The function of the N-terminal region of the Oenococcus oeni phage fOg44 lysin (Lys44) as an export signal was investigated. We observed that when induced in Escherichia coli, Lys44 was cleaved between residues 27 and 28 in a SecA-dependent manner. Lys44 processing could be blocked by a specific signal peptidase inhibitor and was severely reduced by modification of the cleavage site. The lethal effect of Lys44 expression observed in E. coli was ascribed to the presence of its N-terminal 27-residue sequence, as its deletion resulted in the production of a nontoxic, albeit active, product. We have further established that lytic activity in oenococcal cells was dependent on Lys44 processing. An active protein with the molecular mass expected for the cleaved enzyme was detected in extracts from O. oeni-infected cells. The temporal pattern of its appearance suggests that synthesis and export of Lys44 in the infected host progress along with phage maturation. Overall, these results provide, for the first time, experimental evidence for the presence of a signal peptide in a bacteriophage lysin. Database searches and alignment of protein sequences support the prediction that other known O. oeni and Lactococcus lactis phages also encode secretory lysins. The evolutionary significance of a putative phage lysis mechanism relying on secretory lytic enzymes is tentatively discussed, on the basis of host cell wall structure and autolytic capacity.All tailed bacteriophages with double-stranded DNA genomes appear to accomplish lysis of the host cell by the concerted action of a peptidoglycan hydrolase (referred to as endolysin or lysin) and a small hydrophobic protein (holin) presumed to form nonspecific lesions upon oligomerization in the membrane (for a review, see reference 41). The latter function seems essential to allow access of the lytic enzyme to the cell wall compartment since in the phage lysins examined so far, the presence of a signal peptide (SP) that would target them to the translocase of the general secretion pathway (GSP) has never been demonstrated.We have recently described the sequences of the lysin and holin genes from the Oenococcus oeni bacteriophage fOg44 and noted that the N-terminal region of its putative lysin (Lys44) was highly hydrophobic (23). A similar observation was made earlier concerning a related enzyme from the lactococcal phage Tuc2009 (2). In spite of its hydrophobic character, the function of the N-terminal sequence of the Tuc2009 lysin as a possible SP was not considered, presumably because the presence of a holin gene upstream of lys argued for a standard holin-dependent lysin export mechanism. This assumption was strengthened by the observation that the expression of an almost identical lysin in Escherichia coli (LysB from the Lactococcus lactis phage LC3) did not result in a decrease in culture absorbance unless the corresponding holin was simultaneously induced (3).Interestingly, however, during an attempt to overproduce Lys44 in an easily purifiable form (as a histidine-tagged fusi...

Thirty Leuconostoc oenos strains, representing 28 different isolates, were distributed into 20 genomic groups according to PFGE patterns of restriction digests. The 8 bp-specific enzymes Sfi I, Not I and Asc I cleaved the Leuc. oenos DNA in a mean of 17, 11 and four fragments respectively and Sma I produced more than 50 fragments per genome. The strain differentiating capacity of the four enzymes was similar; only two related genomic groups failed to be distinguished by Asc I or Not I. Genomic relationships between Leuc. oenos strains were quantified by numerical analysis of Not I and Sfi I banding patterns. More than half of the strains, including the starters ML34 and PSU-1, formed a major cluster. The average size of the Leuc. oenos genome was estimated as 1.86 Mb. Although similar values were obtained for the genomes of Leuc. mesenteroides, Leuc. pseudomesenteroides, Leuc. gelidum and Leuc. citreum, a significant divergence between wine and non-wine species was inferred from comparisons of genome cleavage frequencies, determined with five different enzymes.

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Twenty‐two Leuconostoc oenos strains from Portuguese wines and seven strains from other sources were induced with mitomycin C. Bacteriophages were detected in the supernatants of 19 different cultures. Analysis of their host‐range and plating efficiencies suggests that phage typing could be useful for strain identification in Leuc. oenos.

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R . SANTOS, G V I E I R A , M . A . SANTOS AND H. P A V E I A . 1996. T h e close relatedness between17 Leiiconostor oenos bacteriophages, induced with mitomycin C from strains isolated in different geographic regions, was inferred from their morphology, DNA homology and protein composition. The genome of all the phages had cohesive end termini and ranged in size from 36.4 to 40.9 kb. According to the restriction patterns obtained by digestion with five enzymes, the phages were divided in six groups. Lysogenization of a spontaneous phage-cured derivative of Leuc. oenos strain PSU-1 was achieved with 16 phages and the analysis of the lysogens showed that the phage DNA integrates in the host chromosome in one or two sites. T h e att B loci were located on the macrorestriction AscI and No11 fragments of the recipient strain. A survey of Leuc. oenos strains with a phage DNA probe confirmed the lysogenic nature of several, but not all of the original phage hosts. These results are discussed in the light of evidence for the instability of some lysogenic PSU-1 derivatives.

R . SANTOS, G V I E I R A , M . A . SANTOS AND H. P A V E I A . 1996. T h e close relatedness between17 Leiiconostor oenos bacteriophages, induced with mitomycin C from strains isolated in different geographic regions, was inferred from their morphology, DNA homology and protein composition. The genome of all the phages had cohesive end termini and ranged in size from 36.4 to 40.9 kb. According to the restriction patterns obtained by digestion with five enzymes, the phages were divided in six groups. Lysogenization of a spontaneous phage-cured derivative of Leuc. oenos strain PSU-1 was achieved with 16 phages and the analysis of the lysogens showed that the phage DNA integrates in the host chromosome in one or two sites. T h e att B loci were located on the macrorestriction AscI and No11 fragments of the recipient strain. A survey of Leuc. oenos strains with a phage DNA probe confirmed the lysogenic nature of several, but not all of the original phage hosts. These results are discussed in the light of evidence for the instability of some lysogenic PSU-1 derivatives.

The genome structure of six bacteriophages of Oenococcus oeni was compared. Two distinct groups with no apparent restriction site conservation were defined. In members of the alpha group (fOgML34, fOg4029, fOg30 and fOg218) a 7.5 kb region containing the origin of DNA packaging (cos) was highly conserved. Stretches of DNA heterogeneity could also be assigned to particular regions and were mostly evident in the right area of the genomes. fOg44 and fOgPSU1 (beta group) were indistinguishable in the left half of their genomes, including cos, but were markedly dissimilar in other regions. Strong labelling signals detected in cross-hybridizations involving members of different groups were confined to fragments centrally located in their physical maps. The attachment site (attP) of fOg44 was assigned to this conserved region. It is suggested that recombination events at this location may have been important in generating the observed diversity of oenophage genomes.