Proteome analysis of heat shock protein expression in Bradyrhizobium japonicum

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Abstract
A set of 19 heat shock proteins (Hsp) was observed – by subtractive two‐dimensional gel electrophoresis – to be induced when Bradyrhizobium japonicum, the nitrogen‐fixing root‐nodule symbiont of soybean, was temperature up‐shifted from 28 °C to 43 °C. Up‐regulated protein spots were excised from multiple two‐dimensional gels. The proteins were concentrated using a funnel‐gel device before being blotted onto poly(vinylidene difluoride) membranes for digestion with trypsin before MS and tandem MS analysis or for Edman sequence determination. Five proteins in the range 8–20 kDa were 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 molecular mass proteins corresponded to GroES1 and GroES2, and five novel proteins were found. Four proteins of ≈ 60 kDa were identified as GroEL2, 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 regulatory groups based on the kinetics of protein appearance.