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1 Centro de Genética e Biologia Molecular and Instituto de Ciência Aplicada e Tecnologia, Edificio ICAT, Universidade de Lisboa, Faculdade de Ciências, Campus da FCUL, Campo Grande, 1749-016 Lisboa, Portugal
2 Centro de Biotecnologia Vegetal and Departamento de Biologia Vegetal, Bloco C2, Universidade de Lisboa, Faculdade de Ciências, Campus da FCUL, Campo Grande, 1749-016 Lisboa, Portugal
Correspondence
Rogério Tenreiro
rptenreiro{at}fc.ul.pt
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain LC51T is AY169967.
A table with fatty acid data and dendrograms depicting the relationships between the fig strains and type strains from several Leuconostoc and Weissella species derived from the independent analysis of phenotypic characteristics, whole-cell protein patterns and genomic data are available as supplementary material in IJSEM Online.
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Cell morphology and motility were tested by phase-contrast microscopy of cells grown in MRS broth. Gram reaction, catalase test, production of gas from glucose and the presence of arginine dihydrolase, in MRS broth without ammonium citrate and with 0.3 % (w/v) arginine, were analysed as described by Gerhardt et al. (1994)
. The optical isomer of lactic acid was determined using a D-/L-lactate dehydrogenase kit (Boehringer Mannheim). All strains were Gram-positive cocci, occurring singly or in pairs, catalase-negative, heterofermentative (produced gas and over 95 % D-lactic acid from glucose) and arginine dihydrolase-negative. These characteristics were in accordance with their inclusion in the genera Leuconostoc (Garvie, 1986; Holt et al., 1994) or Weissella (Collins et al., 1993).
For identification at the species level and differentiation, isolated strains and reference strains from several species of the genera Leuconostoc and Weissella were cultivated in MRS broth at 30 °C for 24 h, except for protein extraction where MRS agar was used. DNA was extracted by the guanidine thiocyanate method (Pitcher et al., 1989) and used in all molecular assays, except for macrorestriction analysis (see below). Dot-blot DNA–DNA hybridization was performed in Hybond-N nylon membranes (Amersham), according to the standard procedures for high-stringency hybridization (68 °C) described by Sambrook et al. (1989), using 40 ng genomic DNA per dot and 32P-labelled DNA of each reference strain as probe (Multiprime DNA labelling system; Amersham). Autoradiographs were digitized and densitometric analysis was performed using the Kodak 1D software package version 3.5. Whole-cell protein extracts were obtained and analysed by SDS-PAGE according to Pot et al. (1994). Densitometric analysis, normalization and numerical analysis of the protein profiles were performed with the BioNumerics software package version 4.0 (Applied Maths). From the several diagnostic tables published for Leuconostoc and Weissella species, a total of 56 different tests was selected for phenotypic characterization, including fermentation of 41 carbohydrates of the API 50CHL system (bioMérieux), production of dextran from sucrose, yellow pigmentation, growth at different temperatures, pH values, NaCl concentrations and in the presence of 10 % ethanol and hydrolysis of starch, gelatin and aesculin. Except for ethanol resistance, which was determined in a stoppered tube, TC24 culture plates (Nunc) were used for phenotypic tests with 1 ml culture medium and following the general procedures described by Gerhardt et al. (1994). Fermentation tests were performed with 0.5 % (w/v) for each carbohydrate and a basal medium equivalent to FT80 medium (Cavin et al., 1989), without glucose, fructose and malic acid and with the pH adjusted to 6.8. All data from the phenotypic characterizations were recorded as positive/negative and numerical analysis was performed with BioNumerics software. Enzymic profiling was obtained with the API ZYM microtubes (bioMérieux), according to the manufacturer's instructions. After 12 h incubation at 37 °C, data were recorded both as positive/negative and on a scale of 0 to 5. Macrorestriction analysis with SmaI (CCCGGG) and AscI (GGCGCGCC) endonucleases was performed using agarose-immobilized unsheared genomic DNA and PFGE running conditions as described by Tenreiro et al. (1994). Genomic DNA was also digested with EcoRI, BamHI, HindIII and PstI frequent-cutting endonucleases and restriction fragments were separated by standard electrophoresis in 0.8 % (w/v) agarose gels. After visualization and digital image recording of restriction profiles, Southern blots were obtained (Sambrook et al., 1989) and ribotyping was performed using the digoxigenin PCR-labelled 16S rRNA gene of Oenococcus oeni CECT 217T as a probe and a non-radioactive hybridization procedure, similar to that described by Alves et al. (2004). As for SDS-PAGE, genomic restriction profiles without hybridization were directly analysed with BioNumerics software. Macrorestriction and ribotyping data were collected as positive/negative from the densitometric analysis performed with Kodak 1D software and numerical analysis was performed with BioNumerics software.
