Proteome analysis of heat shock protein expression in Bradyrhizobium japonicum

A set of 19 heat shock proteins (Hsp) was observed - by subtractive two-dim ensional gel electrophoresis - to be induced when Bradyrhizobium japonicum, the nitrogen-fixing root-nodule symbiont of soybean, was temperature up-sh ifted from 28 degrees C to 43 degrees C. Up-regulated protein spots were ex cised from multiple two-dimensional gels. The proteins were concentrated us ing a funnel-gel device before being blotted onto poly(vinylidene difluorid e) membranes for digestion with trypsin before MS and tandem RIS analysis o r for Edman sequence determination. Five proteins in the range 8-20 kDa wer e identified as the small Hsp (sHsp; HspB, C, D, E and H) and three others showed strong sequence similarity to the sHsp family. Two other low molecul ar mass proteins corresponded to GroES1 and GroES2, and five novel proteins were found. Four proteins of approximate to 60 kDa were identified as GroE L2, GroEL4, and GroEL5 and DnaK. An analysis of the heat shock induction of DnaK, of four of the most strongly induced GroESL proteins and six of the sHsp revealed that the proteins could be placed into four distinct regulato ry groups based on the kinetics of protein appearance.