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1.  Alanine 32 in PilA is important for PilA stability and type IV pili function in Myxococcus xanthus 
Microbiology  2011;157(Pt 7):1920-1928.
Type IV pili (TFP) are membrane-anchored filaments with a number of important biological functions. In the model organism Myxococcus xanthus, TFP act as molecular engines that power social (S) motility through cycles of extension and retraction. TFP filaments consist of several thousand copies of a protein called PilA or pilin. PilA contains an N-terminal α-helix essential for TFP assembly and a C-terminal globular domain important for its activity. The role of the PilA sequence and its structure–function relationship in TFP-dependent S motility remain active areas of research. In this study, we identified an M. xanthus PilA mutant carrying an alanine to valine substitution at position 32 in the α-helix, which produced structurally intact but retraction-defective TFP. Characterization of this mutant and additional single-residue variants at this position in PilA demonstrated the critical role of alanine 32 in PilA stability, TFP assembly and retraction.
PMCID: PMC3167889  PMID: 21493683
2.  PilA localization affects extracellular polysaccharide production and fruiting body formation in Myxococcus xanthus 
Molecular microbiology  2010;76(6):1500-1513.
Myxococcus xanthus is a gram-negative bacterium capable of complex developmental processes involving vegetative swarming and fruiting body formation. Social (S-) gliding motility, one of the two motility systems employed by M. xanthus, requires at least two cell surface structures: type IV pili (TFP) and extracellular polysaccharides (EPS). Extended TFP which are composed of thousands of copies of PilA retract upon binding to EPS and thereby pull the cell forward. TFP also act as external sensor to regulate EPS production. In this study, we generated a random PilA mutant library and identified one derivative, SW1066, which completely failed to undergo developmental processes. Detailed characterization revealed that SW1066 produced very little EPS but wild-type amounts of PilA. These mutated PilA subunits, however, are unable to assemble into functional TFP despite their ability to localize to the membrane. By preventing the mutated PilA of SW1066 to translocate from the cytoplasm to the membrane, fruiting body formation and EPS production was restored to the levels observed in mutant strains lacking PilA. This apparent connection between PilA membrane accumulation and reduction in surface EPS implies that specific cellular PilA localization are required to maintain the EPS level necessary to sustain normal S-motilityin M. xanthus.
PMCID: PMC2935901  PMID: 20444090
Myxococcus xanthus; type four pili; PilA; extracellular polysaccharide
3.  Direct visualization of the interaction between pilin and exopolysaccharides of Myxococcus xanthus with eGFP fused PilA protein 
FEMS microbiology letters  2011;326(1):23-30.
Type IV pili (TFP) and exopolysaccharides (EPS) are important components for social behaviors in Myxococcus xanthus, including gliding motility and fruiting body formation. Although specific interactions between TFP and EPS have been proposed, direct observations of these interactions under native condition have not yet been made. In this study, we found that a truncated PilA protein (PilACt) which only contains the C-terminal domain (amino acids 32-208) is sufficient for EPS binding in vitro. Furthermore, an enhanced green fluorescent protein (eGFP) and PilACt fusion protein was constructed and used to label the native EPS in M. xanthus. Under confocal laser scanning microscope, the eGFP-PilACt-bound fruiting bodies, trail structures and biofilms exhibited similar patterns as the wheat germ agglutinin lectin (WGA)-labeled EPS structures. This study showed that eGFP-PilACt fusion protein was able to efficiently label the EPS of M. xanthus and for the first time provided evidence for the direct interaction between the PilA protein and EPS under native conditions.
PMCID: PMC3454480  PMID: 22092602
Type IV Pilin; Exopolysaccharides; Biofilm; Fruiting body; Confocal laser scanning microscopy; eGFP
4.  Characterization of Four Type IV Pilin Homologues in Stigmatella aurantiaca DSM17044 by Heterologous Expression in Myxococcus xanthus 
PLoS ONE  2013;8(9):e75105.
As prokaryotic models for multicellular development, Stigmatellaaurantiaca and Myxococcus xanthus share many similarities in terms of social behaviors, such as gliding motility. Our current understanding of myxobacterial grouped-cell motilities comes mainly from the research on M. xanthus, which shows that filamentous type IV pili (TFP), composed of type IV pilin (also called PilA protein) subunits, are the key apparatus for social motility (S-motility). However, little is known about the pilin protein in S. aurantiaca. We cloned and sequenced four genes (pilASa1~4) from S. aurantiaca DSM17044 that are homologous to pilAMx (pilA gene in M. xanthus DK1622). The homology and similarities among PilASa proteins and other myxobacterial homologues were systematically analyzed. To determine their potential biological functions, the four pilASa genes were expressed in M. xanthus DK10410 (ΔpilAMx), which did not restore S-motility on soft agar or EPS production to host cells. After further analysis of the motile behaviors in a methylcellulose solution, the M. xanthus strains were categorized into three types. YL6101, carrying pilASa1, and YL6104, carrying pilASa4, produced stable but unretractable surface pili; YL6102, carrying pilASa2, produced stable surface pili and exhibited reduced TFP-dependent motility in methylcellulose; YL6103, carrying pilASa3, produced unstable surface pili. Based on these findings, we propose that pilASa2 might be responsible for the type IV pilin production involved in group motility in S. aurantiaca DSM17044. After examining the developmental processes, it was suggested that the expression of PilASa4 protein might have positive effects on the fruiting body formation of M. xanthus DK10410 cells. Moreover, the formation of fruiting body in M. xanthus cells with stable exogenous TFPSa were compensated by mixing them with S. aurantiaca DSM17044 cells. Our results shed some light on the features and functions of type IV pilin homologues in S. aurantiaca.
