Lactobacillus sicerae sp. nov., a lactic acid bacterium ...

International Journal of Systematic and Evolutionary Microbiology (2014), 64, 2949–2955

DOI 10.1099/ijs.0.059980-0

Lactobacillus sicerae sp. nov., a lactic acid bacterium isolated from Spanish natural cider Ana Isabel Puertas,1 David R. Arahal,2,3 Idoia Ibarburu,1 Patricia Elizaquı´vel,3,4 Rosa Aznar2,3,4 and M. Teresa Duenãs1 Correspondence David R. Arahal [email protected]

1

Department of Applied Chemistry, University of Basque Country (UPV/EHU), Paseo Manuel de Lardizabal 3, 20018 Donostia, Spain

2

Spanish Type Culture Collection (CECT), University of Valencia, Catedra´tico Agustıń Escardino 9, 46980 Paterna, Spain

3

Department of Microbiology and Ecology, University of Valencia, Dr. Moliner 50, 46100 Burjassot, Spain

4

Institute of Agrochemistry and Food Technology (IATA), Spanish Council for Scientific Research (CSIC), Catedra´tico Agustıń Escardino 7, 46980 Paterna, Spain

Strains CUPV261T and CUPV262 were isolated from ropy natural ciders of the Basque Country, Spain, in 2007. Cells are Gram-stain positive, non-spore-forming, motile rods, facultative anaerobes and catalase-negative. The strains are obligately homofermentative (final product DLlactate) and produce exopolysaccharides from sucrose. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the highest similarity to both isolates corresponded to the type strain of Lactobacillus vini (99.1 %), followed by Lactobacillus satsumensis (96.4 %), and Lactobacillus oeni (96.2 %), and for all other established species, 16S rRNA gene sequence similarities were below 96 %. The species delineation of strains CUPV261T and CUPV262 was evaluated through RAPD fingerprinting. In addition, a random partial genome pyrosequencing approach was performed on strain CUPV261T in order to compare it with the genome sequence of Lactobacillus vini DSM 20605T and calculate indexes of average nucleotide identity (ANI) between them. Results permit the conclusion that strains CUPV261T and CUPV262 represent a novel species of the genus Lactobacillus, for which the name Lactobacillus sicerae sp. nov. is proposed. The type strain is CUPV261T (5CECT 8227T5KCTC 21012T).

Lactic acid bacteria (LAB) of the genera Lactobacillus, Oenococcus, Leuconostoc and Pediococcus are involved in the cider making process (Beech & Carr, 1977; Duenãs et al., 1994; Laplace et al., 2001). The fermentation of apple juice is a microbiologically complex process involving the activities of acetic acid bacteria, lactic acid bacteria and yeasts. In Spain, especially in the Basque Country and Asturias, natural cider is usually produced in small cider factories by using traditional techniques, which implies, among other practices, that yeasts and LAB starters are not Abbreviations: ANI, average nucleotide identity; DDH, DNA–DNA hybridization; EPS, exopolysaccharides; LAB, lactic acid bacteria; MLF, malolactic fermentation; RAPD, randomly amplified polymorphic DNA; UPMGA, unweighted pair group method with arithmetic averages. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CUPV261T (5CECT 8227T) is HG794492. Shotgun genome raw sequence data generated for this strain in this study is publicly available at the Sequence Read Archive (SRA) repository under study accession PRJEB5073 and run accession ERR385820. Two supplementary figures and a supplementary table are available with the online version of this paper.

059980 G 2014 IUMS

Printed in Great Britain

added (Salih et al., 1990; Duenãs et al., 1994). Malolactic fermentation (MLF) occurs spontaneously by indigenous LAB present in the musts (Duenãs et al., 1994). This transformation has a great importance from an organoleptic point view, and most of the LAB isolated from cider has this capacity (Beech & Carr, 1977; Duenãs et al., 1994). Several species of the genus Lactobacillus have been isolated from juice and apple cider of different countries. The obligate homofermentative Lactobacillus mali (Carr & Davies, 1970), Lactobacillus delbrueckii subsp. lactis (Marshall & Walkley, 1951; Weiss et al., 1983) and Lactobacillus acidophilus (Laplace et al., 2001) are considerably rare. In contrast, the species most frequently found are the heterofermentative lactobacilli: Lactobacillus collinoides (Carr & Davies, 1972; Duenãs et al., 1995; Claisse & Lonvaud-Funel, 2000; Sauvageot et al., 2000; Garai et al., 2007), Lactobacillus paracollinoides (Whiting & Carr, 1957), Lactobacillus fermentum (Carr & Davies, 1972; Dellaglio et al., 2004), Lactobacillus buchneri, Lactobacillus viridescens, and Lactobacillus hilgardii (Carr & Davies, 1972), Lactobacillus diolivorans, Lactobacillus plantarum and Lactobacillus suebicus (Kleynmans et al., 1989; 2949

