Isolation of acid-tolerant Bradyrhizobium japonicum for hydrogenase activity and protein pattern determination

Abstract

The aim of this research is to isolate acid-tolerant Bradyrhizobium japonicum for use in pinpointing the effects of initial pH on hydrogenase activity and protein patterns. 56 soybean root nodule isolates obtained from 13 soil samples of pH ranging from 4.19-6.89 consisted of 6 fast-growing isolates and 50 slow-growing isolates. Authentication test on soybean plants grown in Leonard jars showed that all isolates were B.japonicum. Analysis of variance indicated that the source of variations in the observed plant dry weight and nodule dry weight was the types of isolates and not pH 4.5 or 6.8 of the N-free plant nutrient solutions. The following four criteria were used to select 4 distinct strains of B.japonicum (#S50, S58, S179, S204): (1) nitrogen fixing ability at pH 4.5 and 6.8 (2) colony morphology (3) RAPD-PCR fingerprint patterns obtained when RPO1 was used as the primer (4) extent of B.japonicum growth in buffered yeast extract mannitol broth (YMB) at pH 9.0. Initial pH had 3 types of effects on hydrogen uptake hydrogenase activity. Type 1 (#S50) exhibited hup- phenotype when initial pH of the medium was 4.0 but exhibited hup+ phenotype when media initial pHs were 6.0-9.0 with maximum specific activity 30.1 U.mg-1 protein at initial pH 8.0. Type 2 (#S58, S179) exhibited hup- phenotype when media initial pHs were 4.0-8.0 but exhibited hup+ phenotype when the initial pH was 9.0 with specific hydrogenase activity of 29.4 U.mg-1 protein. Type 3 (#S204) exhibited hup- phenotype when the initial media pH was in the range of 5.0-7.0 but exhibited hup+ phenotype when the initial pH was 4.0 with specitic hydrogenase activity of 43.9 U.mg-1 protein. The results indicated for the first time that expression of hup genes in free-living B.japonicum depended upon initial pH. SDS-PAGE separation of cellular proteins of isolates #S50, S58, S179, and S204 indicated an increase in quantities of the following polypeptides of isolate S58 (28, 33, 37, 50 kDa) when grownin unbuffered YMB at pH 4.0-8.0 and an increase in quantities of polypeptides 29 and 120 kDa when grown in buffered YMB, pH 6.0-8.0. Isolate #S179 was found to synthesize more of the following polypeptides (25, 28, 33, 37, 50 kDa) when grown in buffered YMB at pH 8.0-9.0. Isolate #S204 contained more 120 kDa polypeptide when grown in buffered YMB at pH 5.0-7.0.