Comparative genome analysis of the candidate functional starter culture strains Lactobacillus fermentum 222 and Lactobacillus plantarum 80 for controlled cocoa bean fermentation processes
© Illeghems et al. 2015
Received: 24 February 2015
Accepted: 12 September 2015
Published: 12 October 2015
Lactobacillus fermentum 222 and Lactobacillus plantarum 80, isolates from a spontaneous Ghanaian cocoa bean fermentation process, proved to be interesting functional starter culture strains for cocoa bean fermentations. Lactobacillus fermentum 222 is a thermotolerant strain, able to dominate the fermentation process, thereby converting citrate and producing mannitol. Lactobacillus plantarum 80 is an acid-tolerant and facultative heterofermentative strain that is competitive during cocoa bean fermentation processes. In this study, whole-genome sequencing and comparative genome analysis was used to investigate the mechanisms of these strains to dominate the cocoa bean fermentation process.
Through functional annotation and analysis of the high-coverage contigs obtained through 454 pyrosequencing, plantaricin production was predicted for L. plantarum 80. For L. fermentum 222, genes encoding a complete arginine deiminase pathway were attributed. Further, in-depth functional analysis revealed the capacities of these strains associated with carbohydrate and amino acid metabolism, such as the ability to use alternative external electron acceptors, the presence of an extended pyruvate metabolism, and the occurrence of several amino acid conversion pathways. A comparative genome sequence analysis using publicly available genome sequences of strains of the species L. plantarum and L. fermentum revealed unique features of both strains studied. Indeed, L. fermentum 222 possessed genes encoding additional citrate transporters and enzymes involved in amino acid conversions, whereas L. plantarum 80 is the only member of this species that harboured a gene cluster involved in uptake and consumption of fructose and/or sorbose.
In-depth genome sequence analysis of the candidate functional starter culture strains L. fermentum 222 and L. plantarum 80 revealed their metabolic capacities, niche adaptations and functionalities that enable them to dominate the cocoa bean fermentation process. Further, these results offered insights into the cocoa bean fermentation ecosystem as a whole and will facilitate the selection of appropriate starter culture strains for controlled cocoa bean fermentation processes.
Lactic acid bacteria (LAB) are Gram-positive bacteria that are key players in the majority of food fermentation ecosystems [1, 2]. The genus Lactobacillus represents the largest group among the LAB, encompassing more than 200 species [3, 4]. As this genus possesses a wide metabolic diversity, lactobacilli occur and have been used as functional starter cultures in a wide variety of fermented food products, including cheese, fermented plant-derived foods, fermented meats, wine and beer production, and sourdoughs . Furthermore, lactobacilli play an important role during the cocoa bean fermentation process .
At the onset of the cocoa bean fermentation process, a wide LAB species diversity is often present [7–12]. However, only a restricted number of Lactobacillus species predominate this fermentation process, consisting mainly of (i) strictly heterofermentative Lactobacillus fermentum [7–22] and (ii) facultative heterofermentative Lactobacillus plantarum [7–12, 14–21, 23]. It has been proposed that L. plantarum is the dominant member at the onset of the cocoa bean fermentation process, while L. fermentum dominates in later stages [7, 8, 18, 21]. This is in accordance with the cocoa-specific functional roles that have been associated with these two species [24–26].
Homolactic fermentation of cocoa pulp-bean mass carbohydrates such as glucose and fructose, resulting from the hydrolysis of sucrose present in the cocoa pulp-bean mass by pulp or yeast invertase, results in lactic acid, while heterolactic fermentation gives lactic acid, acetic acid and/or ethanol, and carbon dioxide . Fructose and citric acid, present in the cocoa pulp, are also used as alternative external electron acceptors by heterofermentative LAB, enhancing their competitiveness and resulting in the production of mannitol and succinate, or lactate and flavor-active compounds such as 2,3-butanediol or acetoin, respectively [24–26].