PMCID: PMC3776727  PMID: 24058653
5.  Cooperative Retraction of Bundled Type IV Pili Enables Nanonewton Force Generation 
PLoS Biology  2008;6(4):e87.
The causative agent of gonorrhea, Neisseria gonorrhoeae, bears retractable filamentous appendages called type IV pili (Tfp). Tfp are used by many pathogenic and nonpathogenic bacteria to carry out a number of vital functions, including DNA uptake, twitching motility (crawling over surfaces), and attachment to host cells. In N. gonorrhoeae, Tfp binding to epithelial cells and the mechanical forces associated with this binding stimulate signaling cascades and gene expression that enhance infection. Retraction of a single Tfp filament generates forces of 50–100 piconewtons, but nothing is known, thus far, on the retraction force ability of multiple Tfp filaments, even though each bacterium expresses multiple Tfp and multiple bacteria interact during infection. We designed a micropillar assay system to measure Tfp retraction forces. This system consists of an array of force sensors made of elastic pillars that allow quantification of retraction forces from adherent N. gonorrhoeae bacteria. Electron microscopy and fluorescence microscopy were used in combination with this novel assay to assess the structures of Tfp. We show that Tfp can form bundles, which contain up to 8–10 Tfp filaments, that act as coordinated retractable units with forces up to 10 times greater than single filament retraction forces. Furthermore, single filament retraction forces are transient, whereas bundled filaments produce retraction forces that can be sustained. Alterations of noncovalent protein–protein interactions between Tfp can inhibit both bundle formation and high-amplitude retraction forces. Retraction forces build over time through the recruitment and bundling of multiple Tfp that pull cooperatively to generate forces in the nanonewton range. We propose that Tfp retraction can be synchronized through bundling, that Tfp bundle retraction can generate forces in the nanonewton range in vivo, and that such high forces could affect infection.
Author Summary
Type IV pili are filamentous appendages borne by a large number of pathogenic and nonpathogenic bacteria. They play crucial roles in basic microbial processes such as surface motility, virulence, and DNA exchange. Neisseria gonorrhoeae, the causative agent of gonorrhea, can extend and retract these long, thin threads—around 6 nm in diameter and up to 30 μm long—to explore and pull on the environment. The retraction of one N. gonorrhoeae pilus filament can exert forces of 50–100 piconewtons, or roughly 10,000 times the bacterium's bodyweight. The bacteria can exert those forces on human cells that they infect, and force has been shown to be an important parameter in their infectivity. We use a micropillar assay system to show that N. gonorrhoeae cells can exert even higher forces by forming bundles of 8–10 filaments that act as coordinated retractable units. The bacteria can thus achieve forces in the nanonewton range (or 100,000 times their bodyweight) making them the strongest microscale elements known to date. This study demonstrates the power and cooperativity of pilus nanomotors and opens new territories for the exploration of force-mediated bacteria–host-cell interactions.
To make its way into cells,Neisseria gonorrhoeae, the pathogen that causes gonorrhea, may exert nanonewton forces through cooperative bundling of pilus fibers.
PMCID: PMC2292754  PMID: 18416602
6.  Isolation and Characterization of a Suppressor Mutation that Restores Myxococcus xanthus Exopolysaccharide Production 
Microbiology (Reading, England)  2009;155(Pt 11):3599-3610.
Myxococcus xanthus, a Gram-negative soil bacterium, undergoes multicellular development when nutrients become limiting. Aggregation, which is part of the developmental process, requires the surface motility of this organism. One component of M. xanthus motility, the social (S) gliding motility, enables the movement of cells in close physical proximity. Previous studies demonstrated that the cell-surface associated exopolysaccharide (EPS) is essential for S motility and the Dif proteins form a chemotaxis-like pathway that regulates EPS production in M. xanthus. DifA, a homologue of methyl-accepting chemotaxis proteins (MCPs) in the Dif system, is required for EPS production, S motility and development. In this study, a spontaneous extragenic suppressor of a difA deletion was isolated in order to identify additional regulators of EPS production. The suppressor mutation was found to be a single base-pair insertion in cheW7 at the che7 chemotaxis gene cluster. Further examination indicated that mutations in cheW7 may lead to the interaction of Mcp7 with DifC (CheW-like) and DifE (CheA-like) to reconstruct a functional pathway to regulate EPS production in the absence of DifA. In addition, the cheW7 mutation was found to partially suppress a pilA mutation in EPS production in a difA+ background. Further deletion of difA from the pilA cheW7 double mutant resulted in a triple mutant that produced wild-type levels of EPS, implying that DifA (MCP-like) and Mcp7 compete for interactions with DifC and DifE in the modulation of EPS production.
