Transcriptional Control of the Lateral-Flagellar Genes of Bradyrhizobium diazoefficiens

Abstract

Bradyrhizobium diazoefficiens, a soybean N₂-fixing symbiont, possesses a dual flagellar system comprising a constitutive subpolar flagellum and inducible lateral flagella. Here, we analyzed the genomic organization and biosynthetic regulation of the lateral-flagellar genes. We found that these genes are located in a single genomic cluster, organized in two monocistronic transcriptional units and three operons, one possibly containing an internal transcription start site. Among the monocistronic units is blr6846, homologous to the class IB master regulators of flagellum synthesis in Brucella melitensis and Ensifer meliloti and required for the expression of all the lateral-flagellar genes except lafA2, whose locus encodes a single lateral flagellin. We therefore named blr6846 lafR (lateral-flagellar regulator). Despite its similarity to two-component response regulators and its possession of a phosphorylatable Asp residue, lafR behaved as an orphan response regulator by not requiring phosphorylation at this site. Among the genes induced by lafR is flbT_L , a class III regulator. We observed different requirements for FlbT_L in the synthesis of each flagellin subunit. Although the accumulation of lafA1, but not lafA2, transcripts required FlbT_L, the production of both flagellin polypeptides required FlbT_L Moreover, the regulation cascade of this lateral-flagellar regulon appeared to be not as strictly ordered as those found in other bacterial species.IMPORTANCE Bacterial motility seems essential for the free-living style in the environment, and therefore these microorganisms allocate a great deal of their energetic resources to the biosynthesis and functioning of flagella. Despite energetic costs, some bacterial species possess dual flagellar systems, one of which is a primary system normally polar or subpolar, and the other is a secondary, lateral system that is produced only under special circumstances. Bradyrhizobium diazoefficiens, an N₂-fixing symbiont of soybean plants, possesses dual flagellar systems, including the lateral system that contributes to swimming in wet soil and competition for nodulation and is expressed under high energy availability, as well as under requirement for high torque by the flagella. The structural organization and transcriptional regulation of the 41 genes that comprise this secondary flagellar system seem adapted to adjust bacterial energy expenditures for motility to the soil's environmental dynamics.

Keywords: Bradyrhizobium; FlbT; LafR; expression; flagella; flbT; lafR.

FIG 1

Control of flagellin expression and motility by lafR in bacteria grown in liquid HMY with arabinose (Ara) or mannitol (Man) as a carbon source. (A) SDS-PAGE of extracellular B. diazoefficiens proteins of the subpolar flagellins (FliC, upper bands) and lateral flagellins (LafA, lower bands) in the wild-type (WT) and the lafR::Km extracts alone or in extracts from the WT and the lafR::Km strains complemented with a WT copy of lafR under the direction of the nptII promoter (pFAJ::lafR). (B) Agarose gel of RNA retrotranscripts amplified by RT-PCR of lafR in the WT or the lafR::Km mutant with the primers indicated in Fig. S2B and listed in Table S2 in the supplemental material compared to sigA as constitutive reference gene. A PCR from genomic DNA was performed as a positive control (+C). (C) Swimming motility in 0.3% (wt/vol) agar-containing AG medium. Left panel: wild type compared to lafR::Km, ΔflbT_L, and ΔlafA mutants, the last one lacking lateral flagellins. Center panel: complementation of motility in the lafR::Km mutant with the pFAJ::lafR plasmid in comparison to the lafR::Km mutant carrying empty vector (pFAJ) or lafR::Km mutant carrying pFAJ::flbT_L. Right panel: complementation of motility in the ΔflbT_L mutant with the pFAJ::flbT_L plasmid in comparison with ΔflbT_L mutant carrying the empty vector (pFAJ). The results of all the complementations may be compared to the motility of the WT carrying the empty vector (WT pFAJ, right). (D) SDS-PAGE of B. diazoefficiens extracellular proteins—the subpolar flagellins (FliC, upper bands) and lateral flagellins (LafA, lower bands)—in the wild type and lafR point mutants D50A (with the Asp50 residue replaced by Ala), D50G (with Asp50 replaced by Gly), and D50E (with Asp50 replaced by Glu). (E) Composite SDS-PAGE of the subpolar (FliC, upper bands) and lateral (LafA, lower bands) flagellins of B. diazoefficiens or the FlaA-D flagellins of E. meliloti (Fla, middle bands). The flagellins are from B. diazoefficiens (Bd) wild-type and lafR::Km mutant either alone or complemented with pFAJ::lafR or with a wild-type copy of rem under the nptII promoter (pFAJ::rem), wild-type E. meliloti (Em WT), and the E. meliloti rem mutant (Δrem) either alone or complemented with pFAJ::rem or pFAJ::lafR. All the bacteria were grown on HMY with arabinose as the carbon source. The gels were run simultaneously in the same equipment.

