Identification and cell cycle control of a novel pilus system in Caulobacter crescentus
2000; Springer Nature; Volume: 19; Issue: 13 Linguagem: Inglês
10.1093/emboj/19.13.3223
ISSN1460-2075
Autores Tópico(s)Biochemical and Structural Characterization
ResumoArticle3 July 2000free access Identification and cell cycle control of a novel pilus system in Caulobacter crescentus Jeffrey M. Skerker Jeffrey M. Skerker Department of Developmental Biology, Beckman Center, Stanford University School of Medicine, Stanford, CA, 94305 USA Search for more papers by this author Lucy Shapiro Corresponding Author Lucy Shapiro Department of Developmental Biology, Beckman Center, Stanford University School of Medicine, Stanford, CA, 94305 USA Department of Developmental Biology, Stanford University Medical Center, Beckman Center, B351, 279 Campus Drive, Palo Alto, CA, 94304-5329 USA Search for more papers by this author Jeffrey M. Skerker Jeffrey M. Skerker Department of Developmental Biology, Beckman Center, Stanford University School of Medicine, Stanford, CA, 94305 USA Search for more papers by this author Lucy Shapiro Corresponding Author Lucy Shapiro Department of Developmental Biology, Beckman Center, Stanford University School of Medicine, Stanford, CA, 94305 USA Department of Developmental Biology, Stanford University Medical Center, Beckman Center, B351, 279 Campus Drive, Palo Alto, CA, 94304-5329 USA Search for more papers by this author Author Information Jeffrey M. Skerker1 and Lucy Shapiro 1,2 1Department of Developmental Biology, Beckman Center, Stanford University School of Medicine, Stanford, CA, 94305 USA 2Department of Developmental Biology, Stanford University Medical Center, Beckman Center, B351, 279 Campus Drive, Palo Alto, CA, 94304-5329 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:3223-3234https://doi.org/10.1093/emboj/19.13.3223 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Pilus assembly in Caulobacter crescentus occurs during a short period of the cell cycle and pili are only present at the flagellar pole of the swarmer cell. Here we report a novel assay to visualize pili by light microscopy that led to the purification of Caulobacter pili and the isolation of a cluster of seven genes, including the major pilin subunit gene pilA. This gene cluster encodes a novel group of pilus assembly proteins. We have shown that the pilA promoter is activated late in the cell cycle and that transcription of the pilin subunit plays an important role in the timing of pilus assembly. pilA transcription is regulated by the global two-component response regulator CtrA, which is essential for the expression of multiple cell cycle events, providing a direct link between assembly of the pilus organelle and bacterial cell cycle control. Introduction Pili are extracellular filaments, found on a wide variety of bacteria, that play an important role in adhesion of pathogenic bacteria to their host, biofilm formation, conjugative DNA transfer, non-flagellar motility and bacteriophage infection (Clewell, 1993; O'Toole and Kolter, 1998; Soto and Hultgren, 1999; Wall and Kaiser, 1999). Pilus filaments range from 2 to 10 nm in diameter and from 0.2 to 6 μm in length (Strom and Lory, 1993; Hultgren et al., 1996). Some bacteria have pili localized to the poles of the cells, and others have pili covering the entire cell surface. These structures are formed by the polymerization of pilin subunits at the base of the growing filament (Lowe et al., 1987). In Gram-negative bacteria, the pilin subunit must be secreted across both inner and outer membranes before being assembled into an extracellular filament. Although several distinct mechanisms of pilus assembly have been described, they all share common requirements: peptidases that process the signal peptide found on prepilin, energy provided by ATP hydrolysis for transport of pilin across the inner membrane, and specialized outer membrane proteins that form channels that allow the pilin subunit to reach the cell surface (Christie, 1997; Soto and Hultgren, 1999). In many bacteria, pili are receptors for bacteriophage, which bind either to the tip or the sides of the pilus filament (Clewell, 1993). Studies of infection of