This review summarizes a collection of lactic acid bacteria that are now undergoing genomic sequencing and analysis. Summaries are presented on twenty different species, with each overview discussing the organisms fundamental and practical significance, environmental habitat, and its role in fermentation, bioprocessing, or probiotics. For those projects where genome sequence data were available by March 2002, summaries include 30 a listing of key statistics and interesting genomic features. These efforts will revolutionize our molecular view of Gram-positive bacteria, as up to 15 genomes from the low GC content lactic acid bacteria are expected to be available in the public domain by the end of 2003. Our collective view of the lactic acid bacteria will be fundamentally changed as we rediscover the relationships and capabilities of these organisms through genomics.

To determine if the food-grade bacterium Lactococcus lactis holds promise as a vaccine antigen delivery vector we have investigated whether this bacterium can be made to produce high levels of a heterologous protein antigen. A regulated expression system has been developed which may be generally suitable for the expression of foreign antigens (and other proteins) in L. lactis. The system utilizes the fast-acting T7 RNA polymerase to transcribe target genes, and provides the first example of the successful use of this polymerase in a Gram-positive bacterium. When the performance of the expression system was characterized using tetanus toxin fragment C (TTFC) up to 22% of soluble cell protein was routinely obtained as TTFC. Mice immunized subcutaneously with L. lactis expressing TTFC were protected from lethal challenge with tetanus toxin. These results show for the first time that L. lactis is able to express substantial quantities of a heterologous protein antigen and that this organism can present this antigen to the immune system in an immunogenic form.

Aims: To test the efficacy of Lactobacillus johnsonii FI9785 in reducing the colonization and shedding of Salmonella enterica serotype Enteritidis, Escherichia coli O78:K80 and Clostridium perfringens in poultry. Methods and Results: Specific pathogen-free chicks (1 day old) were dosed with a single oral inoculum of 1 · 10 9 CFU. Lactobacillus johnsonii FI9785 and 24 h later were challenged in separate experiments with S. Enteritidis (S1400, nal r ) and E. coli O78:K80 (EC34195, nal r ). There were no significant effects against S. Enteritidis whereas colonization of the small intestine by E. coli O78:K80 was reduced significantly. Both S. Enteritidis and E. coli colonized the caeca and colon to levels equivalent to control birds and there was no reduction in shedding as assessed by a semi-quantitative cloacal swabbing technique. Specific pathogen-free chicks (20 day old) were dosed with a single oral inoculum of 1 · 10 9 CFU L. johnsonii FI9785 and 24 h later were challenged with C. perfringens. A single oral dose of L. johnsonii FI9785 was sufficient to suppress all aspects of colonization and persistence of C. perfringens. Conclusions: Lactobacillus johnsonii FI9785 may be given to poultry for use as a competitive exclusion agent to control C. perfringens. Significance and Impact of the Study: Lactobacillus johnsonii FI9785 may be a valuable tool to control the endemic disease of necrotic enteritis, thereby reducing economic losses associated with reduced use of antimicrobials in the poultry industry.

The Lactococcus lactis subsp. lactis 712 lacG gene encoding phospho-beta-galactosidase was isolated from the lactose mini-plasmid pMG820 and cloned and expressed in Escherichia coli and L. lactis. The low phospho-beta-galactosidase activity in L. lactis transformed with high-copy-number plasmids containing the lacG gene contrasted with the high activity found in L. lactis containing the original, low-copy-number lactose plasmid pMG820, and indicated that the original lactose promoter was absent from the cloned DNA. In E. coli the phospho-beta-galactosidase could be overproduced using the strong inducible lambda PL promoter, which allowed a rapid purification of the active enzyme. The complete nucleotide sequence of the L. lactis lacG gene and its surrounding regions was determined. The deduced amino acid sequence was confirmed by comparison with the amino acid composition of the purified phospho-beta-galactosidase and its amino-terminal sequence. This also allowed the exact positioning of the lacG gene and identification of its characteristic Gram-positive translation initiation signals. The homologous expression data and the sequence organization of the L. lactis lacG gene indicate that the gene is organized into a large lactose operon which contains an intergenic promoter located in an inverted repeat immediately preceding the lacG gene. The organization and sequence of the L. lactis lacG gene were compared with those of the highly homologous lacG gene from Staphylococcus aureus. A remarkable bias for leucine codons was observed in the lacG genes of these two species. Heterogramic homology was observed between the deduced amino acid sequence of the L. lactis phospho-beta-galactosidase, that of the functionally analogous E. coli phospho-beta-glucosidase, and that of an Agrobacterium beta-glucosidase (cellobiase).

