A Family of Six Flagellin Genes Contributes to the Caulobacter crescentus Flagellar Filament

The flagellin gene family.We determined the nucleotide sequences of five of the six flagellin genes in the alpha (fljK and fljL) and beta (fljM,fljN, and fljO) regions using the plasmids described in Table 1. The nucleotide sequence of the sixth gene, fljJ, had been determined previously (11), and errors in this sequence were corrected using nucleotide sequence information produced by The Institute for Genome Research. Each region encodes a set of three independently transcribed flagellin genes. A comparison of these sequences to theC. crescentus genome sequence being produced by The Institute for Genome Research indicated that no additional flagellin genes were present elsewhere in the genome. Thus, C. crescentus has six flagellin genes in two clusters of three genes.

To facilitate comparison of members of the flagellin gene family, the nucleotide sequences of the flagellin genes were aligned. There were 455 variable sites in the 822-bp sequence. Most of the variable sites were found in the fljJ gene, which encodes the 29-kDa flagellin. The most similar pair of genes, fljM andfljN, contained 46 differences (5.6%), indicating that there has been considerable nucleotide divergence among all of the genes. Furthermore, transversions outnumbered transitions 25 to 21, suggesting either that the mutational process was approaching saturation or that selection was occurring. A neighbor-joining tree indicated that the three beta flagellin genes clustered withfljK, suggesting that all four genes encode 25-kDa flagellins (Fig. 1).

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Fig. 1.

Neighbor-joining tree indicating phylogenetic relationships among flagellin genes. The same configuration was obtained by maximum-parsimony analysis. Flagellin gene phylogenies were determined using Test Version 4.0b3a of PAUP written by D. L. Swofford.

A comparison of the derived amino acid sequences of the flagellin genes demonstrated considerable variation among the flagellin proteins, with FljJ differing at approximately half of the amino acid positions (Fig.2). The two most similar proteins, FljM and FljN, differed at 14 (5%) of 273 amino acid positions. A neighbor-joining tree identical to that obtained with the nucleotide sequences was obtained when the amino acid sequences were compared. This result provides further evidence that the three beta flagellin genes encode 25-kDa flagellins. This result is consistent with previous observations that the alpha region deletion mutants SC507 and PC7810 produce 25-kDa flagellins but not the 27- or 29-kDa flagellin (25,34).

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Fig. 2.

Comparison of derived amino acid sequences of the six flagellin genes. Dashes indicate deletions in the amino acid sequence relative to one or more of the other sequences. Dots indicate identity with the reference amino acid sequence.

N-terminal amino acid sequences of purified flagellins.The 25- and 27-kDa flagellins were dissociated from intact flagellar filaments as described by Johnson et al. (16) and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the N-terminal amino acid sequences of these purified flagellins were determined, we found that the sequence of the 27-kDa flagellin was identical to that predicted from the sequence of the fljLgene (Fig. 3). However, the N-terminal amino acid sequence of the 25-kDa flagellin appeared to be a mixture of two proteins, one encoded by the fljK gene and one encoded by the fljM or fljN gene or both. Thus, the flagellar filament contains multiple 25-kDa flagellins in addition to the 27- and 29-kDa flagellins. No evidence was obtained for the presence of the FljO flagellin. However, the fljO promoter is expressed at approximately one-fourth of the combined rate of thefljM and fljN promoters (see Table 2 footnote). Consequently, it is possible that the FljO flagellin was present in the flagellar filament at levels too low to be detected.

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Fig. 3.

N-terminal amino acid sequences of purified flagellin proteins. An amino acid pair, e.g., A/S, indicates that two amino acids were found at the same position, suggesting that the protein sample consists of a mixture of two proteins.

