Structural And Functional Analysis Of Ammonium Transporter-encoding Gene AmtB In The Associative Nitrogen-fixing Bacterium Pseudomonas Stutzeri A1501

Pseudomonas stutzeri A1501, isolated from rhizosphere of rice, is an associative nitrogen fixing bacterium, belongs toγsubgroup of Proteobacteria. Ammonium transport system play a key role in balancing the fixed nitrogen gradient between the cytoplasm in cell and ambient environment, which give rise to the great meaning of study the regulation of ammonium transport system encoded genes. A 2684 bp DNA fragment was cloned by PCR amplification using specialized primers, and two amt genes were identified by sequence Blast, the two genes were named amtB1 and amtB2 respectively. Sequence analysis displayed no promoter exists between the two amtB genes and this was confirmed by RT-PCR. Analysis of the predicted three dimension structures of proteins revealed that AmtB1 contained 12 transmembrane domains and AmtB2 contained 11.Polar amtB1 mutant TB12, amtB1 nonpolar mutant TB1, and amtB1 nonpolar mutant TB2 were obtained by using the suicide vector pK18mob or pT18mob. Part of amtB1 or amtB2 was obtained by PCR and then cloned into pT18mob or pK18mob, after transferred into recipient A1501 by tri-parental conjugation, the transformants were isolated on selective media and confirmed by PCR. Growth test revealed that at the high concentration of ammonium mutants share the same growth rate with wild A1501 at high concentration of ammonia, the TB12 showed retard growth at low concentration of ammonium. The ammonium absorption test revealed that TB1 and TB2 showed the same decrease tendency with wild A1501, the TB12 showed the stability of ammonium concentration in culture which confirmed that the amtB encoding ammonium transport system and the two genes complement each other in function. The physiological test showed that the TB12 excreted ammonium after grow up to leg phase. The defects in the ammonium transport proteins have no effect on the nitrogenase activities, and the TB12 remain relatively high level of nitrogenase activity at high concentration of ammonium.The mutants displayed higher competition in colonization on the rice root and growth promotion by comparison to wild type A1501, the mutants account 74% of total bacteria. The constructed plasmid pLAB1 containing amtB1 can recover the TB12 several characters including growth, nitrogen fixation, and ammonium execretion of wild A1501. These results lead us to conclude that amtB encode ammonium transport system and play the key role in sensing and translocating the ammonium between the cell and environment.