Induction of heavy-metal-transporting CPX-type ATPases during acid adaptation in Lactobacillus bulgaricus.

Lactobacillus bulgaricus is a lactic acid bacteria (LAB) that, through the production of lactic acid, gradually acidifies its environment during growth. In the course of this process, L. bulgaricus acquires an improved tolerance to acidity. A survey of the recently established genome sequence shows that this bacterium possesses few of the pH control functions that have been described in other LAB and raises the question of what other mechanisms could be involved in its adaptation to the decreasing environmental pH. In some bacteria other than LAB, ion transport systems have been implicated in acid adaptation. We therefore studied the expression of this type of transport system during acid adaptation in L. bulgaricus by reverse transcription and real-time quantitative PCR and mapped transcription start sites. Intriguingly, the most significantly induced were three ATPases carrying the CPX signature of heavy-metal transporters. Protein homology and the presence of a conserved sequence motif in the promoter regions of the genes encoding these proteins strongly suggest that they are involved in copper homeostasis. Induction of this system is thought to assist in avoiding indirect damage that could result from medium acidification.

AGMIAL: implementing an annotation strategy for prokaryote genomes as a distributed system.

We have implemented a genome annotation system for prokaryotes called AGMIAL. Our approach embodies a number of key principles. First, expert manual annotators are seen as a critical component of the overall system; user interfaces were cyclically refined to satisfy their needs. Second, the overall process should be orchestrated in terms of a global annotation strategy; this facilitates coordination between a team of annotators and automatic data analysis. Third, the annotation strategy should allow progressive and incremental annotation from a time when only a few draft contigs are available, to when a final finished assembly is produced. The overall architecture employed is modular and extensible, being based on the W3 standard Web services framework. Specialized modules interact with two independent core modules that are used to annotate, respectively, genomic and protein sequences. AGMIAL is currently being used by several INRA laboratories to analyze genomes of bacteria relevant to the food-processing industry, and is distributed under an open source license.

The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution.

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) is a representative of the group of lactic acid-producing bacteria, mainly known for its worldwide application in yogurt production. The genome sequence of this bacterium has been determined and shows the signs of ongoing specialization, with a substantial number of pseudogenes and incomplete metabolic pathways and relatively few regulatory functions. Several unique features of the L. bulgaricus genome support the hypothesis that the genome is in a phase of rapid evolution. (i) Exceptionally high numbers of rRNA and tRNA genes with regard to genome size may indicate that the L. bulgaricus genome has known a recent phase of important size reduction, in agreement with the observed high frequency of gene inactivation and elimination; (ii) a much higher GC content at codon position 3 than expected on the basis of the overall GC content suggests that the composition of the genome is evolving toward a higher GC content; and (iii) the presence of a 47.5-kbp inverted repeat in the replication termination region, an extremely rare feature in bacterial genomes, may be interpreted as a transient stage in genome evolution. The results indicate the adaptation of L. bulgaricus from a plant-associated habitat to the stable protein and lactose-rich milk environment through the loss of superfluous functions and protocooperation with Streptococcus thermophilus.

Lactobacillus casei is able to survive and initiate protein synthesis during its transit in the digestive tract of human flora-associated mice.

Live Lactobacillus casei is present in fermented dairy products and has beneficial properties for human health. In the human digestive tract, the resident flora generally prevents the establishment of ingested lactic acid bacteria, the presence of which is therefore transient. The aim of this work was to determine if L. casei DN-114 001 survives during transit and how this bacterium behaves in the digestive environment. We used the human flora-associated (HFA) mouse model. L. casei DN-114 001 was genetically modified by the introduction of erm and lux genes, encoding erythromycin resistance and luciferase, respectively. For this modified strain (DN-240 041), light emission related to luciferase expression could easily be detected in the contents of the digestive tract. When inoculated into the digestive tract of HFA mice, L. casei (DN-240 041) survives but is eliminated with the same kinetics as an inert transit marker, indicating that it does not establish itself. In pure culture of L. casei, luciferase activities were high in the exponential and early stationary growth phases but decreased to become undetectable 1 day after inoculation. Viability was only slightly reduced even after more than 5 days. After transit in HFA mice, luciferase activity was detected even when 5-day-old L. casei cultures were given to the mice. In culture, the luciferase activity could be restored after 0.5 to 7 h of incubation in fresh medium or milk containing glucose, unless protein synthesis was inhibited by the addition of chloramphenicol or rifampin. These results suggest that in HFA mice L. casei DN-240 041, and thus probably L. casei DN-114 001, is able to initiate new protein synthesis during its transit with the diet. The beneficial properties of L. casei-fermented milk for human health might be related to this protein synthesis in the digestive tract.

Production of growth-inhibiting factors by Lactobacillus delbrueckii.