PMCID: PMC2879065  PMID: 19684067
7.  Isolation and characterization of a suppressor mutation that restores Myxococcus xanthus exopolysaccharide production 
Microbiology  2009;155(Pt 11):3599-3610.
Myxococcus xanthus, a Gram-negative soil bacterium, undergoes multicellular development when nutrients become limiting. Aggregation, which is part of the developmental process, requires the surface motility of this organism. One component of M. xanthus motility, the social (S) gliding motility, enables the movement of cells in close physical proximity. Previous studies demonstrated that the cell surface-associated exopolysaccharide (EPS) is essential for S motility and that the Dif proteins form a chemotaxis-like pathway that regulates EPS production in M. xanthus. DifA, a homologue of methyl-accepting chemotaxis proteins (MCPs) in the Dif system, is required for EPS production, S motility and development. In this study, a spontaneous extragenic suppressor of a difA deletion was isolated in order to identify additional regulators of EPS production. The suppressor mutation was found to be a single base pair insertion in cheW7 at the che7 chemotaxis gene cluster. Further examination indicated that mutations in cheW7 may lead to the interaction of Mcp7 with DifC (CheW-like) and DifE (CheA-like) to reconstruct a functional pathway to regulate EPS production in the absence of DifA. In addition, the cheW7 mutation was found to partially suppress a pilA mutation in EPS production in a difA+ background. Further deletion of difA from the pilA cheW7 double mutant resulted in a triple mutant that produced wild-type levels of EPS, implying that DifA (MCP-like) and Mcp7 compete for interactions with DifC and DifE in the modulation of EPS production.
PMCID: PMC2879065  PMID: 19684067
8.  FrzS Regulates Social Motility in Myxococcus xanthus by Controlling Exopolysaccharide Production 
PLoS ONE  2011;6(8):e23920.
Myxococcus xanthus Social (S) motility occurs at high cell densities and is powered by the extension and retraction of Type IV pili which bind ligands normally found in matrix exopolysaccharides (EPS). Previous studies showed that FrzS, a protein required for S-motility, is organized in polar clusters that show pole-to-pole translocation as cells reverse their direction of movement. Since the leading cell pole is the site of both the major FrzS cluster and type IV pilus extension/retraction, it was suggested that FrzS might regulate S-motility by activating pili at the leading cell pole. Here, we show that FrzS regulates EPS production, rather than type IV pilus function. We found that the frzS phenotype is distinct from that of Type IV pilus mutants such as pilA and pilT, but indistinguishable from EPS mutants, such as epsZ. Indeed, frzS mutants can be rescued by the addition of purified EPS, 1% methylcellulose, or co-culturing with wildtype cells. Our data also indicate that the cell density requirement in S-motility is likely a function of the ability of cells to construct functional multicellular clusters surrounding an EPS core.
PMCID: PMC3158785  PMID: 21886839
9.  Pseudomonas aeruginosa Type IV Pilus Expression in Neisseria gonorrhoeae: Effects of Pilin Subunit Composition on Function and Organelle Dynamics▿ †  
Journal of Bacteriology  2007;189(18):6676-6685.
Type IV pili (TFP) play central roles in the expression of many phenotypes including motility, multicellular behavior, sensitivity to bacteriophages, natural genetic transformation, and adherence. In Neisseria gonorrhoeae, these properties require ancillary proteins that act in conjunction with TFP expression and influence organelle dynamics. Here, the intrinsic contributions of the pilin protein itself to TFP dynamics and associated phenotypes were examined by expressing the Pseudomonas aeruginosa PilAPAK pilin subunit in N. gonorrhoeae. We show here that, although PilAPAK pilin can be readily assembled into TFP in this background, steady-state levels of purifiable fibers are dramatically reduced relative those of endogenous pili. This defect is due to aberrant TFP dynamics as it is suppressed in the absence of the PilT pilus retraction ATPase. Functionally, PilAPAK pilin complements gonococcal adherence for human epithelial cells but only in a pilT background, and this property remains dependent on the coexpression of both the PilC adhesin and the PilV pilin-like protein. Since P. aeruginosa pilin only moderately supports neisserial sequence-specific transformation despite its assembly proficiency, these results together suggest that PilAPAK pilin functions suboptimally in this environment. This appears to be due to diminished compatibility with resident proteins essential for TFP function and dynamics. Despite this, PilAPAK pili support retractile force generation in this background equivalent to that reported for endogenous pili. Furthermore, PilAPAK pili are both necessary and sufficient for bacteriophage PO4 binding, although the strain remains phage resistant. Together, these findings have significant implications for TFP biology in both N. gonorrhoeae and P. aeruginosa.
