THE ISOLATION OF NOVEL HEAT-SHOCK GENES IN LACTOCOCCUS-LACTIS USING RNA SUBTRACTIVE HYBRIDIZATION

Lactococcus lactis is subjected to heat shock (hs) during cheese manuf acturing. A number of conserved hs genes have been cloned and studied in this organism, although no regulatory gene, e.g. alternative sigma factor, has been identified. RNA subtractive hybridization was used to identify genes expressed very early when L. lactis MG1363 was shifted from 30 to 43 degrees C. P-32-labeled cDNA synthesized from RNA isola ted from hs cells at 43 degrees C was mixed with an excess vegetative RNA and the mixture was directly used as a probe after a short hybridi zation step. Northern analysis revealed a moderate induction for the p robes used, and low expression was also detected in non-hs cells, demo nstrating the applicability of this technique for the detection of dif ferentially expressed genes. The probes were used to identify genomic library clones containing the corresponding genes. Among the five clon es studied, a cell division operon including a putative ftsZ homolog ( pJAK2) was identified. Additionally, a putative hsp86 homolog (pJAK3), three different transposase encoding genes (pJAK1 and pJAK3), a gene coding for a deoR-like transcriptional repressor (pJAK4) and a putativ e regulatory gene that showed homology to an alkaline shock protein (p JAK5) were characterized.