Escherichia coli by the filamentous DNA phage fd led to the idea that pili may be dynamic structures (Brinton, 1971; Jacobson, 1972). According to this model, phage infection is a two-step process: first, fd binds to the tip of the F-pilus, then the pilus retracts, bringing the phage to the cell surface where interaction with a secondary receptor facilitates DNA injection (Click and Webster, 1997). Subsequent studies of Pseudomonas aeruginosa infection by the DNA phage PO4 also support this model (Bradley, 1974). Caulobacter crescentus is a non-pathogenic, α-purple bacterium that divides asymmetrically (Brun et al., 1994) to generate two progeny cells: a non-motile stalked cell and a motile swarmer cell with several pili and a single flagellum (see Figure 9B). The stalked cell grows and replicates its chromosome, producing a predivisional cell with a new flagellum at the pole opposite the stalk. Pilus assembly occurs later than the assembly of the flagellum; the flagellum is assembled at the incipient swarmer pole of the predivisional cell, while pili are found only on progeny swarmer cells (Sommer and Newton, 1988). When the swarmer cell later differentiates into a stalked cell, the flagellum is released from the pole to be replaced by a new stalk. Pili are also lost at this transition, and it has been suggested that pilus retraction may be responsible for this loss (Lagenaur and Agabian, 1977; Sommer and Newton, 1988). Figure 1.Electron microscopy of φCbK infection of C.crescentus swarmer cells showing that φCbK is a pili-specific bacteriophage. The flagellar pole of two swarmer cells is shown. (A) φCbK infection after 15 s adsorption. φCbK attaches to pili by its long non-contractile tail (attachment site marked with a white arrow). Some of the phage are bound to the cell pole. (B) φCbK infection after 15 min adsorption. φCbK is bound predominantly to the cell pole. Scale bars ∼120 nm. Download figure Download PowerPoint Pilus biogenesis in Caulobacter provides a unique system to study the temporal control of organelle assembly and to understand the mechanisms that restrict its assembly to a specific cellular location. Although evidence is accumulating that many proteins are localized to specific sites in the bacterial cell (Shapiro and Losick, 2000), the mechanisms involved are unknown. In this paper, we report the identification and temporal regulation of a cluster of genes involved in pilus biogenesis in Caulobacter. The pilin subunit was purified using a novel fluorescence assay for visualizing bacterial pili, allowing the isolation of the gene encoding the pilin subunit, pilA. Six adjacent genes required for Caulobacter pilus assembly, cpaA-cpaF, were identified. Although the Caulobacter pilin subunit shares some features with Type IV pilins, the entire cluster of pilus assembly genes most closely resembles a gene cluster recently identified in the human oral pathogen, Actinobacillus actinomycetemcomitans (Inoue et al., 1998; Haase et al., 1999). Transcription of the pilA gene is regulated by the same response regulator, CtrA, that controls multiple cell cycle events, such as DNA replication, DNA methylation and flagellar biogenesis (Quon et al., 1996). The timing of pilus assembly can be altered by expressing the pilA gene from a constitutive promoter, demonstrating that transcriptional regulation of the pilin subunit gene is an important factor in determining the cell cycle pattern of pilus assembly. The identification of CtrA as a positive regulator of pilus biogenesis provides a direct link between pilus assembly and the genetic network that controls cell cycle progression. Results Bacteriophage φCbK binds to the polar pili on Caulobacter swarmer cells Caulobacter-specific bacteriophage include a generalized transducing phage φCr30, which binds to the S-layer surface protein, a small spherical RNA phage φCb5, which binds to the sides of pili, and a large DNA phage, φCbK, which binds specifically to the flagellar pole of swarmer cells (Schmidt, 1966; Agabian-Keshishian and Shapiro, 1971; Edwards and Smit, 1991). Mutants selected for φCbK resistance were found always to be resistant to φCb5, suggesting a relationship between φCbK infection and pili (Lagenaur