A. I. Puertas and others

Marieta et al., 2009). In natural cider from Basque Country, microbiological stabilization is not performed after MLF or before bottling. As a result, LAB becomes predominant in the microbiota, being able to promote detrimental effects, such as the alteration known as oiliness or ropiness (Duenãs et al., 1995). This alteration is the consequence of exopolysaccharides (EPS) produced by some Lactobacillus sp. (Duenãs et al., 1995), Pediococcus parvulus (Duenãs-Chasco et al., 1997), Lactobacillus diolivorans (Duenãs-Chasco et al., 1998), and Oenococcus oeni strains (Ibarburu et al., 2007), increasing the consistency of the beverage, which causes important economic losses in the Basque Country cider industry. However, EPS-producing LAB are becoming industrially important micro-organisms for elaboration of functional food products, since they provide thickening properties and contribute to improve the texture and mouth-feel of the resulting fermented products. In this context, we have previously found that an EPS-producing strain of Lactobacillus suebicus isolated from cider is a potential candidate for the production of functional foods (GaraiIbabe et al., 2010). Moreover, certain EPS produced by LAB have beneficial effects on human health (Ruas-Madiedo et al., 2008).

Genomic DNA of strains CUPV261T and CUPV262 was extracted and purified with the DNeasy Blood & Tissue kit (Qiagen) supplemented with mutanolysin 2 U ml21 (Sigma-Aldrich). DNA templates were amplified by PCR using three primers: flanking primers 616V and 630R (Ehrmann et al., 2003) and internal primer 699R (Arahal et al., 2008) to obtain two large fragments (1466 pb and 1009 pb) of the 16S rRNA gene. The PCR products were purified using a NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) according to the manufacturer’s instructions. Subsequent sequencing reactions were done on an Abi Prism 3730 automated sequencer using the Big Dye Terminator v3.1 cycle sequencing kit, premixed format. Sequencing primers were the same ones used in the amplification reaction but diluted ten-fold (5 pmol). The new sequences were identical (100 % similarity) to each other so for the rest of the text we refer only to the sequence of strain CUPV261T. The isolates were identified using the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012) and the highest level of pairwise similarity was obtained with the type strain of Lactobacillus vini CECT 5924T (99.1 %), followed by Lactobacillus satsumensis NRIC 0604T (96.4 %) and Lactobacillus oeni 59bT (96.2 %).

Strains CUPV261T and CUPV262 were isolated from ropy natural ciders according to the methods described by Duenãs et al. (1994). We chose those colonies that showed ‘ropy’ phenotype in de Man, Rogosa and Sharpe (MRS) agar (Pronadisa). The colonies formed long filaments after being touched with the loop. The strains exhibited a ropy appearance in liquid culture and the cell deposit formed a long string upon agitation. Strains CUPV261T and CUPV262 were preserved frozen at 280 uC in 20 % (v/v) glycerol. Phenotypic characterization of strains was done following standardized methods. Morphological, cultural and biochemical testing according to standard techniques were performed at 28 uC unless otherwise stated. Catalase activity was assessed by bubble production after adding a drop of 10 % (v/v) H2O2 solution. Gas production from glucose and gluconate was determined in MRS broth provided with 2 % (w/v) of the carbohydrate and Durham tubes (Man˜es-La´zaro et al., 2008). Production of dextran from sucrose was assessed on MRS agar in which glucose was replaced with 5 % (w/v) sucrose. The effects of temperature and various initial pH levels were tested in MRS broth for 5 days. Resistance to NaCl and ethanol was examined after 5 days incubation at 28 uC in MRS broth containing 5 % (w/v) NaCl or 10 % (v/v) ethanol. Biochemical characteristics were determined with the API 50CH, rapid ID32 STREP and API ZYM galleries (bioMe´rieux) according to the manufacturer’s instructions. An increase in the viscosity of the medium was observed when cells were grown in a MRS-based broth (without glucose and meat extract at pH 6.2 and containing 0.04 % (w/v) chlorophenol red) supplemented with 2 % (w/v) of glucose, trehalose, fructose or sucrose, but not with galactose, maltose or lactose. Results are reported in the species description and summarized in Table 1.

