Research On Function And Expression Of Dicarboxylate Transport Genes In Pseudomonas Stutzeri

Pseudomonas. stutzeri A1501was isolated from paddy field in South China in 1980, which colonized tightly on rhizoplane of the host plants or invaded the roots of plants for growth and nitrogen fixation. In contrast to Rhizobium-legume symbiosis, the supply of energy to nitrogen-fixing bacteria associated with roots of host plant is not sufficient. In this study, the expression vector of dctBD and mutants of dctPQM were constructed. The function and expression regulation of dicarboxylate acids transport system were studied systematically.Plasmid pLL2922 carrying the dctB region was constructed. The dctB mutant containing plasmid pLL2922 restored growth and nitrogenase activity in media containing succinate or fumarate as sole carbon scources. Introduction of plasmid pLL2922 into the dctB mutant restored growth and nitrogenase activity in succinate and fumarate containing media. The activities of wild type containing pLL2922 was increased 28.9%, 22.7%. 15.6% using succinate, fumarate and malate as the sole carbon sources.Polar and nonpolar mutant of dctP was constructed using suicide plasmid pK18mob. Polar mutant of dctP inactivited of dctQ and dctM which located downstream of dctP. Both polar and nonpolar mutant of dctP could not grow on medium containing succinate, fumarate and malate as the sole carbon sources. nifH-lacZ were introduced into mutants of dctP and dctM. Expression of nifH-lacZ are much lower in mutants than in wild type strain Pseudomonas stutzeri A1501. dctM mutant was acquired using insertional mutagenesis. Growth experiment results showed that dctM mutant could not grow on medium containing succinate, fumarate and malate as the sole carbon sources. It indicate that dctM is essential to dicarboxylate transport. β -Galactosidase activity in mutants of dctP and dctM are 17.0%. 23.3%. 24.9% of wild type in medium containing succinate, fumarate and malate as the sole carbon sources.The results indicated that dct gene are essential to dicarboxylate transport.Expression regulation of dct gene was studied using complemention analysis, RT-PCR and construction of dctPp-lacZ and dctBp-lacZ fusion vector. dctPp-lacZ and dctBp-lacZ fusion vector introducted into dctB mutant, σ⁵⁴ factor gene mutant and ntrBC mutant. Results showed the promoter of dctP was a induceable σ⁵⁴-dependent promoter. Expression of dctP needs DctB, NtrB, NtrC. Glucose inhibit the expression level of dctP. Dicarboxylate acids induce the expression of dctP. The expression level of malate was higher than that of succinate and fumarate. The fragment containing dctPQM and the promoter region of dctP can complement the dctM mutant. The smaller fragment of dctQM could not complement the mutant of dctM. The results showed that dctPQM has a common promoter. RT-PCR results showed that the expression of dctP and dctQ were lower than those in wild type. Results showed that dctB is low level and constitutive expression.Results of experiments and bioinformatics analysis showed that the Dct system consists of a two-component regulatory system (DctBD) and C₄-dicarboxylate transport proteins(DctPQM). The C₄-dicarboxylate transport protein is encoded by structural genes dctPQM in Pseudomonas stutzeri...