et al., 1977). We have extended previous studies of φCbK infection by examining short adsorption times by electron microscopy. Cells fixed immediately after the addition of phage (∼15 s adsorption time) showed φCbK bound to the sides of pili, in addition to the cell pole (Figure 1A). After 15 min of adsorption, φCbK was bound primarily to the cell pole (Figure 1B). φCbK infection may be a two-step process in which initial attachment to pili is followed by pilus retraction and subsequent binding to a cell surface receptor. Thus, φCbK appears to be a pili-specific Caulobacter phage, and provides an explanation for the co-selection of φCbK and φCb5 resistance. We used the fact that pili are an adsorption site for phage φCbK both to purify the pilin subunit and as a genetic selection to identify genes involved in pilus biogenesis. Figure 2.Purification of bNY30a pili and identification of the NA1000 pilA gene. (A) Purified pili (∼2 μg protein) visualized by silver stain. Pili were purified from a hyperpiliated C.crescentus strain bNY30a and examined by 16.5% Tris-tricine SDS-PAGE. One major band (marked with an arrow) of ∼4.8 kDa was identified as the pilin subunit. Molecular weight markers are labeled in kilodaltons. (B) Electron micrograph of purified pili, stained with uranyl acetate. This fraction contains intact pili filaments, of a single diameter. Scale bar ∼100 nm. (C) Comparison of the predicted NA1000 PilA sequence with protein sequence data obtained from purified bNY30a pili. The predicted sequence of NA1000 PilA is shown in bold. The N-terminal sequence data and tryptic peptide sequences are shown in italics. (D) Caulobacter PilA has a Type IV-like leader peptide. Shown are the N-terminal sequences for three Type IVA pilin subunits: Pseudomonas aeruginosa PAK pilin (Pa; DDBJ/EMBL/GenBank accession No. X02402), Moraxella bovis pilin (Mb; DDBJ/EMBL/GenBank accession No. M92155) and Neisseria gonorrhoeae PilE (Ng; DDBJ/EMBL/GenBank accession No. X66834). Two Type IVB pilin subunits, EPEC E.coli BfpA (Ec; DDBJ/EMBL/GenBank accession No. Z12295) and V.cholerae TcpA (Vc; DDBJ/EMBL/GenBank accession No. U09807), are aligned with C.crescentus PilA (Cc) and A.actinomycetemcomitans Flp (Aa; DDBJ/EMBL/GenBank accession No. AB005741) because they have a longer leader peptide and the N-terminal residue is not a phenylalanine. Cleavage of Type IV leader peptides occurs after a conserved glycine (marked with an arrow). A conserved glutamate (asterisk) is found at position +5. Download figure Download PowerPoint Purification of Caulobacter pili using a novel fluorescence assay and identification of the pilA locus encoding the pilin subunit Aided by a novel fluorescence assay (described in Materials and methods) that allows the visualization of pili by light microscopy (see QuickTime movie in the Supplementary data at The EMBO Journal Online), we purified pili filaments. Pure pili allowed us to obtain the N-terminal amino acid sequence and then the gene encoding the major pilin subunit. We used a hyperpiliated strain (bNY30a), closely related to NA1000, as our source of pili and a modification of the purification protocol described by Lagenaur and Agabian (1977). Examination of the purified pili fraction by PAGE revealed one major band at ∼4.8 kDa (Figure 2A) and intact filaments by electron microscopy (Figure 2B). We obtained both N-terminal and internal tryptic peptide sequences of this 4.8 kDa protein and used this information to search the TIGR Caulobacter genome sequence for the gene encoding the pilin subunit. A single open reading frame (ORF) was identified encoding a predicted protein of 59 amino acids, which we named pilA, for the pilin subunit gene (Figure 2C). The predicted start methionine is 14 residues upstream from the first residue obtained by N-terminal sequencing, suggesting that pilin has a leader peptide that is processed after Gly14 to yield a mature protein of 45 amino acids (4360 Da). The size of the purified pilin protein (4.8 kDa) determined by gel electrophoresis closely matches that of the predicted mature pilin subunit. Figure 3.Sequence analysis of a pilus assembly gene cluster. (A) Diagram of an 8 kb StuI fragment that complements