Identification and cell cycle control of a novel pilus system in Caulobacter crescentus

Abstract

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.

Fig. 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.

Fig. 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.

Fig. 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).

Fig. 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).

Fig. 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.

Fig. 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).

Fig. 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.

Fig. 8. Transcription of pilA is cell cycle controlled. (A) Diagram of the P_pilA–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.

Fig. 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.