H. Israelsen et al., CLONING AND PARTIAL CHARACTERIZATION OF REGULATED PROMOTERS FROM LACTOCOCCUS-LACTIS TN917-LACZ INTEGRANTS WITH THE NEW PROMOTER PROBE VECTOR, PAK80, Applied and environmental microbiology, 61(7), 1995, pp. 2540-2547
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.