FliI is a Salmonella typhimurium protein that is needed for flagellar assembly and may be involved in a specialized protein export pathway that proceeds without signal peptide cleavage. FliI shows extensive sequence similarity to the catalytic beta subunit of the F0F1 ATPase (A. P. Volger, M. Homma, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 173:3564-3572, 1991). It is even more similar to the Spa47 protein of Shigella flexneri (M. M. Venkatesan, J. M. Buysse, and E. V. Oaks, J. Bacteriol. 174:1990-2001, 1992) and the HrpB6 protein of Xanthomonas campestris (S. Fenselau, I. Balbo, and U. Bonas, Mol. Plant-Microbe Interact. 5:390-396, 1992), which are believed to play a role in the export of virulence proteins. Site-directed mutagenesis of residues in FliI that correspond to catalytically important residues in the F1 beta subunit resulted in loss of flagellation, supporting the hypothesis that FliI is an ATPase. FliI was overproduced and purified almost to homogeneity. It demonstrated ATP binding but not hydrolysis. An antibody raised against FliI permitted detection of the protein in wild-type cells and an estimate of about 1,500 subunits per cell. An antibody directed against the F1 beta subunit of Escherichia coli cross-reacted with FliI, confirming that the proteins are structurally related. The relationship between three proteins involved in flagellar assembly (FliI, FlhA, and FliP) and homologs in a variety of virulence systems is discussed.

Bacteria swim in liquid environments by means of a complex rotating structure known as the flagellum. Approximately 40 proteins are required for the assembly and functionality of this structure.Rhodobacter sphaeroideshas two flagellar systems. One of these systems has been shown to be functional and is required for the synthesis of the well-characterized single subpolar flagellum, while the other was found only after the genome sequence of this bacterium was completed. In this work we found that the second flagellar system ofR. sphaeroidescan be expressed and produces a functional flagellum. In many bacteria with two flagellar systems, one is required for swimming, while the other allows movement in denser environments by producing a large number of flagella over the entire cell surface. In contrast, the second flagellar system ofR. sphaeroidesproduces polar flagella that are required for swimming. Expression of the second set of flagellar genes seems to be positively regulated under anaerobic growth conditions. Phylogenic analysis suggests that the flagellar system that was initially characterized was in fact acquired by horizontal transfer from a γ-proteobacterium, while the second flagellar system contains the native genes. Interestingly, other α-proteobacteria closely related toR. sphaeroideshave also acquired a set of flagellar genes similar to the set found inR. sphaeroides, suggesting that a common ancestor received this gene cluster.

Summary: The expression of the bacterial flagellar genes follows a hierarchical pattern. In Rhodobacter sphaeroides the flagellar genes encoding the hook and basal body proteins are expressed from σ⁵⁴‐dependent promoters. This type of promoters is always regulated by transcriptional activators that belong to the family of the enhancer‐binding proteins (EBPs). We searched for possible EBPs in the genome of R. sphaeroides and mutagenized two open reading frames (ORFs) (fleQ and fleT), which are in the vicinity of flagellar genes. The resulting mutants were non‐motile and could only be complemented by the wild‐type copy of the mutagenized gene. Transcriptional fusions showed that all the flagellar σ⁵⁴‐dependent promoters with exception of fleTp, required both transcriptional activators for their expression. Interestingly, transcription of the fleT operon is only dependent on FleQ, and FleT has a negative effect. Both activators were capable of hydrolysing ATP, and were capable of promoting transcription from the flagellar promoters at some extent. Electrophoretic mobility shift assays suggest that only FleQ interacts with DNA whereas FleT improves binding of FleQ to DNA. A four‐tiered flagellar transcriptional hierarchy and a regulatory mechanism based on the intracellular concentration of both activators and differential enhancer affinities are proposed.

In this work we characterize the function of the flagellar protein FliL in Rhodobacter sphaeroides . Our results show that FliL is essential for motility in this bacterium and that in its absence flagellar rotation is highly impaired. A green fluorescent protein (GFP)-FliL fusion forms polar and lateral fluorescent foci that show different spatial dynamics. The presence of these foci is dependent on the expression of the flagellar genes controlled by the master regulator FleQ, suggesting that additional components of the flagellar regulon are required for the proper localization of GFP-FliL. Eight independent pseudorevertants were isolated from the fliL mutant strain. In each of these strains a single nucleotide change in motB was identified. The eight mutations affected only three residues located on the periplasmic side of MotB. Swimming of the suppressor mutants was not affected by the presence of the wild-type fliL allele. Pulldown and yeast two-hybrid assays showed that that the periplasmic domain of FliL is able to interact with itself but not with the periplasmic domain of MotB. From these results we propose that FliL could participate in the coupling of MotB with the flagellar rotor in an indirect fashion.

The six copies of the response regulator CheY from Rhodobacter sphaeroides bind to the switch protein FliM. Phosphorylation by acetyl phosphate (AcP) was detected by tryptophan fluorescence quenching in three of the four CheYs that contain this residue. Autophosphorylation with Ac ³² P was observed in five CheY proteins. We also show that all of the cheY genes are expressed simultaneously; therefore, in vivo all of the CheY proteins could bind to FliM to control the chemotactic response. Consequently, we hypothesize that in this complex chemotactic system, the binding of some CheY proteins to FliM, does not necessarily imply switching of the flagellar motor.