Laura Camarena

Response regulator output in bacterial chemotaxis

Chemotaxis responses in Escherichia coli are mediated by the phosphorylated response‐regulator protein P‐CheY. Biochemical and genetic studies have established the mechanisms by which the various components of the chemotaxis system, the membrane receptors and Che proteins function to modulate levels of CheY phosphorylation. Detailed models have been formulated to explain chemotaxis sensing in quantitative terms; however, the models cannot be adequately tested without knowledge of the quantitative relationship between P‐CheY and bacterial swimming behavior. A computerized image analysis system was developed to collect extensive statistics on freeswimming and individual tethered cells. P‐CheY levels were systematically varied by controlled expression of CheY in an E.coli strain lacking the CheY phosphatase, CheZ, and the receptor demethylating enzyme CheB. Tumbling frequency was found to vary with P‐CheY concentration in a weakly sigmoidal fashion (apparent Hill coefficient ∼2.5). This indicates that the high sensitivity of the chemotaxis system is not derived from highly cooperative interactions between P‐CheY and the flagellar motor, but rather depends on nonlinear effects within the chemotaxis signal transduction network. The complex relationship between single flagella rotation and free‐swimming behavior was examined; our results indicate that there is an additional level of information processing associated with interactions between the individual flagella. An allosteric model of the motor switching process is proposed which gives a good fit to the observed switching induced by P‐CheY. Thus the level of intracellular P‐CheY can be estimated from behavior determinations: ∼30% of the intracellular pool of CheY appears to be phosphorylated in fully adapted wild‐type cells.

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.

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 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.

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.

Transcriptional specificity of RpoN1 and RpoN2 involves differential recognition of the promoter sequences and specific interaction with the cognate activator proteins.

The four RpoN factors of Rhodobacter sphaeroides are functionally specialized. In this bacterium, RpoN1 and RpoN2 are specifically required for the transcription of the nitrogen fixation and flagellar genes, respectively. Analysis of the promoter sequences recognized by each of these RpoN proteins revealed some significant differences. To investigate the functional relevance of these differences, the flagellar promoter fliOp was sequentially mutagenized to resemble the nitrogen fixation promoter nifUp. Our results indicate that the promoter sequences recognized by these sigma factors have diverged enough so that particular positions of the promoter sequence are differentially recognized. In this regard, we demonstrate that the identity of the -11-position is critical for promoter discrimination by RpoN1 and RpoN2. Accordingly, purified RpoN proteins with a deletion of Region I, which has been involved in the recognition of the -11-position, did not show differential binding of fliOp and nifUp promoters. Substitution of the flagellar enhancer region located upstream fliOp by the enhancer region of nifUp allowed us to demonstrate that RpoN1 and RpoN2 interact specifically with their respective activator protein. In conclusion, two different molecular mechanisms underlie the transcriptional specialization of these sigma factors.

The Flagellar Muramidase from the Photosynthetic Bacterium Rhodobacter sphaeroides

We have characterized open reading frame RSP0072, which is located within the flgG operon in Rhodobacter sphaeroides. The amino acid sequence analysis of this gene product showed the presence of a soluble lytic transglycosylase domain. The deletion of the N-terminal region (90 amino acids) of the product of RSP0072 yields a leaky nonmotile phenotype, as determined by swarm assays in soft agar. Electron micrographs revealed the lack of flagella in mutant cells. The purified wild-type protein showed lytic activity on extracts of Micrococcus luteus. In contrast, no lytic activity was observed when the residues E57 or E83 were replaced by alanine. Affinity blotting suggests that the protein encoded by RSP0072 interacts with the flagellar rod-scaffolding protein FlgJ, which lacks the muramidase domain present in FlgJ of many bacteria. We propose that the product of RSP0072 is a flagellar muramidase that is exported to the periplasm via the Sec pathway, where it interacts with FlgJ to open a gap in the peptidoglycan layer for the subsequent penetration of the nascent flagellar structure.

Characterization of the flgG operon of Rhodobacter sphaeroides WS8 and its role in flagellum biosynthesis

In this work, we show evidence regarding the functionality of a large cluster of flagellar genes in Rhodobacter sphaeroides. The genes of this cluster, flgGHIJKL and orf-1, are mainly involved in the formation of the basal body, and flgK and flgL encode the hook-associated proteins HAP1 and HAP3. In general, these genes showed a good similarity as compared with those reported for Salmonella enterica. However, flgJ and flgK showed particular features that make them unique among the flagellar sequences already reported. flgJ is only a third of the size reported for flgJ from Salmonella; whereas flgK is about three times larger than any other flgK sequence previously known. Our results indicate that both genes are functional, and their products are essential for flagellar assembly. In contrast, the interruption of orf-1, did not affect motility suggesting that this sequence, if functional, is not indispensable for flagellar assembly. Finally, we present genetic evidence suggesting that the flgGHIJKL genes are expressed as a single transcriptional unit depending on the sigma-54 factor.