Complete genome sequence and comparative analysis of Acetobacter pasteurianus 386B, a strain well-adapted to the cocoa bean fermentation ecosystem
© Illeghems et al.; licensee BioMed Central Ltd. 2013
Received: 18 February 2013
Accepted: 27 July 2013
Published: 1 August 2013
Acetobacter pasteurianus 386B, an acetic acid bacterium originating from a spontaneous cocoa bean heap fermentation, proved to be an ideal functional starter culture for coca bean fermentations. It is able to dominate the fermentation process, thereby resisting high acetic acid concentrations and temperatures. However, the molecular mechanisms underlying its metabolic capabilities and niche adaptations are unknown. In this study, whole-genome sequencing and comparative genome analysis was used to investigate this strain’s mechanisms to dominate the cocoa bean fermentation process.
The genome sequence of A. pasteurianus 386B is composed of a 2.8-Mb chromosome and seven plasmids. The annotation of 2875 protein-coding sequences revealed important characteristics, including several metabolic pathways, the occurrence of strain-specific genes such as an endopolygalacturonase, and the presence of mechanisms involved in tolerance towards various stress conditions. Furthermore, the low number of transposases in the genome and the absence of complete phage genomes indicate that this strain might be more genetically stable compared with other A. pasteurianus strains, which is an important advantage for the use of this strain as a functional starter culture. Comparative genome analysis with other members of the Acetobacteraceae confirmed the functional properties of A. pasteurianus 386B, such as its thermotolerant nature and unique genetic composition.
Genome analysis of A. pasteurianus 386B provided detailed insights into the underlying mechanisms of its metabolic features, niche adaptations, and tolerance towards stress conditions. Combination of these data with previous experimental knowledge enabled an integrated, global overview of the functional characteristics of this strain. This knowledge will enable improved fermentation strategies and selection of appropriate acetic acid bacteria strains as functional starter culture for cocoa bean fermentation processes.
KeywordsAcetobacter pasteurianus Cocoa bean fermentation 454 pyrosequencing
Acetic acid bacteria (AAB) are a group of microorganisms that belong to the family of the Acetobacteraceae of the Alpha-proteobacteria . AAB can be found on (tropical) fruits and flowers [2–4], in fermented foods [1, 3], and as members of the Drosophila gut . Overall, AAB are of industrial interest because of their physiology, which is the case for acetic acid production out of ethanol during vinegar, kombucha, or cocoa bean fermentation [6–8] as well as for fine chemical productions such as those of ascorbic acid and cellulose [9, 10]. Furthermore, AAB can occur as spoilage bacteria, as can be the case in beer, wine, and cider fermentations [1, 3]. One of the key metabolic features of AAB is the conversion of ethanol into acetic acid by two sequential reactions catalyzed by membrane-bound alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes .
Currently, AAB include twelve genera, among which Acetobacter, Gluconobacter, and Gluconacetobacter are the most studied ones [3, 8, 12]. The genus Acetobacter is one of the most interesting from a biotechnological point of view [1, 3], because of its ability to oxidize ethanol into acetate while tolerating high acetic acid concentrations in the environment . Different species within the Acetobacter genus are distinguished, among which Acetobacter pasteurianus, Acetobacter aceti and Acetobacter pomorum are important in industrial vinegar production [3, 14, 15], Acetobacter cerevisiae is present in beer and on grapes [16, 17], and Acetobacter tropicalis and Acetobacter senegalensis are involved in the cocoa bean fermentation process . Acetobacter species are able to oxidize acetate completely (so-called overoxidation) and use ubiquinones of the Q-9 type, the latter being in contrast with species of the AAB genera Gluconacetobacter and Gluconobacter that contain mainly ubiquinones of the Q-10 type . At present, A. pasteurianus IFO 3283 (originating from a fermentation) is the only member of the genus Acetobacter of which the genome has been sequenced completely, including six plasmids . However, draft genomes are available for A. pasteurianus 3P3 (originating from submerged wine vinegar) , A. pasteurianus NBRC 101655 (Thai pineapple) , A. pasteurianus subsp. pasteurianus LMG 1262T (Dutch beer, type strain) , A. aceti NBRC 14818 (ethanol-based vinegar) , A. pomorum DM001 (Drosophila gut) , and A. tropicalis NBRC 101654 (Thai fruit) [2, 24]. In general, Acetobacter species possess relatively small genomes (approximately 3 Mb), including plasmids in particular cases [20, 25–27].