PMCID: PMC2045180  PMID: 17573479
10.  The type IV pilin, PilA, is required for full virulence of Francisella tularensis subspecies tularensis 
BMC Microbiology  2010;10:227.
All four Francisella tularensis subspecies possess gene clusters with potential to express type IV pili (Tfp). These clusters include putative pilin genes, as well as pilB, pilC and pilQ, required for secretion and assembly of Tfp. A hallmark of Tfp is the ability to retract the pilus upon surface contact, a property mediated by the ATPase PilT. Interestingly, out of the two major human pathogenic subspecies only the highly virulent type A strains have a functional pilT gene.
In a previous study, we were able to show that one pilin gene, pilA, was essential for virulence of a type B strain in a mouse infection model. In this work we have examined the role of several Tfp genes in the virulence of the pathogenic type A strain SCHU S4. pilA, pilC, pilQ, and pilT were mutated by in-frame deletion mutagenesis. Interestingly, when mice were infected with a mixture of each mutant strain and the wild-type strain, the pilA, pilC and pilQ mutants were out-competed, while the pilT mutant was equally competitive as the wild-type.
This suggests that expression and surface localisation of PilA contribute to virulence in the highly virulent type A strain, while PilT was dispensable for virulence in the mouse infection model.
PMCID: PMC2941502  PMID: 20796283
11.  Coupling of protein localization and cell movements by a dynamically localized response regulator in Myxococcus xanthus 
The EMBO Journal  2007;26(21):4433-4444.
Myxococcus xanthus cells harbor two motility machineries, type IV pili (Tfp) and the A-engine. During reversals, the two machineries switch polarity synchronously. We present a mechanism that synchronizes this polarity switching. We identify the required for motility response regulator (RomR) as essential for A-motility. RomR localizes in a bipolar, asymmetric pattern with a large cluster at the lagging cell pole. The large RomR cluster relocates to the new lagging pole in parallel with cell reversals. Dynamic RomR localization is essential for cell reversals, suggesting that RomR relocalization induces the polarity switching of the A-engine. The analysis of RomR mutants shows that the output domain targets RomR to the poles and the receiver domain is essential for dynamic localization. The small GTPase MglA establishes correct RomR polarity, and the Frz two-component system regulates dynamic RomR localization. FrzS localizes with Tfp at the leading pole and relocates in an Frz-dependent manner to the opposite pole during reversals; FrzS and RomR localize and oscillate independently. The Frz system synchronizes these oscillations and thus the synchronous polarity switching of the motility machineries.
PMCID: PMC2034494  PMID: 17932488
A-motility; morphogenetic cell movements; Myxococcus xanthus; polarity; response regulator
12.  Disparate Subcellular Localization Patterns of Pseudomonas aeruginosa Type IV Pilus ATPases Involved in Twitching Motility 
Journal of Bacteriology  2005;187(3):829-839.
The opportunistic pathogen Pseudomonas aeruginosa expresses polar type IV pili (TFP), which are responsible for adhesion to various materials and twitching motility on surfaces. Twitching occurs by alternate extension and retraction of TFP, which arise from assembly and disassembly of pilin subunits at the base of the pilus. The ATPase PilB promotes pilin assembly, while the ATPase PilT or PilU or both promote pilin dissociation. Fluorescent fusions to two of the three ATPases (PilT and PilU) were functional, as shown by complementation of the corresponding mutants. PilB and PilT fusions localized to both poles, while PilU fusions localized only to the piliated pole. To identify the portion of the ATPases required for localization, sequential C-terminal deletions of PilT and PilU were generated. The conserved His and Walker B boxes were dispensable for polar localization but were required for twitching motility, showing that localization and function could be uncoupled. Truncated fusions that retained polar localization maintained their distinctive distribution patterns. To dissect the cellular factors involved in establishing polarity, fusion protein localization was monitored with a panel of TFP mutants. The localization of yellow fluorescent protein (YFP)-PilT and YFP-PilU was independent of the subunit PilA, other TFP ATPases, and TFP-associated proteins previously shown to be associated with the membrane or exhibiting polar localization. In contrast, YFP-PilB exhibited diffuse cytoplasmic localization in a pilC mutant, suggesting that PilC is required for polar localization of PilB. Finally, localization studies performed with fluorescent ATPase chimeras of PilT and PilU demonstrated that information responsible for the characteristic localization patterns of the ATPases likely resides in their N termini.
PMCID: PMC545728  PMID: 15659660
13.  Pseudomonas aeruginosa fimL regulates multiple virulence functions by intersecting with Vfr-modulated pathways 
Molecular microbiology  2005;55(5):1357-1378.