transposon mutant Tn24-3. Seven open reading frames were identified on this fragment, including the pilA locus encoding the pilin subunit and three other genes with homology to known pilus assembly proteins. (B) Alignment of the deduced amino acid sequence of Caulobacter CpaA with several members of the prepilin peptidase family. Only the region of the protein that is thought to contain the active site of the peptidase has been aligned. Putative active site residues are marked with an arrow. Invariant residues are marked with an asterisk. The sequences used in this alignment are Chlorobium limicola plasmid pCL1, Pph (Cl; DDBJ/EMBL/GenBank accession No. U77780), Aeromonas hydrophila TapD (Ah; DDBJ/EMBL/GenBank accession No. U20255), P.aeruginosa PilD (Pa; DDBJ/EMBL/GenBank accession No. M32066), Erwinia caratovora OutO (Ec; DDBJ/EMBL/GenBank accession No. X70049), C.crescentus CpaA (Cc) and V.cholerae TcpJ (Vc; DDBJ/EMBL/GenBank accession No. M74708). (C) Caulobacter CpaC is similar to the PulD/pIV family of outer membrane channels. Only the most highly conserved region of this protein family has been aligned. Invariant residues are marked with an asterisk and two functionally important residues are indicated with an arrow. The sequences used in this alignment are Rhizobium sp. NGR234 Y4×J (Rh; DDBJ/EMBL/GenBank accession No. AE000106), C.crescentus CpaC (Cc), P.aeruginosa PilQ (Pa; DDBJ/EMBL/GenBank accession No. L13865), A.actinomycetemcomitans RcpA (Aa; DDBJ/EMBL/GenBank accession No. AF139249), Klebsiella pneumoniae PulD (Kp; DDBJ/EMBL/GenBank accession No. M32613), A.hydrophila SpsD (Ah; DDBJ/EMBL/GenBank accession No. L41682) and coliphage f1 pIV (f1; DDBJ/EMBL/GenBank accession No. V00606). (D) Caulobacter CpaF is a member of the TrbB/VirB11 family of secretion ATPases. Only the most highly conserved region, surrounding the Walker Box (underlined), has been aligned. Invariant residues are marked with an asterisk. The sequences used in this alignment are A.actinomycetemcomitans TadA (Aa; DDBJ/EMBL/GenBank accession No. AF152598), C.crescentus CpaF (Cc), plasmid RP4 TrbB (RP4; DDBJ/EMBL/GenBank accession No. M93696), K.pneumoniae PulE (Kp; DDBJ/EMBL/GenBank accession No. M32613), P.aeruginosa PilB (Pa; DDBJ/EMBL/GenBank accession No. M32066) and Agrobacterium tumefaciens plasmid pTiC58, VirB11 (At; DDBJ/EMBL/GenBank accession No. X53264). Download figure Download PowerPoint Identification of a cpa gene cluster Phage φCbK resistance was used as a genetic tool to identify Caulobacter genes required for pilus assembly. The RNA phage φCb5 produces very cloudy plaques on the wild-type host NA1000, making it difficult to use in a genetic screen (Schmidt and Stanier, 1965). In contrast, φCbK produces clear plaques, which allows easy detection of phage-resistant mutants. We specifically looked for φCbK-resistant mutants that retained motility, to avoid the pleiotropic class of genes that affect multiple polar structures (Fukuda et al., 1976; Wang et al., 1993). From a collection of mini-Tn5lacZ2 insertion mutants, we identified one mutant, Tn24-3, with the appropriate phenotype. The region flanking the transposon insertion site was used to isolate an 8 kb StuI complementing fragment. Sequence analysis of this region identified seven ORFs with appropriate Caulobacter codon bias (Figure 3A). The pilA gene in this cluster encodes the pilin subunit that we identified biochemically, as described in the previous section. Figure 4.Schematic of the mutations used in this study. Six in-frame deletions were made in the Caulobacter pilA-cpaA cluster. The amino acids deleted in each gene are indicated. The mutation in cpaF was a mini-Tn5lacZ2 insertion (Tn24-3). Download figure Download PowerPoint Downstream of pilA are six genes, which we named cpaA-cpaF for Caulobacter pilus assembly. The protein encoded by cpaA is similar to the C-terminal region of prepilin peptidases (Figure 3B), which are bifunctional enzymes that process and methylate substrates required for Type II protein secretion or Type IV pilus assembly (Nunn and Lory, 1991; Russel, 1998). Downstream of cpaA is a group of five closely spaced genes, which most likely form a single transcriptional unit. Three of these