A physical map of the chromosome of Oenocccus oeni PSU-1 was constructed. This represents the fir... more A physical map of the chromosome of Oenocccus oeni PSU-1 was constructed. This represents the first map for a strain of this species. A total of 37 restriction sites for the rare-cutting endonucleases Ascl, Fsel, Notl and Sfil were mapped on the chromosome, which was found to be circular with an estimated size of 1857 kb. Fragment order was determined using several approaches: analysis of partial and double digestions, two-dimensional pulsed-field gel electrophoresis, isolation of linking clones, and Southern hybridization with labelled restriction fragments both from PSU-1 and from O. oeni strain GM. Oenococcal genes alsS/alsD, mleA and mir, two phage attachment sites and recurrent sequences such as IS 1165-like elements and rrn loci were located on the physical map. Specific fragments hybridizing with gene probes from Lactococcus lactis, Leuconostoc mesenteroides and Bacillus subtilis were also identified. The two ribosomal operons have been precisely located and their transcripti...
ABSTRACTThe results reported here have identifiedyueBas the essential gene involved in irreversib... more ABSTRACTThe results reported here have identifiedyueBas the essential gene involved in irreversible binding of bacteriophage SPP1 toBacillus subtilis. First, a deletion in an SPP1-resistant (pha-2) strain, covering most of theyueBgene, could be complemented by a xylose-inducible copy ofyueBinserted atamyE. Second, disruption ofyueBby insertion of a pMutin4 derivative resulted in a phage resistance phenotype regardless of the presence or absence of IPTG (isopropyl-β-d-thiogalactopyranoside). YueB homologues are widely distributed in gram-positive bacteria. The protein Pip, which also serves as a phage receptor inLactococcus lactis, belongs to the same family.yueBencodes a membrane protein of ∼120 kDa, detected in immunoblots together with smaller forms that may be processed products arising from cleavage of its long extracellular domain. Insertional inactivation ofyueBand the surrounding genes indicated thatyueBis part of an operon which includes at least the upstream genesyukE,yukD,...
The intrinsic resistance of Oenococcus oeni cells to the secreted endolysin from oenophage fOg44 ... more The intrinsic resistance of Oenococcus oeni cells to the secreted endolysin from oenophage fOg44 (Lys44) was investigated. Experiments with several antimicrobials support the hypothesis that the full activity of Lys44 requires sudden ion-nonspecific dissipation of the proton motive force, an event undertaken by the fOg44 holin in the phage infection context.
The function of the N-terminal region of the Oenococcus oeni phage fOg44 lysin (Lys44) as an expo... more 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 prov...
Bacteriophage SPP1 targets the host cell membrane protein YueB to irreversibly adsorb and infect ... more Bacteriophage SPP1 targets the host cell membrane protein YueB to irreversibly adsorb and infect Bacillus subtilis . Interestingly, SPP1 still binds to the surface of yueB mutants, although in a completely reversible way. We evaluated here the relevance of a reversible step in SPP1 adsorption and identified the receptor(s) involved. We show that reversible adsorption is impaired in B. subtilis mutants defective in the glucosylation pathway of teichoic acids or displaying a modified chemical composition of these polymers. The results indicate that glucosylated poly(glycerolphosphate) cell wall teichoic acid is the major target for SPP1 reversible binding. Interaction with this polymer is characterized by a fast adsorption rate showing low-temperature dependence, followed by a rapid establishment of an equilibrium state between adsorbed and free phages. This equilibrium is basically determined by the rate of phage dissociation, which exhibits a strong dependence on temperature compati...
... Bac-teriophages in wine were first detected by Somi et al. ... The pelleted phage particles w... more ... Bac-teriophages in wine were first detected by Somi et al. ... The pelleted phage particles were resus-pended in 2-3 ml of TM buffer (0.05 mol I-' Tris.CI, pH 7.8, 0.01 rnol I-' MgS0.+.7H20) and purified in CsCl step gradients as described by Sambrook et nl. (1989). ...
International Journal of Systematic and Evolutionary Microbiology, 2006
Six strains of lactic acid bacteria (LAB) were isolated from a ripe fig. These strains constitute... more Six strains of lactic acid bacteria (LAB) were isolated from a ripe fig. These strains constituted a highly homogeneous, but distinct, cluster that was separate from other LAB species in a polyphasic approach including dot-blot DNA–DNA hybridization, SDS-PAGE whole-cell protein profiling, carbohydrate fermentation ability, growth characteristics, enzymic profiling, pulsed-field gel electrophoresis macrorestriction analysis and RFLPs. Phylogenetic analysis based on 16S rRNA gene sequencing positioned a representative strain, LC51T, in a distinct line of descent within the recently described clade comprising Leuconostoc ficulneum, Leuconostoc fructosum and Leuconostoc durionis; L. ficulneum was its closest neighbour (98 % sequence similarity). DNA–DNA hybridization values and chemotaxonomic and biochemical characteristics, including enzymic profiles detected with API ZYM microtubes, confirmed that this group of strains is distinct from L. ficulneum and represents a novel species withi...
The nucleotide sequence of a DNA fragment previously shown to contain the attachment site (attP) ... more The nucleotide sequence of a DNA fragment previously shown to contain the attachment site (attP) of Oenococcus oeni phage fOg44 (Santos et al., 1998. Arch. Virol. 143, 523–536) has been determined. Sequence analysis indicated that this 6226bp EcoRI fragment harbours ...