To evaluate the relationships among the group of six fig strains and 19 type strains from species (or subspecies) of the genera Leuconostoc and Weissella, separate dendrograms were constructed derived from phenotypic characteristics, whole-cell protein patterns and genomic data (see Supplementary Figs S1–S3 available in IJSEM Online). Although different hierarchical relationships were observed among the type strains of Leuconostoc and Weissella species, the fig strains always formed a homogeneous and distinct cluster in each dendrogram, displaying levels of similarity with the most closely related species that were equivalent to or lower than those found between distinct type strains. As shown in Fig. 1, which results from numerical analysis integrating all available data, the strains isolated from the ripe fig constituted a coherent cluster displaying a low similarity level (27 %) with all type strains included in the study. Although some degree of variability among the six isolates was displayed in the dendrogram, a high similarity was obtained for protein profiles (91 %) and phenotypic tests (only isolate LC50 behaved differently by showing no growth with 6.5 % NaCl), and no differences were observed in macrorestriction profiles and ribotyping patterns. Since the strains were obtained from the same sample, these facts point to a close relationship among them; strain LC51T was selected as the representative strain.
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). PCR amplification was carried out as follows: denaturation at 94 °C for 4 min; 35 cycles of 1 min denaturation at 94 °C, 1 min annealing at 50 °C and 2 min extension at 72 °C; and a final extension step at 72 °C for 5 min. The amplified fragment was purified with the Concert Rapid PCR Purification system (Gibco) and sequenced in an automated DNA capillary sequencer CEQ 2000-XL (Beckman Coulter) by a dye-labelled dideoxy termination method (Dye Terminator Cycle Sequencer start kit; Beckman Coulter). Sequencing reactions were performed using primers pA and pH and the internal primers 519R (5'-GWATTACCGCGGCKGCTG), 907R (5'-CCGTCAATTCMTTTRAGTTT), 926F (5'-AAACTYAAAKGAATTGACGG) and 1392r (5'-ACGGGCGGTGTGTRC) (Lane, 1991), as well as F1B (5'-GAACGCTGGCGGCGTGC) and F2 (5'-TATCCGGATTTATTGGGCGT). The 1494 bp sequence of the LC51T 16S rRNA gene was compared with NCBI/GenBank entries using the BLAST algorithm (Altschul et al., 1997) and a high similarity (98 %) was found with Leuconostoc ficulneum. 16S rRNA gene sequence similarities to other Leuconostoc species were in the range of 92–94 %, whereas only 87–88 % similarity was found to sequences from Weissella species.
The position of strain LC51T inside the Leuconostoc clade was determined by phylogenetic analysis. Previously published 16S rRNA gene sequences of type strains from species of Leuconostoc, Weissella and Oenococcus, as well as Lactobacillus delbrueckii (as outgroup), were obtained from GenBank and aligned with the sequence of strain LC51T using CLUSTAL_X software. Evolutionary distance matrices were calculated (with both Jukes–Cantor and Kimura two-parameter models) and neighbour-joining phylogenetic trees were generated with 1000 replicate bootstrap analysis using the PHYLIP software package (Felsenstein, 1993). The phylogenetic tree obtained with the Kimura two-parameter model is depicted in Fig. 2. Highly congruent tree topologies were also observed with neighbour-joining (Jukes–Cantor model), maximum-likelihood and maximum-parsimony methods. Except for the peripheral clustering of Leuconostoc mesenteroides subsp. mesenteroides relative to the Leuconostoc lactis clade, obtained with the maximum-likelihood method, and the previously observed variable position of the O. oeni type strain relative to the Leuconostoc and Weissella clades (Tanasupawat et al., 2000; Lee et al., 2002), the four phylogenetic trees are in complete agreement, supporting the proposal of LC51T as a representative of a novel species closely related to Leuconostoc ficulneum.