genes, cpaB, cpaD and cpaE, encode proteins of unknown function. The protein encoded by cpaC is related to the PulD/pIV family of proteins, refered to as secretins (Nouwen et al., 1999). These proteins are required for extracellular secretion, filamentous phage assembly and pilus biogenesis, and are thought to function as a gated channel in the outer membrane (Russel et al., 1997). The most highly conserved portion of these secretins (Russel, 1994), containing an invariant glycine and proline residue, has been aligned with CpaC (Figure 3C). The fifth gene in this cluster, cpaF, encodes a protein related to the TrbB/VirB11 family of Walker Box-containing proteins. The TrbB/VirB11 family of proteins are involved in DNA uptake, extracellular secretion and pilus assembly (Hobbs and Mattick, 1993). Mutations in the Walker Box motif abolish their function in vivo, suggesting that ATP binding or hydrolysis is critical to their function (Turner et al., 1993; K.M.Stephens et al., 1995). The biochemical function of the TrbB/Virb11 protein family is not understood, but it is thought that the energy from ATP hydrolysis is used to export proteins across the inner membrane (Russel, 1998). An alignment of CpaF with other members of the TrbB/VirB11 protein family is shown in Figure 3D. pilA, cpaA, cpaB, cpaC, cpaD, cpaE and cpaF are required for Caulobacter pilus assembly To test directly the function of the genes in the pilA-cpa cluster, we constructed in-frame deletion mutants of pilA, cpaA, cpaB, cpaC, cpaD and cpaE in wild-type C.crescentus strain NA1000 (Figure 4). All six chromosomal in-frame deletion strains, as well as the transposon insertion in the cpaF gene, were found to be resistant to the pili-specific phages φCbK and φCb5, but remained sensitive to φCr30 (Figure 5B). These mutant strains all exhibited normal morphology and motility (data not shown) and were found to be pili-less by electron microscopy. Phage sensitivity and piliation could be restored for each mutant strain by a plasmid carrying the corresponding wild-type allele (Figure 5A), demonstrating that the phenotype was due to the effect of a single gene mutation. The degree of complementation, as determined by the percentage of piliated swarmer cells, varied among the different constructs, perhaps due to inappropriate levels of expression of the particular gene product. Taken together, these data indicate that the seven genes contained on the 8 kb StuI complementing fragment are required for pilus assembly in Caulobacter. Figure 5.Complementing clones and electron microscopy data. (A) Diagram of the plasmid clones used to complement each of the mutants shown in Figure 4. (B) Summary of complementation data. All seven mutants were found to be sensitive (S) to the generalized transducing phage φCr30, and resistant (R) to the pili-specific phages φCbK and φCb5, and pili-less as determined by electron microscopy. Phage sensitivity and piliation were restored by a plasmid carrying the corresponding wild-type allele, whereas the vector alone had no effect (not shown). The number of swarmer cells examined for each strain by electron microscopy is shown in parentheses. Download figure Download PowerPoint Figure 6.Identification of the pilA transcription start site. (A) Sequence of the pilA promoter region. The pilA transcription start site is marked with a +1 and an arrow. Three regions which are protected by CtrA∼P (see Figure 7) are underlined, and CtrA binding motifs are marked in bold. A divergently transcribed gene, orfX, is 316 nt upstream of the predicted PilA methionine. (B) Start site of pilA transcription determined by primer extension. Ten micrograms of total RNA obtained from NA1000 or 10 μg of yeast tRNA were hybridized with the pilinrev2 primer and transcribed with Superscript II at 42°C. A sequence ladder generated with the same primer was used to determine the start site of transcription. The sequencing ladder corresponds to the non-coding strand. The doublet start site indicated with small arrows, and designated in bold, was observed with several different RNA preparations and two additional primers (data not shown). Download figure Download PowerPoint Pilin transcription is activated by the