Selection of TnAraOut mutants from nitrogen-fixing and heat tolerant Sinorhizobium fredii S174

Abstract

Sinorhizobium fredii is a fast growing nitrogen-fixing heat tolerant bacterium in soybean root nodules with doubling time less than 6 hours. Another category of soybean rhizobia is a slow-growing Bradyrhizobium japonicum with doubling time more than 6 hours. The aims of the experiments are to isolate slow-growing TnAraOut mutants from fast-growing nitrogen-fixing and heat tolerant S. fredii S174 by biparental mating between S. fredii S174 and E. coli S17-1[lambda]pir (pNJ17). TnAraOut is a transposon on the plasmid pNJ17 with arabinose-inducible promoter (P[subscript BAD]), araC and kanamycin resistant gene. The transposon inserts in the TATA region of -10/-35 promoters rendering the promoters to be arabinose-inducible. Mutants with cell division gene promoter disruption grew normally and yielded large colonies in the presence of arabinose. They grew slowly resulting in small colonies when arabinose was not in the medium. Confirmation of the presenceof TnAraOut in the mutant genomes was carried out by digesting each mutants DNA with SphI, recircularization, then electroporation into E. coli DH5[alpha][lambda]pir and selection for kanamycin resistant colonies. DNA of each kanamycin-resistant colony was isolated and used as target DNA for PCR with P[subscript BADout2] as the primer. PCR products were sequenced for the presence of an inverted repeat sequence of TnAraOut. Experimental results revealed the first full 16S rDNA sequence of S. fredii S174 (1457 nucleotides). In addition, fifteen TnAraOut mutants were obtained. Two TnAraOut mutants (ST49 and ST60) were used to prove the presence of TnAraOut sequence in their genomes. Experimental results revealed the presence of the insertion sequence in the TATA region of the promoters of the mutants. In addition, thermotolerance properties of TnAraOut mutants ST49 and ST60 were found to be comparable to that of the wild type. SDSPAGE of intracellular proteins of cells grown under high temperatures indicated similar protein profiles with increased synthesis of polypeptides 10, 12, 60, and 62 kDa. Nitrogen fixing potential of TnAraOut mutant ST60 was found to be comparable to that of the wild type when Glycine max cultivar SJ4 was used as the soybean host