A Tar aspartate receptor and Rubisco-like protein substitute biotin in the growth of rhizobial strains.

Biotin is a key cofactor of metabolic carboxylases, although many rhizobial strains are biotin auxotrophs. When some of these strains were serially subcultured in minimal medium, they showed diminished growth and increased excretion of metabolites. The addition of biotin, or genetic complementation with biotin synthesis genes resulted in full growth of Rhizobium etli CFN42 and Rhizobium phaseoli CIAT652 strains. Half of rhizobial genomes did not show genes for biotin biosynthesis, but three-quarters had genes for biotin transport. Some strains had genes for an avidin homologue (rhizavidin), a protein with high affinity for biotin but an unknown role in bacteria. A CFN42-derived rhizavidin mutant showed a sharper growth decrease in subcultures, revealing a role in biotin storage. In the search of biotin-independent growth of subcultures, CFN42 and CIAT652 strains with excess aeration showed optimal growth, as they also did, unexpectedly, with the addition of aspartic acid analogues α- and N-methyl aspartate. Aspartate analogues can be sensed by the chemotaxis aspartate receptor Tar. A tar homologue was identified and its mutants showed no growth recovery with aspartate analogues, indicating requirement of the Tar receptor in such a phenotype. Additionally, tar mutants did not recover full growth with excess aeration. A Rubisco-like protein was found to be necessary for growth as the corresponding mutants showed no recovery either with high aeration or aspartate analogues; also, diminished carboxylation was observed. Taken together, our results indicate a route of biotin-independent growth in rhizobial strains that included oxygen, a Tar receptor and a previously uncharacterized Rubisco-like protein.

Expanding the regulatory network that controls nitrogen fixation in Sinorhizobium meliloti: elucidating the role of the two-component system hFixL-FxkR.

In Sinorhizobium meliloti, nitrogen fixation is regulated in response to oxygen concentration through the FixL-FixJ two-component system (TCS). Besides this conserved TCS, the field isolate SM11 also encodes the hFixL-FxkR TCS, which is responsible for the microoxic response in Rhizobium etli. Through genetic and physiological assays, we evaluated the role of the hFixL-FxkR TCS in S. meliloti SM11. Our results revealed that this regulatory system activates the expression of a fixKf orthologue (fixKa), in response to low oxygen concentration. Null mutations in either hFixL or FxkR promote upregulation of fixK1, a direct target of FixJ. Furthermore, the absence of this TCS translates into higher nitrogen fixation values as well as higher expression of fixN1 in nodules. Individual mutations in each of the fixK-like regulators encoded in the S. meliloti SM11 genome do not completely restrict fixN1 or fixN2 expression, pointing towards redundancy among these regulators. Both copies of fixN are necessary to achieve optimal levels of nitrogen fixation. This work provides evidence that the hFixL-FxkR TCS is activated in response to low oxygen concentration in S. meliloti SM11 and that it negatively regulates the expression of fixK1, fixN1 and nitrogen fixation.

FxkR provides the missing link in the fixL-fixK signal transduction cascade in Rhizobium etli CFN42.

Transcriptional control of the fixK gene in Rhizobium etli and R. leguminosarum bv. viciae is governed by a two-component signal transduction system that diverts from the conventional FixL-FixJ cascade that occurs in model rhizobia. Although a fixL gene, encoding a hybrid histidine kinase (hFixL), is present in R. etli, no fixJ, the cognate response regulator, has been identified. In this work, we present evidence that the pRet42f-located open reading frame RHE_PF00530 (fxkR) encodes a novel response regulator indispensable for fixKf activation under microaerobic growth. Moreover, results from complementation assays demonstrate that the activation of fixKf expression requires the presence of both hFixL and FxkR, and that the fxkR ortholog from R. leguminosarum bv. viciae is able to substitute for FxkR transcriptional control in R. etli. In addition, in these two organisms, hFixL- and FxkR-related proteins were identified in other bacteria, located in close proximity to a fixK-related gene. Using reporter fusions, site-directed mutagenesis, and electrophoretic mobility shift assays, we identified the FxkR binding site upstream from the transcriptional start site of fixKf. Similar to our previous observations for fixL and fixKf mutants, a null mutation in fxkR does not affect the symbiotic effectiveness of the strain. Thus, our findings reveal that FxkR is the long-standing missing key regulator that allows the transduction of the microaerobic signal for the activation of the FixKf regulon.

Regulation and symbiotic role of nirK and norC expression in Rhizobium etli.

Rhizobium etli CFN42 is unable to use nitrate for respiration and lacks nitrate reductase activity as well as the nap or nar genes encoding respiratory nitrate reductase. However, genes encoding proteins closely related to denitrification enzymes, the norCBQD gene cluster and a novel nirKnirVnnrRnnrU operon are located on pCFN42f. In this study, we carried out a genetic and functional characterization of the reductases encoded by the R. etli nirK and norCB genes. By gene fusion expression analysis in free-living conditions, we determined that R. etli regulates its response to nitric oxide through NnrR via the microaerobic expression mediated by FixKf. Interestingly, expression of the norC and nirK genes displays a different level of dependence for NnrR. A null mutation in nnrR causes a drastic drop in the expression of norC, while nirK still exhibits significant expression. A thorough analysis of the nirK regulatory region revealed that this gene is under both positive and negative regulation. Functional analysis carried out in this work demonstrated that reduction of nitrite and nitric oxide in R. etli requires the reductase activities encoded by the norCBQD and nirK genes. Levels of nitrosylleghemoglobin complexes in bean plants exposed to nitrate are increased in a norC mutant but decreased in a nirK mutant. The nitrate-induced decline in nitrogenase-specific activity observed in both the wild type and the norC mutant was not detected in the nirK mutant. This data indicate that bacterial nitrite reductase is an important contributor to the formation of NO in bean nodules in response to nitrate.