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.
The physical and genetic maps of the Oenococcus oeni strains GM and PSU-1, which represent two genomic divergent groups on the basis of macrorestriction and ribotyping analysis, were compared. To achieve this comparison, the GM maps were constructed and the PSU-1 maps, already established, were improved. All the recognition sites of the restriction enzymes AscI, I-CeuI, FseI, NotI and SfiI were located in both chromosomes and the position of 26 genetic markers, including two rrn operons and 14 new putative oenococcal genes, were allocated to the restriction fragments generated by the five enzymes. The comparative analysis of O. oeni GM and PSU-1 genomes revealed extensive conservation of loci order. As for the differences encountered in the locations of restriction sites, they seem to be a reflection of the differences in restriction fragment sizes, explainable by insertion/deletion events and point mutations. No evidence for major genomic rearrangements was found. The genomic conservation between the two strains is in agreement and suggests homogeneity within the species, which was not unexpected in view of the restricted ecological niche of O. oeni. Further comparisons of physical maps, both of O. oeni strains and related species, will certainly help to assess whether O. oeni is really an homogeneous species and physical mapping is suitable for taxonomic purposes, both at the supra-and intraspecific levels.
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.
Constitutive mutants for L-arabinose utilization were isolated from Bacillus subtilis 168T+ and showed resistance to D-fucose, a nonmetabolizable analog of L-arabinose. The mutations that conferred the constitutive phenotype (Arac) were mapped between cysB and hisA. All the mutants showed an isomerase activity which was reduced to 50 to 70% in the presence of L-arabinose and to 10% in the presence of glucose.The L-arabinose pathway in Bacillus subtilis has been described; its enzymes are inducible, and the isomerase activity is subject to catabolite repression (10). The corresponding structural genes-araA, araB, and araD-coding for L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate-4-epimerase, respectively, were mapped between argA and leuA; strains with mutations in another gene, named araC and located in the region between cysB and hisA, do not exhibit any isomerase or ribulokinase activities (13; H. Paveia, Ph.D. thesis, University of Lisbon, Lisbon, Portugal, 1987).We isolated five mutants that have a constitutive phenotype for L-arabinose utilization (Arac) and that still retain carbon catabolite repression of L-arabinose isomerase activity. PBS1 transduction analysis of the corresponding mutations [ara(Con)] revealed that they are located very close to the ara mutations used to define araC.The strains used in this study are listed in Table 1. All the mutations were made isogenic by congression in BR151, with selection for Trp+ and scoring for the Arac phenotype. Transformation was carried out as described by Bott and Wilson (4).The minimal medium used for selection of recombinants was MM (2) containing 0.1% glucose; auxotrophic requirements were added to a final concentration of 50 jig/ml. For the preparation of crude cellular extracts, strains were grown in salts C (12) supplemented with 1% casein hydrolysate; to this medium 0.4% L-arabinose, glucose, or D-fucose was added whenever necessary.The methods used in this study were previously described: mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (1) and PBS1 transduction analysis (8), isolation of Arac mutants by the filter paper (7) and D-fucose resistance (3) techniques, L-arabinose isomerase assay (6,14), and determination of amount of protein (11).For the preparation of crude cellular extracts, cells grown overnight at 37°C with vigorous shaking in the indicated media were collected by centrifugation, washed in 0.9% NaCl, suspended in Tris hydrochloride (50 mM) (pH 7.5)-EDTA (1 mM Isolation of Arac mutants. Five Arac mutants were isolated, purified, and numbered. Strains carrying mutations ara-31(Con), ara-32(Con), and ara-33(Con) were isolated by the filter paper technique, while those carrying ara-35(Con) and ara-37(Con) were selected by the D-fucose resistance method. After ara(Con) mutations were made isogenic, the phenotypes of the constructed strains were rechecked by both methods.Mapping of ara(Con) mutations. PBS1 lysates prepared for each isogenic Arac mutant were used to transduce B. subtilis QB936, BD111, and IGCg301...
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