The Hook Gene ( flgE ) Is Expressed from the flgBCDEF Operon in Rhodobacter sphaeroides : Study of an flgE Mutant

Abstract

In this work we identified the flgE gene encoding the flagellar hook protein from Rhodobacter sphaeroides. Our results show that this gene is part of a flagellar cluster that includes the genes flgB, flgC, flgD, flgE, and flgF. Two different types of mutants in the flgE gene were isolated, and both showed a Fla ؊ phenotype, indicating the functionality of this sequence. Complementation studies of these mutant strains suggest that flgE is included in a single transcriptional unit that starts in flgB and ends in flgF. In agreement with this possibility, a specific transcript of approximately 3.5 kb was identified by Northern blot. This mRNA is large enough to represent the complete flgBCDEF operon. FlgE showed a relatively high proline content; in particular, a region of 12 amino acids near the N terminus, in which four prolines were identified. Cells expressing a mutant FlgE protein lacking this region showed abnormal swimming behavior, and their hooks were curved. These results suggest that this region is involved in the characteristic quaternary structure of the hook of R. sphaeroides and also imply that a straight hook, or perhaps the rigidity associated with this feature, is important for an efficient swimming behavior in this bacterium.Salmonella enterica serovar Typhimurium swims toward favorable environments in response to changes in the surrounding medium using its flagella; these appendages consist basically of a helical filament driven by a rotary motor. When flagella rotate in counterclockwise direction, the filaments coalesce in a bundle that functions as a propeller to push the bacterial cell body in a linear trajectory. On the contrary, when flagella reverse the sense of rotation the bundle is no longer stable, and the uncoordinated movement of each flagellum causes the cell to tumble. As revealed by early studies of electron microscopy, the flagellum consists of a filament, a curved hook, and a basal body (6). The filament and the hook are each composed of repeats of a single protein, flagellin and hook protein, respectively. These polypeptides do not share extensive similarity at the level of its primary sequence but both have the ability to self-assemble, and the resulting structures are capable of displaying polymorphic transitions; this capability has been suggested to be important in the motility of certain species of bacteria (12,16,22).The structure of the filament has been the subject of extensive study during the last few decades, and various structural models have been proposed (18,21,33,37). In contrast, the hook structure has been less well characterized; however, since the hook protein shares important features with flagellin, it has been suggested that hook and flagellin subunits have a similar folding pattern (20,34,35,36).The detailed knowledge about the structure and function of the flagellum in enterobacteria contrasts strongly with the limited data on these aspects that exist for other bacterial groups. However, it seems clear that as far as structure and function are concerned, ...

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

“…Motility was not observed for the transconjugant cells when they were examined in swarming plates containing 0.25% agar or directly with the light microscope (data not shown). In agreement with previous observations, the motility the LC1 strain was recovered when a plasmid expressing the flgE1 gene was introduced into this strain (4). The FlgE2 protein expressed in the LC1 strain carrying the pRKflgE2 plasmid was detected by Western blotting (Fig.…”

“…To generate the flgE2::aadA allele, we amplified the flgE2 gene with oligonucleotides flgE2F (5Ј-CCGATGGCGGTGCGAAACAAC-3Ј) and flgE2R (5Ј-GGTCGCATTCA TCTGCTGCAC-3Ј) using chromosomal DNA from WS8. The 2,080-bp PCR product was cloned in pTZ19R; the aadA gene was amplified by PCR as described previously (4), and the product was cloned in the SalI site of the flgE2 gene. The resultant allele, flgE2::aadA, was subcloned into pJQ200mp18 and introduced into SP18 fla2ϩ by conjugation.…”

A molecular brake, not a clutch, stops the Rhodobacter sphaeroides flagellar motor

“…This is consistent with electron micrographs showing that the R. sphaeroides hook is straight, not curved like E. coli (27). Recent models of the flagellar motor have proposed elastic links between stators and the cell wall, each with a stiffness of approximately 200 pNnm/rad 2 (24,28).…”

Posttranscriptional Control of the Salmonella enterica Flagellar Hook Protein FlgE

“…The finding that FlgE was posttranscriptionally regulated in response to flagellar assembly led us to examine a possible role of mRNA processing in the expression of the 9.9-kbp class 2 flgBCDEFGHIJKL operon. Also, cleavage of the flgBCDEF GHIJ operon has been implicated in posttranscriptional regulation of the flgE gene in Rhodobacter sphaeroides (4,18).…”

“…The stoichiometry of the FlgE protein is 5 to 20 times higher than that of other flgB-L operon proteins. Northern blot analysis of Rhodobacter sphaeroides flgE suggested a role for mRNA processing in the regulation of flgB-J operon expression and identified two different flgE-containing transcripts (4).…”