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1.
De La Fuente L Montanes E Meng Y Li Y Burr TJ Hoch HC Wu M 《Applied and environmental microbiology》2007,73(8):2690-2696
Xylella fastidiosa, a bacterium responsible for Pierce's disease in grapevines, possesses both type I and type IV pili at the same cell pole. Type IV pili facilitate twitching motility, and type I pili are involved in biofilm development. The adhesiveness of the bacteria and the roles of the two pili types in attachment to a glass substratum were evaluated using a microfluidic flow chamber in conjunction with pilus-defective mutants. The average adhesion force necessary to detach wild-type X. fastidiosa cells was 147 +/- 11 pN. Mutant cells possessing only type I pili required a force of 204 +/- 22 pN for removal, whereas cells possessing only type IV pili required 119 +/- 8 pN to dislodge these cells. The experimental results demonstrate that microfluidic flow chambers are useful and convenient tools for assessing the drag forces necessary for detaching bacterial cells and that with specific pilus mutants, the role of the pilus type can be further assessed. 相似文献
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3.
Although flagella are the best-understood means of locomotion in bacteria [1], other bacterial motility mechanisms must exist as many diverse groups of bacteria move without the aid of flagella [2-4]. One unusual structure that may contribute to motility is the type IV pilus [5,6]. Genetic evidence indicates that type IV pili are required for social gliding motility (S-motility) in Myxococcus, and twitching motility in Pseudomonas and Neisseria [6,7]. It is thought that type IV pili may retract or rotate to bring about cellular motility [6,8], but there is no direct evidence for the role of pili in cell movements. Here, using a tethering assay, we obtained evidence that the type IV pilus of Myxococcus xanthus functions as a motility apparatus. Pili were required for M. xanthus cells to adhere to solid surfaces and to generate cellular movement using S-motility. Tethered cells were released from the surface at intervals corresponding to the reversal frequency of wild-type cells when gliding on a solid surface. Mutants defective in the control of directional movements and cellular reversals (frz mutants) showed altered patterns of adherence that correlate reversal frequencies with tethering. The behavior of the tethered cells was consistent with a model in which the pili are extruded from one cell pole, adhere to a surface, and then retract, pulling the cell in the direction of the adhering pili. Cellular reversals would result from the sites of pili extrusion switching from one cell pole to another and are controlled by the frz chemosensory system. 相似文献
4.
Assessing Adhesion Forces of Type I and Type IV Pili of Xylella fastidiosa Bacteria by Use of a Microfluidic Flow Chamber 总被引:1,自引:0,他引:1
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Leonardo De La Fuente Emilie Montanes Yizhi Meng Yaxin Li Thomas J. Burr H. C. Hoch Mingming Wu 《Applied microbiology》2007,73(8):2690-2696
Xylella fastidiosa, a bacterium responsible for Pierce's disease in grapevines, possesses both type I and type IV pili at the same cell pole. Type IV pili facilitate twitching motility, and type I pili are involved in biofilm development. The adhesiveness of the bacteria and the roles of the two pili types in attachment to a glass substratum were evaluated using a microfluidic flow chamber in conjunction with pilus-defective mutants. The average adhesion force necessary to detach wild-type X. fastidiosa cells was 147 ± 11 pN. Mutant cells possessing only type I pili required a force of 204 ± 22 pN for removal, whereas cells possessing only type IV pili required 119 ± 8 pN to dislodge these cells. The experimental results demonstrate that microfluidic flow chambers are useful and convenient tools for assessing the drag forces necessary for detaching bacterial cells and that with specific pilus mutants, the role of the pilus type can be further assessed. 相似文献
5.
Friedrich A Rumszauer J Henne A Averhoff B 《Applied and environmental microbiology》2003,69(7):3695-3700
The extreme thermophile Thermus thermophilus HB27 exhibits high frequencies of natural transformation. Although we recently reported identification of the first competence genes in Thermus, the molecular basis of DNA uptake is unknown. A pilus-like structure is assumed to be involved. Twelve genes encoding prepilin-like proteins were identified in three loci in the genome of T. thermophilus. Mutational analyses, described in this paper, revealed that one locus, which contains four genes that encode prepilin-like proteins (pilA1 to pilA4), is essential for natural transformation. Additionally, comZ, a new competence gene with no similarity to known genes, was identified. Analysis of the piliation phenotype revealed wild-type piliation of a pilA1-pilA3Deltakat mutant and a comZ mutant, whereas a pilA4 mutant was found to be completely devoid of pilus structures. These findings, together with the significant similarity of PilA4 to prepilins, led to the conclusion that the T. thermophilus pilus structures are type IV pili. Furthermore, the loss of the transformation and piliation phenotype in the pilA4 mutant suggests that type IV pili are implicated in natural transformation of T. thermophilus HB27. 相似文献
6.
