Research Of The Operon Matpqmab Which Involved In Malonate Metabolism In Sinorhizobium Meliloti

Tripartite ATP-independent periplasmic (TRAP) transporters form a distinct class of ESR-dependent (extracytoplasmic solute receptor,ESR)secondary transporters where the driving force for solute accumulation is an electrochemical ion gradient and not ATP hydrolysis. They are present in a diverse range of prokaryotes, both bacteria and archaea. TRAP transporters are a new family of periplasmic solute transport systems which composed of three protein components or domains; an extracellular solute receptor and two distinct integral membrane proteins of unequal size.In this study, we mainly make a preliminary research and its expression regulation of the matPQMAB gene cluster in Sinorhizobium meliloti Rm1021 genomic. Genome bioinformatics analysis of S.meliloti Rm1021 has been showed that four consecutive gene SMa0157, SMa0155, SMa0151, SMa0150 which on the large symbiotic plasmid A (pSymA) constitute a possible operon matPQMAB, may be form a TRAP transport systems which involved in malonate transportation. These four genes has been designated as matP, matQ, matMA, matB. In addition in the downstream of the matPQMAB operon there has a transcription regulation factor GtrA, may be able to regulate the expression of the operon.In order to prove the matPQMAB operon forms a TRAP transporter which involved in malonate transportation, as well as the expression and regulated by GtrA. We constructed the plasmid insertion mutants of gtrA, matP, matQ, matMA, matB, and a series of phenotypic analysis of self- and the symbiotic phenotype.The mutants of matP, matQ, matMA, matB can not grow in the M9 minimum medium with the sodium malonate as the sole carbon source in free-living conditions,but the wild-type Rm1021 can grow normally, this indicate that the four genes truly constitute a malonate metabolism transport system, and also is the unique metabolic pathway. The gtrA mutant just cause self-growth delay in the same conditions. In addition, the nitrogen fixation nodulation efficiency will reduce when the gtrA mutant inoculate the Alfalfa. By the gel shift assay, we found GtrA protein can combine with the promoter region of the matPQMAB operon,these results show that GtrA can be able to interact with matPQMAB operon directly and regulate its expression.In addition, we have a study on the functional characteristics of the matB gene from the matPQMAB operon. We cloned the matB gene from the Rm1021 genome and expressed and purified in vitro from Escherichia coli. The purified MatB has the activities of malonyl-CoA synthetase, showing that its Km value is 710μmoL and Vmax is 0.209μmoL/min/mg.