The new sequence was compared with public sequences in the EMBL gene databases using the BLAST program (National Center for Biotechnology Information; http:// www.ncbi.nlm.nih.gov/). Related sequences were further analysed using the ARB program package (Ludwig et al., 2004; http://www.arb-home.de) using alternative treeing methods (maximum-parsimony, maximum-likelihood and distance matrix) and data subsets. Automated sequence alignments were corrected manually using the ARB_EDIT sequence editor and alignments retrieved from SILVA and LTP latest updates were also used as references (Pruesse et al., 2007; Yarza et al., 2010). The analysis of the 16S rRNA gene sequence showed that strain CUPV261T forms a very stable pair with Lactobacillus vini CECT 5924T and both of them with the pair formed by the type strains of Lactobacillus ghanensis and Lactobacillus nagelii. Bootstrap support to these nodes was maximal and the same topology was retrieved in different reconstructions (Fig. 1 and Figs S1 and S2, available in the online Supplementary Material). Other related species placed in the vicinity with high confidence are Lactobacillus satsumensis, Lactobacillus oeni, Lactobacillus cacaonum, Lactobacillus mali, Lactobacillus hordei, Lactobacillus capillatus, Lactobacillus sucicola, Lactobacillus aquaticus and Lactobacillus uvarum. In addition, genotypic differentiation was approached by randomly amplified polymorphic DNA (RAPD) comparing the profiles with those of some reference strains: Lactobacillus mali CECT 4149, Lactobacillus nagelii CECT 5983T, Lactobacillus satsumensis CECT 7371, Lactobacillus uvarum CECT 7335T, Lactobacillus vini CECT 5924T, Lactobacillus vini CECT 7072 and Lactobacillus vini CECT 7073. Analysis was performed using three universal primers: M13 (59-GAGGGTGGCGGTTCT-39) (Pinto et al., 2005), P1 (59-ACGCGCCCT-39) and P2 (59-ATGTAACGCC-39)

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International Journal of Systematic and Evolutionary Microbiology 64

Lactobacillus sicerae sp. nov.

Table 1. Differential traits of strain CUPV261T and strains of closely related species Strains: 1, CUPV261T; 2, Lactobacillus vini CECT 5924T; 3, Lactobacillus ghanensis L489T; 4, Lactobacillus nagelii CECT 5983T; 5, Lactobacillus satsumensis CECT 7371; 6, Lactobacillus oeni CECT 7334T; 7, Lactobacillus uvarum CECT 7335T; 8, Lactobacillus mali CECT 4149. All data are from this study unless otherwise indicated. All strains were motile, catalase-negative and produced acid from D-mannose. Strain CUPV262 gave the same results as CUPV261T except for fermentation of lactose (negative for CUPV262). +, Positive; 2, negative; W, weakly positive; ND, no data available. Characteristic

1

EPS formation from sucrose Lactic acid isomer Growth with/at: 5 % NaCl 15 uC 45 uC Acid production from: L-Arabinose D-Ribose D-Galactose L-Sorbose L-Rhamnose D-Mannitol D-Sorbitol Methyl a-D-mannopyranoside Methyl a-D-glucopyranoside N-Acetylglucosamine Amygdalin Aesculin ferric citrate Cellobiose Maltose Lactose Melibiose Melezitose b-Gentiobiose Turanose D-Tagatose D-Arabitol DNA G+C content (mol%)