CtrA response regulator and CtrA∼P binds the pilA promoter We examined Caulobacter piliation by electron microscopy using the negative stain uranyl acetate. Consistent with previous work (Sommer and Newton, 1988), we found that 35% of swarmer cells (n = 440) were piliated and that predivisional cells were rarely piliated (1.4%, n = 278). Piliated swarmer cells had 1-7 pili, with an average of 2.6 pili per cell (data not shown). Pili are lost at the swarmer to stalked cell transition and reappear only in the progeny swarmer cell, after cell division (Sommer and Newton, 1988). To understand the mechanism that controls the timing of pilus assembly, we have examined the promoter of the pilA gene and its time of expression during the cell cycle. The start site of pilA transcription was identified by primer extension analysis using a primer located 30 nucleotides downstream of the start codon of the pilA gene (Figure 6A). The start site was localized to two adjacent nucleotides, a G and T (Figure 6B). Inspection of the promoter region revealed a potential CtrA binding site overlaping the −35 region (Figure 6A), a configuration observed in many genes known to be regulated by CtrA (Quon et al., 1996). To test directly whether transcription of the pilA gene is regulated by CtrA, we constructed a pilA promoter fusion to lacZ (PpilA-lacZ). This construct (pJS70) extended from a BamHI site (−230 nt) to just downstream of the ATG start codon (+90 nt). An ORF in the opposite direction to pilA is just upstream of the BamHI site (orfX) so we believe that this fragment contains the entire pilin promoter (Figure 6A). β-galactosidase activity was measured at 28°C in wild-type C.crescentus strain NA1000, and in ctrA401ts (LS2195), a temperature-sensitive lethal allele of ctrA, each carrying pJS70. At the permissive temperature (28°C), ctrA401ts is a partial loss of function mutation (Quon et al., 1996). Promoter activity of PpilA-lacZ in ctrA401ts (882 ± 136 Miller units) is 17% of wild type (5294 ± 1200 Miller units), demonstrating that CtrA is a positive regulator of pilA transcription. Using a similar promoter fragment, we performed DNase I protection analysis using purified CtrA protein, which was phosphorylated in vitro (Reisenauer et al., 1999). We found that CtrA∼P, but not unphosphorylated CtrA, binds to the pilA promoter in three regions (Figure 7), coinciding with four consensus CtrA binding sites (Figure 6A). Taken together, these results demonstrate that CtrA directly regulates pilA transcription. Figure 7.DNase I protection of the pilin promoter with purified CtrA∼P. Purified CtrA was phosphorylated with MBP-EnvZ fusion protein in vitro in a reaction mixture containing ATP. A sequence ladder generated using the pilinrev2 primer was used to identify the protected bases. (A) In the absence of ATP, no protected regions were observed. CtrA concentrations in the footprint reactions are indicated. (B) Three distinct regions of the promoter were protected with CtrA∼P and are marked with a solid black line. These protected regions coincide with CtrA binding motifs in the pilA promoter shown in Figure 6A. Download figure Download PowerPoint Figure 8.Transcription of pilA is cell cycle controlled. (A) Diagram of the PpilA-lacZ fusion construct, pJS70. Wild-type C.crescentus strain NA1000 carrying pJS70 was synchronized and allowed to proceed through the cell cycle. The synthesis of β-galactosidase was monitored by pulse labeling cells with [35S]methionine for 5 min at the times indicated and immunoprecipitating with anti-β-galactosidase antibody. Labeled β-galactosidase was resolved on a 10% SDS-PAGE gel. As a control, the flagellin subunits were immunoprecipitated from the same labeled cell extracts. (B) The β-galactosidase and the 25 kDa flagellin bands were quantitated using a phosphoimager and were normalized to the value of the most intense band in each series. A diagram of the Caulobacter cell cycle is shown. The pattern of pilA transcription coincides with the pattern of 25 kDa flagellin gene transcription and is similar to the timing of pilus assembly observed by electron microscopy. Download figure Download PowerPoint Transcription of the pilA gene occurs only in late