Virulence of Pseudomonas aeruginosa involves the co-ordinate expression of a range of factors including type IV pili (tfp), the type III secretion system (TTSS) and quorum sensing. Tfp are required for twitching motility, efficient biofilm formation, and for adhesion and type III secretion (TTS)-mediated damage to mammalian cells. We describe a novel gene (fimL) that is required for tfp biogenesis and function, for TTS and for normal biofilm development in P. aeruginosa. The predicted product of fimL is homologous to the N-terminal domain of ChpA, except that its putative histidine and threonine phosphotransfer sites have been replaced with glutamine. fimL mutants resemble vfr mutants in many aspects including increased autolysis, reduced levels of surface-assembled tfp and diminished production of type III secreted effectors. Expression of vfr in trans can complement fimL mutants. vfr transcription and production is reduced in fimL mutants whereas cAMP levels are unaffected. Deletion and insertion mutants of fimL frequently revert to wild-type phenotypes suggesting that an extragenic suppressor mutation is able to overcome the loss of fimL. vfr transcription and production, as well as cAMP levels, are elevated in these revertants, while Pseudomonas quinolone signal (PQS) production is reduced. These results suggest that the site(s) of spontaneous mutation is in a gene(s) which lies upstream of vfr transcription, cAMP, production, and PQS synthesis. Our studies indicate that Vfr and FimL are components of intersecting pathways that control twitching motility, TTSS and autolysis in P. aeruginosa.
PMCID: PMC1266277  PMID: 15720546
14.  Identification of Surprisingly Diverse Type IV Pili, across a Broad Range of Gram-Positive Bacteria 
PLoS ONE  2011;6(12):e28919.
In Gram-negative bacteria, type IV pili (TFP) have long been known to play important roles in such diverse biological phenomena as surface adhesion, motility, and DNA transfer, with significant consequences for pathogenicity. More recently it became apparent that Gram-positive bacteria also express type IV pili; however, little is known about the diversity and abundance of these structures in Gram-positives. Computational tools for automated identification of type IV pilins are not currently available.
To assess TFP diversity in Gram-positive bacteria and facilitate pilin identification, we compiled a comprehensive list of putative Gram-positive pilins encoded by operons containing highly conserved pilus biosynthetic genes (pilB, pilC). A surprisingly large number of species were found to contain multiple TFP operons (pil, com and/or tad). The N-terminal sequences of predicted pilins were exploited to develop PilFind, a rule-based algorithm for genome-wide identification of otherwise poorly conserved type IV pilins in any species, regardless of their association with TFP biosynthetic operons ( Using PilFind to scan 53 Gram-positive genomes (encoding >187,000 proteins), we identified 286 candidate pilins, including 214 in operons containing TFP biosynthetic genes (TBG+ operons). Although trained on Gram-positive pilins, PilFind identified 55 of 58 manually curated Gram-negative pilins in TBG+ operons, as well as 53 additional pilin candidates in operons lacking biosynthetic genes in ten species (>38,000 proteins), including 27 of 29 experimentally verified pilins. False positive rates appear to be low, as PilFind predicted only four pilin candidates in eleven bacterial species (>13,000 proteins) lacking TFP biosynthetic genes.
We have shown that Gram-positive bacteria contain a highly diverse set of type IV pili. PilFind can be an invaluable tool to study bacterial cellular processes known to involve type IV pilus-like structures. Its use in combination with other currently available computational tools should improve the accuracy of predicting the subcellular localization of bacterial proteins.
PMCID: PMC3244431  PMID: 22216142
15.  Pseudomonas aeruginosa PilY1 Binds Integrin in an RGD- and Calcium-Dependent Manner 
PLoS ONE  2011;6(12):e29629.
PilY1 is a type IV pilus (tfp)-associated protein from the opportunistic pathogen Pseudomonas aeruginosa that shares functional similarity with related proteins in infectious Neisseria and Kingella species. Previous data have shown that PilY1 acts as a calcium-dependent pilus biogenesis factor necessary for twitching motility with a specific calcium binding site located at amino acids 850–859 in the 1,163 residue protein. In addition to motility, PilY1 is also thought to play an important role in the adhesion of P. aeruginosa tfp to host epithelial cells. Here, we show that PilY1 contains an integrin binding arginine-glycine-aspartic acid (RGD) motif located at residues 619–621 in the PilY1 from the PAK strain of P. aeruginosa; this motif is conserved in the PilY1s from the other P. aeruginosa strains of known sequence. We demonstrate that purified PilY1 binds integrin in vitro in an RGD-dependent manner. Furthermore, we identify a second calcium binding site (amino acids 600–608) located ten residues upstream of the RGD. Eliminating calcium binding from this site using a D608A mutation abolished integrin binding; in contrast, a calcium binding mimic (D608K) preserved integrin binding. Finally, we show that the previously established PilY1 calcium binding site at 851–859 also impacts the protein's association with integrin. Taken together, these data indicate that PilY1 binds to integrin in an RGD- and calcium-dependent manner in vitro. As such, P. aeruginosa may employ these interactions to mediate host epithelial cell binding in vivo.
PMCID: PMC3248442  PMID: 22242136
16.  Uncovering the Mystery of Gliding Motility in the Myxobacteria 
Annual Review of Genetics  2011;45:21-39.