To better understand the exact role of cocoa-derived microorganisms, their whole-genome sequence can be investigated, as has been done for the cocoa-specific Acetobacter pasteurianus 386B strain . Whole-genome sequencing of particular strains of the LAB species L. fermentum and L. plantarum originating from other sources (including fermented food ecosystems; Additional files 1 and 2) has been performed previously, thereby elucidating a plethora of specific functionalities and niche adaptations. For instance, genome sequence analysis of particular L. fermentum strains has revealed adaptations to specific host systems, such as the presence of adhesins  and probiotic properties [29–31]. Further, comparative genomics has enabled insights into the genomic features that are unique for L. fermentum strains . Genome sequencing of particular L. plantarum strains elucidated niche adaptations [33, 34], probiotic properties [35–38], prophage occurrence [39–41], general metabolic properties [42–46], and bacteriocin production . Therefore, it can be expected that whole-genome sequence analysis of the cocoa-derived L. fermentum 222 and L. plantarum 80 strains, isolates from a spontaneous Ghanaian cocoa bean fermentation process and proposed as interesting functional starter culture strains [7, 25, 26], will provide detailed insight into their genetic potential, metabolic capacities and functionalities as well as cocoa niche adaptations. Next to their central carbohydrate metabolism, their proteolytic system and amino acid conversion pathways have to be analysed, as these are important functionalities of LAB in various food fermentation ecosystems [48–50].
Results and discussion
454 pyrosequencing, genome sequence assembly, and annotation
General features of the reads, contigs, and draft genome sequences of Lactobacillus fermentum 222 and L. plantarum 80
L. fermentum 222
L. plantarum 80
Number of reads
Average read length
Median read length
Number of contigs
Depth of coverage
Draft genome sequences
Estimated genome size (Mb)a
Total contig length (bp)
G+C content (%)
Number of CDS
Coding density (%)
Average gene length (bp)
Number of rRNA operonsb
Number of tRNAs
Genome architecture and genetic potential
Mechanisms of Lactobacillus fermentum 222 and Lactobacillus plantarum 80 involved in stress responses. The locus tags are depicted for each mechanism as well as the gene names when appropriate
L. fermentum 222
L. plantarum 80
Heat-shock operon hrcA-grpE-dnaKJ
LP80_658; LP80_2456; LP80_2821
LP80_47; LP80_193; LP80_2125
Clp ATPases and proteases
clpL (LFER_111); clpB (LFER_572); clpP (LFER_1165, LFER_1522); clpE (LFER_1495); clpX (LFER_1563)
clpB (LP80_2653); clpC (LP80_170, LP80_2748); clpE (LP80_2599); clpP (LP80_1719, LP80_2338); clpX (LP80_923); clpQY; (LP80_1233-LP80_1234); lon (LP80_936)
Metabolic pathway reconstruction
Pathway analysis of Lactobacillus fermentum 222
Central carbohydrate metabolism
Pathways that enable the use of oxygen or glycerol as alternative external electron acceptors were not found in the genome sequence of L. fermentum 222. Although this strain is able to use fructose as an alternative external electron acceptor  and the genome sequence contained a gene coding for a fructose permease (frw), a gene encoding a mannitol dehydrogenase could not be found. However, a fructose-specific PEP-dependent PTS (fru) was present, which is associated with mannitol production from fructose in homofermentative LAB . Based on the genome sequence annotation, no further evidence was found that this transporter was involved in mannitol production.
Proteolytic system and amino acid conversion pathways
The genome sequence of L. fermentum 222 harboured genes encoding a peptide transporter (dtpT) and several amino acid ATP-binding cassette (ABC) transporters specific for glutamine (gln), methionine (met), and several other amino acids that could not be further specified based on the genome sequence annotation (Fig. 2; Additional file 3). Further, a plethora of genes encoding peptidases were retrieved, such as pepF and pepO encoding endopeptidases, pepI, pepP, pepQ, and pepX encoding proline-specific peptidases, pepD and pepV encoding dipeptidases, pepT encoding a tripeptidase, and pepC, pepN, and pepM encoding aminopeptidases (Additional file 3). The acquisition of a large variety of peptidases by Lactobacillales is related to their adaptation to nutritionally rich environments , which might also be the case for members of the cocoa bean fermentation process, as cocoa pulp consists of 0.5–0.7 % proteins .
