Regulation of expression of the pilA gene in Myxococcus xanthus.

Type IV pili are required for social gliding motility in Myxococcus xanthus. In this work, the expression of pilin (the pilA gene product) during vegetative growth and fruiting-body development was examined. A polyclonal antibody against the pilA gene product (prepilin) was prepared, along with a pilA-lacZ fusion, and was used to assay expression of pilA in M. xanthus in different mutant backgrounds. pilA expression required the response regulator pilR but was negatively regulated by the putative sensor kinase pilS. pilA expression did not require pilB, pilC, or pilT. pilA was also autoregulated; a mutation which altered an invariant glutamate five residues from the presumed prepilin processing site eliminated this autoregulation, as did a deletion of the pilA gene. Primer extension and S1 nuclease analysis identified a sigma54 promoter upstream of pilA, consistent with the homology of pilR to the NtrC family of response regulators. Expression of pilA was found to be developmentally regulated; however, the timing of this expression pattern was not entirely dependent on pilS or pilR. Finally, pilA expression was induced by high nutrient concentrations, an effect that was also not dependent on pilS or pilR.     

Genetic and functional evidence that Type IV pili are required for social gliding motility in Myxococcus xanthus

The social gliding behaviour of Myxococcus xanthus has previously been associated with the presence of polar pili. A Tn 5 transposon insertion was isolated which introduces a defect in social gliding and is genetically linked to a known sgl locus; this insertion was found also to cause a piliation defect. A 2.7 kb section of DNA was isolated from either side of this transposon and sequenced, revealing three genes which encode amino acid sequences with substantial similarity to components of the Type IV pilus biogenesis pathway in Pseudomonas aeruginosa . The myxococcal pilA gene encodes a putative pilin precursor with a short signal sequence and processing site similar to those of other Type IV pilins. Myxococcal pilS and pilR encode amino acid sequences with similarity to PilS and PilR of P. aeruginosa , as well as to other members of the NtrB/C family of two-component regulators. Mutations within pilR and pilA that have no polar effect were demonstrated to be responsible for pilus and social motility defects. These results indicate that the pili of M. xanthus belong to the Type IV family of pili, and demonstrate that these pili are actually required for social motility.     

The Myxococcus xanthuspilT locus is required for social gliding motility although pili are still produced

Social gliding motility in Myxococcus xanthus depends on the presence of Type IV pili. To begin to examine the role of pili in social motility, 17 mutants were identified which had lost social motility, but still expressed pili. Four of these mutants carry point mutations which mapped to a locus upstream of the recently identified pilS , pilR , and pilA genes. Sequencing of this locus revealed a gene with homology to pilT from Pseudomonas aeruginosa . Sequencing of the four point mutations revealed that they occurred within the M. xanthus pilT locus. A markerless deletion within M. xanthus pilT , similar to the four point mutations, disrupted social gliding behaviour but did not interfere with pilus formation or pilus-dependent cell–cell agglutination. Using time-lapse videomicroscopy, residual social motility was observed in dsp ⁻ strains (known to be deficient in fibril but not pilus production); this was not observed in a Δ pilT dsp ⁻ double mutant. Two genes flanking pilT  were also sequenced, and found to have homology to pilB and pilC from P. aeruginosa . Markerless deletions within these genes caused both pilus and social-motility defects. These results indicate that M. xanthus pilB and pilC are required for pilus biogenesis, while pilT is required for assembled pili to play their role in social motility. Thus, pilB , pilT , pilC , pilS , pilR and pilA form a contiguous cluster of pil genes required for social motility.     

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

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.     

Myxococcus xanthus dif genes are required for biogenesis of cell surface fibrils essential for social gliding motility

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.     

Effect of mechanical removal of pili on gliding motility of Myxococcus xanthus.

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.     

Markerless deletions of pil genes in Myxococcus xanthus generated by counterselection with the Bacillus subtilis sacB gene.

In-frame deletions of pilA and pilS were constructed in Myxococcus xanthus with a plasmid integration-excision strategy facilitated by sacB. sacB conferred sucrose sensitivity upon its M. xanthus host only when it lay in the same orientation as adjacent M. xanthus genes. Gene orientation also affected the efficiency of sucrose counterselection in the sucrose-sensitive strains. The deltapilA mutant lacked pili and social motility, while the deltapilS mutant showed no defect in either phenotype.     

Role of the Eikenella corrodens pilA locus in pilus function and phase variation

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.     

Characterization of type IV pilus genes in plant growth-promoting Pseudomonas putida WCS358.

In a search for factors that could contribute to the ability of the plant growth-stimulating Pseudomonas putida WCS358 to colonize plant roots, the organism was analyzed for the presence of genes required for pilus biosynthesis. The pilD gene of Pseudomonas aeruginosa, which has also been designated xcpA, is involved in protein secretion and in the biogenesis of type IV pili. It encodes a peptidase that processes the precursors of the pilin subunits and of several components of the secretion apparatus. Prepilin processing activity could be demonstrated in P. putida WCS358, suggesting that this nonpathogenic strain may contain type IV pili as well. A DNA fragment containing the pilD (xcpA) gene of P. putida was cloned and found to complement a pilD (xcpA) mutation in P. aeruginosa. Nucleotide sequencing revealed, next to the pilD (xcpA) gene, the presence of two additional genes, pilA and pilC, that are highly homologous to genes involved in the biogenesis of type IV pili. The pilA gene encodes the pilin subunit, and pilC is an accessory gene, required for the assembly of the subunits into pili. In comparison with the pil gene cluster in P. aeruginosa, a gene homologous to pilB is lacking in the P. putida gene cluster. Pili were not detected on the cell surface of P. putida itself, not even when pilA was expressed from the tac promoter on a plasmid, indicating that not all the genes required for pilus biogenesis were expressed under the conditions tested. Expression of pilA of P. putida in P. aeruginosa resulted in the production of pili containing P. putida PilA subunits.     

