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For this study, an in-depth review of the classification of Lactococcus lactis phages was performed. Reference phages as well as unclassified phages from international collections were analyzed by stringent DNA-DNA hybridization studies, electron microscopy observations, and sequence analyses. A new classification scheme for lactococcal phages is proposed that reduces the current 12 groups to 8. However, two new phages (Q54 and 1706), which are unrelated to known lactococcal phages, may belong to new emerging groups. The multiplex PCR method currently used for the rapid identification of phages from the three main lactococcal groups (936, c2, and P335) was improved and tested against the other groups, none of which gave a PCR product, confirming the specificity of this detection tool. However, this method does not detect all members of the highly diverse P335 group. The lactococcal phages characterized here were deposited in the Félix d'Hérelle Reference Center for Bacterial Viruses and represent a highly diverse viral community from the dairy environment.

We report the genetic organisation of six prophages present in the genome of Lactococcus lactis IL1403. The three larger prophages (36-42 kb), belong to the already described P335 group of temperate phages, whereas the three smaller ones (13-15 kb) are most probably satellites relying on helper phage(s) for multiplication. These data give a new insight into the genetic structure of lactococcal phage populations. P335 temperate phages have variable genomes, sharing homology over only 10-33% of their length. In contrast, virulent phages have highly similar genomes sharing homology over >90% of their length. Further analysis of genetic structure in all known groups of phages active on other bacterial hosts such as Escherichia coli, Bacillus subtilis, MYCOBACTERIUM: and Streptococcus thermophilus confirmed the existence of two types of genetic structure related to the phage way of life. This might reflect different intensities of horizontal DNA exchange: low among purely virulent phages and high among temperate phages and their lytic homologues. We suggest that the constraints on genetic exchange among purely virulent phages reflect their optimal genetic organisation, adapted to a more specialised and extreme form of parasitism than temperate/lytic phages.

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The genes for biosynthesis of the branched-chain amino acids leucine, isoleucine, and valine in Lactococcus lactis subsp. lactis NCDO2118 were characterized by cloning, complementation in Escherichia coli and Bacilus subtilis, and nucleotide sequence analysis. Nine structural genes are clustered on a 12-kb DNA fragment in the order leuABCD ilvDBNCA. Upstream of these genes, the nucleotide sequence suggests the existence of regulation by transcriptional attenuation. Between the leuD and ilvD genes is an unexpected gene, encoding a protein which belongs to the ATP-binding cassette protein superfamily.The branched-chain amino acid (BCAA) pathway, by which leucine, isoleucine, and valine are synthesized, has been widely studied in bacteria, fungi, and plants (8,31,59,60). Regulation of the expression of BCAA genes is complex because of the common steps for synthesis of the three amino acids, and the pathway is often presented as a model for organization and regulation studies. However, the sequences of all of the genes from the same organism have never been reported. Organization of the genes of the BCAA pathway has been characterized for Escherichia coli (59), Salmonella typhimurium (59), Bacillus subtilis (37, 62, 63), Corynebacterium glutamicum (9), and Staphylococcus aureus (44). On the E. coli chromosome, the genes are located in three clusters (3). The largest, at 85 min, is organized into one large and two small transcription units, comprising the ilvGMEDA, ilvY, and ilvC genes, respectively (36, 64). Another cluster, at 2 min, is composed of two transcription units, comprising the ilvIH and leuACBD genes (17,56), and the last cluster, at 82 min, groups the ilvBN genes in a single transcription unit (65). A similar organization is found in other enterobacteriaceae (12). In B. subtilis, ilvBNC and leuACBD genes are found in one chromosomal region (37) and ilvAD genes are found in another (46). Three noncontiguous chromosomal fragments cloned from C. glutamicum carry five BCAA genes, ilvCBN, ilvA, and ilvE (9). In S. aureus, eight structural genes are clustered in the order ilvABCD leuABCD, as found by genetic mapping (44). Here we report the cloning, characterization, and sequences of L. lactis subsp. lactis BCAA genes. Similar analyses of the genes of the tryptophan and histidine biosynthesis pathways are described in the accompanying reports (5, 10). MATERIALS AND METHODSThe bacterial strains and plasmids used are described in Table 1. Media and growth conditions and DNA cloning and manipulation procedures are described in an accompanying report (5). The reported sequence was determined for both strands. Restrictionless B. subtilis strains mutated in the ilvA gene were constructed by transforming MT119-competent cells with pHV438 (41) r-m-Cmr clone was designated IL2685. A restrictionless B. subtilis ilvD4 leuB6 double mutant was constructed by congression, using GSY276 DNA to transform 1012 competent cells to methionine independence. Transformants were further tested for isoleucine and leucine requirements...