Aspartate transaminase-encoding genes were found (aspC), enabling L. fermentum 222 to produce oxaloacetate from aspartate. This oxaloacetate might contribute to the pyruvate metabolism through the pathway associated with citrate metabolism (see above). Indeed, it has been shown for other Lactobacillus species that diacetyl and acetoin can be produced through aspartate catabolism . Genes encoding aspartase (aspA), fumarase (fum), and succinate dehydrogenase (sdh) were found as well, offering an alternative pathway for aspartate catabolism (Fig. 2; Additional file 3). This pathway may contribute to NAD+ regeneration, and, therefore, offer a more efficient degradation of carbohydrates .
Genes encoding the transaminases araT and bcaT, which catalyse the hydrolysis of branched-chain amino acids as well as aromatic amino acids, were retrieved (Fig. 2; Additional file 3). The concomitant α-oxoacids produced might be further reduced to hydroxyacids by the 2-hydroxyacid dehydrogenases (hycDH) present, thereby contributing to NAD+ regeneration and thus allowing more efficient energy production through heterofermentation, as hypothesised before . Genes associated with the conversion of α-oxoacids by oxidative decarboxylation into carboxylic acids or by decarboxylation into aldehydes were not found. Neither were enzymes involved in decarboxylation of histidine and aromatic amino acids found, the latter indicating that L. fermentum 222 is unable of biogenic amine production. Further, evidence was found for the presence of other amino acid conversion pathways, such as the occurrence of genes encoding cystathionine β-lyase (cbl) that is involved in methionine catabolism, serine deaminase (sda) that enables the production of pyruvate and ammonia from serine, and serine hydroxymethyltransferase (shm) that is involved in the production of the flavour compound acetaldehyde (Fig. 2; Additional file 3).
The genome sequence of L. fermentum 222 contained an operon encoding the arginine deiminase (ADI) pathway, consisting of genes encoding arginine deiminase (arcA), ornithine transcarbamoyltransferase (arcB), carbamate kinase (arcC), an arginine/ornithine antiporter (arcD), and a transaminase (arcT; Fig. 2; Additional file 3). This pathway might provide L. fermentum 222 with additional energy, provided through amino acid conversions, as well as protection against acid stress conditions [63, 64] and has been shown to be of importance for competitiveness in food fermentation processes [64–66].
Further, a gene encoding ornithine cyclodeaminase (ocd) was present, enabling direct biosynthesis of proline from ornithine, thereby producing ammonia (Fig. 2; Additional file 3). The production of additional ammonia may further protect against acid stress, whereas proline can act as an osmotic protectant .
Pathway analysis of Lactobacillus plantarum 80
Central carbohydrate metabolism
The genome sequence of L. plantarum 80 contained all genes encoding the enzymes involved in the homo- and heterolactic fermentation pathways (Fig. 2; Additional file 3) as well as various genes encoding carbohydrate uptake systems, enabling this strain to switch between both metabolic pathways, depending on the energy source available. Metabolic pathway analysis revealed that pyruvate can be converted into acetyl-CoA by a pyruvate dehydrogenase complex (pdhAC, lpdA) or by pyruvate-formate lyase (pfl), the latter yielding formate (Fig. 2; Additional file 3). Further, a gene encoding pyruvate oxidase (pox) was present, enabling L. plantarum 80 to convert pyruvate into carbon dioxide and acetyl phosphate under oxic conditions. Genes encoding α-acetolactate synthase (als) and α-acetolactate decarboxylase (aldB) were found, but diacetyl reductase- or 2,3-butanediol dehydrogenase-encoding genes were absent, indicating that acetoin would be the only flavour compound that can be formed out of pyruvate by L. plantarum 80, although 2,3-butanediol nor acetoin were found during monoculture fermentations with L. plantarum 80 in cocoa pulp simulation medium for LAB .
