Georges Dreyfus

A Complete Set of Flagellar Genes Acquired by Horizontal Transfer Coexists with the Endogenous Flagellar System in Rhodobacter sphaeroides

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 sphaeroides has 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 of R. sphaeroides can 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 of R. sphaeroides produces 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 gamma-proteobacterium, while the second flagellar system contains the native genes. Interestingly, other alpha-proteobacteria closely related to R. sphaeroides have also acquired a set of flagellar genes similar to the set found in R. sphaeroides, suggesting that a common ancestor received this gene cluster.

The flagellar hierarchy of Rhodobacter sphaeroides is controlled by the concerted action of two enhancer‐binding proteins

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.

Electrochemical gradient induced displacement of the natural ATPase inhibitor protein from mitochondrial ATPase as detected by antibodies against the inhibitor protein

Summary Antibodies against the natural ATPase inhibitor protein of bovine heart mitochondria (1) block the inhibitory action of the protein on ATP hydrolysis by soluble or particulate F 1 -ATPase. By immunodiffusion tests, inhibitor protein may be detected in Mg-ATP and State 3 submitochondrial particles. The binding of 121 I-labeled antibodies to submitochondrial particles that possess their ATPase in the inhibited state is several times lower than in particles that had been exposed to electrochemical gradients and which show higher rates of ATPase activity. The results indicate that electrochemical gradients induce a change in position of the inhibitor protein in relation to F 1 -ATPase which results in the appearance of the catalytic properties of the enzyme.

The four different σ54 factors of Rhodobacter sphaeroides are not functionally interchangeable

The σ54 factor is highly conserved in a large number of bacterial species. From the complete genome sequence of Rhodobacter sphaeroides, it was possible to identify four different sequences encoding potentially functional σ54 factors. In this work, we provide evidence that one of these copies (rpoN2) is specifically required to express the flagellar genes in this bacterium. A mutant strain carrying a lesion in the rpoN2 gene was unable to swim even though the RpoN1 and RpoN3 proteins were present in the cytoplasm. The possibility that the different copies of the σ54 factor might be specific for the transcription of a particular subset of σ54 promoters was reinforced by the fact that a mutant strain carrying a lesion in rpoN1 showed a severe growth defect in nitrogen‐free culture medium, even though the rpoN2 and rpoN4 genes were actively transcribed from a plasmid or from the chromosome. Different mech‐anisms that might be responsible for this specificity are discussed.

The muramidase EtgA from enteropathogenic Escherichia coli is required for efficient type III secretion

Enteropathogenic Escherichia coli (EPEC) is an important cause of infectious diarrhoea. It colonizes human intestinal epithelial cells by delivering effector proteins into the host cell cytoplasm via a type III secretion system (T3SS) encoded within the chromosomal locus of enterocyte effacement (LEE). The LEE pathogenicity island also encodes a lytic transglycosylase (LT) homologue named EtgA. In the present work we investigated the significance of EtgA function in type III secretion (T3S). Purified recombinant EtgA was found to have peptidoglycan lytic activity in vitro. Consistent with this function, signal peptide processing and bacterial cell fractionation revealed that EtgA is a periplasmic protein. EtgA possesses the conserved glutamate characteristic of the LT family, and we show here that it is essential for enzymic activity. Overproduction of EtgA in EPEC inhibits bacterial growth and induces cell lysis unless the predicted catalytic glutamate is mutated. An etgA mutant is attenuated for T3S, red blood cell haemolysis and EspA filamentation. BfpH, a plasmid-encoded putative LT, was not able to functionally replace EtgA. Overall, our results indicate that the muramidase activity of EtgA is not critical but makes a significant contribution to the efficiency of the T3S process.

The Flagellar Protein FliL Is Essential for Swimming in Rhodobacter sphaeroides

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.

Chemotactic Control of the Two Flagellar Systems of Rhodobacter sphaeroides Is Mediated by Different Sets of CheY and FliM Proteins

Rhodobacter sphaeroides expresses two different flagellar systems, a subpolar flagellum (fla1) and multiple polar flagella (fla2). These structures are encoded by different sets of flagellar genes. The chemotactic control of the subpolar flagellum (fla1) is mediated by three of the six different CheY proteins (CheY6, CheY4, or CheY3). We show evidence that CheY1, CheY2, and CheY5 control the chemotactic behavior mediated by fla2 flagella and that RSP6099 encodes the fla2 FliM protein.

Characterization of the α and β-subunits of the F0F1-ATPase from the alga Polytomella spp., a colorless relative of Chlamydomonas reinhardtii

The isolation and partial characterization of the oligomycin-sensitive F0F1-ATP synthase/ATPase from the colorless alga Polytomella spp. is described. Purification was performed by solubilization with dodecyl-β-d-maltoside followed by Sepharose Hexyl ammonium chromatography, a matrix that interacts with the F1 sector of mitochondrial ATPases. The α-subunit, which migrates on SDS-polyacrylamide gels with an apparent molecular mass of 55 kDa, was identified by the N-terminal sequencing of 47 residues. This subunit exhibited a short extension at its N-terminus highly similar to the one described for the unicellular alga Chlamydomonas reinhardtii (Nurani, G. and Franzen L.-G. (1996) Plant Mol. Biol. 31, 1105–1116). In whole mitochondria, the α-subunit was susceptible to limited proteolytic digestion induced by heat. An endogenous protease removed the first 22 residues of the mature α-subunit. Subunit β was also identified by N-terminal sequencing of 31 residues. This subunit of 63 kDa exhibited a higher apparent molecular mass than α, as judged by its mobility on denaturing polyacrylamide gel electrophoresis. This β-subunit is 7–8 kDa larger than the β-subunits of other mitochondrial ATPases. It is suggested that the β-subunit from Polytomella spp. may have a C-terminal extension similar to that described for the green alga C. reinhardtii (Franzen, L.-G. and Falk, G. (1992) Plant Mol. Biol. 19, 771–780). In addition, it was found that the C-terminal extension of the β-subunit of C. reinhardtii showed homology with the endogenous ATPase inhibitors from various sources and with the ϵ-subunit from the F0F1-ATP synthase from Escherichia coli, which is considered to be a functional homolog of the inhibitor proteins. The data reported here provide the first biochemical evidence for a close relationship between the colorless alga Polytomella spp. and its photosynthetic counterpart C. reinhardtii. It is also suggested that the C-terminal extensions of the β-subunits of the ATP synthases from these algae, may play a regulatory role in these enzymes.

Biochemical Study of Multiple CheY Response Regulators of the Chemotactic Pathway of Rhodobacter sphaeroides

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.

Interaction of FliI, a component of the flagellar export apparatus, with flagellin and hook protein.

FliI is a key component of the flagellar export apparatus in Salmonella typhimurium. It catalyzes the hydrolysis of ATP which is necessary for flagellar assembly. Affinity blotting experiments showed that purified flagellin and hook protein, two flagellar axial proteins, interact specifically with FliI. The interaction of either of the two proteins with FliI, increases the intrinsic ATPase activity. The presence of either flagellin or hook protein stimulates ATPase activity in a specific and reversible manner. A Vmax of 0.12 nmol Pi min-1 microgram-1 and a Km for MgATP of 0.35 mM was determined for the unstimulated FliI; the presence of flagellin increased the Vmax to 0.35 nmol Pi min-1 microgram-1 and the Km for MgATP to 1.1 mM. The stimulation induced by the axial proteins was fully reversible suggesting a direct link between the catalytic activity of FliI and the export process.