INTRODUCTION

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The flagellum is the structure responsible for the motility of many bacteria. Some of its structural features include the basal body, the hook, and the helical filament. In Salmonella enterica serovar Typhimurium, biosynthesis of the flagellum depends on the expression of more than 40 genes. The products of these genes are required for several flagellar processes, including assembly, export, and transcriptional control (for recent reviews, see references 1 and 16).

The expression of these genes follows a hierarchical pattern that is highly regulated. At the top of the hierarchy is the flhDC operon, encoding two proteins which form the heterotetrameric positive transcriptional regulator of the class II genes. Global regulators, such as the cyclic AMP receptor protein, DnaA, and the nucleoid-associated protein H-NS, influence the expression level of this operon and consequently the formation of flagella (18, 21, 23). The expression of class II genes is dependent on the RNA polymerase-⁷⁰ holoenzyme (E⁷⁰) and FlhD-FlhC. Proteins involved in the formation of the hook and the basal body complex (HBB), as well as the regulatory proteins FlgM and FliA, belong to this class (11). FliA is a specific sigma factor (²⁸) required for the expression of class III genes, while FlgM is an anti-sigma factor that inhibits FliA activity. The release of FliA from the inhibitory action of FlgM occurs when the HBB structure is completed, allowing FlgM export out of the cell. FliA is then free to associate with the RNA polymerase core enzyme in order to transcribe class III genes (10, 15).

The flagellar genetic system of Rhodobacter sphaeroides is poorly understood. Detailed analyses of some structural components of the flagellum have been described, but nothing is known about the factors that regulate gene expression. Recently, genetic evidence has suggested the location of functional flagellar promoters in this organism. Complementation studies have indicated the presence of promoters at the fliN-fliO intercistronic region (7), upstream of the flgBCDEF operon (T. Ballado, L. Camarena, B. González-Pedrajo, E. Silva-Herzog, and G. Dreyfus, unpublished data) and upstream of motA (6). However, no physical evidence supporting these results has been reported.

In this work we show evidence that a ⁵⁴ promoter is located at the fliN-fliO intercistronic region and is responsible for the transcription of the fliOPQR flhB operon. In addition, primer extension experiments revealed transcription start sites upstream of flgB and fliK. In these two cases, a sequence similar to that of the ⁵⁴ promoter was also identified a few base pairs upstream of the transcription start sites. These results indicate that E⁵⁴ is responsible for the expression of genes encoding structural components of the flagellar export apparatus, the motor, the hook, and the basal body proteins. We also determined that mutations in the fliM, fliK, and fliR genes did not affect the expression of other flagellar genes dependent on ⁵⁴. In contrast, fliC mRNA was reduced in fliM or flgE strains. These results allow us to propose a regulatory hierarchy controlling the expression of the flagellar genes in R. sphaeroides.

	MATERIALS AND METHODS

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Bacterial strains and growth conditions. R. sphaeroides cells were grown in Sistrom's succinate-basal salt medium at 30°C (20). Heterotrophic growth conditions were achieved by growing 10-ml cultures in 250-ml Erlenmeyer flasks with strong shaking (300 rpm) in the dark. Phototrophic conditions were achieved by growing cultures in completely filled screw-cap tubes under continuous illumination. Cultures were harvested at an optical density at 600 nm of 0.5 ± 0.05 (mean ± standard deviation). When required, spectinomycin (15 µg/ml), kanamycin (25 µg/ml), or tetracycline (1 µg/ml) was added to the culture medium. Escherichia coli strains were grown aerobically at 37°C on Luria-Bertani medium. Antibiotics were added at the following concentrations: ampicillin, 100 µg/ml; tetracycline, 10 µg/ml; and kanamycin, 50 µg/ml.

Recombinant DNA techniques. Routine genetic manipulations were performed as described elsewhere (2). Restriction enzymes, alkaline phosphatase, T4 ligase, and T4 polynucleotide kinase were purchased from GIBCO-BRL. Plasmid DNA was isolated from E. coli using Qiagen columns and procedures. Sequencing was carried out using a Thermosequenase kit (Amersham) on single-stranded DNA.