Genes associated with uptake and conversion mechanisms of citrate, such as a gene encoding citrate lyase (citDEF), oxaloacetate decarboxylating malate dehydrogenase (mae), a citrate transporter (citT), and accessory proteins (citGX), were all retrieved in the genome sequence of L. plantarum 80 (Fig. 2; Additional file 3). However, these were not clustered together (Fig. 3), as genes encoding transposases (LP80_92, LP80_93), an ABC transporter (mtsABC), a fumarase (fumC) and a fumarate reductase (frdC) were found between them. Further, a gene encoding a malate dehydrogenase (mdh) was found, which might enable L. plantarum 80 to use citrate as an alternative external electron acceptor by converting citrate into succinate by the reductive tricarboxylic acid cycle, next to its ability to convert citrate into pyruvate, which has been shown experimentally . Lactobacillus plantarum 80 might be able to use oxygen as an alternative external electron acceptor, as a NADH-dependent oxidase-encoding gene (nox) was found. Genes associated with the ability to use glycerol or fructose as alternative external electron acceptors were absent.
The genome sequence of L. plantarum 80 harboured genes encoding a respiration machinery, which is an energetically favourable metabolic pathway that leads to less oxidative and acid stress in LAB . Indeed, genes encoding a NADH-dependent dehydrogenase, a bd-type cytochrome (cydABCD), an ATP synthase (see above), and a heme-dependent catalase (katA; see above) were found (Fig. 2; Additional file 3). Although several L. plantarum strains possess a similar electron transport chain [69, 70], there are no experimental data indicating that L. plantarum 80 could perform aerobic respiration when provided with exogenous sources of heme and menaquinones.
Proteolytic system and amino acid conversion pathways
Genes encoding extracellular proteases were not found in the genome sequence of L. plantarum 80, which seems to be a general feature for L. plantarum strains . Genes encoding a DtpT proton motive force-dependent peptide transporter and an Opp transporter (oppABCDF) were found as well as peptidase-encoding genes, including the genes pepD, pepE, and pepV encoding dipeptidases, the genes pepN and pepC encoding aminopeptidases, the genes pepX, pepR, and pepP encoding proline-specific peptidases, and the genes pepF, pepQ, pepO, and pepR encoding endopeptidases (Fig. 2; Additional file 3). This elaborate peptide uptake and degradation machinery might be an important mechanism for L. plantarum 80 to thrive in the protein-containing fermenting cocoa pulp-bean mass. Further, several genes encoding various amino acid transporters were present, such as livABCDE, enabling L. plantarum 80 to take up a variety of amino acids from the environment.
The genome sequence of L. plantarum 80 contained genes encoding an asparaginase (ans), an aspartate transaminase (aspC), and an aspartase (aspA; Fig. 2; Additional file 3). This pathway enables production of oxaloacetate and/or fumarate, which can subsequently be used for energy production and/or NAD+ regeneration by the enzymes encoded in the citrate operon (Fig. 3). Although a gene encoding glutamate decarboxylase was found (gadB), this pathway was unlikely to be operational, as the genes encoding the glutamate-γ-aminobutyric acid antiporter (gadC) and the transcriptional regulator (gadR) were not found, which are required for glutamate decarboxylation activity in other LAB .
