Transcriptional Control of the Lateral-Flagellar Genes of Bradyrhizobium diazoefficiens

Abstract

Bradyrhizobium diazoefficiens, a soybean N 2 -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 N 2 -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, flagella, expression, lafR, flbT, FlbT, LafR F lagellum-driven swimming motility-a characteristic trait of many bacterial species-is essential for the colonization of diverse niches in environments such as seas, freshwaters, sediments, soils, and the organs of plant or animal hosts. This form of bacterial locomotion requires the propulsion provided by flagella, as well as a guidance system mediated by chemotaxis (1).Flagella are complex organelles formed by three main structures: a basal body that anchors the flagellum to the cell envelope...

Induction by Bradyrhizobium diazoefficiens of Different Pathways for Growth in D-mannitol or L-arabinose Leading to Pronounced Differences in CO2 Fixation, O2 Consumption, and Lateral-Flagellum Production

“…In agreement with our previous observations ( Covelli et al, 2013 ; Mongiardini et al, 2017 ) and the results presented in Supplementary Figure S1B , we found the lateral-flagellar–filament structural flagellins Bll6865 and Bll6866, as well as the lateral-flagellar–hook protein Bll6858 in the AraDP ( Table 2 ), but we failed to detect the majority of the other lateral-flagellar components. Nevertheless, we recently found the transcripts for these proteins produced in the HMY-Ara but not in the HMY-Mtl, under the control of the lafR transcriptional regulator ( Mongiardini et al, 2017 ). Because flagellins and the flagellar-hook protein are the most abundant flagellar gene products, the lack of detection of the other proteins might have resulted from a low abundance or from a tight association with the flagellar structure that perhaps we did not manage to disassemble during the extraction.…”

“…Since the amount of biomass produced per mole of ATP ( y x/ATP ) was the same for both culture conditions and the stoichiometric balances failed to reveal any substantial differences in the efficiency of ATP production per C-mole of substrate, the results would indicate that the cultures in the HMY-Ara consumed a larger proportion of the catabolic energy produced for simple maintenance—encompassing processes different from the production of biomass or C-products—than did those in the HMY-Mtl. One of these maintenance processes requiring energy may be the generation and use of the lateral flagella, which appendages are present in the HMY-Ara cultures but are scanty in the HMY-Mtl cultures ( Mongiardini et al, 2017 ; see also Supplementary Figure S1B ). To evaluate the contribution of the lateral flagella to qO 2max in HMY-Ara, we repeated the above measurements in both culture media with a B. diazoefficiens USDA 110 lafR ::Km insertional mutant lacking the class IB regulator of lateral-flagellum synthesis and therefore unable to produce any lateral flagellar structures, including the flagellar motor ( Mongiardini et al, 2017 ).…”

“…The wild-type B. diazoefficiens strain employed in this work is a spontaneous Sm resistant derivative from the type-strain USDA 110. The lafR ::Km mutant strain is a USDA 110 insertional mutant in which the Km resistance cassette from the plasmid pUC4K was inserted into the coding region of lafR ( Mongiardini et al, 2017 ). We grew the bacteria for inoculum in liquid HM salts–yeast-extract medium (HMY) supplemented with either 0.5% (w/v) Mtl or 0.5% (w/v) Ara ( Mongiardini et al, 2017 ) as the C sources (HMY-Mtl or HMY-Ara cultures, respectively), taking into account that the C-mole of each substrate is very similar (30.3 and 30.0 g.C-mole -1 for Mtl and Ara, respectively).…”

“…Normally, bacteria with dual flagellar systems express one for swimming in liquid medium and the other for swarming over wet surfaces ( Partridge and Harshey, 2013 ); but B. diazoefficiens , together with Shewanella putrefaciens , are the only bacteria known to produce both functional systems simultaneously in liquid medium ( Bubendorfer et al, 2014 ; Quelas et al, 2016a ). In the example of B. diazoefficiens , the primary (subpolar) flagellar system is expressed constitutively, while the secondary (lateral) system is fully expressed with Ara but is undetectable with Mtl as the carbon source ( Covelli et al, 2013 ; Mongiardini et al, 2017 ). Once expressed, both flagellar systems are active in liquid medium and integrate their functions in an emergent swimming performance that cannot be explained by the additive effects of each flagellar system ( Quelas et al, 2016a ).…”

Physical Properties of Carbon Nanomaterials and Nanoceria Affect Pathways Important to the Nodulation Competitiveness of the Symbiotic N2‐Fixing Bacterium Bradyrhizobium diazoefficiens

“…B. diazoefficiens possesses a dual flagellar system with a constitutively expressed thick flagellum and several thin lateral flagella that are synthesized only in certain environmental conditions such as using arabinose as a carbon source, in a high-viscosity culture medium, or meeting obstacles in the swimming path. [44] Out of ≈40 genes known to be directly involved in flagellar structure and motor function in B. diazoefficiens, [45] 36 genes were upregulated by CeO 2 NPs and MWCNTs. However, the number of genes affected by ENMs in RE-supplemented media was lower than in minimal medium without RE (Figure 3).…”