A physical map of the chromosome of Oenocccus oeni PSU-1 was constructed. This represents the fir... more A physical map of the chromosome of Oenocccus oeni PSU-1 was constructed. This represents the first map for a strain of this species. A total of 37 restriction sites for the rare-cutting endonucleases Ascl, Fsel, Notl and Sfil were mapped on the chromosome, which was found to be circular with an estimated size of 1857 kb. Fragment order was determined using several approaches: analysis of partial and double digestions, two-dimensional pulsed-field gel electrophoresis, isolation of linking clones, and Southern hybridization with labelled restriction fragments both from PSU-1 and from O. oeni strain GM. Oenococcal genes alsS/alsD, mleA and mir, two phage attachment sites and recurrent sequences such as IS 1165-like elements and rrn loci were located on the physical map. Specific fragments hybridizing with gene probes from Lactococcus lactis, Leuconostoc mesenteroides and Bacillus subtilis were also identified. The two ribosomal operons have been precisely located and their transcripti...
ABSTRACTThe results reported here have identifiedyueBas the essential gene involved in irreversib... more ABSTRACTThe results reported here have identifiedyueBas the essential gene involved in irreversible binding of bacteriophage SPP1 toBacillus subtilis. First, a deletion in an SPP1-resistant (pha-2) strain, covering most of theyueBgene, could be complemented by a xylose-inducible copy ofyueBinserted atamyE. Second, disruption ofyueBby insertion of a pMutin4 derivative resulted in a phage resistance phenotype regardless of the presence or absence of IPTG (isopropyl-β-d-thiogalactopyranoside). YueB homologues are widely distributed in gram-positive bacteria. The protein Pip, which also serves as a phage receptor inLactococcus lactis, belongs to the same family.yueBencodes a membrane protein of ∼120 kDa, detected in immunoblots together with smaller forms that may be processed products arising from cleavage of its long extracellular domain. Insertional inactivation ofyueBand the surrounding genes indicated thatyueBis part of an operon which includes at least the upstream genesyukE,yukD,...
The intrinsic resistance of Oenococcus oeni cells to the secreted endolysin from oenophage fOg44 ... more The intrinsic resistance of Oenococcus oeni cells to the secreted endolysin from oenophage fOg44 (Lys44) was investigated. Experiments with several antimicrobials support the hypothesis that the full activity of Lys44 requires sudden ion-nonspecific dissipation of the proton motive force, an event undertaken by the fOg44 holin in the phage infection context.
The function of the N-terminal region of the Oenococcus oeni phage fOg44 lysin (Lys44) as an expo... more 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 prov...
Bacteriophage SPP1 targets the host cell membrane protein YueB to irreversibly adsorb and infect ... more Bacteriophage SPP1 targets the host cell membrane protein YueB to irreversibly adsorb and infect Bacillus subtilis . Interestingly, SPP1 still binds to the surface of yueB mutants, although in a completely reversible way. We evaluated here the relevance of a reversible step in SPP1 adsorption and identified the receptor(s) involved. We show that reversible adsorption is impaired in B. subtilis mutants defective in the glucosylation pathway of teichoic acids or displaying a modified chemical composition of these polymers. The results indicate that glucosylated poly(glycerolphosphate) cell wall teichoic acid is the major target for SPP1 reversible binding. Interaction with this polymer is characterized by a fast adsorption rate showing low-temperature dependence, followed by a rapid establishment of an equilibrium state between adsorbed and free phages. This equilibrium is basically determined by the rate of phage dissociation, which exhibits a strong dependence on temperature compati...
... Bac-teriophages in wine were first detected by Somi et al. ... The pelleted phage particles w... more ... Bac-teriophages in wine were first detected by Somi et al. ... The pelleted phage particles were resus-pended in 2-3 ml of TM buffer (0.05 mol I-' Tris.CI, pH 7.8, 0.01 rnol I-' MgS0.+.7H20) and purified in CsCl step gradients as described by Sambrook et nl. (1989). ...
International Journal of Systematic and Evolutionary Microbiology, 2006
Six strains of lactic acid bacteria (LAB) were isolated from a ripe fig. These strains constitute... more Six strains of lactic acid bacteria (LAB) were isolated from a ripe fig. These strains constituted a highly homogeneous, but distinct, cluster that was separate from other LAB species in a polyphasic approach including dot-blot DNA–DNA hybridization, SDS-PAGE whole-cell protein profiling, carbohydrate fermentation ability, growth characteristics, enzymic profiling, pulsed-field gel electrophoresis macrorestriction analysis and RFLPs. Phylogenetic analysis based on 16S rRNA gene sequencing positioned a representative strain, LC51T, in a distinct line of descent within the recently described clade comprising Leuconostoc ficulneum, Leuconostoc fructosum and Leuconostoc durionis; L. ficulneum was its closest neighbour (98 % sequence similarity). DNA–DNA hybridization values and chemotaxonomic and biochemical characteristics, including enzymic profiles detected with API ZYM microtubes, confirmed that this group of strains is distinct from L. ficulneum and represents a novel species withi...
The nucleotide sequence of a DNA fragment previously shown to contain the attachment site (attP) ... more The nucleotide sequence of a DNA fragment previously shown to contain the attachment site (attP) of Oenococcus oeni phage fOg44 (Santos et al., 1998. Arch. Virol. 143, 523–536) has been determined. Sequence analysis indicated that this 6226bp EcoRI fragment harbours ...
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