To investigate the role of type IV pili in the virulence of phytopathogenic bacteria, four mutant strains for pilus biogenesis-related genes were generated in Pseudomonas syringae pv. tabaci 6605. PilA encodes the pilin protein as a major subunit of type IV pili, and the pilO product is reported to be required for pilus assembly. The fimU and fimT genes are predicted to produce minor pilins. Western blot analysis revealed that pilA, pilO, and fimU mutants but not the fimT mutant failed to construct type IV pili. Although the swimming motility of all mutant strains was not impaired in liquid medium, they showed remarkably reduced motilities on semisolid agar medium, suggesting that type IV pili are required for surface motilities. Virulence toward host tobacco plants and hypersensitive response-inducing ability in nonhost Arabidopsis leaves of pilA, pilO, and fimU mutant strains were reduced. These results might be a consequence of reduced expression of type III secretion system-related genes in the mutant strains. Further, all mutant strains showed enhanced expression of resistance-nodulation-division family members mexA, mexB, and oprM, and higher tolerance to antimicrobial compounds. These results indicate that type IV pili are an important virulence factor of this pathogen. 相似文献
7.
Nishiyama M Vetsch M Puorger C Jelesarov I Glockshuber R 《Journal of molecular biology》2003,330(3):513-525
The outer membrane protein FimD represents the assembly platform of adhesive type 1 pili from Escherichia coli. FimD forms ring-shaped oligomers of 91.4 kDa subunits that recognize complexes between the pilus chaperone FimC and individual pilus subunits in the periplasm and mediate subunit translocation through the outer membrane. Here, we have identified a periplasmic domain of FimD (FimD(N)) comprising the N-terminal 139 residues of FimD. Purified FimD(N) is a monomeric, soluble protein that specifically recognizes complexes between FimC and individual type 1 pilus subunits, but does not bind the isolated chaperone, or isolated subunits. In addition, FimD(N) retains the ability of FimD to recognize different chaperone-subunit complexes with different affinities, and has the highest affinity towards the FimC-FimH complex. Overexpression of FimD(N) in the periplasm of wild-type E.coli cells diminished incorporation of FimH at the tip of type 1 pili, while pilus assembly itself was not affected. The identification of FimD(N) and its ternary complexes with FimC and individual pilus subunits opens the avenue to structural characterization of critical type 1 pilus assembly intermediates. 相似文献
8.
Natural competence for DNA transformation by Legionella pneumophila and its association with expression of type IV pili
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We have recently described the expression of two pili of different lengths on the surface of Legionella pneumophila (B. J. Stone and Y. Abu Kwaik, Infect. Immun. 66:1768-1775, 1998). Production of long pili requires a functional pilEL locus, encoding a type IV pilin protein. Since type IV pili in Neisseria gonorrhoeae are associated with competence for DNA transformation, we examined the competence of L. pneumophila for DNA transformation under conditions that allowed the expression of type IV pili. We show that L. pneumophila is naturally competent for DNA transformation by isogenic chromosomal DNA and by plasmid DNA containing L. pneumophila DNA. Many different L. pneumophila loci are able to transform L. pneumophila after addition of plasmid DNA, including gspA, ppa, asd, and pilEL. The transformation frequency is reduced when competing DNA containing either L. pneumophila DNA or vector sequences is added to the bacteria, suggesting that uptake-specific sequences may not be involved in DNA uptake. Competence for DNA transformation correlates with expression of the type IV pili, and a pilEL mutant defective in expression of type IV pili is not competent for DNA transformation. Complementation of the mutant for competence is restored by the reintroduction of a cosmid that restores production of type IV pili. Minimal competence is restored to the mutant by introduction of pilEL alone. We conclude that competence for DNA transformation in L. pneumophila is associated with expression of the type IV pilus and results in recombination of L. pneumophila DNA into the chromosome. Since expression of type IV pili also facilitates attachment of L. pneumophila to mammalian cells and protozoa, we designated the type IV pili CAP (for competence- and adherence-associated pili). 相似文献
9.
