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Lactococcus lactis is of great importance for the nutrition of hundreds of millions of people worldwide. This paper describes the genome sequence of Lactococcus lactis subsp. cremoris MG1363, the lactococcal strain most intensively studied throughout the world. The 2,529,478-bp genome contains 81 pseudogenes and encodes 2,436 proteins. Of the 530 unique proteins, 47 belong to the COG (clusters of orthologous groups) functional category "carbohydrate metabolism and transport," by far the largest category of novel proteins in comparison with L. lactis subsp. lactis IL1403. Nearly one-fifth of the 71 insertion elements are concentrated in a specific 56-kb region. This integration hot-spot region carries genes that are typically associated with lactococcal plasmids and a repeat sequence specifically found on plasmids and in the "lateral gene transfer hot spot" in the genome of Streptococcus thermophilus. Although the parent of L. lactis MG1363 was used to demonstrate lysogeny in Lactococcus, L. lactis MG1363 carries four remnant/satellite phages and two apparently complete prophages. The availability of the L. lactis MG1363 genome sequence will reinforce its status as the prototype among lactic acid bacteria through facilitation of further applied and fundamental research.Lactococcus lactis, a mesophilic fermentative bacterium producing lactic acid from sugar (hexose) fermentation, is an important industrial microorganism with extensive and diverse uses in food fermentation. Strains of L. lactis are used as defined mixtures or in undefined combinations with other lactic acid bacteria (LAB) in the production of fermented milk products. The organism has adapted to growth in milk under stringent human selection for better performance with respect to taste, flavor, and texture of dairy products, and this process continues today (57,98,99). In 1985, the "dairy streptococci" were reclassified into two L. lactis subspecies, Lactococcus lactis subsp. lactis (previously Streptococcus lactis) and Lactococcus lactis subsp. cremoris (previously Streptococcus cremoris), to distinguish them from the streptococci sensu stricto, which contain a number of notorious human pathogens (82, 83).The strain used in this study, L. lactis subsp. cremoris MG1363, is the international prototype for LAB genetics, and the knowledge gained from fundamental research on this strain has been exploited for a wide variety of biotechnological applications. The large and unstable complement of plasmid DNA of the parent strain, L. lactis NCDO712, was eliminated by employing UV treatment and protoplast-curing strategies in the early 1980s (41). The resultant plasmid-free strain, L. lactis MG1363, is robust and genetically amenable, which has facilitated the analysis of introduced lactococcal and heterologous DNA. Sophisticated systems have been developed for the expression of proteins and peptides in this strain, and it has been used as a cell factory for a wide variety of heterologous products (e.g., antimicrobials, including bacteriocins [50], bacteriop...

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A gene of Lactococcus lactis subsp. cremoris MG1363 encoding a peptidoglycan hydrolase was identified in a genomic library of the strain in pUC19 by screening Escherichia coli transformants for cell wall lysis activity on a medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells. In cell extracts of L. lactis MG1363 and several halo-producing E. coli transformants, lytic bands of similar sizes were identified by denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gels containing L. lactis or M. lysodeikticus cell walls. Of these clearing bands, corresponding to the presence of lytic enzymes with sizes of 46 and 41 kDa, the 41-kDa band was also present in the supernatant of an L. lactis culture. Deletion analysis of one of the recombinant plasmids showed that the information specifying lytic activity was contained within a 2,428-bp EcoRV-Sau3A fragment. Sequencing of part of this fragment revealed a gene (acmA) that could encode a polypeptide of 437 amino acid residues. The calculated molecular mass of AcmA (46,564 Da) corresponded to that of one of the lytic activities detected. Presumably, the enzyme is synthesized as a precursor protein which is processed by cleavage after the Ala at position 57, thus producing a mature protein with a size of 40,264 Da, which would correspond to the size of the enzyme whose lytic activity was present in culture supernatants of L. lactis. The N-terminal region of the mature protein showed 60% identity with the N-terminal region of the mature muramidase-2 of Enterococcus hirae and the autolysin of Streptococcus faecalis. Like the latter two enzymes, AcmA contains C-terminal repeated regions. In AcmA, these three repeats are separated by nonhomologous intervening sequences highly enriched in serine, threonine, and asparagine. Genes specifying identical activities were detected in various strains of L. lactis subsp. lactis and L. lactis subsp. cremoris by the SDS-polyacrylamide gel electrophoresis detection assay and PCR experiments. By replacement recombination, an acmA deletion mutant which grew as long chains was constructed, indicating that AcmA is required for cell separation.

A system for generating chromosomal insertions in lactococci is described. It is based on the conditional replication of lactococcal pWV01-derived Ori ؉ RepA ؊ vector pORI19, containing lacZ␣ and the multiple cloning site of pUC19. Chromosomal AluI fragments of Lactococcus lactis were cloned in pORI19 in RepA ؉ helper strain Escherichia coli EC101. The frequency of Campbell-type recombinants, following introduction of this plasmid bank into L. lactis (RepA ؊ ), was increased by combining the system with temperature-sensitive pWV01 derivative pVE6007. Transformation of L. lactis MG1363(pVE6007) with the pORI19 bank of lactococcal chromosomal fragments at the permissive temperature allowed replication of several copies of a recombinant plasmid from the bank within a cell because of the provision in trans of RepA-Ts from pVE6007. A temperature shift to 37؇C resulted in loss of pVE6007 and integration of the pORI19 derivatives at high frequencies. A bank of lactococcal mutants was made in this way and successfully screened for the presence of two mutations: one in the monocistronic 1.3-kb peptidoglycan hydrolase gene (acmA) and one in the hitherto uncharacterized maltose fermentation pathway. Reintroduction of pVE6007 into the Mal ؊ mutant at 30؇C resulted in excision of the integrated plasmid and restoration of the ability to ferment maltose. The integration plasmid (pMAL) was rescued by using the isolated plasmid content of a restored Mal ؉ colony to transform E. coli EC101. Nucleotide sequencing of the 564-bp chromosomal fragment in pMAL revealed an internal part of an open reading frame of which the translated product showed significant homology with ATP-binding proteins MalK of E. coli, Salmonella typhimurium, and Enterobacter aerogenes and MsmK of Streptococcus mutans. This combined use of two types of conditional replicating pWV01-derived vectors represents a novel, powerful tool for chromosomal gene inactivation, targeting, cloning, and sequencing of the labelled gene.Considerable effort in recent years has focussed on the development of gene tagging and targeting techniques by insertions in the lactococcal genome to facilitate chromosomal gene analysis and gene cloning. Chromosomal integration and gene inactivation in Lactococcus lactis have been achieved by using the conjugative transposable elements Tn916, Tn919, and Tn1545 (9). A gene targeting and cloning system based on the Tn919 and Tn916 family of transposons was used for the cloning of streptococcal genes (11,12,38). However, it was subsequently shown (17) that this system could not be successfully applied to lactococci. Although chromosomal mutations in the genes involved in citrate metabolism have been obtained in L. lactis subsp. lactis biovar diacetylactis 18-16 by using Tn919, the activity of this transposon is apparently strain dependent and transposition appears to be site specific in L. lactis MG1363 (17). Recently, a potentially useful system based on the lactococcal insertion sequence IS946 was studied and random integration in L. lactis w...