+

2

DL

DLd

2 + +

+ 2 +

2 2 + 2 2 2 2 + 2 2 2 2 2

+ + 2 2 2 2 2 2 2 + + + + + 2 2 2 + 2 2 + 39.4a

W

+ 2 2 2 2 + 2 37.5

2

3* a

4

5

6D

7

8

+

+

2

+

+

+

DL

DL

L

L

L

+ + +

+ + +

+ + +

ND

2 + + + + + + 2 + + + + + + 2 + 2 + 2 + 2 37.7*

2 2 + 2 2 + + 2 +

2 2 2 + 2 + + 2 + + 2 2 2 2 2 2 2 2 2 2 2 37.2

ND W

+ 2 2 + + + + 2 ND

2 + + + + 2 2 2 2 2 2 2 ND

37.8

b

c

W

+ + + 2 2 2 2 + + + 2 40.0c

d

+ ND

2 2 2 2 2 + 2 2 + W

+ + + 2 2 2 2 + + 2 2 36.1d

e

L

2 + 2 2 2 2 2 2 + + 2 2 + + + + + + 2 + + 2 2 2 32.5e

*Data from Nielsen et al. (2007). DData from Man˜es-La´zaro et al. (2009). dData from: a, Rodas et al. (2006); b, Edwards et al. (2000); c, Endo & Okada (2005); d, Man˜es-La´zaro et al. (2008); e, Kaneuchi et al. (1988).

(Simpson et al., 2002). The banding patterns of each strain obtained after the three amplifications were combined, and analysed with BioNumerics 2.5 software (Applied Maths) using the Dice coefficient and the unweighted pair group method with arithmetic averages (UPGMA) (Fig. 2). Strains CUPV261T and CUPV262 clustered together at 83.17 % similarity, suggesting they pertain to the same species but are not clones; and joined with the reference strains at 20.51 % similarity, supporting they belong to a separate species. The calculated global cophenetic correlation value for the global analysis was 92 indicating a good level of reliability. Although the results of RAPD fingerprinting were clear, we wanted to reinforce the genetic evidence that strain CUPV261T and the type strain of Lactobacillus vini belong http://ijs.sgmjournals.org

to different species in spite of sharing 99.12 % 16S rRNA sequence similarity. It has been recently proposed that DNA–DNA hybridization (DDH) determinations, the gold standard of prokaryotic species definition, may be substituted by a comparison between sequenced genomes of the case strains, by using the average nucleotide identity (ANI) index applied to a minimum of 20 % of the randomly sequenced complete genome (Richter & Rossello´-Mo´ra, 2009). Therefore, a random partial genome pyrosequencing approach was performed on strain CUPV261T as previously reported (Lucena et al., 2012a) using a GS-Junior (Roche) sequencing instrument at the Central Service of Support to Experimental Research (SCSIE) of the University of Valencia. Subsequent assembly produced 1.73 Mb (978 contigs); this represents 66 % of the estimated genome size. Calculation of ANI according to MUMmer (ANIm) and 2951


T 86 Lactobacillus aquaticus IMCC1736 (DQ664203) Lactobacillus uvarum 8T (AY681126)

0.01

○●

97

Lactobacillus sucicola NRIC 0736T (AB433982)

○●

○●

90 ○●

83

100

○●

Lactobacillus capillatus YIT 11306T (AB365976) Lactobacillus hordei UCC128T (EU074850) Lactobacillus mali KCTC 3596T (M58824)

○●

○●

Lactobacillus cacaonum LMG 24285T (AM905389) Lactobacillus satsumensis NRIC 0604T (AB154519)

83

○●

90

Lactobacillus oeni 59bT (AY681127)

○●

100 ○●

100

○●

Lactobacillus sicerae CECT 8227T (HG794492) Lactobacillus vini CECT 5924T (AJ576009) Lactobacillus ghanensis L489T (DQ523489)

100

○●

Lactobacillus nagelii NRIC 0559T (AB162131) Lactobacillus hayakitensis KBL13T (AB267406)

92

○●

Lactobacillus salivarius ATCC 11741T (AF089108) Lactobacillus aviarius subsp. aviarius DSM 20655T (M58808)

80 ●

100 ○●

100 ○●

Lactobacillus acidipiscis FS60-1T (AB023836) Lactobacillus pobuzihii NBRC 103219T (AB326358) Lactobacillus saerimneri GDA154T (AY255802)

Lactobacillus ceti 142-2T (AM292799)

○●

100 ○●

Lactobacillus agilis DSM 20509T (M58803)

100 81

○●

Lactobacillus equi YIT 0455T (AB048833) Lactobacillus ruminis NBRC 102161T (AB326354) Lactobacillus animalis NBRC 15882T (AB326350) 100

○●

Lactobacillus murinus LMG 14189T (AJ621554)

●

Lactobacillus apodemi ASB1T (AJ871178) Lactobacillus algidus JCM 10491T (AB033209)

Fig. 1. Neighbour-joining phylogenetic tree based on almost complete 16S rRNA gene sequences of strain CUPV261T (5CECT 8227T) and closely related species. GenBank accession numbers are given in parentheses. Bootstrap values .70 % confidence are shown at branching points (percentage of 1000 resamplings). Open circles and filled circles indicate nodes coincident in the maximum-likelihood and maximum-parsimony trees, respectively. Bar, 0.01 substitutions per position.