Bacterial gliding motility is the smooth movement of cells on solid surfaces unaided by flagella or pili. Many diverse groups of bacteria exhibit gliding, but the mechanism of gliding motility has remained a mystery since it was first observed more than a century ago. Recent studies on the motility of Myxococcus xanthus, a soil myxobacterium, suggest a likely mechanism for gliding in this organism. About forty M. xanthus genes were shown to be involved in gliding motility, and some of their protein products were labeled and localized within cells. These studies suggest that gliding motility in M. xanthus involves large multiprotein structural complexes, regulatory proteins, and cytoskeletal filaments. In this review, we summarize recent experiments that provide the basis for this emerging view of M. xanthus motility. We also discuss alternative models for gliding.
PMCID: PMC3397683  PMID: 21910630
Myxococcus xanthus; proton motive force; cytoskeleton; protein localization; model
17.  Retraction of enteropathogenic E. coli type IV pili promotes efficient host cell colonization, effector translocation and tight junction disruption 
Gut Microbes  2012;3(3):267-271.
Type IV pili (Tfp) play a primary role in mediating the adherence of pathogenic bacteria to their hosts. The pilus filament can retract with an immense force. However, the role of this activity in microbial pathogenesis has not been rigorously explored. Experiments performed on volunteers suggested that the retraction capacity of enteropathogenic Escherichia coli (EPEC) Tfp is required for full virulence. Here we review our recent study1 in which we showed that the retraction capacity of the EPEC Tfp facilitates tight-junction disruption and actin-rich pedestal formation by promoting efficient bacterial protein effector translocation into epithelial host cells. We also present new data using live imaging confocal microscopy suggesting that EPEC adheres to monolayers in microcolonies and that Tfp retraction facilitates significant changes in the microcolony shape, which may be critical for efficient effector delivery. Our studies hence suggest novel insights into the role of pili retraction in EPEC pathogenesis.
PMCID: PMC3427219  PMID: 22572833
enteropathogenic E. coli; type IV pili; bundle forming pili; tight junctions; polarized epithelial cells
18.  Myxococcus xanthus dif Genes Are Required for Biogenesis of Cell Surface Fibrils Essential for Social Gliding Motility 
Journal of Bacteriology  2000;182(20):5793-5798.
Myxococcus xanthus social (S) gliding motility has been previously reported by us to require the chemotaxis homologues encoded by the dif genes. In addition, two cell surface structures, type IV pili and extracellular matrix fibrils, are also critical to M. xanthus S motility. We have demonstrated here that M. xanthus dif genes are required for the biogenesis of fibrils but not for that of type IV pili. Furthermore, the developmental defects of dif mutants can be partially rescued by the addition of isolated fibril materials. Along with the chemotaxis genes of various swarming bacteria and the pilGHIJ genes of the twitching bacterium Pseudomonas aeruginosa, the M. xanthus dif genes belong to a unique class of bacterial chemotaxis genes or homologues implicated in the biogenesis of structures required for bacterial surface locomotion. Genetic studies indicate that the dif genes are linked to the M. xanthus dsp region, a locus known to be crucial for M. xanthus fibril biogenesis and S gliding.
PMCID: PMC94702  PMID: 11004179
19.  Dynamics of Neisseria gonorrhoeae Attachment: Microcolony Development, Cortical Plaque Formation, and Cytoprotection▿ §  
Infection and Immunity  2007;75(10):4743-4753.
Neisseria gonorrhoeae is the bacterium that causes gonorrhea, a major sexually transmitted disease and a significant cofactor for human immunodeficiency virus transmission. The retactile N. gonorrhoeae type IV pilus (Tfp) mediates twitching motility and attachment. Using live-cell microscopy, we reveal for the first time the dynamics of twitching motility by N. gonorrhoeae in its natural environment, human epithelial cells. Bacteria aggregate into microcolonies on the cell surface and induce a massive remodeling of the microvillus architecture. Surprisingly, the microcolonies are motile, and they fuse to form progressively larger structures that undergo rapid reorganization, suggesting that bacteria communicate with each other during infection. As reported, actin plaques form beneath microcolonies. Here, we show that cortical plaques comigrate with motile microcolonies. These activities are dependent on pilT, the Tfp retraction locus. Cultures infected with a pilT mutant have significantly higher numbers of apoptotic cells than cultures infected with the wild-type strain. Inducing pilT expression with isopropyl-β-d-thiogalactopyranoside partially rescues cells from infection-induced apoptosis, demonstrating that Tfp retraction is intrinsically cytoprotective for the host. Tfp-mediated attachment is therefore a continuum of microcolony motility and force stimulation of host cell signaling, leading to a cytoprotective effect.
PMCID: PMC2044525  PMID: 17682045
20.  Contribution of Moraxella catarrhalis Type IV Pili to Nasopharyngeal Colonization and Biofilm Formation▿  
Infection and Immunity  2007;75(12):5559-5564.
