Identification And Expression Of A LuxI-Type AHL Synthase Gene In Mesorhizobium Tianshanense And Its Effect On Symbiotic Nodulation

The ability of rhizobia to symbiotically fix nitrogen from the atmosphere when forming 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 symbiotic process in a number of rhizobium species, such as nodulation efficiency, symbiosome development, exopolysaccharide production, and nitrogen fixation. To date there have been no detailed studies on quorum-sensing regulatory systems in Mesorhizobium genus, a moderately growing rhizobium.In this study, high-cell-density cultures of eight type strains of the genus Mesorhizobium were extracted with ethyl acetate and subjected to TLC detection of AHLs content by using ultrasensitive bioassay strain JZA1. The AHL profiles of these eight strains were quite different. While, an M.tianshanense strain CCBAU 3306, a moderately growing root nodule bacterium that forms nodules on at least eight different plant species including Glycyrrhiza, produced eight different AHL molecules and showed the greatest diversity of AHL signaling molecule production among the Mesorhizobium strains tested. Further studies showed that accumulation of AHL activity in supernatants of CCBAU 3306 displayed a typical cell density-dependent pattern, with low AHL activity at low cell density and higher activities as the bacterial culture grew. The lower AHL activity observed after the highest activity may due to the alkaline hydrolysis by high pH in the culture.In an effort to identify the genes involved in AHL production in M.tianshanesne, a genomic expression library of M.tianshanense was constructed on a high-copy-number vector pEZSeq-Kan and electroporated into E.coli. A novel and simple screening method were developed so that we could pick up 3 positive clones which showed strong AHL activity with high efficiency. Restriction enzyme digestion for these 3 clones indicated a biggest insertion in a positive clone pHMZ9B1. DNA sequencing of pHMZ9B1 revealed a 4,061-bp insertion. One 665-bp ORF and an 819-bp ORF upstream of it are similar to several Luxl-type or LuxR-type transcriptional activators respectively, therefore they are...