Weissella ghanensis sp. nov., isolated from a Ghanaian cocoa fermentation

During a study on lactic acid bacteria (and their species diversity) in spontaneous heap fermentations of Ghanaian cocoa beans, two strains, designated 215^T and 194B, were isolated. A phylogenetic analysis based on 16S rRNA gene sequences demonstrated that these strains represented a distinct lineage close to the genus Weissella and showing only 92.1 % 16S rRNA gene sequence similarity with respect to their closest neighbour, Weissella soli LMG 20113^T. Whole-cell protein electrophoresis, fluorescent amplified fragment length polymorphism fingerprinting of whole genomes and physiological and biochemical tests confirmed the unique taxonomic position of the two novel isolates. On the basis of the results of the morphological and biochemical tests and 16S rRNA gene sequence analysis, strains 215^T and 194B represent the most peripheral lineage of the genus Weissella, for which we propose the name Weissella ghanensis sp. nov. The type strain is 215^T (=LMG 24286^T=DSM 19935^T).

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Abbreviations: FAFLP, fluorescent amplified fragment length polymorphism; RAPD, randomly amplified polymorphic DNA

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strain 215^T and strain 194B are AM882997 and AM882998, respectively.

An extended neighbour-joining phylogenetic tree (based on 16S rRNA gene sequences) and whole-cell protein profiles for the novel strains, type strains of recognized Weissella species and other closely related taxa are available as supplementary figures with the online version of this paper.

The genus Weissella was created by Collins et al. (1993), in the course of a phylogenetic analysis of some unknown Leuconostoc-like lactic acid bacteria, to accommodate five heterofermentative Lactobacillus species and a species formerly classified as Leuconostoc paramesenteroides. Novel species of the genus Weissella have mainly been isolated from fermented foods: Weissella hellenica strains were isolated from fermented sausages (Collins et al., 1993), Weissella kimchii (a later heterotypic synonym of Weissella cibaria) (Ennahar & Cai, 2004) and Weissella koreensis strains were isolated from kimchi (Choi et al., 2002; Lee et al., 2002), and Weissella thailandensis strains were isolated from fermented fish (Tanasupawat et al., 2000). The exceptions are Weissella soli strains, which were isolated from soil (Magnusson et al., 2002), and some W. cibaria strains that were detected in both fermented food and clinical samples (Björkroth et al., 2002).

The population dynamics of spontaneous heap fermentations of cocoa beans in Ghana during the main cropping season of 2005 (October–December) were studied by Camu et al. (2007). The levels of lactic acid bacteria species diversity of different fermentation heaps and different fermentation times were signalled by the relative intensities of bands obtained using a culture-independent technique, namely 16S rRNA gene sequence-based PCR-denaturing gradient gel electrophoresis. This analysis detected an unidentified bacterium that appeared in five of the seven heap fermentations studied, from the beginning of the fermentation up to 48 h after the start of the fermentation process. The 16S rRNA gene sequence of this bacterium was determined. Only three isolates (215^T, 194B and 225) that had 16S rRNA gene sequences identical to that of the unidentified bacterium were obtained (and subsequently cultivated) from the same fermenting cocoa bean heap in New Tafo, Ghana (Camu et al., 2007). Isolate 225 lost viability during subsequent cultivation. To check clonality using randomly amplified polymorphic DNA (RAPD; Chen et al., 2001), DNA from each isolate was prepared by alkaline lysis (Coenye et al., 2002) and the DNA solutions were stored at –20 °C. Fingerprinting using RAPD was performed with primers RAPD-270 (5'-TGCGCGCGGG-3') and RAPD-272 (5'-AGCGGGCCAA-3'), as described previously (Mahenthiralingam et al., 1996). Isolates 215^T and 194B were found to be non-clonal strains (data not shown).

