Changes in protein profiles of Burkholderia sp. S172, Sinorhizobium fredii S173, S174 and Bradyrhizobium japonicum S76, S78, S162, S178 when cultured at high temperatures

Abstract

Comparisons of RAPD-PCR DNA fingerprints when either RPO1 or CRL-7 was used as the primer revealed that three fast-growing isolates (S172, S173, and S174) and three slow-growing isolates (S76, S78, and S162) were distinct strains. Isolates S162 and S178 were found to be the same strain. Identification of the bacterial strains by Gram staining, ability to nodulate 7 soybean cultivars (SJ 4, SJ 5, CM 2, CM 60, ST 1, ST 2, ST3) and for the fast-growing bacteria, by additional negative staining and observation for flagella under the electron microscope and comparisons of 16S rDNA sequences with corresponding sequences deposited at GenBank showed strain S172 was Burkholderia sp. S172, and strains S173, S174 were tentatively identified as Sinorhizobium fredii S173 and Sinorhizobium fredii S174. The slow-growing strains S76, S78 and S162 (S178) were identified as Bradyrhizobium japonicum S76, S 78 and S162 (S178). Duncan's Multiple Range Test showed inoculation of each of the above-mentioned strains onto germinating seeds of the seven soybean cultivars grown in Leonard jars with nitrogen-free medium pH 5.0 or 6.8 resulted in high nitrogen-fixing potential of strains S76 and S78 when nodulated soybean. cultivars CM 2, CM 60 (average plant dry weight 2.17 g.plant¯¹). Similarly, strains S162, and S178 showed high nitrogen-fixing potential when nodulated soybean cultivars ST 2 (average plant dry weight 2.24 g.plant¯¹). Burkholderia sp. S172 was not found to nodulate the seven soybean cultivars used in the experiments but was found to yield comparable soybean dry weight with that of the positive control in Leonard jars when pH of the nitrogen-free medium was either 5.0 or 6.8 (average plant dry weight 1.51 g.plant¯¹) Burkholderia sp. S172 was found to promote the most growth in soybean cv. CM 60. This is the first report on the identification of Burkholderia sp. from soybean rhizosphere which promoted soybean growth. Sinorhizobium fredii S173, S174 were not found to nodulate soybean cultivars ST 1, ST 2 and CM 2, ST 1, ST 3 respectively. The two bacterial strains were found to yield high plant dry weight for soybean cultivars SJ 4 and SJ 5 in Leonard jars with nitrogen-free medium pH 5.0 or pH 6.8. (average plant dry weight 2.07 g.plant¯¹) Growth of the seven bacterial strains in yeast extract mannitol broth at 30℃, 35℃. 40℃ and 45℃ showed Burkholderia sp. S172, Sinorhizobium fredii S173, S174 grew equally well over a broad temperature range (30℃-45℃) while Bradyrhizobium japonicum S76, S78 and S162 and S178 were thermotolerant Analysis of changes in protein profiles showed different responses in term of quantity of proteins when the seven bacterial strains were cultured at high temperatures. More 40 kDa polypeptide was found in Burkholderia sp. S172 when cultured at 40℃ and 45℃. More 43 kDa polypeptide was found in B. japonicum S162(S178) when cultured at high temperatures. S. fredii S173, S. fredii S174, and B. japonicum S162(S178) were found with more 55, 38, and 25 kDa polypeptides when cultured at high temperatures. No increase was observed for polypeptides with similar molecular weight as those of the heat shock proteins Clp (100 kDa), DnaK (70 kDa), GroEL (60 kDa) and GroES (10 kDa). Neither new protein bands nor decrease in protein quantity was observed in protein profiles of the seven bacterial strains cultured at high temperatures.