CLONING AND CHARACTERIZATION OF MULTIPLE GROEL CHAPERONIN-ENCODING GENES IN RHIZOBIUM-MELILOTI

Heat-shock treatment of Rhizobium meliloti cells causes major enhancem ent in the synthesis of several proteins with apparent molecular weigh ts in the range of 58 60 kDa. Using the polymerase chain reaction and degenerate oligodeoxyribonucleotide primers for conserved regions of t he 60-kDa heat-shock protein (HSP60) or GroEL protein family, a 0.6-kb probe for the R. meliloti hsp60 gene was prepared. Southern blot anal ysis of R. meliloti DNA digested with different restriction enzymes an d hybridized to R. meliloti hsp60 probes indicated the presence of bet ween four and five hsp60 or groEL in this species. From the cloning an d sequencing of several of these fragments, we have been able to deduc e the complete nucleotide sequences of three groEL in R. meliloti. The deduced amino acid (aa) sequences of these proteins show extensive si milarity to each other (78 85% aa identity) and to other GroEL homolog ues. In the upstream regions of two of the groEL, but not the third, o pen reading frames corresponding to GroES proteins were also identifie d. Analysis of various prokaryotic GroEL sequences suggests that the m ultiple groEL of R. meliloti have evolved by means of gene duplication events within this or a related group of organisms. Results presented in this paper also show that some of the groEL in R. meliloti are loc ated on the two megaplasmids present in these cells. The presence of m ultiple GroEL homologues in R. meliloti suggests a possible role of th e GroEL or HSP60 chaperonins in the nodulation (symbiosis) and nitroge n fixation processes.