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, and DNA was extracted as described above. Non-radioactive filter hybridization was performed with a positively charged nylon membrane (Roche) using 30 ng DNA per dot and the Dig High-Prime DNA Labelling and Detection Starter kit II (Roche). Labelling of genomic DNA, stringent hybridization and washes, and luminescent detection were performed strictly according to the supplier's procedures. Results were observed by autoradiographic exposure and analysed with Kodak 1D software. As shown in Table 1, hybridization levels higher than 85 % were observed among the six fig strains, indicating that these strains belong to a single species. The lower relatedness values obtained with Leuconostoc ficulneum (7–24 %) confirm that the fig strains represent a novel species.
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using a Waters column Microbondapack 10C18, an isocratic flow of 0.2 M NH4PO4/acetonitrile and non-methylated phage lambda DNA as the calibration reference. The G+C content of DNA of strain LC51T was 44.5 mol%, which is almost identical to the value of 44 mol% described for Leuconostoc durionis (Leisner et al., 2005) and close to the values of 42.6 and 43.4 mol% for Leuconostoc ficulneum and Leuconostoc fructosum, respectively (Antunes et al., 2002).
To analyse fatty acid composition, strains LC51T, DSM 13613T and DSM 20349T were grown in MRS agar plates at 30 °C for 48 h and fatty acid methyl esters were obtained by alkaline hydrolysis as described by Embley & Wait (1994). The fatty acid methyl esters were separated by GC (FID Perkin Elmer 8700) and identified by GC-MS (Perkin Elmer Autosystem XL GC interfaced with a Perkin Elmer Turbo MS; software version 4.1). The identity of the components was determined by comparison of GC-MS data with corresponding data of NIST and of Wiley mass spectral libraries and of laboratory-synthesized components and commercially available standards from a homemade library. The double-bond position of unsaturated fatty acid methyl esters was determined by GC-MS of their dimethyl disulphide adducts. Data shown (%) are mean values of two injections of each sample. The major fatty acids of strain LC51T were 11,12-octadecenoic acid (18 : 1
11, 18 : 1
7c) and hexadecanoic acid (16 : 0), corresponding to 40.7 and 31.2 %, respectively. The unsaturated acids 9,10-octadecenoic acid (18 : 19, 18 : 19c) and 9,10-hexadecenoic acid (16 : 19, 16 : 17c) were also detected, at 13.6 and 9.8 %, respectively. As described by Antunes et al. (2002), these fatty acids were also observed, although in different relative amounts, in the type strains of Leuconostoc ficulneum and Leuconostoc fructosum (see Supplementary Table S1 available in IJSEM Online), thus reinforcing the discriminatory potential of fatty acid composition for this clade of Leuconostoc species. Compared to the results of Antunes et al. (2002), our data revealed smaller amounts of 18 : 111 (18 : 17c) and larger amounts of 18 : 19 (18 : 19c) and 16 : 0 in the type strains of Leuconostoc ficulneum and Leuconostoc fructosum, corresponding to an overall increase in the saturated fatty acids. Similar differences were also found for the proportions of fatty acids in Leuconostoc pseudomesenteroides and Weissella paramesenteroides reported by Limonet et al. (2002) and Antunes et al. (2002). Nevertheless, an overall consistency was observed regarding the major identified fatty acids and quantitative differences may be explained by adaptation to different incubation conditions, since the growth temperature used in our study and by Limonet et al. (2002) was higher (30 °C) than that used in other studies.
