Lactobacillus plantarum is a ubiquitous microorganism that is able to colonize several ecological niches, including vegetables, meat, dairy substrates and the gastro-intestinal tract. An extensive phenotypic and genomic diversity analysis was conducted to elucidate the molecular basis of the high flexibility and versatility of this species. First, 185 isolates from diverse environments were phenotypically characterized by evaluating their fermentation and growth characteristics. Strains clustered largely together within their particular food niche, but human fecal isolates were scattered throughout the food clusters, suggesting that they originate from the food eaten by the individuals. Based on distinct phenotypic profiles, 24 strains were selected and, together with a further 18 strains from an earlier low-resolution study, their genomic diversity was evaluated by comparative genome hybridization against the reference genome of L. plantarum WCFS1. Over 2000 genes were identified that constitute the core genome of the L. plantarum species, including 121 unique L. plantarum-marker genes that have not been found in other lactic acid bacteria. Over 50 genes unique for the reference strain WCFS1 were identified that were absent in the other L. plantarum strains. Strains of the L. plantarum subspecies argentoratensis were found to lack a common set of 24 genes, organized in seven gene clusters/operons, supporting their classification as a separate subspecies. The results provide a detailed view on phenotypic and genomic diversity of L. plantarum and lead to a better comprehension of niche adaptation and functionality of the organism.

Lactobacillus plantarum belongs to the Lactobacillus caseiPediococcus phylogenetic group of the genus Lactobacillus (Hammes & Vogel, 1995) and in particular to the L. plantarum phylogenetic subgroup (Hammes & Hertel, 2003). This species is characterized by many metabolic activities that allow it to colonize different environments such as dairy products, pickled vegetables, fish products, silage, wine as well as mammal intestinal tracts (Vescovo et al., 1993). The species is highly heterogeneous (Dellaglio et al., 1975). Several studies, which have tried to elucidate intraspecific relationships, led to the description of two novel species: Lactobacillus pentosus and Lactobacillus paraplantarum (Zanoni et al., 1987;Curk et al., 1996). These three closely related species cannot be distinguished by 16S rRNA gene sequence analysis because they show 99 % sequence similarity. They also have very similar fermentation abilities: L. pentosus can be differentiated from L. plantarum only by its capacity to ferment glycerol and xylose (Zanoni et al., 1987). Some exceptions, however, exist: some L. plantarum strains are able to metabolize glycerol as with L. pentosus and not all L. pentosus strains can metabolize xylose . Fermentation differences between L. plantarum and L. paraplantarum are even less helpful: the main difference is that strains of the latter species metabolize methyl a-D-glucopyranoside, whereas 66 % of L. plantarum strains cannot .With regard to molecular techniques, L. plantarum, L. pentosus and L. paraplantarum can be rapidly distinguished using PCR amplification targeting the 16S/23S rRNA gene spacer region (Berthier & Ehrlich, 1998) or the recA gene as described by Torriani et al. (2001). Moreover, a polyphasic approach with a combination of RAPD (randomly amplified polymorphic DNA)-PCR, Southern hybridization and phenotypic traits not only distinguished the three species L. plantarum, L. paraplantarum and L. pentosus, but also revealed the presence of two groups of strains in the species L. plantarum (Bringel et al., 2001): 90 % of these were closely linked to the L. plantarum type strain, ATCC 14917 T , and 10 % (14 strains) formed a distinct group. We report here the analyses performed on those 14 'atypical' strains and, on the basis of collected data, a novel subspecies of L. plantarum is proposed.