A gene encoding the α-oxoglutarate-dependent aminotransferase BcaT was found in the genome sequence of L. plantarum 80, enabling the production of α-oxoacids from branched-chain amino acids. Further degradation of the α-oxoacids into hydroxyacids by this strain is feasible, as the genome sequence harboured a gene encoding a 2-hydroxyacid dehydrogenase (Fig. 2; Additional file 3). Enzymes involved in the conversion of α-oxoacids by oxidative decarboxylation into carboxylic acids or by decarboxylation into aldehydes were not found in the genome sequence. Further, the aromatic aminotransferase AraT was present in the genome sequence, which enables L. plantarum 80 to transaminate aromatic amino acids. In addition, aromatic amino acid decarboxylation enzymes were not found, indicating that L. plantarum 80 is not able to produce biogenic amines.
Genes encoding cystathionine β-lyase (metC) and cysteine kinase (cysK) were found in the same locus, which indicated that the methionine and cysteine catabolic pathways of L. plantarum 80 are linked, as has been shown previously for other LAB [72, 73]. Further, genes associated with the catabolism of serine and threonine, i.e., a serine deaminase (sda) and a serine hydroxymethyltransferase (shm), were retrieved in the genome sequence of L. plantarum 80 (Fig. 2; Additional file 3). Genes involved in histidine decarboxylation or the ADI pathway were absent.
Comparative genome sequence analysis
Comparative genomics of Lactobacillus fermentum 222
The genome sequence of L. fermentum 222 harboured 182 strain-specific genes, i.e., genes without a homolog in other known L. fermentum genome sequences (Additional file 1). Most of these genes encoded hypothetical proteins or were disrupted, although some had a predicted function. The strain-specific genes included two putative citrate transporters (LFER_11 and LFER_1857, discussed above) and a gene cluster (LFER_810-LFER_816) containing a diacetyl reductase-encoding gene (LFER_814; Fig. 2; Additional file 3). The complete cit gene cluster that was not interrupted by other genes or transposable elements (discussed above) and the occurrence of two unique additional (putative) citrate transporters might be the result of specific adaptations that enable this strain to consume citrate in an efficient way during the cocoa bean fermentation process [25, 74]. Further, a gene cluster involved in amino acid uptake and catabolism, consisting of genes coding for an amino acid transporter (LFER_135), an aspartase (LFER_136, discussed above), an amino acid permease (LFER_137), and an aminotransferase (LFER_138, discussed above) was unique for L. fermentum 222, providing evidence that amino acid conversion pathways might be an important feature for functional starter culture strains that have to improve the cocoa bean fermentation process.
Comparative genomics of Lactobacillus plantarum
A total of 223 strain-specific genes were found in the genome sequence of L. plantarum 80 compared to the currently available L. plantarum genome sequences (Additional file 2). Among these strain-specific genes was a gene cluster (LP80_2381-LP80_2387) consisting of the PTS-encoding sorABCDE genes, a dehydrogenase-encoding gene (sorF), and a gene encoding the transcriptional regulator (sorR). In Lactobacillus casei, this gene cluster is involved in transport of sorbose and subsequent production of fructose 6-phosphate, which can be channeled into the glycolytic pathway . This mechanism can also be used to transport fructose, as shown for L. casei and Klebsiella pneumoniae . These genes might thus offer L. plantarum 80 unique features to be able to dominate the fructose-containing cocoa pulp-bean mass during cocoa bean fermentation. Furthermore, a unique cluster of four genes was found, consisting of an oxidoreductase (LP80_2995), a ribose 5-phosphate isomerase (LP80_2996), a fructose-specific PTS (LP80_2997), and a transcriptional regulator (LP80_2998). These traits underline the fructophilic character of L. plantarum 80 during cocoa bean fermentation processes .
Genomics-based pathway reconstruction of the carbohydrate metabolism and amino acid conversion mechanisms of candidate functional LAB starter culture strains for the cocoa bean fermentation process revealed important insights into their lifestyle, niche adaptations, and metabolic capacities. Although some studies report on comparative genomics of LAB [32, 59, 82], this is the first extensive (comparative) genome sequence analysis of these species, revealing their core- and pan-genome. Identification of the genetic potential of cocoa-derived candidate functional starter cultures obtained from this and other studies  in metagenomic data sets will allow further insights into the contribution of functional starter culture strains to cocoa bean fermentation processes . This information is crucial for an improved and rationalised selection of functional starter culture strains for the cocoa bean fermentation process.
