Dataset Information

Mutational analysis of the Rhizobium lupini H13-3 and Sinorhizobium meliloti flagellin genes: importance of flagellin A for flagellar filament structure and transcriptional regulation.

ABSTRACT: Complex flagellar filaments are unusual in their fine structure composed of flagellin dimers, in their right-handed helicity, and in their rigidity, which prevents a switch of handedness. The complex filaments of Rhizobium lupini H13-3 and those of Sinorhizobium meliloti are composed of three and four flagellin (Fla) subunits, respectively. The Fla-encoding genes, named flaA through flaD, are separately transcribed from sigma(28)-specific promoters. Mutational analysis of the fla genes revealed that, in both species, FlaA is the principal flagellin and that FlaB, FlaC, and FlaD are secondary. FlaA and at least one secondary Fla protein are required for assembling a functional flagellar filament. Western analysis revealed a ratio close to 1 of FlaA to the secondary Fla proteins (= FlaX) present in wild-type extracts, suggesting that the complex filament is assembled from FlaA-FlaX heterodimers. Whenever a given mutant combination of Fla prevented the assemblage of an intact filament, the biosynthesis of flagellin decreased dramatically. As shown in S. meliloti by reporter gene analysis, it is the transcription of flaA, but not of flaB, flaC, or flaD, that was down-regulated by such abortive combinations of Fla proteins. This autoregulation of flaA is unusual. We propose that any combination of Fla subunits incapable of assembling an intact filament jams the flagellar export channel and thus prevents the escape of an (as yet unidentified) anti-sigma(28) factor that antagonizes the sigma(28)-dependent transcription of flaA.

SUBMITTER: Scharf B

PROVIDER: S-EPMC95416 | biostudies-literature | 2001 Sep

REPOSITORIES: biostudies-literature

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Publications

Mutational analysis of the Rhizobium lupini H13-3 and Sinorhizobium meliloti flagellin genes: importance of flagellin A for flagellar filament structure and transcriptional regulation.

Scharf B B Schuster-Wolff-Bühring H H Rachel R R Schmitt R R

Journal of bacteriology 20010901 18

Complex flagellar filaments are unusual in their fine structure composed of flagellin dimers, in their right-handed helicity, and in their rigidity, which prevents a switch of handedness. The complex filaments of Rhizobium lupini H13-3 and those of Sinorhizobium meliloti are composed of three and four flagellin (Fla) subunits, respectively. The Fla-encoding genes, named flaA through flaD, are separately transcribed from sigma(28)-specific promoters. Mutational analysis of the fla genes revealed ...[more]

PMID: 11514517

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Project description:Symbiotic nitrogen fixation (SNF) is an energetically expensive process performed by bacteria during endosymbiotic relationships with plants. The bacteria require the plant to provide a carbon source for the generation of reductant to power SNF. While C4-dicarboxylates (succinate, fumarate, and malate) appear to be the primary, if not sole, carbon source provided to the bacteria, the contribution of each C4-dicarboxylate is not known. We address this issue using genetic and systems-level analyses. Expression of a malate-specific transporter (MaeP) in Sinorhizobium meliloti Rm1021 dct mutants unable to transport C4-dicarboxylates resulted in malate import rates of up to 30% that of the wild type. This was sufficient to support SNF with Medicago sativa, with acetylene reduction rates of up to 50% those of plants inoculated with wild-type S. melilotiRhizobium leguminosarum bv. viciae 3841 dct mutants unable to transport C4-dicarboxylates but expressing the maeP transporter had strong symbiotic properties, with Pisum sativum plants inoculated with these strains appearing similar to plants inoculated with wild-type R. leguminosarum This was despite malate transport rates by the mutant bacteroids being 10% those of the wild type. An RNA-sequencing analysis of the combined P. sativum-R. leguminosarum nodule transcriptome was performed to identify systems-level adaptations in response to the inability of the bacteria to import succinate or fumarate. Few transcriptional changes, with no obvious pattern, were detected. Overall, these data illustrated that succinate and fumarate are not essential for SNF and that, at least in specific symbioses, l-malate is likely the primary C4-dicarboxylate provided to the bacterium.IMPORTANCE Symbiotic nitrogen fixation (SNF) is an economically and ecologically important biological process that allows plants to grow in nitrogen-poor soils without the need to apply nitrogen-based fertilizers. Much research has been dedicated to this topic to understand this process and to eventually manipulate it for agricultural gains. The work presented in this article provides new insights into the metabolic integration of the plant and bacterial partners. It is shown that malate is the only carbon source that needs to be available to the bacterium to support SNF and that, at least in some symbioses, malate, and not other C4-dicarboxylates, is likely the primary carbon provided to the bacterium. This work extends our knowledge of the minimal metabolic capabilities the bacterium requires to successfully perform SNF and may be useful in further studies aiming to optimize this process through synthetic biology approaches. The work describes an engineering approach to investigate a metabolic process that occurs between a eukaryotic host and its prokaryotic endosymbiont.

Dataset Information

Mutational analysis of the Rhizobium lupini H13-3 and Sinorhizobium meliloti flagellin genes: importance of flagellin A for flagellar filament structure and transcriptional regulation.

Publications

Mutational analysis of the Rhizobium lupini H13-3 and Sinorhizobium meliloti flagellin genes: importance of flagellin A for flagellar filament structure and transcriptional regulation.

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