Analysis of genomic dna fragment involved in acid- aluminium tolerance in bradyrhizobium japonicum

Abstract

Bradyrhizobium japonicum is one of Rhizobia species which generally form root nodule on soybean plant (Glycine max). In association with leguminous plant, root nodule bacteria (RNB) fix atmospheric nitrogen (N2) into NH3 which can be used for plant growth. Soil acidity is a significant problem affecting agricultural production in many areas of the world. Lowering pH of the soil can affect soil organisms including the microflora. Among the biological system affected by the decrease in soil pH are those between RNB and leguminous plants. It could affect the association result between them in fixing nitrogen. In the previous work, screening indigenous B. japonicum strains which tolerant to acid-aluminium has been reported. The potential strains were strains BJ11, BJ38, and KDR15. These tolerant strains could become a source of genetic material exploration which involved in acid-Al tolerance in B. japonicum. Transposon delivery was carried out through conjugation between Escherichia coli S17-1 (λ pir) carrying pUTmini-Tn5Km1 and acid-Al tolerant B. japonicum at 1:1 ratio. Frequency of transconjugation was in the range of 10-8–10-6 cells per recipients. A mutant AAS11.2 did not able to grow on the Ayanaba medium (pH 4.5) supplemented with 50 μM aluminium. AAS11.2 was able to form root nodule on siratro plant indicating that the gene involved in acid-Al tolerance was not related with nodulation. A 0.8 kb of the genomic DNA fragment flanking transposon involved in acid-Al tolerance was successfully isolated by inverse polymerase chain reaction (inverse PCR) from AAS11.2 genome. This fragment was subsequently cloned into pGEM-T Easy (∼3 kb) to yield a recombinant plasmid, designated as pGEMT-11 (∼3.8 kb), and sequenced. DNA sequenced analysis revealed that the genomic DNA fragment sequence had high homology with yhfK gene encoding putative inner membrane protein from Salmonella typhimurium (79% identity and 84% similarity, E-value= 1.0xe-100) which function as efflux transporter.