Acetobacter pasteurianus strains are used for vinegar fermentations worldwide [28–30] and also occur in beer as spoilers . Further, it has been shown that this species plays an essential role in the fermentation of cocoa pulp-bean mass, the first step in chocolate production [31–33]. Spontaneous cocoa bean fermentation is characterized by a succession of microbial activities carried out by yeasts (in particular Hanseniaspora opuntiae/uvarum and Saccharomyces cerevisiae), involved in depectinization and ethanol formation; lactic acid bacteria (LAB, in particular Lactobacillus fermentum), involved in citric acid and fructose conversion and lactic acid production; and AAB (in particular A. pasteurianus), involved in the oxidation of ethanol produced by the yeasts into acetic acid and further overoxidation of acetic acid and lactic acid produced by LAB into carbon dioxide and water [6, 34].
Acetobacter pasteurianus 386B originates from a spontaneous cocoa bean heap fermentation carried out in Ghana and has been characterized as an ethanol-oxidizing, lactic acid-oxidizing, and acetic acid-producing strain [18, 35]. Furthermore, A. pasteurianus 386B is a thermotolerant strain with high resistance to ethanol and acetic acid [36, 37]. These functional properties make it an ideal starter culture strain for cocoa bean fermentations . In this study, we present the complete genome sequence and analysis of A. pasteurianus 386B to obtain insights into the genomic features of this interesting starter culture strain [37, 38]. A better understanding of the molecular mechanisms underlying its metabolic capabilities will lead to detailed insights into the mechanisms of niche adaptation of this strain. Furthermore, comparison of A. pasteurianus 386B with other sequenced members of the Acetobacteraceae will address the unique functional properties of this strain as well as the common characteristics of the Acetobacteraceae.
Results and discussion
454 Pyrosequencing and sequence annotation
General features of the Acetobacter pasteurianus 386B chromosome and plasmids
EMBL accession no.
Features of the Acetobacter pasteurianus 386B genome
Total size (bp)
G+C content (%)
No. of protein-coding genes
Coding density (%)
Average gene length (bp)
No. of rRNA operons
5 × 16S-23S-5S
No. of tRNAs
No. of transposases
General architecture of the A. pasteurianus 386B genome
A plot of the calculated G/C skew [(G–C)/(G + C)] indicated a bidirectional replication mechanism of the chromosome (Figure 1A), which was confirmed by the biased distribution of architecture imparting sequences (AIMS) on the leading and lagging strands , dividing the chromosome of A. pasteurianus 386B into two replichores of similar sizes (Figure 1B). This enabled the prediction of the origin of chromosomal replication (oriC), located near the dnaA-coding region (APA386B_66), as well as a replication termination (dif) region at position 1,590,515 on the chromosomal map . The sequence of the 32-bp dif region was aligned with the consensus sequence of Gamma-proteobacterial dif sites . The dif region, positioned opposite of the oriC, is involved in replication termination and defines the leading and lagging strands during replication, together with oriC. The occurence of the G/C skew, replichores, and biased distribution of AIMS supports the accuracy of the sequence assembly, as they represent a common general architecture of a genome.