Four strains isolated from Indian dairy products and initially identified as Lactobacillus delbrueckii could not be assigned to a definite subspecies because molecular identification and phenotypic traits did not agree with those of recognized subspecies of L. delbrueckii. Hybridization of total DNA (78-86 % against type strains of the other three subspecies), AFLP and RAPD-PCR fingerprints, phylogenetic analysis based on 16S rRNA gene sequences and sequence analysis of two coding genes (recA and hsp60), together with phenotypic profiles, indicated that the four strains form a coherent cluster and represent a novel subspecies, for which the name Lactobacillus delbrueckii subsp. indicus subsp. nov. is proposed. The type strain is NCC725 T (=LMG 22083 T =DSM 15996 T ).Lactobacillus delbrueckii is the type species of the genus Lactobacillus and comprises three subspecies, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus delbrueckii subsp. lactis and Lactobacillus delbrueckii subsp. bulgaricus (Weiss et al., 1983). Strains belonging to the three subspecies show a high degree of DNA-DNA relatedness but can be differentiated on the basis of different phenotypic traits (Weiss et al., 1983;Kandler & Weiss, 1989).In a previous study regarding the evolutionary structure of the species L. delbrueckii (Germond et al., 2003), a number of lactobacilli originating from five dairy fermented products from India were analysed. Among those lactobacilli, four strains isolated from traditional Indian dairy fermented (type Dahi) products were identified as L. delbrueckii on the basis of molecular and phenotypic evidence, i.e. hybridization with a probe specific for L. delbrueckii, pY85 (Delley et al., 1990), typing based on digested genomic DNA probed with ribosomal or ISL7 sequences, specific PCR amplification and the exclusive production of D-lactate from lactose. However, they could not be assigned to one of the known subspecies of L. delbrueckii because they presented atypical characteristics such as the ability of L. delbrueckii subsp. bulgaricus to degrade lactose constitutively, but no amplification of the gene encoding b-galactosidase using specific primers for L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis.We report here the polyphasic analysis performed to define the taxonomic position of the four strains and describe a novel subspecies for L. delbrueckii.The bacterial strains NCC725 T , NCC665, NCC757 and NCC780 and the type strains of L. delbrueckii subsp. delbrueckii NCC621 T (=ATCC 9649 T =LMG 6412 T ), L. delbrueckii subsp. lactis NCC946 T (=ATCC 12315 T =LMG 7942 T ) and L. delbrueckii subsp. bulgaricus NCC641 T (=ATCC 11842 T =LMG 6901 T ) were grown in de ManRogosa-Sharpe (MRS) or acidified MRS (pH 5?4) medium at 37 u C.Sugar metabolism was evaluated using the API-50CH detection kit. All strains tested fermented glucose, fructose and mannose. The ability to metabolize lactose distinguished the four strains from L. delbrueckii subsp. delbrueckii. These four strains can also be differenti...

The development of molecular tools and in particular the use of 16S rRNA gene sequencing has had a profound effect on the taxonomy of many bacterial groups. Gram-positive organisms that encompass the genera Lactobacillus and Clostridium within the Firmicutes are examples of taxa that have undergone major revisions based on phylogenetic information. A consequence of these reorganizations is that a number of organisms are now recognized as being misclassified. Previous studies have demonstrated that Lactobacillus catenaformis and Lactobacillus vitulinus are phylogenetically unrelated to Lactobacillus sensu stricto, being placed within the Clostridia rRNA cluster XVII. Based on the phenotypic, chemotaxonomic and phylogenetic data presented, it is proposed that L. catenaformis and L. vitulinus be reclassified in two new genera, named respectively Eggerthia gen. nov., with the type species Eggerthia catenaformis gen. nov., comb. nov. (type strain DSM 20559T = ATCC 25536T = CCUG 48174T = CIP 104817T = JCM 1121T) and Kandleria gen. nov., with the type species Kandleria vitulina gen. nov., comb. nov. (type strain LMG 18931T = ATCC 27783T = CCUG 32236T = DSM 20405T = JCM 1143T).

Lactobacillus casei,Lactobacillus paracasei and Lactobacillusrhamnosus form a closely related taxonomic group (the L. casei group) within the facultatively heterofermentative lactobacilli. Strains of these species have been used for a long time as probiotics in a wide range of products, and they represent the dominant species of nonstarter lactic acid bacteria in ripened cheeses, where they contribute to flavour development. The close genetic relationship among those species, as well as the similarity of biochemical properties of the strains, hinders the development of an adequate selective method to identify these bacteria. Despite this being a hot topic, as demonstrated by the large amount of literature about it, the results of different proposed identification methods are often ambiguous and unsatisfactory. The aim of this study was to develop a more robust species-specific identification assay for differentiating the species of the L. casei group. A taxonomy-driven comparative genomic analysis was carried out to select the potential target genes whose similarity could better reflect genome-wide diversity. The gene mutL appeared to be the most promising one and, therefore, a novel species-specific multiplex PCR assay was developed to rapidly and effectively distinguish L. casei, L. paracasei and L. rhamnosus strains. The analysis of a collection of 76 wild dairy isolates, previously identified as members of the L. casei group combining the results of multiple approaches, revealed that the novel designed primers, especially in combination with already existing ones, were able to improve the discrimination power at the species level and reveal previously undiscovered intraspecific biodiversity.

