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

Abstract

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.Many bacteria move by using flagella as the locomotive organelle. Flagella alternate between clockwise and counterclockwise rotation or in some cases between rotation and brief stop periods, which allow the bacterial cell to swim in a linear trajectory or to reorient (1,14,15). Bacterial taxis is achieved by modifying the frequency of reorientation events. The bias in the frequency of reorientation is modified by the interaction of the phosphorylated form of the response regulator CheY-P with the switch protein (FliM), which is part of the motor-switch structure located at the base of the flagellum. The concentration of CheY-P is regulated by the chemotactic system (3).A limited number of bacteria possess dual flagellar systems and are able to express two different flagellum types: a polar flagellum for swimming and lateral flagella for swarming; these microorganisms include Vibrio parahaemolyticus (27), Vibrio alginolyticus (8), Aeromonas spp. (26), Azospirillum brasilense (18), Rhodospirillum centenum (17), Helicobacter mustelae (19), and Plesiomonas shigelloides (7). The best-characterized examples are those that belong to the Vibrio genus. The ability to synthesize two different types of flagella allows these microorganisms to populate various niches (16).Rhodobacter sphaeroides is a facultative nonsulfur photosynthetic bacterium. Its genome sequence revealed many intriguing features related to motility and chemotaxis (13), including the presence of two flagellar gene sets. The genes that belong to the first set (fla1) are expressed constitutively and allow the strong swimming previously reported for the WS8 wild-type strain (1,10,21,22). The genes of the second flagellar set are not expressed in the wild-type WS8 strain; however, strains that express it can be isolated. In contrast with the case for other dual-flagellum systems, the fla2 genes of R. sphaeroides produce polar flagella that allow swimming (21).Several chemotactic genes are also reiterated (two cheB genes, three cheR genes, four cheA and cheW genes, and six cheY genes). In spite of this, only some of these gene copies are required when the cell is swimming with the fla1 flagellum. For example, it has been reported that only CheY6 and either CheY3 or CheY4 are required for chemotaxis mediated by the fla1 flagellum (23), despite the fact that the six cheY genes are expressed (6). The presence of a second functional flagellum in R. sphaeroides suggests that some of the chemotactic genes could be involved in its tactic control. To test this hypothesis, we investigated...

The Master Regulators of the Fla1 and Fla2 Flagella of Rhodobacter sphaeroides Control the Expression of Their Cognate CheY Proteins

“…The expression of three chemotaxis proteins encoded in cheOp1 (Fig. 1) was evaluated by Western blotting using total cell extracts obtained from AM1 (25) and its derivative, EA1 (ΔctrA::aadA) (21). The proteins CheY2 and CheY5 were detected in AM1 but not in the absence of ctrA ( Fig.…”

“…WS8N is the wild-type strain that constitutively expresses the Fla1 flagellum but is unable to express the fla2 genes given that the two-component system CckA/ChpT/CtrA is inactive. The AM1 strain is a WS8N derivative that is unable to express the fla1 genes, given that it carries a mutation in the flagellar master activator fleQ and also carries a point mutation in cckA that activates the CckA/ChpT/CtrA two-component system (21,25); therefore, this strain has a Fla1 Ϫ Fla2 ϩ phenotype. cheOp1 expression is dependent on the master regulator of the fla2 genes.…”

In Rhodobacter sphaeroides, Chemotactic Operon 1 Regulates Rotation of the Flagellar System 2

“…This result is consistent with the idea that the proteins encoded by cheOp 1 control the chemotactic response of Fla2. The same mutations in the genetic background where only the Fla1 flagellum is expressed have no significant effect on chemotaxis, as reported previously (data not shown) (11,15,24,25,33,50). In order to demonstrate that the observed phenotypes in the Fla1 Ϫ Fla2 ϩ background were due only to the absence of each of the cheOp 1 genes, we carried out a complementation test introducing the wild-type gene cloned into pRK415 (19).…”

“…2A) contains 11 genes, including one unassigned open reading frame and genes encoding the following putative proteins: three response regulators, one sensor kinase, one methyltransferase, one adaptor protein, two membrane chemoreceptors, one cytoplasmic chemoreceptor, and one receptor deamidase. We mutated each gene, with the exception of the cheY genes, which were mutated previously (11). Mutants were generated by the amplification of the desired gene followed by the insertion of a nonpolar Spc r cassette (11).…”

“…Cells expressing Fla2 flagella form a ring in swimming plates containing different organic acids. This chemotactic response is mediated only by CheY1, CheY2, and CheY5 (11). Given that these proteins are encoded by cheOp 1 , we hypothesized that this locus must control Fla2 flagella.…”