The Role Of A LuxR-Type Regulator MrtR In Mesorhizobium Tianshanense And Its Effect On Physiological Function

The ability of rhizobia to symbiotically fix nitrogen from the atmosphere whenforming nodules on their plant hosts requires various signal transduction pathways.LuxR-LuxI-type quorum-sensing has been implicated as one of the player in the symbioticprocess in a number ofrhizobium species, such as nodulation efficiency, symbiosomedevelopment, exopolysaccharide production, and nitrogen fixation. To date there have beenno detailed studies on quorum-sensing regulatory systems in Mesorhizobium genus, amoderately growing rhizobium.In this study, to investigate whether LuxR. homologs MrtR obtained from our screensin E. coli are involved in autoinducer production in M. tianshanense, null mutations ofmrtR were constructed by replacing each gene with a tetracycline-resistant cassette.Cell-free culture supernatants of these strains were then assayed for AHL activity and AHLcontent. As noted above, the wild-type supernatant contained high AHL activity andproduced at least seven different AHLs on a TLC analysis. Strikingly, no AHL was detectedin the supernatant of the mrtR mutants from both liquid assays and TLC assays. There aretwo possibilities for why a deletion in mrtI/mrtR abolishes all AHL production under ourtesting conditions. One is that MrtI is responsible for synthesizing all these AHLs.Alternatively, MrtI-MrtR are situated at the top of a cascade of other quorum-sensingregulators, similar to the case of CinR-CinI in R. leguminosarum bv. Viciae.To investigate mrtR expression, an mrtR-lacZ transcriptional fusion was created bycloning an intact 5_-end mrtR fragment (including the putative promoter region) into thesuicide vector pVIK112 (15) and then transforming it into wild-type or the mrtI mutant M.tianshanense to select for Campbell-type integration by homologous recombination. Thisconstruct preserved a functional copy of the mrtR gene. The result shows that mrtRexpression is dependent on AHLs. In the mrtI mutant background, mrtR was induced byadding the supernatant containing AHLs, while in the wild-type background, lacZexpression reached high levels at high cell density. Taken together, these data suggest thatin M. tianshanense, the expression of both mrtR genes is autoregulated, which is a hallmark of quorum-sensing regulation.To ensure that the loss of AHL production in the mrtR mutant was not due to a polareffect of the mrtR mutation on downstream mrtI expression, a plasmid containing aPlac-controlled mrtR fusion was introduced to provide the MrtR proteins in trans.Overexpression of MrtR. in the mrtR null mutant recovered AHL production in the resultingstrain. Meanwhile, a mrtR-lacZ with deleted mrtR strain was constructed by pVIK112 andwas used to measure the in-frame mrtR expression level, as the mrtR both absent andpresent.Compared with the wild-type strains, mrtR quorum sensing- deficient mutant wasdefective in root hair adherence. These data provide strong evidence that quorum sensingplays a critical role in the M.tianshanense symbiotic process, but the exactly mechanism ofthis is not clear now.