Bacterial strains and growth conditions
Lactobacillus fermentum 222 and L. plantarum 80 were originally isolated from a spontaneous cocoa bean heap fermentation process carried out in Ghana . The strains were stored at −80 °C in de Man-Rogosa-Sharpe (MRS) medium (Oxoid, Basingstoke, United Kingdom), supplemented with 25 % (v/v) glycerol as a cryoprotectant. To obtain cell pellets, the pure strains were grown overnight in MRS medium at 37 °C, followed by cell harvesting through centrifugation (21,036 × g, 15 min, 4 °C) of 2-ml cultures.
DNA extraction and 454 pyrosequencing
Total genomic DNA was extracted from four cell pellets of both L. fermentum 222 and L. plantarum 80, using a previously described protocol with modifications . A thawed pellet was washed in 1 ml of TES buffer [50 mM Tris base, 1 mM ethylene diamine tetra-acetic acid (EDTA), 6.7 % (m/v) sucrose, pH 8.0] and centrifuged at 2,795 × g for 10 min at 4 °C. The resulting pellet was collected and 300 μl of STET buffer [50 mM Tris base, 50 mM EDTA, 8 % (m/v) sucrose, 5 % (m/v) Triton X-100, pH 8.0] was added. The suspension was incubated in the presence of lysozyme (VWR International, Darmstadt, Germany), which was dissolved in TES buffer in a final concentration of 5 U/μl, at 37 °C for 1 h. Thereafter, a chemical treatment with a preheated (37 °C) solution of 20 % (m/v) sodium dodecyl sulphate (SDS; VWR International) in TE buffer (50 mM Tris base, 1 mM EDTA, pH 8.0) and a mechanical treatment with a pinch of glass beads (150–210 μm; Sigma-Aldrich, Steinheim, Germany) was applied, by vortexing the lysate for 60 s and subsequently incubating the mixture at 37 °C for 10 min, followed by a 10-min treatment at 65 °C to inactivate the enzymes. This lysate was extracted with one volume of phenol/chloroform/isoamylalcohol (49.5:49.5:1.0; Sigma-Aldrich). Phases were separated by centrifugation (18,900 × g, 5 min) using Phase Lock Gel™ tubes (Eppendorf AG, Hamburg, Germany). DNA was precipitated with 1 ml of isopropanol and 70 μl of NaCl (5 M) on ice and was collected by centrifugation (22,000 × g, 30 min, 4 °C). The resulting DNA pellet was washed with 500 μl of a 70 % (v/v) ethanol solution, dried under vacuum, and resuspended in 50 μl of TE buffer. Two μl RNase (10 mg/ml; Sigma-Aldrich) was added, followed by incubation at 37 °C for 10 min. For each strain, the four solutions were pooled and the DNA precipitation and washing steps were repeated. To confirm the identity of the bacterial strains grown, the near full-length 16S rRNA gene was amplified based on the genomic DNA extracted, purified, and sequenced, as described previously . The quality of the genomic DNA was assessed by gel electrophoresis; its quantity was estimated by a fluorescence-based method using the Quant-iT dsDNA assay kit (Invitrogen, Carlsbad, CA, USA) and the DTX800 multimode detector (Beckman Coulter, Pasadena, CA, USA). Next, the isolated genomic DNA was used as template for shotgun pyrosequencing on a genome sequencer (GS) FLX system (Roche Applied Science, Mannheim, Germany) using Titanium chemistry, which was performed by a commercial facility (VIB Nucleomics Core, Leuven, Belgium). A DNA library was constructed for each strain according to the GS FLX rapid library preparation kit (Roche Applied Science). The optimal DNA copy per bead ratio was determined by an emulsion PCR titration, using a GS FLX Titanium SV emPCR kit (Lib-L; Roche Applied Science). A final emulsion PCR for the sequencing runs was performed using the GS FLX Titanium LV emPCR kit (Lib-L; Roche Applied Science). One pyrosequencing run was carried out with the DNA libraries, with each strain occupying half a PicoTiterPlate.