Flagellar filaments containing multiple flagellins have been found in a variety of other eubacterial genera, including Yersinia(18), Vibrio (20, 24),Helicobacter (17), Campylobacter(12), Agrobacterium (4), andRhizobium (1, 27). However, in contrast to that in the C. crescentus flagellar filament, the physical arrangement of these multiple flagellins within the corresponding flagellar filaments is not known. In C. crescentus, the most divergent flagellin, FljJ, is found in small quantities in the region closest to the flagellar hook (6). Thus, FljJ is used to initiate flagellin subunit assembly and then other flagellins are assembled once a short segment is completed. These observations suggest that the divergent FljJ flagellin is a specialized flagellin that is designed to facilitate the first steps of filament assembly and attachment to the hook. Similarly, the moderately diverged FljL flagellin is located between the FljJ flagellin and the 25-kDa flagellins that form the main part of the filament. Thus, it may serve as an adapter between the two disparate flagellins. Mutants that do not produce the FljJ and FljL flagellins assemble a shortened flagellum and exhibit reduced motility (25, 33). Thus, an abnormal flagellar filament is produced in the absence of these two flagellins.

Origin of the 22-kDa flagellin.The origin of the 22-kDa flagellin protein has been a mystery since it was first observed (13). To determine whether the 22-kDa flagellin was derived from one or more of the six flagellin genes, we purified the 22-kDa flagellin from the insoluble red material produced by the SC3845 mutant strain cells as described by Smit et al. (37) and determined its N-terminal amino acid sequence using an automated amino acid sequencer (Beckman Coulter, Fullerton, Calif.). The results were identical to those obtained with the 25-kDa flagellins (Fig. 3), indicating that the 22-kDa flagellin was derived from at least two of the 25-kDa flagellin genes. To confirm this result, we isolated the 22-kDa flagellin from SC3794, a strain that contains a deletion of the alpha region. The N-terminal sequence of this 22-kDa flagellin was identical to that of the FljM and FljN flagellins, and the sequence corresponding to the deleted fljK gene was not present. This result proves that both the fljK gene and at least one of the beta region genes are the source of the 22-kDa flagellins.

Regulation of flagellin gene expression.Previous studies have demonstrated that the fljK and fljL genes are expressed from ς⁵⁴ promoters and that they require both the RpoN protein, encoding ς⁵⁴, and the FlbD activator protein for expression (2, 29). However, inspection of the regions upstream from the beta cluster flagellin genes revealed no sequences that resemble a ς⁵⁴ promoter. This result is consistent with the observation that flagellin gene expression does occur at a low level in rpoN and flbD mutants (26; J. Malakooti and B. Ely, unpublished data). To determine experimentally whether ς⁵⁴ is required for expression of the beta cluster flagellin genes, we cloned each of the promoters in front of a lacZ reporter gene and measured β-galactosidase levels in various genetic backgrounds (3). Expression of the fljMNO genes was only 10 to 20% of that measured with the fljK gene in a wild-type background (Table2). However, when expression in anrpoN mutant background was compared, the fljM,fljN, and fljO promoters were expressed at 20 to 50% of the rate measured for their expression in wild-type cells. In contrast, the fljK and fljL genes were expressed at less than 2% of the rate measured in wild-type cells. Similar results were obtained when flagellin promoter expression was measured in a flbD mutant (Table 2). Thus, neither RpoN nor FlbD is required for fljMNO expression, and expression of these beta region flagellar genes can account for the residual flagellin synthesis observed in rpoN and flbD mutants. As withfljK, expression of the fljMNO promoters was not affected by the presence of other flagellar mutations (Table 2).

Previous studies have demonstrated that flbT mutants overproduce the 25-kDa flagellin proteins (15, 30). The FlbT protein must be involved in flagellin mRNA turnover, since flagellin mRNA is more stable in an flbT mutant strain than in a wild-type strain (23). When expression of thefljM, fljN, and fljO promoters was measured in an flbT mutant background, expression was 45 to 90% of that measured in wild-type cells. Since expression from thefljK and fljL promoters is reduced 10-fold inflbT mutants, the beta cluster flagellin genes are likely to be responsible for most of the overproduction of flagellin observed inflbT mutants.

Nucleotide sequence accession numbers.The flagellin gene nucleotide sequences in Fig. 2 have been assigned GenBank accession numbers AF089835 and AF040268.