Are the Three Chaperonin Operons of Rhizobium leguminosarum Essential?

Abstract

R. leguminosarum contains three operons which are homologous to the essential groE operon of E. coli, cpn-1, cpn-2 and cpn-3. The groE molecular chaperones GroES and GroEL help the folding of some proteins in normally growing cells, and protect cells against damage caused by stresses such as heat shock. In E. coli, both genes are essential. We therefore are studying whether the homologous genes are also required by R. leguminosarum. We have studied the requirement for these three operons by cloning an nptII gene into each one, and attempting to isolate strains where the chromosomal gene is insertionally inactivated. Two approaches have been used: forcing recombination by introduction of a non-compatible plasmid, and selection for single cross-overs of a non-replicating plasmid followed by selection for loss of the plasmid (which carries a sacB gene and hence confers sucrose sensitivity) by a second cross-over event. Our conclusions are as follows: 1. cpn-1 is essential. Although single cross-overs can be obtained, the second double cross-over is never seen. In a control experiment where the nptII gene was inserted downstream of the cpn-1 operon, double cross-overs readily occurred. Southern analysis confirmed that all the strains produced were as expected. No recombinants were found using the plasmid incompatibility method. Thus cpn-1 codes for the housekeeping chaperones, as predicted from the analysis of regulation of this operon. 2. cpn-2 is an enigma. Expression of this operon cannot be found under any conditions which we have studied, but repeated attempts to construct knock-out mutants have failed. Only a very few apparent single cross-over events were found, and those which have so far been analysed have proven to be in the cpn-1 operon. Moreover, single cross-overs with the nptII gene downstream of the operon have also proved impossible to identify. RT-PCR has ruled out transcription of a gene on the opposite strand. 3. cpn-3 appears to be non-essential as knock-outs have been obtained in the cpn10 gene of this operon. As this operon is Nif A regulated, it will be of interest to determine the Nod and Fix phenotype of this strain.