Molecular characterization of the pH‐inducible and growth phase‐dependent promoter P170 of Lactococcus lactis

Abstract

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.

Inactivation of the Stress- and Starvation-Inducible gls24 Operon Has a Pleiotrophic Effect on Cell Morphology, Stress Sensitivity, and Gene Expression in Enterococcus faecalis

“…Whereas Ϫ10 and Ϫ35 boxes close to the consensus of A -dependent promoters have been found nearby the transcriptional start site in P1, P2 is characterized by less-conserved sequences in these regions. In L. lactis, a promoter upregulated during transition into stationary phase (P170) has recently been described (42). P170 controls the expression of a monocistronic gene orfX (encoding a protein of unknown function) and may be induced by a hithertounknown transcriptional factor.…”

Specific Delayed-Type Hypersensitivity Responses to ESAT-6 Identify Tuberculosis-Infected Cattle

“…PSM631 (Biotechnological Institute, Horsholm, Denmark), a mutant strain of MG1363 (9), was transformed with pAMJ752ESAT-6. A growth-phase-dependent promoter (P170) up-regulated the synthesis of ESAT-6 at a constant pH of 6.0 during the transition to stationary phase (17), and a signal sequence from the usp45 gene promoted secretion (26). Compared to the monomeric molecule, dimeric ESAT-6 contains six additional amino acids: aspartic acid, threonine, arginine and serine in the N-terminal end and arginine and serine between the two monomers.…”

A Plasmid Selection System in Lactococcus lactis and Its Use for Gene Expression in L. lactis and Human Kidney Fibroblasts

“…As a reporter gene, we used nucB fused to the optimized SP3mut2 signal peptide sequence. The reporter gene was transcribed from the pH-and growth-phase-regulated P170 promoter (28). We analyzed the level of nuclease production using this reporter gene in two vectors, both containing the theta-type citrate replicon but harboring either the hom-thrB-complementing marker (pJAG6) or the Ery resistance marker (pSMBI109).…”

“…A suitable choice of host and production conditions is important for the manufacture of a pharmaceuticalgrade product. We have recently developed a plasmid expression system for use in the lactic acid bacterium Lactococcus lactis (28). In this paper, we present a nonantibiotic alternative for selection of plasmid-harboring bacterial cells for use in heterologous gene expression.…”