Conjugal mating. Plasmid DNA was mobilized into R. sphaeroides cells by conjugation according to procedures previously reported (5).

Site-directed mutagenesis. Site-directed mutagenesis was performed according to the method of Kunkel (14) with a uracil-containing single-stranded DNA as the template. The oligonucleotides used for mutagenesis were 5'-CTGCAACATCCGTGACGCCCGCCCGCG-3', 5'-CTGCAACATCCGTGCTGCCCGCCCGCG-3', 5'-GTCCCCCTCCGCTACAACATCCGTGCCG-3', and 5'-GTCCCCCTCCGCAACAACATCCGTGCCG-3'.

RNA isolation and Northern blot analysis. Total RNA was isolated from heterotrophic cultures as described previously (24). For Northern blotting, 20 µg of each RNA sample was separated electrophoretically on agarose-formaldehyde gels and transferred by capillary action onto nylon membranes with a pore size of 0.45 µm. Filter hybridizations were performed as described previously (2). The DNA probe used was a 1.1-kb PstI-BglII fragment from fliC, labeled with [-³²P]dCTP by nick translation.

Primer extension analysis. Reactions were performed as described previously (2). Total RNA (50 µg for fliO and flgB reactions and 100 µg for fliK reactions) was annealed with a specific primer at 42°C in the presence of 50% formamide. Oligonucleotides used as primers for cDNA synthesis were 5' end labeled with T4 polynucleotide kinase and 20 µCi of [-³²P]ATP at 37°C for 30 min. Unincorporated nucleotides were removed by chromatography. The primer elongation reactions were carried out with avian myeloblastosis virus reverse transcriptase (Promega). Unlabeled primers were used to generate a nucleotide sequence ladder.

-Glucuronidase activity assay.

	RESULTS

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Transcriptional organization of the flagellar cluster fliHIJKLMNOPQR flhB. A large flagellar cluster was previously identified which contained 12 flagellar genes from fliH to fliR and flhB (3, 7). The transcriptional organization of this region was inferred from a complementation study on a strain carrying a polar insertion in the fliM gene (fliM::uidA-aadA). This study indicated the presence of a promoter downstream of fliN (7).

View this table: [in this window] [in a new window]   TABLE 1.   -Glucuronidase specific activities expressed from chromosomal 'uidA fusions to fli promoters

Deletion mapping of a transcriptionally active flagellar region. To identify the promoter controlling transcription of fliR, deletion analysis of a 4.4-kb EcoRI fragment carrying the fliR::uidA-aad allele was performed. This fragment, which incorporates sequences from the middle of fliM to the middle of flhB, was cloned into the pRK415 vector in an orientation opposite to that of the pRK415 promoters.

View larger version (10K): [in this window] [in a new window]   FIG. 1.   -Glucuronidase activity from fliOp-uidA fusions. The upper part shows some of the relevant restriction sites within the 4.6-kb EcoRI fragment. Arrows indicate the direction of the uidA coding region. Broken arrows indicate the direction of the pRK415 promoters. Abbreviations for restriction sites: E, EcoRI; Sc, SacII; Ss, SstI; Sa, SalI; Bg, BglII. -Glucuronidase activities are given as indicated in Table 1. GUS, -glucuronidase; ND, not determined.

Site-directed mutagenesis of the ⁵⁴ consensus region. It is known that the most conserved regions of the promoters recognized by E⁵⁴ are the dinucleotides GG and GC located near positions 24 and 12 upstream from the transcription start site. Changes in these bases interfere with the ability of ⁵⁴ to promote transcription (4).

View larger version (13K): [in this window] [in a new window]   FIG. 2.   -Glucuronidase expression of wild-type and mutated fliOp promoters. The dark box represents the 54 promoter sequence. The conserved dinucleotides GG and GC are shown in bold. Specific changes made in each construct are shown in the left column. -Glucuronidase activities are given as indicated in Table 1. GUS; -glucuronidase; WT, wild type.