AIMS: The detection of growth-inhibiting factors produced by Lactobacillus delbrueckii. METHODS AND RESULTS: A bioscreen assay was developed to study the effect of Lact. delbrueckii culture supernatant fluids on the growth of phylogenically or functionally related bacteria in broth cultures. Several growth-inhibiting factors could be distinguished based on differential effects on different test strains, separation by ultrafiltration and sensitivity to heat, proteinase treatment or catalase addition. CONCLUSION: Lactobacillus delbrueckii strain VI1007 was found to produce at least three growth-inhibiting factors, other than lactic acid, when grown under microaerobic conditions in MRS broth. These included H2O2 and a bacteriocin-like, heat- and proteinase-sensitive bactericidal molecule or complex with a molecular weight greater than 50 kDa. A third factor inhibited the growth of Streptococcus thermophilus. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay system used allows the detection of subtle interactions between strains, that are likely to be of ecological importance in mixed cultures but would go unnoticed in classical agar diffusion tests.

tRNATrp as a key element of antitermination in the Lactococcus lactis trp operon.

The expression of the trp operon of Lactococcus lactis is regulated in response to tryptophan availability by a mechanism of transcription antitermination. We present evidence in support of a previously described model involving tRNATrp as a key element in the sensing of tryptophan levels and the realization of the regulatory response to tryptophan limitation. In agreement with this model, two sites of presumed direct interaction between the trp leader transcript and tRNATrp are found to be of crucial importance for efficient antitermination. These correspond to the specifier codon, which presumably interacts with the anticodon in the tRNA, and a sequence complementary to, and presumably interacting with, the acceptor stem of the tRNA. Through these interactions, uncharged tRNATrp is believed to stabilize an antiterminator conformation of the trp leader transcript, thus allowing transcription and expression of the structural genes of the operon. For the first time, we present direct evidence that it is the ratio of uncharged to charged tRNA that is important for the regulation of antitermination, rather than the absolute amount of uncharged tRNA. In addition, our results indicate that the codon-anticodon interaction, although contributing largely to the efficiency of the regulatory response, is not strictly indispensable, which suggests the existence of additional interactions between mRNA and tRNA. Finally, we describe a possible additional level of regulation, superimposed and dependent on tRNA-mediated anti-termination control, that is based on the processing of the trp leader transcript. Together with the regulation mechanisms described earlier for the Escherichia coli and Bacillus subtilis trp operons, this constitutes the third different mechanism of transcript elongation control found to be involved in the regulation of an operon of which the structural genes are highly conserved.

Identification of int and attP on the genome of lactococcal bacteriophage Tuc2009 and their use for site-specific plasmid integration in the chromosome of Tuc2009-resistant Lactococcus lactis MG1363.

The DNA sequence of the int-attP region of the small-isometric-headed lactococcal bacteriophage Tuc2009 is presented. In this region, an open reading frame, int, which potentially encodes a protein of 374 amino acids, representing the Tuc2009 integrase, was identified. The nucleotide sequence of the bacteriophage attachment site, attP, and the sequences of attB, attL, and attR in the lysogenic host Lactococcus lactis subsp. cremoris UC509 were determined. A sequence almost identical to the UC509 attB sequence was found to be present in the plasmid-free Tuc2009-resistant L. lactis subsp. cremoris MG1363. This site could be used for the site-specific integration of a plasmid carrying the Tuc2009 int-attP region in the chromosome of MG1363, thereby demonstrating that the application of chromosomal insertion vectors based on bacteriophage integration functions is not limited to the prophage-cured original host strain of the phage.

Identification of the putative repressor-encoding gene cI of the temperate lactococcal bacteriophage Tuc2009.

The putative repressor-encoding gene cI of the temperate lactococcal bacteriophage Tuc2009 was cloned and sequenced. In the inferred amino-acid sequence, two domains can be recognized, one of which shows homology to DNA-binding domains of various regulatory proteins, while the other is thought to be involved in oligomerisation.

Molecular characterization of lactococcal bacteriophage Tuc2009 and identification and analysis of genes encoding lysin, a putative holin, and two structural proteins.

Bacteriophage Tuc2009 is a temperate bacteriophage with a small isometric head and is isolated from Lactococcus lactis subsp. cremoris UC509. The phage genome is packaged by a headful mechanism, giving rise to circularly permuted molecules with terminal redundancy. The unit genome size is approximately 39 kb. A map of the phage genome on which several determinants could be localized was constructed: pac, the site of initiation of DNA packaging; lys (1,287 bp), specifying the phage lysin; S (267 bp), specifying a putative holin; and mp1 (522 bp) and mp2 (498 bp), each specifying one of the phage's structural proteins. lys, S, mp1, and mp2 were further characterized. lys and S are partially overlapping and appear to be part of one operon. The lysin shows homology to the lysins of the Streptococcus pneumoniae phages Cp-9, Cp-1, and Cp-7. The putative holin, which is thought to be involved in the release of lysin from the cytoplasm, contains two strongly hydrophobic presumptive transmembrane domains and a highly charged C-terminal domain.

