CELL CYCLE CONTROL OF A HOLDFAST ATTACHMENT GENE IN CAULOBACTER CRESCENTUS

Cell division of the gram-negative bacterium Caulobacter crescentus gives rise to two distinct progeny cells, a sessile cell (containing a stalk) and a motile cell (containing a single polar flagellum) (5). Stalked cells are often found attached to surfaces by means of an adhesive holdfast found at the tip of the stalk. Staining properties and enzyme sensitivity studies indicate that the holdfast is a complex polysaccharide (20), with acidic components such as uronic acids (33). It has been proposed that the appearance of the holdfast is temporally regulated during the cell cycle and that the holdfast first appears at the base of the flagellum in the swarmer pole of the predivisional cell (26). The presence of the holdfast at the tip of the stalk is thought to result from the growth of the stalk at the site previously occupied by the flagellum during the differentiation of swarmer to stalked cells. How the spatial and temporal regulation of holdfast expression is achieved is unclear. A cluster of four genes (hfaABDC) involved in the attachment of the holdfast to the cell was previously identified by Tn5 insertion mutagenesis (16). The exact role of each of these genes is unknown. The C-terminal region of HfaA is similar to the C termini of pilus tip proteins, such as the PapG adhesin from Escherichia coli and the SmfG adhesin from Serratia marcescens (16). These adhesins interact with host cell polysaccharides and with a protein anchor in the pilus, thus mediating the attachment of these bacteria to host cell surfaces. It is possible that HfaA functions analogously in C. crescentus (16). HfaB is similar to proteins that function in transcriptional activation and may activate the transcription of hfaC (16). The sequence of HfaD contains three putative membrane-spanning regions, and it has been suggested that it acts as the membrane-associated protein anchor between HfaA and the cell (17). HfaC is similar to ATP-binding transport-related proteins (17). A sequence identical to the consensus for ς54-dependent promoters was found upstream of the hfaA transcription start site, suggesting that its transcription may be subject to cell cycle regulation like other known ς54 promoters in C. crescentus (16). ς54 is present in both gram-negative and gram-positive members of the Eubacteria and is required for the expression of a wide variety of genes, including those involved in nitrogen fixation, pilus production, dicarboxylic acid transport, and xylene catabolism in these bacteria (21). In C. crescentus, ς54 does not appear to be required for general metabolic functions but is needed for the biosynthesis of two polar organelles, the flagellum and the stalk (6). The hfaA gene was initially characterized in C. crescentus CB2A (16, 23). In this study, we extend this work to investigate the transcriptional regulation of hfaA in strain CB15. We show that hfaA is not transcribed by a ς54 promoter. We find that the transcription of hfaA is cell cycle regulated and that hfaA is maximally expressed in the swarmer compartment of the predivisional cell. The use of fluorescently labeled lectin to detect the holdfast on live cells indicates that neither the flagellar poles of predivisional cells nor swarmer cells possess a holdfast. However, the holdfast was readily detectable at the tips of short stalks. This is consistent with the hypothesis that the holdfast appears at the tips of nascent stalks during the differentiation of swarmer to stalked cells.