5' end mapping of the flagellar mRNA dependent on ⁵⁴ promoters. Putative ⁵⁴ promoter sequences have been identified upstream of the flagellar genes flgB, fliK, and motA (GenBank accession number U86454) (6; Ballado et al., unpublished). The nucleotide sequence of each of these regions was aligned with the sequence of the functional ⁵⁴ promoter in the fliN-fliO intercistronic region. The nucleotide residues GGCA and TTGC are present in all sequences (data not shown). In order to obtain evidence that these sequences correspond to functional flagellar promoters, we examined if specific mRNA transcripts were produced from these putative promoters.

View larger version (33K): [in this window] [in a new window]   FIG. 3.   Determination of the fliO, flgB, and fliK transcriptional start sites by reverse transcriptase-mediated primer extension. Extension analysis carried out with total RNA isolated from WS8 wild-type cells is indicated as "chr." The assay carried out with total RNA from WS8 cells transformed with the pRK415 plasmid containing the putative 54 promoter cloned in opposite orientation to the vector promoters is indicated as "pl." Nucleotide sequences upstream of the observed transcriptional start sites are shown. The asterisk indicates the nucleotide proposed to be the transcriptional start site. The conserved regions including the 24 and 12 positions are underlined.

Expression of the flagellar fliO promoter is not dependent on growth conditions. It has been shown for several bacterial groups that flagellar synthesis is affected by environmental conditions (9, 13). In the case of R. sphaeroides, flagellar control signals might be related to the cell cycle and possibly independent of environmental conditions. To determine if growth conditions affect the expression of fliOp, WS8 cells carrying the fusion fliOp-'uidA in pRK415 were grown heterotrophically or photoheterotrophically. No significant difference in the level of -glucuronidase activity was observed, suggesting that the expression of some of the components involved in flagellar synthesis is not significantly affected by these growth conditions (Fig. 1). Since our results are focused on only two extreme conditions, i.e., light and oxygen, it remains to be investigated if other environmental conditions modify the expression level of the flagellar promoters.

Is flagellar gene expression in R. sphaeroides hierarchical? In enteric bacteria, the best-characterized checkpoint in flagellar assembly is controlled by the protein FlgM (10, 15). To test if this checkpoint exists in R. sphaeroides, we investigated whether a functional HBB structure was necessary for flagellin gene expression.

View larger version (14K): [in this window] [in a new window]   FIG. 4.   Northern blot analysis of fliC transcripts. Total RNA extracted from WS8 wild-type cells (lane 1), NG1 cells (fliM::uidA-aadA) (lane 2), and LC1 cells (flgE::aadA) (lane 3) was probed with a 32P-labeled fliC fragment as described in Materials and Methods. The nucleotide sequence of the 5' end of the fliC gene, including its control region, is shown (accession number AF274346). The sequence resembling the 28 promoter, the putative start codon of fliC, and the ribosomal binding site are shown in bold.

	DISCUSSION

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Structural components of the flagellum of R. sphaeroides have been recently characterized (3, 6, 7, 8, 19; Ballado et al., unpublished). However, still nothing is known about the factors that regulate the expression of the genes involved in motility. In this work, we identified several promoters responsible for the transcription of the flagellar genes involved in the formation of the basal body, the switch, and the hook.

This work showed that fliOp is a functional ⁵⁴ promoter. Our results support the idea that this promoter is responsible for the expression of fliO, fliP, fliQ, fliR, and probably flhB. Additional studies are in progress to define whether this operon includes flhB and beyond.