The widely used plasmid-free Lactococcus lactis strain MG1363 was derived from the industrial dairy starter strain NCDO712. This strain carries a 55.39 kb plasmid encoding genes for lactose catabolism and a serine proteinase involved in casein degradation. We report the DNA sequencing and annotation of pLP712, which revealed additional metabolic genes, including peptidase F, D-lactate dehydrogenase and a-keto acid dehydrogenase (E3 complex). Comparison of pLP712 with other large lactococcal lactose and/or proteinase plasmids from L. lactis subsp. cremoris SK11 (pSK11L, pSK11P) and the plant strain L. lactis NCDO1867 (pGdh442) revealed their close relationship. The plasmid appears to have evolved through a series of genetic events as a composite of pGdh442, pSK11L and pSK11P. We describe in detail a scenario by which the metabolic genes relevant to the growth of its host in a milk environment have been unified on one replicon, reflecting the evolution of L. lactis as it changed its biological niche from plants to dairy environments. The extensive structural instability of pLP712 allows easy isolation of derivative plasmids lacking genes for casein degradation and/or lactose catabolism. Plasmid pLP712 is transferable by transduction and conjugation, and both of these processes result in significant molecular rearrangements. We report the detailed molecular analysis of insertion sequence element-mediated genetic rearrangements within pLP712 and several different mechanisms, including homologous recombination and adjacent deletion. Analysis of the integration of the lactose operon into the chromosome highlights the fluidity of the MG1363 integration hotspot and the potential for frequent movement of genes between plasmids and chromosomes in Lactococcus.

Nisin, a 34 residue lantibiotic produced by strains of Lactococcus lactis subsp. lactis, exerts antimicrobial activity against Gram-positive bacteria at the cytoplasmic membrane. The structural aspects of nisin which facilitate membrane interaction and permeabilization have been investigated in planar lipid bilayers and liposomes with proteolytic fragments and site-directed variants. N-Terminal nisin fragments N1-12 and N1-20 had little effect on phospholipid mobility, on macroscopic electrical conductance, or on calcein release from liposomes. By contrast, the I30W nisin A variant induced a time-dependent reduction in lipid mobility, indicative of nisin-membrane surface interactions, as well as a decline in membrane capacitance, rise in conductance, and calcein release from liposomes. In these respects I30W nisin A is similar to native nisin. Charge substitutions were also engineered to generate K12L and H27K nisin A variants, both of which were similar to I30W nisin A with respect to an overall reduction in phospholipid mobility. While the K12L nisin A variant elicited a higher increase in membrane capacitance and electrical conductance than I30W nisin A, the H27K nisin A variant elicited weaker effects. These results point to a substantial role for intramembrane charged residues in controlling ion flow through nisin-doped membranes. Native nisin and variants elicit an enhanced release of calcein from liposomes composed of the negatively-charged phospholipids cardiolipin and phosphatidylserine, compared with phospholipid bearing no net charge, suggesting that an electrostatic attraction encourages the initial nisin-membrane association. The results are discussed in the context of other recently proposed models for nisin action.

IS905 is a multicopy insertion sequence identified in Lactococcus lactis. It is 1313 bp long, bounded by 28-bp imperfect inverted repeats, and encodes a putative transposase of 391 amino acids. One end of IS905 contains sequences that are potentially promoter active. It displays sequence homology to the IS256 class of elements.

CluA is a 136 kDa surface-bound protein encoded by the chromosomally located sex factor of Lactococcus lactis MG1363 and is associated with cell aggregation linked to high-frequency transfer of the sex factor. To further investigate the involvement of CluA in these phenomena, the cluA gene was cloned on a plasmid, downstream from the lactococcal nisA promoter. In a sex-factor-negative MG1363 derivative, nisin-controlled CluA expression resulted in aggregation, despite the absence of the other genes of the sex factor. Therefore, CluA is the only sex factor component responsible for aggregation. The direct involvement of CluA in the establishment of cell-to-cell contact for aggregate formation was observed by electron microscopy using immunogold-labelled CluA antibodies. Inactivation of cluA in an MG1363 background led to a dramatic decrease in sex factor conjugation frequency compared to the parental strain. Increasing levels of CluA expressed in trans in the cluA-inactivated donor strain facilitated a gradual restoration of conjugation frequency, reaching that of the parental strain. In conclusion, CluA is essential for efficient sex factor transfer in conjugation of L. lactis.