The taxonomic status of the species Lactobacillus durianis and Lactobacillus vaccinostercus is briefly summarized and experimental evidence concerning their similarity is presented. Highly similar 16S rRNA gene sequences (99.8 % similarity over 1523 bp), partial recA gene sequences (99.5 % similarity over 600 bp) and partial hsp60 gene sequences (99.1 % similarity over 924 bp) suggest that the two species are closely related. Moreover, a high DNA–DNA binding level (87 %) and similar genomic DNA G+C contents (41–44 mol% for both species) as well as similar biochemical characteristics support the evidence that they constitute a single species. Consequently, according to Rules 38 and 42 of the Bacteriological Code, the name Lactobacillus vaccinostercus, the oldest legitimate name, must be maintained and the name Lactobacillus durianis should be considered a later heterotypic synonym.

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The relatedness of the species Lactobacillus ingluviei and Lactobacillus thermotolerans was investigated by comparing partial sequences of the 16S rRNA gene (99?9 % similarity over 1504 bp), the hsp60 gene (98?8 % similarity over 954 bp) and the recA gene (98?5 % similarity over 452 bp) and by determining DNA-DNA binding levels (79±3 %) and genomic DNA G+C contents (50 and 49 mol%, respectively). These data, in addition to their similar biochemical characteristics, suggest that the two taxa constitute a single species. According to Rules 38 and 42 of the Bacteriological Code, they should be united under the name Lactobacillus ingluviei, with the name Lactobacillus thermotolerans as a later heterotypic synonym.Lactobacillus ingluviei Baele et al. 2003 was described as follows on the basis of the characteristics of seven strains isolated from pigeon crops and intestines. Cells are Grampositive (rapidly decolourizing in the Gram-stain procedure), non-motile, very short, plump rods that mostly occur singly or in pairs. Better growth is obtained at 42 uC than at 37 or 30 u C; no growth occurs at 25 u C. Acid is produced from L-arabinose, D-fructose, methyl b-xyloside, ribose, sucrose and D-xylose; fermentation of D-glucose, maltose, D-mannose, D-raffinose, gluconate and aesculin is variable. The DNA G+C content is 49 mol%. This species, based on 16S rRNA gene sequence analysis, belongs to the Lactobacillus reuteri phylogenetic group and highest sequences similarities were obtained to the sequences of Lactobacillus fermentum and Lactobacillus mucosae.Lactobacillus thermotolerans Niamsup et al. 2003 was described on the basis of morphological, physiological and biochemical characteristics of five thermotolerant strains isolated from chicken faeces in Thailand. Cells are Grampositive, non-motile, non-spore-forming, catalase-negative rods, 162-3 mm in size, which occur singly, in pairs or as short chains. Obligately heterofermentative, they produce both D-and L-lactic acid isomers. Growth is observed up to 50 u C, but not at 15 u C (the optimum temperature is 42 u C). meso-Diaminopimelic acid is not present in the cell wall and the DNA G+C content of the type strain is 50?5 mol%.A comparative sequence analysis based on 16S rRNAencoding genes obtained from GenBank (accession numbers AF333975 for L. ingluviei KR3 T and AF317702 for L. thermotolerans G 35 T ) revealed very high similarity; only two nucleotides were found to be different (99?87 % similarity over a total of 1504 bp). Moreover, the physiological traits reported by the authors, the habitats from which these bacteria have been isolated and the observation that the two species were described in the same year (and no reciprocal comparison was therefore made) suggest the necessity of clarifying their taxonomic relationship with additional genetic approaches.L. ingluviei LMG 20380 T and L. thermotolerans DSM 14792 T (=LMG 22056 T ) were grown in MRS broth at 37 u C under anaerobiosis and bacterial cultures were checked for purity.In order to look at the difference b...