The taxonomic positions of strain 215^T and strain 194B were first investigated by means of 16S rRNA gene sequence analysis, as described by Vancanneyt et al. (2004) but with the following modification: the PCR-amplified 16S rRNA gene was purified using a NucleoFast 96 PCR Clean-up kit (Macherey-Nagel). Sequencing reactions were purified using a Montage SEQ96 Sequencing Reaction Clean-up kit (Millipore). Electrophoresis of sequence reaction products was performed by using an ABI Prism 3100 genetic analyser (Applied Biosystems). A BLAST search and phylogenetic analysis based on 16S rRNA gene sequence similarity showed that strain 215^T was related to members of the genus Weissella, the highest sequence similarity (92.1 %) being found with respect to W. soli LMG 20113^T. The 16S rRNA gene sequences obtained (continuous stretches of 1541 bp and 1538 bp) and sequences of the type strains from all species of the genus Weissella were aligned using CLUSTAL_X (version 1.81; Thompson et al., 1997) and clustered using the BioNumerics software package, version 5.10 (Applied Maths). The maximum-parsimony and maximum-likelihood tree-building methods revealed similar topologies to those obtained in a phylogenetic tree constructed using the neighbour-joining approach (Fig. 1), which was calculated using bootstrapping based on 200 replications. The neighbour-joining analysis revealed strain 215^T as a distinct phylogenetic lineage close to the genus Weissella. A broader phylogenetic analysis including all of the phylogenetically closest neighbours of the genus Weissella, namely the genera Pediococcus, Oenococcus and Leuconostoc, is shown in Supplementary Fig. S1 (available in IJSEM Online). This analysis demonstrated that strains 215^T and 194B can be considered as representing the most peripheral branch of the genus Weissella. On the basis of the data from additional analyses described below, these strains were not assigned to a novel genus but were recognized as representing a novel species of the genus Weissella. This peripheral position could be the result of differences in the rate of evolution of strains 215^T and 194B with respect to Weissella species. The large divergence in 16S rRNA gene sequence (>7.4 %) demonstrates unambiguously that the two strains represent a novel species that is closely related to the recognized species of the genus Weissella.

(35K): Fig. 1. Phylogenetic neighbour-joining tree, based on 16S rRNA gene sequences, showing the phylogenetic relationships of strains 215T and 194B with respect to members of the genus Weissella. Bootstrap percentages (based on 200 replications) are shown at branch points. Pediococcus damnosus DSM 20331T was used as the outgroup. Bar, 2 % sequence divergence.

SDS-PAGE of whole-cell proteins and fluorescent amplified fragment length polymorphism (FAFLP) techniques were used to characterize these novel bacteria in more detail and were performed as described by Pot et al. (1994) and Franz et al. (2006), respectively. Protein profiles for Weissella reference strains were taken from Björkroth et al. (2002). To complete the protein profile reference data for all Weissella species, protein profiles for W. koreensis LMG 21853^T and W. soli LMG 20113^T were generated. The whole-cell protein profiles of strains 215^T and 194B formed a distinct branch within the dendrogram obtained (see Supplementary Fig. S2, available in IJSEM Online). Similarly, reference FAFLP data for all type strains were supplemented with data for W. koreensis LMG 21853^T, W. soli LMG 20113^T, W. thailandensis LMG 19821^T and Weissella kandleri LMG 18979^T. The unique taxonomic position of strains 215^T and 194B was again confirmed by numerical analysis of the FAFLP fingerprints (Fig. 2).

(28K): Fig. 2. FAFLP patterns and dendrogram based on UPGMA linkage of Dice coefficients of strains 215T and 194B and reference strains from all of the recognized species of the genus Weissella.

The DNA G+C content of strain 215^T was determined, as described by Mesbah et al. (1989), using a Waters Breeze HPLC system and an XBridge Shield RP18 column. The solvent used was 0.02 M NH₄H₂PO₄ (pH 4.0) with 1.5 % (v/v) acetonitrile. Escherichia coli LMG 2093 DNA was used as the control and non-methylated lambda phage DNA (Sigma) was used as the calibration reference. The DNA G+C content of strain 215^T was found to be 40.0 mol%, which falls within the range for the genus Weissella (37–47 mol%; Collins et al., 1993).

Cell and colony morphology were investigated after growth on MRS agar (pH 5.4, Oxoid) and 48 h aerobic incubation at 28 °C, unless stated otherwise. Standard biochemical tests and growth experiments were determined in MRS broth (pH 5.4, Oxoid). All tests were performed in triplicate, unless stated otherwise. Growth was tested at 4, 10, 15, 37 and 45 °C, in the presence of 5, 6, 7 and 8 % NaCl and at pH 3.9 at 37 °C. The production of gas from 2 % glucose and 2 % gluconate in modified MRS broth (pH 5.4, without the addition of triammonium citrate) was investigated using Durham tubes. Arginine hydrolysis was evaluated in a medium containing 0.5 % tryptone, 0.5 % yeast extract, 0.3 % L-arginine, 0.05 % glucose, 0.2 % K₂HPO₄ (pH 7.0), with methyl orange as an indicator. Metabolites from glucose were determined in duplicate by means of HPLC (Waters apparatus). The proportions of D- and L-lactate were determined enzymically (R-Biopharm). Carbohydrate-fermentation tests were performed using the API 50 CHL system (bioMérieux). Other physiological and biochemical characteristics were determined using the API 20E system (bioMérieux); both strips were analysed in duplicate, according to the manufacturer's instructions. For the detection of glucose metabolites and D/L lactate isomers, strains were grown for 96 h at 30 °C in MRS broth (pH 5.4, Oxoid). The results of this characterization are given in the species description.

