A complete set of flagellar genes acquired by horizontal transfer coexists with the endogenous flagellar system in Rhodobacter sphaeroides

Abstract

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.

FIG. 1.

Western blot analysis with anti-FlgE2 antibody of cell lysates of various R. sphaeroides strains. (A) Heterotrophic growth conditions. Lane 1, LC1/pRKflgE2; lane 2, WS8; lane 3, LC1. (B) Photoheterotrophic growth conditions. Lane 4, WS8 (OD₆₀₀, 0.5); lane 5, SP18 (OD₆₀₀, 0.5); lane 6, SP18 fla2+ (OD₆₀₀, 0.5); lane 7, SP18 fla2+ (OD₆₀₀, 1).

FIG. 2.

Cluster and operon arrangement of fla2 genes of R. sphaeroides. The arrows represent genes and their directions of transcription. When possible, gene designations were assigned on the basis of similarity with a gene having a known function. The lines and colors show the possible operon organization, using the criterion of a maximal distance of 30 bp between stop and initiation codons of two contiguous genes. Chromosome and plasmid localizations of the fla2 genes are indicated at the bottom.

FIG. 3.

Swimming patterns of various R. sphaeroides strains in stab tubes with 0.25% agar. The tubes were incubated with constant illumination for 36 h (A), 3 days (B), 7 days (C), and 10 days (D). The strains used were WS8 (wild type), SS1 (flhA2::uidA-aadA), SP18 (flgC1::kan), and SS2 (flgC1::kan flhA2::uidA-aadA).

FIG. 4.

Electron microscopy images of the two different types of flagella assembled by R. sphaeroides. (A) Representative hook-filament structure found in the WS8 strain. (B) Representative hook-filament structure found in the SP18 fla2+ strain. (C) Intertwined filaments frequently found in preparations of the SP18 fla2+ strain. (D) Representative strain WS8 cell showing a subpolar flagellum. (E) Representative strain SP18 fla2+ cell showing several polar flagella. The bars in panels A and B indicate the hook.

FIG. 5.

In vivo fluorescent immunodetection of the hook protein (FlgE2). (A and C) Strain SP18 fla2+ incubated with anti-FlgE2 and fluorescent anti-mouse antibodies. (B and D) SP18 fla2+ cells incubated with only the fluorescent anti-mouse antibody. (A and B) Sections of the fields of view from composite images of fluorescent and DIC images. (C and D) Representative cells from (from top to bottom) the DIC image, the fluorescent image, and the composite of the DIC and fluorescent images.

FIG. 6.

Maximum likelihood species and flagellar gene phylogenies. (A) Phylogram inferred from the concatenated FliG, FliP, FlhA, FlhB, and FliF flagellar protein alignments for 13 α-proteobacteria, 9 β-proteobacteria, and 11 γ-proteobacteria, resulting in a supermatrix of 2,122 residues after removal of gapped sites (for FlhA, residues 1 to 688; for FlhB, residues 689 to 1044; for FliF, residues 1045 to 1553; for FliG, residues 1554 to 1883; for FliP, residues 1884 to 2122). (B) Hypothetical phylogenetic relationships of species inferred from concatenation of 10 core proteins (for CarB, residues 1 to 284; for FtsH, residues 285 to 876; for UvrA, residues 877 to 1808; for NusA, residues 1809 to 2293; for DnaB, residues 2294 to 2737; for RplC, residues 2738 to 2935; for RplF, residues 2936 to 3108; for Lgt, residues 3109 to 3348; for PhoB, residues 3349 to 3572; for ClpP, residues 3573 to 3766) that resulted in a supermatrix of 3,766 aligned residues after removal of gapped sites. Most bipartitions in these phylogenies had >95% bootstrap support (based on 100 pseudoreplicates); the exceptions were the bipartitions whose bootstrap values are indicated.