Moraxella catarrhalis is a gram-negative mucosal pathogen of the human respiratory tract. Although little information is available regarding the initial steps of M. catarrhalis pathogenesis, this organism must be able to colonize the human mucosal surface in order to initiate an infection. Type IV pili (TFP), filamentous surface appendages primarily comprised of a single protein subunit termed pilin, play a crucial role in the initiation of disease by a wide range of bacteria. We previously identified the genes that encode the major proteins involved in the biosynthesis of M. catarrhalis TFP and determined that the TFP expressed by this organism are highly conserved and essential for natural transformation. We extended this initial study by investigating the contribution of TFP to the early stages of M. catarrhalis colonization. TFP-deficient M. catarrhalis bacteria exhibit diminished adherence to eukaryotic cells in vitro. Additionally, our studies demonstrate that M. catarrhalis cells form a mature biofilm in continuous-flow chambers and that biofilm formation is enhanced by TFP expression. The potential role of TFP in colonization by M. catarrhalis was further investigated using in vivo studies comparing the abilities of wild-type M. catarrhalis and an isogenic TFP mutant to colonize the nasopharynx of the chinchilla. These results suggest that the expression of TFP contributes to mucosal airway colonization. Furthermore, these data indicate that the chinchilla model of nasopharyngeal colonization provides an effective animal system for studying the early steps of M. catarrhalis pathogenesis.
PMCID: PMC2168369  PMID: 17908808
21.  MasABK Proteins Interact with Proteins of the Type IV Pilin System to Affect Social Motility of Myxococcus xanthus 
PLoS ONE  2013;8(1):e54557.
Gliding motility is critical for normal development of spore-filled fruiting bodies in the soil bacterium Myxococcus xanthus. Mutations in mgl block motility and development but one mgl allele can be suppressed by a mutation in masK, the last gene in an operon adjacent to the mgl operon. Deletion of the entire 5.5 kb masABK operon crippled gliding and fruiting body development and decreased sporulation. Expression of pilAGHI, which encodes type IV pili (TFP) components essential for social (S) gliding, several cryptic pil genes, and a LuxR family protein were reduced significantly in the Δmas mutant while expression of the myxalamide operon was increased significantly. Localization and two-hybrid analysis suggest that the three Mas proteins form a membrane complex. MasA-PhoA fusions confirmed that MasA is an integral cytoplasmic membrane protein with a ≈100 amino acid periplasmic domain. Results from yeast two-hybrid assays showed that MasA interacts with the lipoprotein MasB and MasK, a protein kinase and that MasB and MasK interact with one another. Additionally, yeast two-hybrid analysis revealed a physical interaction between two gene products of the mas operon, MasA and MasB, and PilA. Deletion of mas may be accompanied by compensatory mutations since complementation of the Δmas social gliding and developmental defects required addition of both pilA and masABK.
PMCID: PMC3546991  PMID: 23342171
22.  Regulation of cohesion-dependent cell interactions in Myxococcus xanthus. 
Journal of Bacteriology  1993;175(11):3636-3647.
Myxococcus xanthus has two nearly independent genetic systems, A and S, which appear to mediate adventurous (single-cell) movement and social (group) movement, respectively. In addition to a notable reduction in group movement, social motility mutants exhibit decreased biofilm formation, cell cohesion, dye binding, fibril production, and fruiting body formation. The stk-1907 allele, containing transposon Tn5 insertion omega DK1907, was introduced into wild-type cells and many social motility mutants. This allele, which was epistatic to most social motility mutations, caused wild-type and most mutant cells to exhibit increased group movement, cell cohesion, dye binding, and production of cell surface fibrils. The presence of the stk-1907 allele in dsp mutants, which almost completely lack cell surface fibrils, did not result in these phenotypic changes; therefore, stk-1907 is hypostatic to dsp mutations. Those mutants which exhibited increased group movement and cell cohesion with the stk-1907 allele also had increased fruiting body formation, but no significant changes in spore production were observed. These results suggest that fibrils may mediate cell cohesion, dye binding, and group movement. Additionally, the results suggest that the dsp locus contains genes involved in subunit synthesis, transport, and/or assembly of fibrils. The wild-type and mutant alleles of stk were cloned and studied in merodiploids. The mutant allele is recessive, suggesting that Tn5 omega DK1907 caused a null mutation in a gene which acts as a negative regulator of fibril synthesis. The stk-1907 allele appears to cause utilization of the A motility system for group movement, possibly because of increased fibril production.
PMCID: PMC204765  PMID: 8501067
23.  Effect of mechanical removal of pili on gliding motility of Myxococcus xanthus. 
Journal of Bacteriology  1992;174(16):5406-5413.
Gliding motility of Myxococcus xanthus is governed by both the adventurous (A) and the social (S) motility gene systems. The presence of pili has previously been shown to be correlated with a genetically intact S-motility system (D. Kaiser, Proc. Natl. Acad. Sci. USA 76:5952-5956, 1979). The purpose of the present work was to study the direct effect of mechanical removal of pill on the social motility of M. xanthus. Depiliation resulted in (i) a loss of streaming motility of A- S+ mutants, i.e., strains which are able to move by virtue of the S-motility system only, (ii) no effect on motility in A+ S- mutants, i.e., strains capable of movement by the A-motility system only, and (iii) a retardation of streaming speed in the wild-type strain (A+ S+). Cell-cell cohesion, another characteristic of social behavior, was not affected by mechanical removal of pill. The observation that mechanical depiliation perturbed the motility of strains which rely on the S-motility system strongly supports a role for pili in social motility of M. xanthus.