BLAST

(ANIb) between Lactobacillus vini DSM 20605T (AHYZ01000001–AHYZ01000220) and assembled data of strain CUPV261T was performed on JSpecies as described in Richter & Rossello´-Mo´ra (2009) and references therein. The low values obtained (ANIb 87.50–87.65 % and ANIm 88.69–88.71 %) were well-below the proposed boundary (96 %) for genomic species definition. The same conclusion can be obtained from the predicted DDH value (37.7 %, well-below the 70 % threshold) using BLAST+ and the recommended formula (identities/high-score segment pair’s length) of the genome-to-genome distance calculator (GGDC2.0) (Meier-Kolthoff et al., 2013). The genomic assembled sequence of strain CUPV261T also allowed the determination of its DNA G+C content, which was 37.5 mol%.

Colonies grown on MRS agar plates at 28 uC, 5 % CO2 after 6–7 days, were rubbery and bright, slightly convex, sometimes displayed a faint halo around, and measured just over 1 mm in diameter. Cells were rods occurring singly, in couples or in short chains. Bacterial suspensions were vitrified and imaged by cryo-transmission electron microscopy (cryo-TEM) and rods with a mean size of 0.52±0.1062.71±0.72 mm and an apical flagellum were observed (Fig. 3). One drop of the 48 h culture in MRS broth was vitrified by rapid freezing in liquid ethane using a Vitrobot Mark IV (FEI). This vitrified sample grid was transferred through 655 Turbo Pumping Station (Gatan) to a 626 DH Single Tilt Liquid Nitrogen Cryo-holder (Gatan), where it was maintained at approximately 2180 uC. Copper grid (300 mesh Quantifoils) was hydrophilized by

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Lactobacillus sicerae sp. nov. P2

P1

10

M13 50

RAPDs

L. satsumensis CECT 7371 L. nagelii CECT 5983T L. mali CECT 4149 L. vini CECT 7072 L. vini CECT 7073 L. vini CECT 5924T L. uvarum CECT 7335T Lactobacillus sicerae CUPV261T Lactobacillus sicerae CUPV262

Fig. 2. Dendrogram derived from UPGMA cluster analysis based on the combined similarity matrix obtained from the M13, P1 and P2 RAPD fingerprinting. The tracks show the processed band patterns corresponding to RAPD M13, P1, and P2 after conversion, normalization, and subtraction of the background.

glow-discharge treatment. The sample was examined in a transmission electron microscope (TECNAI G2 20 TWIN; FEI) operating at an accelerating voltage of 200 KeV in a bright-field and low-dose image mode. Strains CUPV261T and CUPV262 were Gram-stain positive, facultatively anaerobic, non-spore-forming and catalasenegative lactobacilli. The strains grew in MRS broth at pH 3.7, 4.5 or 8.0 (28 uC). Growth occurred at 15–45 uC, but not at 5 uC (pH 5.5). Cells could grow with 10 % (v/v) ethanol, but not with 5 % (w/v) NaCl. The strains are obligately homofermentative: they form DL-lactate from glucose but not gas from glucose, and they do not ferment gluconate. The isomers of lactic acid were determined by the

L-lactic

kit (ENOLOGY; BioSystems) and by HPLC (Velasco et al., 2009), with the molar ratios of D- : L-lactic acid being 1 : 0.85. When malate and glucose were available, the bacteria were able to transform L-malic acid into L-lactic acid, and citrate utilization was also observed. In addition, cellular fatty acid composition was obtained at CECT following previously described procedures (Lucena et al., 2012b). The fatty acid profiles of strains CUPV261T and Lactobacillus vini CECT 5924T contained the same fatty acids but with different relative abundance allowing their differentiation (Table 2). Differences were pronounced for C16 : 0, C19 : 1v7c/v6c, C18 : 1v9c and C14 : 0. In addition, Lactobacillus vini CECT 5924T contained a larger fraction of saturated fatty acids than strain CUPV261T.

