CLONING AND SEQUENCING OF THE LACTOCOCCUS-LACTIS SUBSP LACTIS-GROESL OPERON

The operon (groESL) coding for the Lactococcus lactis subsp. lactis heat-shock proteins GroEL and GroES, has been isolated and its complete nucleotide (nt) sequence determined. A set of degenerate PCR primers, deduced from amino acids which are conserved in a number of prokaryotic GroELs, were synthesized and used to amplify a 957-bp fragment. This PCR fragment was used as a probe to isolate a 5.0-kb EcoRI chromosomally derived fragment. A region of this 5.0-kb EcoRI fragment was sequenced and revealed that the groES gene was located 5' to groEL. This sequence was then used to design a set of inverse PCR primers and a 2.5-kb HindIII fragment was cloned which contained the region 5' to groEL. The complete nt sequence of the groESL operon was determined from overlapping fragments. It revealed that the groESL operon was preceded by a stem-loop structure and the promoter appears similar to most L. lactis subsp. lactis and other Gram+ bacterial promoters. Northern analysis demonstrated that the groESL operon is under tight regulation and a dramatic induction of mRNA synthesis occurs within 15 min after heat shock.