Metastable regulation of type 1 piliation in Escherichia coli and isolation and characterization of a phenotypically stable mutant.

Type 1 piliation in Escherichia coli exhibits phase variation due to the inversion of a small, ca. 300-base-pair, element that regulates pilA (fimA), the gene that encodes the structural subunit of pili (Abraham et al., Proc. Natl. Acad. Sci. USA 82:5724-5727, 1985). We have used the inversion as an assay to characterize a stably piliated mutant. The mutant strain did not exhibit the pilA ON and pilA OFF colonial variants characteristic of the wild type; rather, every clone produced a level of pilA expression intermediate between ON and OFF wild-type populations. The mutant phenotype was conferred by a lesion at a previously undescribed locus between hemA and trpA, which we have termed pilG. Examination of the pilA promoter region in four pilG mutant populations indicated that the phenotypic stability conferred by the pilG mutation was not due to an inability to carry out the inversion. Rather, all pilG mutant populations consisted of approximately equal mixtures of ON and OFF individuals. We suggest that pilG mutants may undergo such rapid switching of the pilA promoter that populations exhibit an intermediate level of pilA expression and phenotypic stability.     

Genetic population structure of the soil bacterium Myxococcus xanthus at the centimeter scale

Myxococcus xanthus is a gram-negative soil bacterium best known for its remarkable life history of social swarming, social predation, and multicellular fruiting body formation. Very little is known about genetic diversity within this species or how social strategies might vary among neighboring strains at small spatial scales. To investigate the small-scale population structure of M. xanthus, 78 clones were isolated from a patch of soil (16 by 16 cm) in Tübingen, Germany. Among these isolates, 21 genotypes could be distinguished from a concatemer of three gene fragments: csgA (developmental C signal), fibA (extracellular matrix-associated zinc metalloprotease), and pilA (the pilin subunit of type IV pili). Accumulation curves showed that most of the diversity present at this scale was sampled. The pilA gene contains both conserved and highly variable regions, and two frequency-distribution tests provide evidence for balancing selection on this gene. The functional domains in the csgA gene were found to be conserved. Three instances of lateral gene transfer could be inferred from a comparison of individual gene phylogenies, but no evidence was found for linkage equilibrium, supporting the view that M. xanthus evolution is largely clonal. This study shows that M. xanthus is surrounded by a variety of distinct conspecifics in its natural soil habitat at a spatial scale at which encounters among genotypes are likely.     

ctr1, a gene involved in a signal transduction pathway of the gliding motility in the cyanobacterium Synechocystis sp. PCC 6803

We generated random Tn5 mutations in Synechocystis sp. PCC 6803 in search for genes involved in the signal transduction cascade for the cyanobacterial gliding motility. One of the non-gliding Tn5 mutants, S1-105, had an insertional inactivation in the slr1044 gene encoding a putative methyl-accepting chemotaxis protein. Interposon mutation on the slr1044 (named ctr1) in the bacterium also eliminated gliding motility. In the interposon mutant, the expression of pilA1 was 5-fold decreased compared with that of wild-type and thick pili, that are believed to be the motor for gliding, could not be observed by an electron microscope. Therefore, we suggest that the Ctr1 protein functions as a transducer that regulates the expression of pilA1, and thus is required for the biogenesis of thick pili.     

Cyanobacterial hybrid kinase Sll0043 regulates phototaxis by suppressing pilin and twitching motility protein

The unicellular cyanobacterium Synechocystis sp. PCC 6803 glides toward a light source through the interplay of positive phototaxis genes and proteins. In genetic analysis, the complete disruption of the hybrid sensory kinase sll0043 produced negative phototaxis. Furthermore, Sll0043 was found to be a hub protein by in silico prediction of protein-protein interaction, in which Sll0043 was predominantly linked to seven two-component proteins with high confidence. To understand the regulation and networking of positive phototaxis proteins, the proteomic profile of the sll0043 mutant was compared to that of wild-type. In the sll0043 mutant, 18 spots corresponding to 15 unique proteins were altered by 1.3 to 59 fold; the spots were identified by 2-DE/MALDI-MS analysis. Down-regulated proteins in the sll0043 null-mutant included chaperonins, superoxide dismutase, and phycocyanin beta-subunit. In contrast, nine proteins involved in photosynthesis, translation, regulatory function, and other functions were up-regulated. In particular, a twitching motility protein (PilT1) was induced over 2-fold in sll0043 mutant. Moreover, semi-quantitative and quantitative RT-PCR analysis revealed that pilin (pilA1), pili motor (pilT1), and pili switch gene (pilT2) were significantly increased in sll0043 mutant. These results suggest that the hybrid kinase Sll0043 regulates positive phototaxis by suppressing the expression of pili biosynthesis and regulatory genes and through the interplay with positive phototaxis/motility two-component proteins.