A sequence similar to that of the ⁵⁴ consensus promoter was identified upstream of the fliK gene. The functionality of this promoter sequence was supported by the identification of the fliK 5'-end mRNA, the start site of which is within the expected range for a functional ⁵⁴ promoter (Fig. 3). On the other hand, the polar effect exerted by the mutation fliK::TnphoA over fliM expression suggests that fliK and fliM belong to the same operon (Table 1). Together, these results support the idea that the ⁵⁴ promoter located upstream of fliK is responsible for transcribing the fliK, fliL, fliM, and fliN genes as a single transcriptional unit. The inclusion of the fliN gene in this operon was previously proposed from a complementation study of a strain carrying the fliM::uidA-aadA mutation (7).

A third transcriptional start site defined in this work was located upstream of flgB. A sequence similar to that of the ⁵⁴ consensus promoter is at a proper distance from the flgB mRNA initiation site. Consequently, we suggest that this sequence is the functional ⁵⁴ promoter responsible for the expression of the flgB, flgC, flgD, flgE, and flgF genes. The internal organization of this region has been recently elucidated, and genetic evidence supports the idea that these genes form an operon (Ballado et al., unpublished).

The data presented here allow us to propose that in R. sphaeroides the expression of the flagellar genes, whose products are involved in the assembly of the basal body, the switch, and the hook structures, is dependent on E⁵⁴. This is in contrast with the situation found in both S. enterica serovar Typhimurium and E. coli, where the flagellar genes involved in the synthesis of these structures are dependent on E⁷⁰ and the FlhD-FlhC activator complex. At a glance, R. sphaeroides flagellar gene expression would appear to be similar to that observed in Caulobacter crescentus, where the genes involved in the formation of the rod and the hook are dependent on E⁵⁴ (22). However, several of the genes reported here as being dependent on ⁵⁴ are dependent on E⁷³ in C. crescentus. Moreover, two flagellin genes of C. crescentus are dependent on E⁵⁴ (22), whereas the regulatory region of fliC of R. sphaeroides shows a sequence resembling that of a ²⁸ promoter, as occurs in enteric bacteria (E. coli and Salmonella). Therefore, the flagellar genetic system of R. sphaeroides seems to combine features from these two systems.

As mentioned before, in S. enterica serovar Typhimurium the flagellar genes follow a hierarchical order of expression; therefore, the transcription of the genes placed at a low level of the hierarchy is dependent on the expression of the genes at a higher level. In this regard, in R. sphaeroides the activity of fliOp and the expression of the fliM gene, both of which were tested as the activity of the 'uidA reporter gene, have been shown to be insensitive to the presence of other mutations in flagellar genes, such as fliR, fliM, and flgE (this work and data not shown). Therefore, we suggest that all these genes belong to the same transcriptional class and may be assigned to class II. Interestingly, genetic evidence has suggested that the motA gene may be dependent on E⁵⁴ (6). Therefore, motA could be expressed simultaneously with the genes encoding the structural components of the basal body and the hook. Additional studies are required to define whether the mot genes, together with the fli and the flg genes, can be grouped within the same transcriptional class.

According to current knowledge of the ⁵⁴ factor (17), the existence of an activator protein must be considered. This protein may represent the flagellar class I in R. sphaeroides. Although its identity remains to be elucidated, it could be suggested that this protein would belong to the family of ⁵⁴-dependent activator proteins.

Finally, the class III genes could be transcribed by E²⁸, as seems to be the case for the fliC gene (Fig. 4). The reduction in the amount of the fliC mRNA observed in the flgE and fliM strains may represent the control that class II genes exert over the expression of ²⁸-dependent genes. This negative control does not seem to be absolute and allows a certain level of fliC expression even in the absence of a functional HBB structure. Supporting the idea that class III genes are expressed from E²⁸ promoters, we have found that fliD, which is expected to belong to this class, also shows a ²⁸ consensus promoter a few base pairs upstream of the putative initiation codon (data not shown).

In summary, our results support the idea that in R. sphaeroides the expression of the flagellar genes is controlled according to a hierarchical pattern. Class II genes are dependent on E⁵⁴, whereas class III genes seem to be dependent on E²⁸. The control that class II genes exert over the expression of class III genes does not seem to be tight, and the molecular bases underlying this weak regulation are under investigation in our laboratory.