The Oenococcus oeni clpX homologue is a heat shock gene preferentially expressed in exponential growth phase

Using degenerated primers from conserved regions of previously studied clpX gene products, we cloned the clpX gene of the malolactic bacterium Oenococ cus oeni. The clpX gene was sequenced, and the deduced protein of 413 amino acids (predicted molecular mass of 45,650 Da) was highly similar to previo usly analyzed clpX gene products from other organisms. An open reading fram e located upstream of the clpX gene was identified as the tig gene by simil arity of its predicted product to other bacterial trigger factors. ClpX was purified by using a maltose binding protein fusion system and was shown to possess an ATPase activity, Northern analyses indicated the presence of tw o independent 1.6-kb monocistronic clpX and tig mRNAs and also showed an in crease in clpX mRNA amount after a temperature shift from 30 to 42 degrees C. The clpX transcript is abundant in the early exponential growth phase an d progressively declines to undetectable levels in the stationary phase. Th us, unlike hsp18, the gene encoding one of the major small heat shock prote ins of Oenococcus oeni, clpX expression is related to the exponential growt h phase and requires de novo protein synthesis. Primer extension analysis i dentified the 5' end of clpX mRNA which is located 408 nucleotides upstream of a putative AUA start codon. The putative transcription start site allow ed identification of a predicted promoter sequence with a high similarity t o the consensus sequence found in the housekeeping gene promoter of gram-po sitive bacteria as well as Escherichia coli.