Cloning of Genomic DNA Fragment Involve in Acid-Aluminium Tolerance in Bradyrhizobium japonicum 38 Through Transposom Mutagenesis

Abstract

An acid-aluminium sensitive mutant of Bradyrhizobium japonicum 38. designated as AAS38, was generated by mini-Tn5 transposon mutagenesis. The experiment was carried out to identify acid-aluminium tolerance gene (AAT) in B. japonicum. Transposon delivery was carried out through conjugation between Escherichia coli S17-I (λ pir) carrying pUTmini-Tn5Kml and acid-Al tolerant B. japonicum with different of mating time. Frequency of transconjugation was in the range of 10-7 -10-6. A mutant AAS38 was not able to grow on the Ayanaba medium (pH 4.5) containing 50 µM aluminium. However, this mutant formed root nodule of soybean and Siratro plants indicating the gene involved in acid-Al tolerance was not related with nodulation. A 0.8 kb of the genomic DNA fragment flanking the transposon involved in acid-aluminium tolerance was successfully isolated by inverse polymerase chain reaction (Inverse PCR) from AAS38 genome. This fragment was subsequently cloned into pGEM-T Easy (~3 kb) to yield a recombinant plasmid, designated as pGEMT-38 (~3.8 kb), and sequenced. DNA sequence analysis revealed that the genomic DNA fragment had high homology to inner membrane protein from Salmonella typhimurium (80% identity and 86% similarity, E-value= 8xe-62 predicted function as efflux transporter.