The human pathogen Eikenella corrodens expresses type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying pilus structure and function in the clinical isolate E. corrodens VA1. Earlier work defined the pilA locus which includes pilA1, pilA2, pilB, and hagA. Both pilA1 and pilA2 predict a type IV pilin, whereas pilB predicts a putative pilus assembly protein. The role of hagA has not been clearly established. That work also confirmed that pilA1 encodes the major pilus protein in this strain and showed that the phase variation involves a posttranslational event in pilus formation. In this study, the function of the individual genes comprising the pilA locus was examined using a recently developed protocol for targeted interposon mutagenesis of S-phase variant VA1-S1. Different pilA mutants were compared to S-phase and L-phase variants for several distinct aspects of phase variation and type IV pilus biosynthesis and function. S-phase cells were characterized by surface pili, competence for natural transformation, and twitching motility, whereas L-phase cells lacked these features. Inactivation of pilA1 yielded a mutant that was phenotypically indistinguishable from L-phase variants, showing that native biosynthesis of the type IV pilus in strain VA1 is dependent on expression of pilA1 and proper export and assembly of PilA1. Inactivation of pilA2 yielded a mutant that was phenotypically indistinguishable from S-phase variants, indicating that pilA2 is not essential for biosynthesis of functionally normal pili. A mutant inactivated for pilB was deficient for twitching motility, suggesting a role for PilB in this pilus-related phenomenon. Inactivation of hagA, which may encode a tellurite resistance protein, had no effect on pilus structure or function. 相似文献
10.
Ng SY Wu J Nair DB Logan SM Robotham A Tessier L Kelly JF Uchida K Aizawa S Jarrell KF 《Journal of bacteriology》2011,193(4):804-814
The structure of pili from the archaeon Methanococcus maripaludis is unlike that of any bacterial pili. However, genetic analysis of the genes involved in the formation of these pili has been lacking until this study. Pili were isolated from a nonflagellated (ΔflaK) mutant and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to consist primarily of subunits with an apparent molecular mass of 17 kDa. In-frame deletions were created in three genes, MMP0233, MMP0236, and MMP0237, which encode proteins with bacterial type IV pilin-like signal peptides previously identified by in silico methodology as likely candidates for pilus structural proteins. Deletion of MMP0236 or MMP0237 resulted in mutant cells completely devoid of pili on the cell surface, while deletion of the third pilin-like gene, MMP0233, resulted in cells greatly reduced in the number of pili on the surface. Complementation with the deleted gene in each case returned the cells to a piliated state. Surprisingly, mass spectrometry analysis of purified pili identified the major structural pilin as another type IV pilin-like protein, MMP1685, whose gene is located outside the first pilus locus. This protein was found to be glycosylated with an N-linked branched pentasaccharide glycan. Deletion and complementation analysis confirmed that MMP1685 is required for piliation. 相似文献
11.
Collins RF Frye SA Balasingham S Ford RC Tønjum T Derrick JP 《The Journal of biological chemistry》2005,280(19):18923-18930
Type IV pili are cell surface organelles found on many Gram-negative bacteria. They mediate a variety of functions, including adhesion, twitching motility, and competence for DNA uptake. The type IV pilus is a helical polymer of pilin protein subunits and is capable of rapid polymerization or depolymerization, generating large motor forces in the process. Here we show that a specific interaction between the outer membrane secretin PilQ and the type IV pilus fiber can be detected by far-Western analysis and sucrose density gradient centrifugation. Transmission electron microscopy of preparations of purified pili, to which the purified PilQ oligomer had been added, showed that PilQ was uniquely located at one end of the pilus fiber, effectively forming a "mallet-type" structure. Determination of the three-dimensional structure of the PilQ-type IV pilus complex at 26-angstroms resolution showed that the cavity within the protein complex was filled. Comparison with a previously determined structure of PilQ at 12-angstroms resolution indicated that binding of the pilus fiber induced a dissociation of the "cap" feature and lateral movement of the "arms" of the PilQ oligomer. The results demonstrate that the PilQ structure exhibits a dynamic response to the binding of its transported substrate and suggest that the secretin could play an active role in type IV pilus assembly as well as secretion. 相似文献
12.
