The alternative factor 54 is required for the biogenesis of both the flagellum and the stalk in Caulobacter crescentus. The DNA sequence downstream of the 54 gene (rpoN) has been determined, revealing three open reading frames (ORFs) encoding peptides of 203, 208, and 159 amino acids. ORF208 and ORF159 are homologous to ORFs found downstream of rpoN in other microorganisms. The organization of this region in C. crescentus is similar to that in other bacteria, with the exception of an additional ORF, ORF203, immediately downstream from rpoN. There is a single temporally regulated promoter that drives the expression of both rpoN and ORF203. Promoter probe analysis indicates the presence of another promoter downstream from ORF203 which exhibits a temporal control that is different from that of the rpoN promoter. Mutational analysis was used to address the function of the proteins encoded by these three downstream ORFs. The mutations have no effect on the transcription of previously known 54 -dependent flagellar promoters except for a slight effect of an ORF159 mutation on transcription of fljK.The aquatic bacterium Caulobacter crescentus exhibits cellular differentiation as a consequence of temporal and positional expression of genetic information (4). This results in an asymmetric predivisional cell that divides to give a motile swarmer cell and a sessile stalked cell. The asymmetry of the predivisional cell is exemplified by the biosynthesis of two different polar structures, the stalk at the pole previously occupied by the flagellum in the swarmer cell, and subsequently the flagellum at the incipient swarmer pole of the predivisional cell. The biogenesis of both these polar structures requires the factor 54 , encoded by the rpoN gene (5). An rpoN::Tn5 mutant of C. crescentus displays a pleiotrophic phenotype, exhibiting defects in cell division and lacking flagella and stalks.54 is required for the transcription of many of the genes encoding flagellar proteins. However, the role of 54 in stalk biosynthesis and cell division is still unknown.In most gram-negative bacteria where the rpoN region has been sequenced, at least two conserved open reading frames (ORFs) are found downstream of rpoN (25). The conserved genes downstream of rpoN have been implicated in modulating the activity of 54 through an unknown mechanism. In Klebsiella pneumoniae, these ORFs are thought to negatively affect 54 activity, as mutations in either of these ORFs result in an increase in the expression of 54 -dependent nif genes (26). In Pseudomonas aeruginosa, ORF2 mutants display the same nutritional requirements for growth on minimal medium as the rpoN mutant (16). This suggests that ORF2 functions as a coinducer of some 54 -controlled genes. Initial sequencing of regions upstream and downstream of rpoN in C. crescentus indicated the presence of ORFs similar to ones genetically linked to rpoN in other bacteria (5). As rpoN functions as a global regulator of cell differentiation in C. crescentus, we wanted to investigate whether the genes ...

Attachment to surfaces by the prosthecate bacteriumCaulobacter crescentus is mediated by an adhesive organelle, the holdfast, found at the tip of the stalk. Indirect evidence suggested that the holdfast first appears at the swarmer pole of the predivisional cell. We used fluorescently labeled lectin and transmission electron microscopy to detect the holdfast in different cell types. While the holdfast was readily detectable in stalked cells and at the stalked poles of predivisional cells, we were unable to detect the holdfast in swarmer cells or at the flagellated poles of predivisional cells. This suggests that exposure of the holdfast to the outside of the cell occurs during the differentiation of swarmer to stalked cells. To investigate the timing of holdfast synthesis and exposure to the outside of the cell, we have examined the regulation of a holdfast attachment gene, hfaA. The hfaA gene is part of a cluster of four genes (hfaABDC), identified in strain CB2A and involved in attachment of the holdfast to the polar region of the cell. We have identified the hfaA gene in the synchronizable C. crescentus strain CB15. The sequence of the CB2A hfaA promoter suggested that it was regulated by ς54. We show that the transcription of hfaAfrom either strain is not dependent on ς54. Using ahfaA-lacZ fusion, we show that the transcription ofhfaA is temporally regulated during the cell cycle, with maximal expression in late-predivisional cells. This increase in expression is largely due to the preferential transcription ofhfaA in the swarmer pole of the predivisional cell.

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