SummaryIn a previous study, we described the use of transposon Tn917-LTV1 for identi®cation of environmentally regulated promoters in Lactococcus lactis. Here, we report the molecular analysis of one of these promoters, P170, that is upregulated at low pH during the transition to stationary phase. The minimal DNA region required for both promoter activity and pH regulation was mapped to a 51 bp fragment located 7 bp upstream of the transcriptional start site. This fragment lacked the consensus À35 promoter region, but it contained an`extended' À10 promoter region. When a 28 bp segment, containing the consensus À35 region and 22 bp upstream of this in a constitutive promoter, was replaced with the corresponding sequence of P170, the hybrid promoter became regulated by pH and growth phase. This demonstrates that the P170 segment contains a cis-acting sequence involved in the control of promoter regulation. Transcriptional analysis showed that P170 is responsible for the transcription of a monocistronic gene orfX encoding a polypeptide homologous to a hypothetical protein from Bacillus subtilis. Analysis of total RNA from L. lactis grown at constant pH con®rmed that transcription from P170 was induced between pH 6.5 and pH 6.0, but only when the culture entered stationary phase. Deletion analysis and chemical mutagenesis of P170 de®ned a speci®c region within the untranslated mRNA leader that is able to modulate the expression level directed by the P170 promoter. Deletion of a 72 bp HaeIII fragment from this leader region resulted in a 150-to 200-fold increase in the level of gene expression, without affecting the regulation. The functionality was con®rmed by introducing this modulating element downstream of other lactococcal promoters.

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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 ␤-galactosidase on plates at 30؇C. Pulsed-field gel electrophoresis revealed Tn917-LTV1 insertions in at least 13 loci in 15 strains analyzed. Integrants in which ␤-galactosidase expression was regulated by temperature or pH and/or arginine concentration were isolated. In most cases, the regulation observed on plates was reproducible in liquid medium. One integrant, PA170, produces ␤-galactosidase at pH 5.2 but not at pH 7.0, produces more ␤-galactosidase at 15؇C than at 30؇C, and has increased ␤-galactosidase activity in the stationary phase. DNA fragments potentially carrying promoters from selected Lactococcus lactis integrants were cloned in Escherichia coli. A new promoter probe vector, pAK80, containing promoterless ␤-galactosidase genes from Leuconostoc mesenteroides subsp. cremoris and the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid replication region was constructed, and the lactococcal fragments were inserted. Plasmid pAK80 was capable of detecting and discriminating even weak promoters in Lactococcus lactis. When inserted in pAK80, the promoter cloned from PA170 displayed a regulated expression of ␤-galactosidase analogous to the regulation observed in PA170.

The enzyme pyruvate formate-lyase (PFL) from Lactococcus lactis was produced in Escherichia coli and purified to obtain anti-PFL antibodies that were shown to be specific for L. lactis PFL. It was demonstrated that activated L. lactis PFL was sensitive to oxygen, as in E. coli, resulting in the cleavage of the PFL polypeptide. The PFL protein level and its in vivo activity and regulation were shown by Western blotting, enzyme-linked immunosorbent assay, and metabolite measurement to be dependent on the growth conditions. The PFL level during anaerobic growth on the slowly fermentable sugar galactose was higher than that on glucose. This shows that variation in the PFL protein level may play an important role in the regulation of metabolic shift from homolactic to mixed-acid product formation, observed during growth on glucose and galactose, respectively. During anaerobic growth in defined medium, complete activation of PFL was observed. Strikingly, although no formate was produced during aerobic growth of L. lactis, PFL protein was indeed detected under these conditions, in which the enzyme is dispensable due to the irreversible inactivation of PFL by oxygen. In contrast, no oxygenolytic cleavage was detected during aerobic growth in complex medium. This observation may be the result of either an effective PFL deactivase activity or the lack of PFL activation. In E. coli, the PFL deactivase activity resides in the multifunctional alcohol dehydrogenase ADHE. It was shown that in L. lactis, ADHE does not participate in the protection of PFL against oxygen under the conditions analyzed. Our results provide evidence for major differences in the mechanisms of posttranslational regulation of PFL activity in E. coli and L. lactis.

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