Identification Of 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Gene In Nitrogen-fixing Pseudomonas Stutzeri A1501

Pseudomonas stutzeri A1501 is an excellent plant growth promoting rhizobacteria(PGPR)and was isolated from rice paddies in southern China. The bacterium fixes nitrogen, secretes phytohormones and enhances the resistance of rice to multiple stresses. Ethylene is a gaseous plant hormone which prompts fruit mature and flower wilt, but excessive ethylene restrains plant growth. Stresses conditions such as drought, water, salt and heavy metal can induce the plant ethylene. Bacteria that contain 1-aminocyclopropane-1-carboxylate (ACC) deaminase, encoded by acdS, improve plant growth and seed production by sequestering the ethylene precursor ACC from plants. So far, ACCD has not been found in plant, even though this enzyme exists in many PGPR bacteria. ACCD significantly decreases plant ethylene levels, especially when plantsare under environmental stress. In this study, we used bioinformatics and laboratory experiments to analyse the genome of P. stutzeri A1501 and to study the function of acdS gene product ACC deaminase. We identified the function of ACCD and discussed the mechanism of prompting growth under salt and heavy metals. Our study has provided theoretical basis to study mechanism interaction between bacteria and plant.The P. stutzeri AcdS is 71.27 % identical to its orthlogs from other Pseudomonas species. The acdS gene and its downstream cAMP binding protein coding gene form a co-transcriptional unit. The G+C content (53.7 %) of acdS is lower than A1501 chromosome G+C content (63.8 %), speculating that this gene may come from horizontal transfer. The result is consistent with previous studies, where it was showed that acdS is not the main part of the microbial chromosome DNA, more as exists in plasmid. We constructed an inactivating acdS insertion mutant of P. stutzeri, which had no detectable ACC deaminase activity, and successfully complemented the mutation using acdS cloned on a plasmid vector. To investigate role of acdS for resistance to salt and heavy metals, we compared the growth and nitrogenase activity of the wild-type and the acdS mutant. The acdS mutation did not affect growth under LB medium. However, the mutant showed slower growth in LB medium containing 0.8 M NaCl. The survival rate of the acdS mutant was also decreased in LB medium containing 1.0 M NaCl or 13.2 mM NiCl2. The nitrogenase activity of the acdS mutant was similar to that of the wild-type under nitrogen-fixing conditions (microaerobic, low nitrogen) in the free-living state. However, in the presence of 0.2 M NaCl nitrogenase activity decreased to about 50 % in the wild-type and to 70 % in the acdS mutant. From the published A1501 genome sequence, we selected nifH, nifA, rpoN and glnA, and examined their expression under nitrogen-fixing conditions in the presence of 0.2M NaCl. All the genes were down-regulated in the acdS mutant. The acdS-deletion might have affected first the growth of the strain and later the expression of nitrogenase. Biolog data revealed that the acdS mutant utilize fewer carbon sources than the wild-type. The acdS mutation seems to have affected the carbon metabolic networks.Wild-type P. stutzeri promoted plant growth in the presence of≥0.12M NaCl, 0.3 mM CoCl2, 0.3 mM CuSO4, 0.3 mM ZnCl2, or 0.3 mM NiCl2, the plant height, root length, root fresh weight, root dry weight increase 19～45 %,30～47 %,22～68 %,28～43 %。But the acdS mutant did not, presumably because it failed to reduce the ethylene concentration.