CLONING AND PARTIAL CHARACTERIZATION OF REGULATED PROMOTERS FROM LACTOCOCCUS-LACTIS TN917-LACZ INTEGRANTS WITH THE NEW PROMOTER PROBE VECTOR, PAK80

Transposon Tn917-LTV1 was used to produce a collection of Lactococcus lactis strains with fusion of a promoterless lacZ gene to chromosomal loci. Screening 2,500 Tn917-LTV1 integrants revealed 222 that express beta-galactosidase on plates at 30 degrees C. Pulsed-field gel electro phoresis revealed Tn917-LTV1 insertions in at least 13 loci in 15 stra ins analyzed. Integrants in which beta-galactosidase expression was re gulated by temperature or pH and/or arginine concentration were isolat ed. In most cases, the regulation observed on plates was reproducible in liquid medium, One integrant, PA170, produces beta-galactosidase at pH 5.2 but not at pH 7.0, produces more beta-galactosidase at 15 degr ees C than at 30 degrees C, and has increased beta-galactosidase activ ity in the stationary phase. DNA fragments potentially carrying promot ers from selected Lactococcus lactis integrants were cloned in Escheri chia coli. A new promoter probe vector, pAK80, containing promoterless beta-galactosidase genes from Leuconostoc mesenteroides subsp. cremor is and the Lactococcus lactis subsp. lactis biovar diacetylactis citra te plasmid replication region was constructed, and the lactococcal fra gments were inserted. Plasmid pAK80 was capable of detecting and discr iminating even weak promoters in Lactococcus lactis. When inserted in pAK80, the promoter cloned from PA170 displayed a regulated expression of beta-galactosidase analogous to the regulation observed in PA170.