Fig. 3. Photographs taken by Cryo-TEM of strain CUPV261T cultured in MRS broth. Arrows indicate a long apical flagellum. Bars, 500 nm. http://ijs.sgmjournals.org

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Table 2. Comparison of cellular fatty acid contents (%) of strain CECT 8227T and Lactobacillus vini CECT 5924T (cells grown on MA, 48 h, 28 6C) Data from this study (mean values of at least three replicates, carried out according to the Microbial Identification System, Microbial ID). Cellular fatty acid Saturated C12 : 0 C14 : 0 C16 : 0 C18 : 0 Unsaturated C18 : 1v9c Sum In Feature 3 (C16 : 1v7c/C16 : 1v6c) 7 (C19 : 1v7c/C19 : 1v6c) 8 (C18 : 1v7c/C18 : 1v6c)

CECT 8227T

Lactobacillus vini CECT 5924T

1.1 5.3 12.4 2.8

1.0 10.0 26.4 2.0

40.0

30.7

3.9 29.9 4.6

5.1 17.2 7.6

D-mannose,

methyl a-D-mannopyranoside, arbutin, salicin, maltose (weak), lactose (strain CUPV261T), sucrose, trehalose and D-tagatose, but not from glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, Dadonitol, methyl b-D-xylopyranoside, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl a-Dglucopyranoside, N-acetylglucosamine, amygdalin, aesculin, cellobiose, melibiose, inulin, melezitose, raffinose, starch, glycogen, xylitol, b-gentiobiose, turanose, D-lyxose, Dfucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, 2-ketogluconate or 5-ketogluconate. Results in rapid ID 32 STREP and API ZYM tests are given in Table S1. The type strain, CUPV261T (5CECT 8227T5KCTC 21012T), was isolated from a ropy natural cider of the Basque Country, Spain, in 2007. The DNA G+C content of the type strain is 37.5 mol% and the predominant cellular fatty acids are C18 : 1v9c, C19 : 1v7c/v6c and C16 : 0. An additional strain of the species is CUPV262.

Acknowledgements Unlike the closest phylogenetic neighbour, strains CUPV261T and CUPV262 were unable to utilize pentoses when tested in API 50 CHL galleries and in MRS-chlorophenol red fermentation broth supplemented with 2 % (w/v) pentoses, following incubation. Table 1 contains further descriptive traits that permit a readily distinction of the novel isolates from related species including, among others, the ability to form EPS from sucrose, the isomer of lactic acid produced in fermentation, and the profile of fermentative substrates. In addition, strains CUPV261T and CUPV262 showed less reactivity compared to Lactobacillus vini CECT 5924T in miniaturized systems rapid ID32 STREP and API ZYM (Table S1). Based on the data provided, strains CUPV261T and CUPV262 represent a novel species of the genus Lactobacillus, for which the name Lactobacillus sicerae sp. nov. is proposed. Description of Lactobacillus sicerae sp. nov. Lactobacillus sicerae (si9ce.rae. L. fem. gen. n. sicerae of cider). Cells are Gram-stain positive, non-spore-forming rods measuring 0.52±0.1062.71±0.72 mm. Cells are found singly, in pairs and in short chains, and are motile with polar flagella (Fig. 3). Facultatively anaerobic and catalasenegative. Colonies on MRS agar after 6 days incubation at 28 uC are rubbery and bright, slightly convex, white, sometimes surrounded by a faint halo, and measure just over 1 mm in diameter. Growth occurs from 15 to 45 uC, at pH 3.7, pH 8.0, and with 10 % ethanol. Growth does not occur at 5 uC, and 5 % NaCl inhibits growth. Produces EPS from sucrose. Obligately homofermentative. DL-Lactic acid is the sole product from glucose fermentation (ratio 1 : 0.85). Both malic and citric acids are utilized in the presence of glucose. Aesculin and arginine are not hydrolysed. Acid is produced from D-galactose, D-glucose, D-fructose, 2954

This study was supported by grants from the Spanish Ministry of Science and Innovation (grant nos AGL2009-12998-C03, AGL201240084-C03 and CSD2007-00063), the Basque Government (no. IT335-10) and the Generalitat Valenciana (PROMETEO/2012/040). A. I. P. acknowledges the ‘Gobierno Vasco, Dpto. Agricultura, Pesca y Alimentacioń’ for the fellowship. We also thank Microscopy: Polymer Characterization Service (SGIker) from the University of the Basque Country.

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