The morphological and biochemical characteristics of strain LC51T and the other five fig isolates are presented in the species description. As shown in Table 2, which summarizes the physiological differences between the novel strains and the most closely related species, both the Leuconostoc ficulneum/Leuconostoc fructosum and the Leuconostoc fallax clades are characterized by an overall inability to ferment carbohydrates when compared to the Leuconostoc mesenteroides clade. Although some differences were observed in the acid production profiles of the novel strains and Leuconostoc ficulneum, the weak or delayed response observed for them strongly impairs their use as discriminating characters. In fact, a similar situation was also observed for Leuconostoc mesenteroides subsp. mesenteroides and Leuconostoc pseudomesenteroides, as reported by Antunes et al. (2002) and Leisner et al. (2005). However, the use of API ZYM microtubes in our polyphasic study pointed to their usefulness for diagnostic purposes in this group of LAB using the growth conditions and procedures already described. From the comparison of enzyme profiles, seven enzyme activities were shown to have diagnostic potential. As reported in Table 2, Leuconostoc ficulneum and Leuconostoc fructosum displayed an identical profile, but the novel strains, as well as Leuconostoc durionis, Leuconostoc fallax and Leuconostoc pseudomesenteroides, could be distinguished from Leuconostoc mesenteroides subsp. mesenteroides. Although application of the API ZYM system in the genus Leuconostoc has only been reported for two isolates of Leuconostoc gasicomitatum (Björkroth et al., 2000), its use in the characterization and discrimination of other groups of LAB has already been described (Arora et al., 1990; Collins et al., 1999; Lawson et al., 2000; Medina et al., 2001).
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) and current recommendations for species definition (Stackebrandt et al., 2002). Taking into account the common origin and the phylogenetic proximity with Leuconostoc ficulneum, the name Leuconostoc pseudoficulneum sp. nov. is proposed.
Description of Leuconostoc pseudoficulneum sp. nov.
Leuconostoc pseudoficulneum [pseu.do.fi.cul'ne.um. Gr. adj. pseudes false; L. neut. adj. ficulneum of the fig-tree and also a bacterial specific epithet; N.L. neut. adj. pseudoficulneum not the true (Leuconostoc) ficulneum].
Cells are Gram-positive, ovoid and 0.7–1.1x2.2–3.0 µm, occurring singly or in pairs. Cells are non-motile and non-spore-forming. Colonies are small, smooth, round, convex, opaque and greyish-white. Facultatively anaerobic. Heterofermentative; the lactate isomer is D(–). Catalase and cytochrome oxidase activities are not detected. Arginine dihydrolase is not produced. The optimum temperature for growth is approximately 30 °C; growth occurs at 37 °C, but not at 4 °C. The optimum pH for growth is between 6.5 and 7.0, but cells can grow at pH 4.8 and 8.5. Growth occurs in 3 and 5 % NaCl, but not in 10 % NaCl. Most strains, including the type strain, grow in 6.5 % NaCl. No growth is observed in the presence of 10 % ethanol. The predominant cellular fatty acids are 11,12-octadecenoic acid (18 : 111, 18 : 17c) and hexadecanoic acid (16 : 0). No yellow pigment is produced. Does not produce dextran from sucrose. Gelatin, starch and aesculin are not hydrolysed. Acid is produced from D-fructose, D-glucose and D-mannitol. Acid is not produced from D-adonitol, amygdalin, L-arabinose, D-arabitol, arbutin, cellobiose, dulcitol, aesculin, erythritol, D-fucose, D-galactose, 
-gentiobiose, D-gluconate, glycerol, glycogen, inositol, inulin, lactose, D-lyxose, D-maltose, D-mannose, D-melezitose, D-melibiose, D-raffinose, L-rhamnose, D-ribose, salicin, D-sorbitol, L-sorbose, sucrose, D-trehalose, D-turanose, xylitol or D-xylose. Alkaline and acid phosphatases, phosphohydrolase and 
-chymotrypsin activities are detected in API ZYM microtubes.
The type strain is LC51T (=DSM 15468T=CECT 5759T), isolated from a ripe fig collected in the Alentejo region, Portugal. The G+C content of the type strain is 44.5 mol% (as determined by HPLC).
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ACKNOWLEDGEMENTS |
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