van Schaik EJ Giltner CL Audette GF Keizer DW Bautista DL Slupsky CM Sykes BD Irvin RT 《Journal of bacteriology》2005,187(4):1455-1464
The opportunistic pathogen Pseudomonas aeruginosa produces multifunctional, polar, filamentous appendages termed type IV pili. Type IV pili are involved in colonization during infection, twitching motility, biofilm formation, bacteriophage infection, and natural transformation. Electrostatic surface analysis of modeled pilus fibers generated from P. aeruginosa strain PAK, K122-4, and KB-7 pilin monomers suggested that a solvent-exposed band of positive charge may be a common feature of all type IV pili. Several functions of type IV pili, including natural transformation and biofilm formation, involve DNA. We investigated the ability of P. aeruginosa type IV pili to bind DNA. Purified PAK, K122-4, and KB-7 pili were observed to bind both bacterial plasmid and salmon sperm DNA in a concentration-dependent and saturable manner. PAK pili had the highest affinity for DNA, followed by K122-4 and KB-7 pili. DNA binding involved backbone interactions and preferential binding to pyrimidine residues even though there was no evidence of sequence-specific binding. Pilus-mediated DNA binding was a function of the intact pilus and thus required elements present in the quaternary structure. However, binding also involved the pilus tip as tip-specific, but not base-specific, antibodies inhibited DNA binding. The conservation of a Thr residue in all type IV pilin monomers examined to date, along with the electrostatic data, implies that DNA binding is a conserved function of type IV pili. Pilus-mediated DNA binding could be important for biofilm formation both in vivo during an infection and ex vivo on abiotic surfaces. 相似文献
13.
Kingella kingae is a gram-negative bacterium that colonizes the respiratory tract and is a common cause of septic arthritis and osteomyelitis. Despite the increasing frequency of K. kingae disease, little is known about the mechanism by which this organism adheres to respiratory epithelium and seeds joints and bones. Previous work showed that K. kingae expresses long surface fibers that vary in surface density. In the current study, we found that these fibers are type IV pili and are necessary for efficient adherence to respiratory epithelial and synovial cells and that the number of pili expressed by the bacterium correlates with the level of adherence to synovial cells but not with the level of adherence to respiratory cells. In addition, we established that the major pilin subunit is encoded by a pilA homolog in a conserved region of the chromosome that also contains a second pilin gene and a type IV pilus accessory gene, both of which are dispensable for pilus assembly and pilus-mediated adherence. Upon examination of the K. kingae genome, we identified two genes in physically separate locations on the chromosome that encode homologs of the Neisseria PilC proteins and that have only a low level homology to each other. Examination of mutant strains revealed that both of the K. kingae PilC homologs are essential for a wild-type level of adherence to both respiratory epithelial and synovial cells. Taken together, these results demonstrate that type IV pili and the two PilC homologs play important roles in mediating K. kingae adherence. 相似文献
14.
The Cpx envelope stress response affects expression of the type IV bundle-forming pili of enteropathogenic Escherichia coli
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The Cpx envelope stress response mediates adaptation to potentially lethal envelope stresses in Escherichia coli. The two-component regulatory system consisting of the sensor kinase CpxA and the response regulator CpxR senses and mediates adaptation to envelope insults believed to result in protein misfolding in this compartment. Recently, a role was demonstrated for the Cpx response in the biogenesis of P pili, attachment organelles expressed by uropathogenic E. coli. CpxA senses misfolded P pilus assembly intermediates and initiates increased expression of both assembly and regulatory factors required for P pilus elaboration. In this report, we demonstrate that the Cpx response is also involved in the expression of the type IV bundle-forming pili of enteropathogenic E. coli (EPEC). Bundle-forming pili were not elaborated from an exogenous promoter in E. coli laboratory strain MC4100 unless the Cpx pathway was constitutively activated. Further, an EPEC cpxR mutant synthesized diminished levels of bundle-forming pili and was significantly affected in adherence to epithelial cells. Since type IV bundle-forming pili are very different from chaperone-usher-type P pili in both form and biogenesis, our results suggest that the Cpx envelope stress response plays a general role in the expression of envelope-localized organelles with diverse structures and assembly pathways. 相似文献
15.
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. 相似文献
16.
