model to address this issue. Via mutating the 28 potential sigma factors in strain OH11, we have identified one protein RpoNOH11 (sigma 54) that is indispensable for T4P formation and TM. We further showed that RpoNOH11 not only regulates the transcription of pilA, but also another crucial gene chpA that encodes a hybrid two-component transduction system. The L. enzymogenes RpoNOH11 was found to directly bind to the promoter of chpA to control its transcription, which is found to be essential for the T4P-mediated TM. To our knowledge, such a transcriptional regulation performed by RpoN in control of bacterial TM has never been reported. Finally, we showed that L. enzymogenes OH11 could also produce biofilm that is likely employed by this strain to infect fungal pathogens. Mutation of rpoN OH11, pilA and chpA all led to a significant decrease in biofilm formation, suggesting that the dual transcriptional regulation of pilA and chpA by RpoNOH11 plays a key role for RpoNOH11 to modulate the biofilm formation in L. enzymogenes. Overall, this study identified chpA as a new target of RpoN for controlling the T4P-mediated Twitching Motility and biofilm formation in L. enzymogenes OH11.
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chpa controls Twitching Motility and broadly affects gene expression in the biological control agent lysobacter enzymogenes
Current Microbiology, 2017Co-Authors: Mimi Zhou, Danyu Shen, Fengquan Liu, Guoliang QianAbstract:Lysobacter enzymogenes (L. enzymogenes) is an agriculturally important Gram-negative bacterium that employs T4P (type IV pili)-driven Twitching Motility to exhibit its antifungal function. Yet, it is still unclear how this bacterium regulates its Twitching Motility. Here, by using strain OH11 as the working model organism, we showed that a hybrid two-component system ChpA acts as a positive regulator in controlling Twitching Motility in L. enzymogenes. ChpA is a hybrid TCS (two-component transduction system) contains 7 domains including those for auto-phosphorylation and phosphate group transfer, as well as a phosphate receiver (REC) domain. Mutation of chpA completely abolished the wild-type Twitching Motility, as evidenced by the absence of mobile cells at the margin of the mutant colonies. Further studies of domain-deletion and phenotypic characterization reveal that domains responsible for phosphorylation and phosphotransfer, but not the REC domain, were indispensable for ChpA in regulating Twitching Motility. Transcriptome analyses of the chpA knockout strain indicated that ChpA was extensively involved in controlling expression of a wide variety of genes (totaling 243). The products of these differentially expressed genes were involved in multiple physiological and biological functions in L. enzymogenes. Thus, we have not only identified a new regulator controlling Twitching Motility in L. enzymogenes, but also provided the first report demonstrating the broad impact of the conserved ChpA in gene regulation in Gram-negative bacteria.