Lysozyme expression in Lactococcus lactis.

Three lysozyme-encoding genes, one of eukaryotic and two of prokaryotic origin, were expressed in Lactococcus lactis subsp. lactis. Hen egg white lysozyme (HEL) could be detected in L. lactis lysates by Western blotting. No lysozyme activity was observed, however, presumably because of the absence of correctly formed disulphide bonds in the L. lactis product. The functionally related lysozymes of the E. coli bacteriophages T4 and lambda were produced as biologically active proteins in L. lactis. In both cases, the highest expression levels were obtained using configurations in which the bacteriophage lysozyme genes had been translationally coupled to a short open reading frame of lactococcal origin. Both enzymes, like HEL, may prevent the growth of food-spoilage bacteria.

Gene expression in Lactococcus lactis.

Lactic acid bacteria are of major economic importance, as they occupy a key position in the manufacture of fermented foods. A considerable body of research is currently being devoted to the development of lactic acid bacterial strains with improved characteristics, that may be used to make fermentations pass of more efficiently, or to make new applications possible. Therefore, and because the lactococci are designated 'GRAS' organisms ('generally recognized as safe') which may be used for safe production of foreign proteins, detailed knowledge of homologous and heterologous gene expression in these organisms is desired. An overview is given of our current knowledge concerning gene expression in Lactococcus lactis. A general picture of gene expression signals in L. lactis emerges that shows considerable similarity to those observed in Escherichia coli and Bacillus subtilis. This feature allowed the expression of a number of L. lactis-derived genes in the latter bacterial species. Several studies have indicated, however, that in spite of the similarities, the expression signals from E. coli, B. subtilis and L. lactis are not equally efficient in these three organisms.

A possible contribution of mRNA secondary structure to translation initiation efficiency in Lactococcus lactis.

Gene expression signals derived from Lactococcus lactis were linked to lacZ-fused genes with different 5'-nucleotide sequences. Computer predictions of mRNA secondary structure were combined with lacZ expression studies to direct base-substitutions that could possibly influence gene expression. Mutations were made such that the DNA sequence upstream of the ATG start codon was not changed. Moreover, care was taken that the substitutions, which were all within the first six codons, neither affected the amino acid sequence of the gene product nor introduced codons rarely used in L. lactis. The results suggest that mRNA secondary structure contributes to the efficiency of translation initiation in L. lactis.

Distance-dependent translational coupling and interference in Lactococcus lactis.

The possibility of raising the expression level of a heterologous gene in Lactococcus lactis by exploiting the principle of translational coupling was investigated. For this purpose, the Escherichia coli lacZ gene was transcriptionally fused to a short open reading frame (ORF) of lactococcal origin. A Shine-Dalgarno (SD) sequence was introduced at the boundary of the two ORFs. In a series of otherwise identical plasmids, the relative positions of the translational stop codon of the upstream ORF and the translational start codon of the downstream ORF (lacZ) were varied. The expression of lacZ gradually increased as the stop and start codons were placed in closer proximity. A concomitant switch from translational interference to translational coupling was observed. Best results were obtained with partially overlapping stop and start codons. It is concluded that the principle of translational coupling offers good possibilities to increase the level of heterologous gene expression in L. lactis.

Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease.

The Bacillus subtilis nprE gene lacking its own promoter sequence was inserted in the lactococcal expression vector pMG36e. Upon introduction of the recombinant plasmid into Lactococcus lactis subsp. lactis strain MG1363, neutral protease activity could be visualized by the appearance of large clearing zones around colonies grown on milk agar plates. By measuring the activities of the neutral protease and the intracellular enzyme lactate dehydrogenase in culture supernatants and cell fractions, it was demonstrated that the neutral protease was actively secreted into the growth medium. This was corroborated by using the Western blot (immunoblot) technique, which showed the presence of the mature form of the neutral protease in the culture supernatant. On the basis of these results, it is concluded that the B. subtilis neutral protease gene was expressed in L. lactis and that the gene product was secreted into the growth medium and was apparently correctly processed to produced a biologically active protein. The secretion of this particular enzyme may be helpful in achieving accelerated cheese ripening.

Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis.

A pair of vectors for expression of heterologous genes in Lactococcus lactis was constructed. In addition to an origin of replication that has a broad host range, these vectors contain a multiple cloning site flanked by gene expression signals originating from L. lactis subsp. cremoris Wg2. The two vectors, about 3.7 kilobase pairs in size, differ only in the type of antibiotic resistance they confer to their hosts. pMG36 carries a kanamycin resistance marker, which was replaced by an erythromycin resistance marker in pMG36e. As an example of the use of these vectors, the hen egg white lysozyme-coding sequence was inserted. A fusion protein of the expected size was detected in a transformed L. lactis subsp. lactis strain by using Western blotting (immunoblotting).