Shaffer CL Gaddy JA Loh JT Johnson EM Hill S Hennig EE McClain MS McDonald WH Cover TL 《PLoS pathogens》2011,7(9):e1002237
Colonization of the human stomach by Helicobacter pylori is an important risk factor for development of gastric cancer. The H. pylori cag pathogenicity island (cag PAI) encodes components of a type IV secretion system (T4SS) that translocates the bacterial oncoprotein CagA into gastric epithelial cells, and CagL is a specialized component of the cag T4SS that binds the host receptor α5β1 integrin. Here, we utilized a mass spectrometry-based approach to reveal co-purification of CagL, CagI (another integrin-binding protein), and CagH (a protein with weak sequence similarity to CagL). These three proteins are encoded by contiguous genes in the cag PAI, and are detectable on the bacterial surface. All three proteins are required for CagA translocation into host cells and H. pylori-induced IL-8 secretion by gastric epithelial cells; however, these proteins are not homologous to components of T4SSs in other bacterial species. Scanning electron microscopy analysis reveals that these proteins are involved in the formation of pili at the interface between H. pylori and gastric epithelial cells. ΔcagI and ΔcagL mutant strains fail to form pili, whereas a ΔcagH mutant strain exhibits a hyperpiliated phenotype and produces pili that are elongated and thickened compared to those of the wild-type strain. This suggests that pilus dimensions are regulated by CagH. A conserved C-terminal hexapeptide motif is present in CagH, CagI, and CagL. Deletion of these motifs results in abrogation of CagA translocation and IL-8 induction, and the C-terminal motifs of CagI and CagL are required for formation of pili. In summary, these results indicate that CagH, CagI, and CagL are components of a T4SS subassembly involved in pilus biogenesis, and highlight the important role played by unique constituents of the H. pylori cag T4SS. 相似文献
17.
Type IV pilus genes pilA and pilC of Pseudomonas stutzeri are required for natural genetic transformation, and pilA can be replaced by corresponding genes from nontransformable species
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Graupner S Frey V Hashemi R Lorenz MG Brandes G Wackernagel W 《Journal of bacteriology》2000,182(8):2184-2190
Pseudomonas stutzeri lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation-deficient mutants were isolated from a P. stutzeri JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of Pseudomonas aeruginosa, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The pilC mutant had no pili and was defective in twitching motility. Further sequencing revealed that pilC is clustered in an operon with genes homologous to pilB and pilD of P. aeruginosa, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a pilA gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of pilA abolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of (3)H-labeled P. stutzeri DNA that were bound to competent parental cells and taken up were strongly reduced in the pilC and pilA mutants. Remarkably, the cloned pilA genes from nontransformable organisms like Dichelobacter nodosus and the PAK and PAO strains of P. aeruginosa fully restored pilus formation and transformability of the P. stutzeri pilA mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium P. stutzeri function in DNA uptake for transformation and that their role in this process is not confined to the species-specific pilin. 相似文献
18.
Effects of bfp mutations on biogenesis of functional enteropathogenic Escherichia coli type IV pili
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Enteropathogenic Escherichia coli expresses a type IV fimbria known as the bundle-forming pilus (BFP) that is required for autoaggregation and localized adherence (LA) to host cells. A cluster of 14 genes is sufficient to reconstitute BFP biogenesis in a laboratory strain of E. coli. We have undertaken a systematic mutagenesis of the individual genes to determine the effect of each mutation on BFP biogenesis and LA. Here we report the construction and analysis of nonpolar mutations in six genes of the bfp cluster, bfpG, bfpB, bfpC, bfpD, bfpP, and bfpH, as well as the further analysis of a previously described bfpA mutant strain that is unable to express bundlin, the pilin protein. We found that mutations in bfpB, which encodes an outer membrane protein; bfpD, which encodes a putative nucleotide-binding protein; and bfpG and bfpC, which do not have sequence homologues in other type IV pilus systems, do not affect prebundlin expression or processing but block both BFP biogenesis and LA. The mutation in bfpP, the prepilin peptidase gene, does not affect prebundlin expression but blocks signal sequence cleavage of prebundlin, BFP biogenesis, and LA. The mutation in bfpH, which is predicted to encode a lytic transglycosylase, has no effect on prebundlin expression, prebundlin processing, BFP biogenesis, or LA. For each mutant for which altered phenotypes were detected, complementation with a plasmid containing the corresponding wild-type allele restored the wild-type phenotypes. We also found that association of prebundlin or bundlin with sucrose density flotation gradient fractions containing both inner and outer membrane proteins does not require any accessory proteins. These studies indicate that many bfp gene products are required for biogenesis of functional type IV pili but that mutations in the individual genes do not lead to the identification of new phases of pilus assembly. 相似文献
19.
Myxococcus xanthus tgl mutants lack social motility and type IV pili but can be transiently stimulated to swarm and to make pili by contacting tgl+ cells. The absence of pili in tgl mutants is shown not to be due to the absence of pilin. The rate of pilus elongation after Tgl stimulation is shown to be similar to the rate of pilus elongation in wild-type cells, using a new more rapid assay for stimulation. 相似文献