In spite of the peripheral position of strains 215^T and 194B in the phylogenetic tree based on 16S rRNA gene sequences (Fig. 1), the biochemical characteristics observed for the two strains (i.e. their cell and colony morphology, motility, catalase activity, heterofermentative metabolism, arginine hydrolysis and growth temperatures) and their DNA G+C content conformed to the expected profile of members of the genus Weissella (Collins et al., 1993). The two novel strains could be clearly distinguished from homofermentative strains (e.g. pediococci) by the formation of gas from carbohydrates. The ability of strains 215^T and 194 to hydrolyse arginine and to form DL-lactate served to differentiate them from strains of the genus Leuconostoc. The novel isolates could be differentiated from recognized Weissella species on the basis of phylogeny, whole-cell protein profiles, AFLP results and biochemical data (Table 1). Thus, strains 215^T and 194 represent a novel species of the genus Weissella, for which the name Weissella ghanensis sp. nov. is proposed.

Table 1. Differential characteristics of Weissella ghanensis sp. nov. and other species of the genus Weissella Taxa: 1, strains 215T and 194B; 2, W. halotolerans; 3, W. minor; 4, W. viridescens; 5, W. soli; 6, W. kandleri; 7, W. koreensis; 8, W. cibaria; 9, W. confusa; 10, W. thailandensis; 11, W. hellenica; 12, W. paramesenteroides. Data partially adapted from Collins et al. (1993), Tanasupawat et al. (2000), Björkroth et al. (2002), Choi et al. (2002), Lee et al. (2002), Magnusson et al. (2002) and Ennahar & Cai (2004). +, Positive for at least 90 % of the strains; –, negative for at least 90 % of the strains; d, positive for 11–89 % of strains; NT, not tested.

Description of Weissella ghanensis sp. nov.
Weissella ghanensis (gha.nen'sis. N.L. fem. adj. ghanensis pertaining to Ghana).

Cells are Gram-positive, catalase-negative and facultatively anaerobic. They do not form spores. Gliding motility is not observed. Cells are small rods (1.0 µm wide and 2.0–4.0 µm long) and appear singly, in pairs or in short chains. Colonies are beige, opaque, smooth, circular or irregular with low convex elevation and entire margins and are 1.5–2.0 mm in diameter. Growth is obtained at 15–37 °C. No growth is observed in MRS broth supplemented with 5 % NaCl. For strain 215^T, lactic acid (D/L, 90 : 10), acetic acid, ethanol and gas formation are detected after growth on glucose, indicating the heterofermentative nature of this strain. Strain 194B produces different end metabolites. Both lactic acid and acetic acid are detected as end products of glucose metabolism. Mainly D-lactate (D/L, 95 : 5) is produced and no gas production is observed. Strain 215^T and strain 194B both produce slime from sucrose and are able to hydrolyse arginine. Acid is produced from glucose, fructose, mannose, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, trehalose and gentiobiose, but not from glycerol, erythritol, D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, adonitol, methyl β-D-xylopyranoside, galactose, sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl α-D-mannopyranoside, methyl α-D-glucopyranoside, lactose, melibiose, inulin, melezitose, raffinose, starch, glycogen, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. Strain 215^T produces acid from sucrose; strain 194B does not produce acid from sucrose.

The type strain, 215^T (=LMG 24286^T=DSM 19935^T), was isolated from Ghanaian cocoa bean heaps undergoing fermentation. The DNA G+C content of the type strain is 40.0 mol%. The two available strains (215^T and 194B) both originated from the same cocoa bean heap undergoing fermentation in New Tafo, Ghana. However, Camu et al. (2007) detected this organism in five out of seven spontaneous heap fermentations of cocoa beans studied, indicating that this species is common in Ghanaian cocoa bean heaps that are undergoing fermentation.