Genome sequence assembly and annotation
Reads were assembled using Newbler version 2.7 (Roche Applied Science) with default parameters. Automated gene prediction and annotation of the assembled contigs longer than 500 bp was carried out using a local installation of the bacterial genome sequence annotation system GenDB version 2.2, as described previously . Briefly, GLIMMER 2.1  and CRITICA  were applied to predict the gene sequences present. Ribosomal binding sites were identified with RBSfinder  and tRNA genes were searched for using tRNAscan-SE . The proteins deduced were functionally characterised by REGANOR  using automated searches in public databases, including SWISS-PROT , Pfam , KEGG , and TIGRFAM . Additionally, SignalP , helix-turn-helix , and TMHMM  were applied to find signal peptides, helix-turn-helix DNA binding motifs, and transmembrane regions, respectively. Each gene was functionally classified by assigning a cluster of orthologous groups (COG) and a gene ontology (GO) number [98, 99]. To further supplement the annotation, plasmids were identified using the PATRIC database plasmid_seq, virulence factors were searched for by BLASTn (based on nucleotide sequence) analysis using the human pathogenic bacteria virulence factor database (VFDB; ), antibiotic resistance genes were searched for by BLASTn analysis using the antibiotic resistance gene annotation database (ARG-ANNOT; ), CRISPRs were searched for with CRISPRFinder  and CRT , prophage sequences were identified by PHAST analysis , and BAGEL3  was used to find genes encoding bacteriocins. Based on these annotations, metabolic pathways were reconstructed.
The annotated genome sequences were deposited in the DDBJ/EMBL/GenBank database as sequencing project PRJEB5182 with EMBL accession numbers CBZV010000001-CBZV010000073 for L. fermentum 222 and sequencing project PRJEB5195 with EMBL accession numbers CBZW010000001-CBZW010000067 for L. plantarum 80.
Comparative genome sequence analysis
Comparative analysis of the genome sequences of L. fermentum 222 and L. plantarum 80 with available complete and draft genome sequences of strains of the same species (Additional files 1 and 2) was performed by the EDGAR framework  using default parameters. This included identification and classification of orthologous genes, identification of strain-specific genes, and calculation of the pan- and core-genomes. Also within this framework, phylogenetic analyses were performed using PHYLIP , thereby relying on an alignment of the core genes of a selection of complete and draft genome sequences of members of the species L. fermentum (Additional file 1) and L. plantarum (Additional file 2), that was generated using MUSCLE  and GBLOCKS . As the publicly available draft genome sequence of L. fermentum FTDC 8312 was not annotated, its genome sequence was retrieved from the NCBI data repository, followed by automated annotation using the GenDB platform as described above. For the phylogenetic analysis of the species L. fermentum, L. plantarum 80 was used as an outgroup; for the phylogenetic analysis of the species L. plantarum, L. fermentum 222 was used as an outgroup.
The authors thank Dr. Jochen Blom and Prof. Dr. Alexander Goesmann (Institute of Bioinformatics and Systems Biology, Justus-Liebig-University, Giessen, Germany) for performing the EDGAR computational analyses and the VIB Nucleomics core for carrying out the 454 pyrosequencing. Part of this research was financed by the Research Council of the Vrije Universiteit Brussel (OZR, GOA, SRP, and IOF projects). The authors further acknowledge their finances from the Research Foundation Flanders (FWO-Vlaanderen) and the Hercules foundation. KI was the receiver of a post-graduate grant of the Agency for Innovation by Science and Technology (IWT). The funders had no role in the design of the study, the collection and analysis of the data, the writing of the manuscript, or the decision to submit the manuscript for publication.
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