PMCID: PMC206379  PMID: 1353759
24.  Transcriptional regulation of type 4 pilin genes and the site-specific recombinase gene, piv, in Moraxella lacunata and Moraxella bovis. 
Journal of Bacteriology  1997;179(23):7298-7305.
Moraxella lacunata and Moraxella bovis use type 4 pili to adhere to epithelial tissues of the cornea and conjunctiva. Primer extension analyses were used to map the transcriptional start sites for the genes encoding the major pilin subunits (tfpQ/I) and the DNA invertase (piv), which determines pilin type expression. tfpQ/I transcription starts at a sigma54-dependent promoter (tfpQ/Ip2) and, under certain growth conditions, this transcription is accompanied by weaker upstream transcription that starts at a potential sigma70-dependent promoter (tfpQ/Ip1). piv is expressed in both M. lacunata and M. bovis from a putative sigma70-dependent promoter (pivp) under all conditions assayed. Sigma54-dependent promoters require activators in order to initiate transcription; therefore, it is likely that tfpQ/Ip2 is also regulated by an activator in Moraxella. Primer extension assays with RNA isolated from Escherichia coli containing the subcloned pilin inversion region from M. lacunata showed that pivp is used for the expression of piv; however, tfpQ/Ip2 is not used for the transcription of tfpQ/I. Transcription from tfpQ/Ip2 was activated in E. coli when the sensor (PilS) and response regulator (PilR) proteins of type 4 pilin transcription in Pseudomonas aeruginosa were expressed from a plasmid. These results suggest that the expression of the type 4 pilin in M. lacunata and M. bovis is regulated not only by a site-specific DNA inversion system but also by a regulatory system which is functionally analogous to the PilS-PilR two-component system of P. aeruginosa.
PMCID: PMC179679  PMID: 9393693
25.  Gliding Motility in Bacteria: Insights from Studies of Myxococcus xanthus 
Gliding motility is observed in a large variety of phylogenetically unrelated bacteria. Gliding provides a means for microbes to travel in environments with a low water content, such as might be found in biofilms, microbial mats, and soil. Gliding is defined as the movement of a cell on a surface in the direction of the long axis of the cell. Because this definition is operational and not mechanistic, the underlying molecular motor(s) may be quite different in diverse microbes. In fact, studies on the gliding bacterium Myxococcus xanthus suggest that two independent gliding machineries, encoded by two multigene systems, operate in this microorganism. One machinery, which allows individual cells to glide on a surface, independent of whether the cells are moving alone or in groups, requires the function of the genes of the A-motility system. More than 37 A-motility genes are known to be required for this form of movement. Depending on an additional phenotype, these genes are divided into two subclasses, the agl and cgl genes. Videomicroscopic studies on gliding movement, as well as ultrastructural observations of two myxobacteria, suggest that the A-system motor may consist of multiple single motor elements that are arrayed along the entire cell body. Each motor element is proposed to be localized to the periplasmic space and to be anchored to the peptidoglycan layer. The force to glide which may be generated here is coupled to adhesion sites that move freely in the outer membrane. These adhesion sites provide a specific contact with the substratum. Based on single-cell observations, similar models have been proposed to operate in the unrelated gliding bacteria Flavobacterium johnsoniae (formerly Cytophaga johnsonae), Cytophaga strain U67, and Flexibacter polymorphus (a filamentous glider). Although this model has not been verified experimentally, M. xanthus seems to be the ideal organism with which to test it, given the genetic tools available. The second gliding motor in M. xanthus controls cell movement in groups (S-motility system). It is dependent on functional type IV pili and is operative only when cells are in close proximity to each other. Type IV pili are known to be involved in another mode of bacterial surface translocation, called twitching motility. S-motility may well represent a variation of twitching motility in M. xanthus. However, twitching differs from gliding since it involves cell movements that are jerky and abrupt and that lack the organization and smoothness observed in gliding. Components of this motor are encoded by genes of the S-system, which appear to be homologs of genes involved in the biosynthesis, assembly, and function of type IV pili in Pseudomonas aeruginosa and Neisseria gonorrhoeae. How type IV pili generate force in S-motility is currently unknown, but it is to be expected that ongoing physiological, genetic, and biochemical studies in M. xanthus, in conjunction with studies on twitching in P. aeruginosa and N. gonorrhoeae, will provide important insights into this microbial motor. The two motility systems of M. xanthus are affected to different degrees by the MglA protein, which shows similarity to a small GTPase. Bacterial chemotaxis-like sensory transduction systems control gliding motility in M. xanthus. The frz genes appear to regulate gliding movement of individual cells and movement by the S-motility system, suggesting that the two motors found in this bacterium can be regulated to result in coordinated multicellular movements. In contrast, the dif genes affect only S-system-dependent swarming.
PMCID: PMC103748  PMID: 10477310

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