FIG 2

Operons of the lateral-flagellar gene cluster, indicating the transcription directions according to Rhizobase (http://genome.annotation.jp/rhizobase/Bradyrhizobium). (A) The genes identified in the cluster are classified by function as regulators (R, gray), unknown (?, white), hook and hook-filament junction (H and HJ, violet), export apparatus (EA, green), motor (M, orange), MS ring (pink), flagellins (F, red), basal body (B, blue), L-ring and P-ring (LRi and PRi, turquoise), distal and proximal rods (Dr and Pr, light blue), and C-ring (CR, light pink). Below this scheme, the operon structure is indicated according to: bioinformatics prediction (upper light-pink line), Čuklina et al. (34) (middle light-pink line), and our own results from RT-PCR (bottom light-pink line). Above the scheme, the positions of the deduced lafR-dependent promoters are shown as black arrows, and the positions of the intergenic amplicons predicted according to the RT-PCR strategy outlined in Fig. S4 in the supplemental material are shown as black segments numbered from 1 to 8. For the sake of simplicity, the L subscripts in the figure have been omitted from the name of each locus. (B) Sequence alignment of the conserved motifs found upstream from the transcription start sites (designated as +1) of the genes motA (row a), fliF_L (row b), and flgF_L1 (row c), the latter being located at the 5′ ends of operons I, II, and III, respectively (see panel A). The consensus sequence that may be deduced is indicated at the bottom of the panel.

FIG 3

Effects of mutations in lafR and flbT_L on the mRNA accumulation of selected lateral-flagellar genes. (A) Transcription expression level in the wild-type strain relative to that of the lafR::Km mutant plus the standard deviations (SD), as determined by qRT-PCR from at least three independent biological replicas for the indicated genes (locus tags), the latter being representative of the different transcriptional units. Mono., monocistronic transcripts. The relative expression of lafR was evaluated with the primers indicated in Fig. S2B in the supplemental material, which amplify the 5′ end of lafR both in the wild-type and in the lafR::Km mutant. Stars, statistically significant differences (P < 0.05) from a threshold interval of 0.5 to 2.0 according to the Student t test. (B) Transcription expression level in the wild-type strain relative to that of the ΔflbT_L strain (LafR⁺/FlbT_L⁻, left) or that of lafR::Km carrying the plasmid pFAJ::flbT_L (LafR⁻/FlbT_L^C, right) ± the SD, as determined by qRT-PCR from at least three independent biological replicas for the indicated genes, the latter having been selected to indicate the differential influence of flbT_L on lafA1 expression. Stars, statistically significant differences (P < 0.05) from a threshold interval of 0.5 to 2.0 according to the Student t test. For the sake of simplicity, the L subscripts in the figure have been omitted from the name of each locus.

FIG 4

Control of flagellin expression by flbT_L in bacteria grown in liquid HMY medium with arabinose (Ara) or mannitol (Man) as carbon source. (A) SDS-PAGE of the B. diazoefficiens extracellular subpolar (FliC) and lateral (LafA) flagellins in the wild-type, the ΔflbT_L strain, the ΔflbT_L strain complemented with the plasmid pFAJ:: flbT_L that carries a wild-type copy of flbT_L under the direction of the nptII promoter, or the lafR::Km strain complemented with the plasmid pFAJ::flbT_L or pFAJ::lafR. (B) Western blots of the cellular B. diazoefficiens proteins FliC and LafA, as visualized by an anti-lafA polyclonal antiserum, from the wild-type strain either alone or complemented with the plasmid pFAJ::flbT_L or from the lafR::Km strain complemented with the plasmid pFAJ::flbT_L. The polyclonal anti-lafA serum exhibited some cross-reaction against FliC, whose activity in this experiment served as an internal standard.

FIG 5

β-Galactosidase activities of pCB::PlafA1 and pCB::PlafA2 lacZ fusions within three genetic backgrounds. In the figure, the β-galactosidase activity in Miller units is plotted on the ordinate for each of the genetic backgrounds denoted on the abscissa. The two clonal fusions are indicated in brackets below the figure. Each mean value is from two independent clones measured in duplicate. Error bars indicate the SD.

FIG 6

Scheme of the regulation of the lateral-flagellar genes that may be deduced from the present results. Cultivation with arabinose as the carbon source induces the expression of lafR, whereas cultivation with mannitol as the carbon source does not. LafR activates the transcription (Txn) of operons I, II, and III without any special hierarchical order among them, while the monocistronic lafA2 is transcribed independently of LafR. Operon I contains flbT_L, whose locus, upon activation, acts as a translation (Tln) inducer of the monocistronic lafA2 and appears to stabilize (Stb) the lafA1 transcript from a promoter within operon II. In addition to the effects of arabinose and mannitol, evidence from the literature indicates that prolonged exposure to moderate oxidative stress also induces lafR and the lateral flagellar regulon (25), whereas situations of O₂ limitation as the bacteroid state (23) or iron limitation (24) repress them. We also observed that viscosity and tortuousity of the medium induce lateral flagella (20) and that microoxia was reported as inhibiting lateral flagella genes expression (23). Therefore, the signal to which the expression of lateral-flagellar genes responds might be related to the energy status of the cell, apart from the specific carbon source available.