Inter‐strain relationships among wine leuconostocs and their divergence from other Leuconostoc species, as revealed by low frequency restriction fragment analysis of genomic DNA

Abstract

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.

Physical map of the genome of Oenococcus oeni PSU-1 and localization of genetic markers

“…The CHEF system was used with 1 ' / o (w/v) agarose gels (Seakem GTG, FMC) and 0 5 x TBE (45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8.0). Three run-types were applied for resolution of fragments in different ranges ( < 150 kb, 150-750 kb, >750 kb), as previously described (Tenreiro et al, 1994). The TAFE system was used with 1 '/O (w/v) agarose gels (Seakem LE, FMC) and 1 x TAE (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.0).…”

“…A macrorestriction analysis previously performed on 30 strains of 0. oeni (Tenreiro et al, 1994) showed that A d , NotI and SP, generating an average number of fragments between 4 and 17, were suitable enzymes for physical mapping. In strain PSU-1 these enzymes cleaved the chromosome into 4,12 and 16 restriction fragments, respectively (the 12th NotI fragment, of 6 kb, had not been previously detected).…”

“…As two linking clones, lc-4 and lc-36 (Table 4), identified the linkages N2-N5 and N5-N6, the clockwise linkage N6-NS-NZ-Nl was also deduced. To identify other linkages between NotI bands, macrorestriction fragments obtained with this enzyme from a strain with a different profile (Tenreiro et al, 1994), 0. oeni GM, were used as probes. Hybridization with fragment NIGM (690 kb) indicated that N3 is adjacent to N1, whereas a composite probe containing fragments NtlGM and NSGM (both 63 kb) hybridized with five NotI (Nl, N2, N3, N8 and N11) and three AscI (Al, A2 and A3) PSU-1 fragments ( Table 4).…”

“…In lactic acid bacteria, such maps are only available for strains of Lactococcus lactis and Streptococcus thermophilus (Davidson et al, 1996;Le Bourgeois et al, 1992;Roussel at al., 1994Roussel at al., , 1997Tulloch et al, 1991). This paper presents a physical map of 0. oeni PSU-1, a strain commonly used as starter in wine-making and for which the macrorestriction profile with endonucleases that recognize GC-rich sequences was already known (Tenreiro et al, 1994). Oenococcal genetic markers, as well as putative genes inferred from hybridization studies with probes from heterologous Gram-positive bacteria, were located on the map constructed.…”

“…Recent research concerning 0. oeni has been focused in five main fields: (i) bioenergetics of organic acid transport and elucidation of metabolic pathways (Ramos & Santos, 1996;Salema et al, 1996;Veiga da Cunha et al, 1993); (ii) analysis of evolutionary divergence and tachytely (Martinez-Murcia et al, 1993 ;Morse et al, 1996;Zavaleta et al, 1996) ; (iii) assessment of intraspecific variability and development of reliable typing methods (Kelly et al, 1993 ;Tenreiro et al, 1994; t Present address: Departamento de BotAnica e Engenharia BioldgicaMicrobiologia, lnstituto Superior de Agronomia, 1399 Lisboa, Portugal.…”