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.

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 Lactococcus lactis pfl gene, encoding pyruvate formate-lyase (PFL), has been cloned and characterized. The deduced amino acid sequence of the L. lactis PFL protein showed high similarity to those of other bacterial PFL proteins and included the conserved glycine residue involved in posttranslational activation of PFL. The genetic organization of the chromosomal pfl region in L. lactis showed differences from other characterized pfl loci, with an upstream open reading frame independently transcribed in the same orientation as the pfl gene. The gene coding for PFL-activase (act), normally found downstream of pfl, was not identified in L. lactis. Analysis of pfl expression showed a strong induction under anaerobiosis at the transcriptional level independent of the growth medium used. During growth with galactose, pfl showed the highest levels of expression. Constructed L. lactis pfl strains were unable to produce formate under anaerobic growth. Higher levels of diacetyl and acetoin were produced anaerobically in the constructed Lactococcus lactis subsp. lactis biovar diacetylactis pfl strain.Pyruvate has a key role in the metabolic network of Lactococcus lactis. In this industrially relevant organism, sugars are metabolized predominantly to generate energy and as a carbon source. The catabolism of sugars leads to pyruvate, whose further metabolism determines the nature of the fermentation (see reference 5 for a review). Lactate is the major end product responsible for acidification, while other minor metabolites, mainly diacetyl, are responsible for aroma in fermented milk products. Several genes involved in pyruvate metabolism have been studied for their role in the production of diacetyl (Fig.

An important criterion for the selection of a probiotic bacterial strain is its ability to adhere to the mucosal surface. Adhesion is usually mediated by proteins or other components located on the outer cell surface of the bacterium. In the present study we characterized the adhesive properties of two classical intracellular enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase (ENO) isolated from the outer cell surface of the probiotic bacterium Lactobacillus plantarum 299v. None of the genes encoded signal peptides or cell surface anchoring motifs that could explain their extracellular location on the bacterial surface. The presence of the glycolytic enzymes on the outer surface was verified by western blotting using polyclonal antibodies raised against the specific enzymes. GAPDH and ENO showed a highly specific binding to plasminogen and fibronectin whereas GAPDH but not ENO showed weak binding to mucin. Furthermore, a pH dependent and specific binding of GAPDH and ENO to intestinal epithelial Caco-2 cells at pH 5 but not at pH 7 was demonstrated. The results showed that these glycolytic enzymes could play a role in the adhesion of the probiotic bacterium L. plantarum 299v to the gastrointestinal tract of the host. Finally, a number of probiotic as well non-probiotic Lactobacillus strains were analyzed for the presence of GAPDH and ENO on the outer surface, but no correlation between the extracellular location of these enzymes and the probiotic status of the applied strains was demonstrated.

Mutations ofEscherichia coli from sensitivity to nalidixic acid resistance were studied by fluctuation analysis.The mutant distributions in replicate cultures were not significantly affected either by the age of the carbon-starved preculture used for inocula or by the inoculum size. The data from 23 fluctuation tests (48 cultures each) were pooled. The mean number of mutations per culture was estimated to be 0.71 from the fraction of cultures without mutants or 0.74 and 0.77 by maximum-likelihood estimation based on the two models under consideration. When the pooled data were compared with the theoretical expectations, the fits were unsatisfactory (P < 0.005). The lack of fit was caused mainly by too high a frequency of cultures with between 17 and 32 mutants and too high a frequency of cultures with more than 128 mutants. Possible reasons for the lack of fit and its implications with respect to estimation of mutation rates from fluctuation tests are discussed.The statistical analysis of bacterial mutations came into prominence with the publication of Luria and Delbruck (16); the system they studied was that of resistance to bacteriophage Ti in Escherichia coli. When a culture of sensitive E. coli is plated in the presence of an excess of the phage, although virtually all the bacteria are killed, a small number survive and give rise to colonies on the plate. The problem analyzed by Luria and Delbruck was whether the survivors were the result of mutations that occurred prior to the plating, i.e., during the growth of the culture, or whether they occurred after plating, i.e., as a direct consequence of the bacteria being exposed to the phage. These two principal conceptions may be labelled the pre-and postadaptive theories, respectively (7). Luria and Delbruck studied the problem using mathematical models. If the mutation occurred postadaptively, the distribution of mutants in a number of replicate (parallel) cultures should be a Poisson distribution, provided that all bacteria have a small, finite probability of mutating to become resistant after the exposure to the phage. The preadaptive mutation theory leads to a quite different prediction. Although the replicate cultures are the same with respect to the total size of population, some may have experienced a mutation at an early generation and thereby yield a large number of mutants, while with others, a mutation may have occurred just prior to the plating so that only one or a few mutants are present in these cultures. The difference in the predictions of the pre-and postadaptive mutation theories could therefore be reduced to a prediction of the variance of the number of mutants in replicate cultures: the postadaptive theory predicted that the variance would equal the mean, whereas the preadaptive mutation theory predicted that the variance would be much larger, owing to a few cultures yielding a large number of mutants.Luria (45)45932809. from each culture. They did not correct for sampling error, nor did they deduce the theoretical distribution expected fr...