predivisional and swarmer cells The cell cycle pattern of pilA transcription was determined in synchronized wild-type cultures containing a PpilA-lacZ fusion on a low copy number plasmid (pJS70). The cells were pulse labeled with [35S]methionine during the cell cycle. Immunoprecipitation with anti-β-galactosidase antibodies was used to determine the timing of pilA transcription (Figure 8A). We compared pilA transcription to the time of expression of the well-characterized flagellin genes (Figure 8B). We found that pilA transcription is cell cycle regulated and its pattern of expression is similar to the timing of pilus assembly determined by electron microscopy. Figure 9.Cell cycle control of a novel cluster of pilus biogenesis genes. (A) The Caulobacter pilus assembly gene cluster is similar to the flp-rcp-tadA region found in the human oral pathogen A.actinomycetemcomitans. The gene order and predicted function of the Caulobacter (Cc) and Actinobacillus (Aa) clusters are conserved. (B) Model for the cell cycle control of Caulobacter pilus assembly. DNA replication and temporally controlled transcription events are shown above a schematic of the cell cycle. Morphological changes are indicated below. CtrA∼P represses the initiation of DNA replication, activates the expression of the Class II flagellar (fla) genes, and late in the cell cycle, activates the transcription of the pilA gene. The genes encoding putative components of the pilus secretion machinery (cpaA-cpaF) are transcriptionally induced before the pilA gene. Download figure Download PowerPoint To determine whether the time of pilA transcription affects the timing of pilus assembly, the pilA gene was expressed throughout the cell cycle by replacing its native promoter with the constitutive Ptac promoter. This Ptac-pilA construct (pJS96), on a high copy plasmid (∼20 copies per cell), was mated into wild-type C.crescentus strain NA1000 and the percentage of piliated swarmers and predivisional cells was determined by electron microscopy (Table I). Constitutive expression of pilA resulted in a higher percentage of swarmer cells with visible pili. In addition, we observed a significant increase in the percentage of piliated predivisional cells, indicating a shift in the timing of pilus assembly to an earlier point in the cell cycle. These results demonstrate that the time of transcription of the pilin subunit gene plays an important role in controlling the time of pilus assembly. Table 1. Constitutive transcription of pilA alters timing of pili formation NA1000+ vector(pJS71) NA1000+ Ptac−pilA (pJS96) Swarmers 38%a 78% Predivisionals 4% 21% Wild-type C.crescentus NA1000 carrying either vector only (pJS71) or Ptac−pilA (pJS96) were examined by electron microscopy and the percentage of piliated swarmer cells and piliated predivisional cells (with a flagellum) was determined. a Percentage piliation (n = 100). Discussion In this paper, we provide both biochemical and genetic data demonstrating that a 4.8 kDa protein (4360 Da predicted) is the major subunit of Caulobacter pili. The pilin subunit, encoded by the pilA gene, has a leader peptide similar to that found in Type IV pilin (see Figure 2D), which is characterized by a net positive charge, cleavage after a glycine residue, and a conserved glutamate at position +5 (Strom and Lory, 1993). The presence of a Type IV-like leader peptide suggests that Caulobacter PilA is processed by a specific prepilin peptidase. The gene downstream of pilA, cpaA, encodes a protein with homology to the C-terminal region of prepilin peptidases. Prepilin peptidase PilD, first identified in P.aeruginosa, cleaves prepilin after a conserved glycine residue and methylates the resulting N-terminal phenylalanine (Nunn and Lory, 1991). Based on our N-terminal sequence data, the N-terminal residue of mature PilA is an alanine. It is known that prepilin peptidase can methylate an alanine residue (Strom and Lory, 1991); however, we do not know whether PilA is methylated. Structure-function studies of P.aeruginosa PilD and the Vibrio cholerae homolog TcpJ suggest that the methylase and peptidase activities reside in separate parts of
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