sigma(54) Promoters control expression of genes encoding the hook and basal body complex in Rhodobacter sphaeroides

Abstract

Gene expression of the flagellar system is tightly controlled by external stimuli or intracellular signals. A general picture of this regulation has been obtained from studies of Salmonella enterica serovar Typhimurium. However, these regulatory mechanisms do not apply to all bacterial groups. In this study, we have investigated regulation of the flagellar genetic system in Rhodobacter sphaeroides. Deletion analysis, site-directed mutagenesis, and 5'-end mapping were conducted in order to identify the fliO promoter. Our results indicate that this promoter is recognized by the factor sigma(54). Additionally, 5'-end mapping of the flgB and fliK transcripts suggests that these mRNAs are also transcribed from sigma(54) promoters. Finally, we showed evidence that suggests that fliC transcription is not entirely dependent on the presence of a complete basal body-hook structure. Our results are discussed in the context of a possible regulatory hierarchy controlling flagellar gene expression in R. sphaeroides.

FIG. 1

β-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. β-Glucuronidase activities are given as indicated in Table 1. GUS, β-glucuronidase; ND, not determined.

FIG. 2

β-Glucuronidase expression of wild-type and mutated fliOp promoters. The dark box represents the ς⁵⁴ promoter sequence. The conserved dinucleotides GG and GC are shown in bold. Specific changes made in each construct are shown in the left column. β-Glucuronidase activities are given as indicated in Table 1. GUS; β-glucuronidase; WT, wild type.

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 ς⁵⁴ 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.

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 ς²⁸ promoter, the putative start codon of fliC, and the ribosomal binding site are shown in bold.