Phylogenetic analysis and comparative genomics
Comparative analysis of the five available genome sequences of strains of the species A. pasteurianus revealed that there were 2,019 shared orthologous proteins, representing 68% of the predicted proteins from A. pasteurianus 386B. This may correspond with the core genome of the species Acetobacter pasteurianus. Furthermore, A. pasteurianus 386B contained 122 strain-specific genes, of which 95 had no known function, which may be related with niche adaptations. The 27 unique genes with an assigned function (Additional file 3) may contribute to the performance of this strain as a starter culture in the cocoa bean fermentation process. For instance, the presence of an endopolygalacturonase in the genome sequence of A. pasteurianus 386B (APA386B_1663; Additional file 3) indicates a possible role of this strain in pectin breakdown, an important metabolic process in the beginning of cocoa bean fermentations [44, 45]. This is the first report of an endopolygalacturonase gene in an A. pasteurianus strain. Indeed, the closest relative possessing such a gene is A. tropicalis NBRC 101654 ; A. tropicalis has been isolated from spontaneous cocoa bean fermentation processes as well . Furthermore, a PCR assay indicated that this polygalacturonase gene was not widespread amongst A. pasteurianus strains isolated from spontaneous cocoa bean fermentations (Additional file 4). This suggests that expression of this gene might contribute to the capability of A. pasteurianus 386B to dominate cocoa bean fermentations.
Comparative analysis of Acetobacter pasteurianus 386B and members of the Acetobacteraceae family
Acetic acid bacterium strain
Number of shared genes
Percentage of shared genes
Acetobacter pasteurianus NBRC 101655
Acetobacter pasteurianus IFO 3283
Acetobacter pasteurianus LMG 1262T
Acetobacter pasteurianus 3P3
Acetobacter pomorum DM001
Acetobacter tropicalis NBRC 101654
Acetobacter aceti NBRC 14818
Gluconacetobacter diazotrophicus Pal5
Gluconacetobacter medellinensis NBRC 3288
Gluconobacter oxydans 621H
Granulibacter bethesdensis CGDNIH1
Acidiphilium multivorum AIU301
Acidiphilium cryptum JF-5
Intracellular metabolism of sugars and sugar derivatives
Genome analysis showed that A. pasteurianus 386B possessed genes encoding metabolic pathways involved in the de novo synthesis of all nucleotides, amino acids, phospholipids and many vitamins, such as biotin, folic acid, pantothenate, pyridoxine, riboflavin, and thiamine. Ammonia, involved in the activity of glutamate synthase (APA386B_893 - APA386B_894) and glutamine synthetase (APA386B_2129), could be taken up by a specific ammonia transporter (APA386B_239). Furthermore, the genome of A. pasteurianus 386B contained genes to synthesize and use trehalose, which can protect the cell from high osmolarity and/or can be used as an energy source in bacteria and yeast . The pathway consisted of trehalose-6-phosphate synthase (otsA; APA386B_1724), trehalose-6-phosphate phosphatase (otsB; APA386B_1723), and trehalase (treA; APA386B_104). In addition, genes coding for the mechanosensitive channels MscL (APA386B_2572) and MscS (APA386B_1440) were present in the genome sequence of A. pasteurianus 386B, which generally play an important role in osmotolerance .
Membrane-bound dehydrogenases and respiratory chain
A second group of membrane-bound dehydrogenases contains flavines as cofactor. The genes coding for flavine adenine dinucleotide (FAD)-dependent sorbitol dehydrogenase (APA386B_1096 - APA386B_1098) were present in the A. pasteurianus 386B genome, which points to the ability of this strain to produce fructose from sorbitol. However, it has been shown previously that this dehydrogenase is also responsible for the conversion of mannitol, an important intermediate of the cocoa bean fermentation process, into fructose . As the major polyol dehydrogenase was not present in this strain, it is likely that the FAD-dependent sorbitol dehydrogenase was responsible for the oxidation of mannitol into fructose, as experimentally shown in A. pasteurianus 386B (F. Moens, T. Lefeber, and L. De Vuyst, unpublished observations). In addition, A. pasteurianus 386B possessed six membrane-bound oxidoreductases with unknown function (Figure 5B; Additional file 5). These oxidoreductases are also present in the genomes of A. pasteurianus IFO 3283, Ga. diazotrophicus Pal5, and G. oxydans 621H (Additional file 6), and could be involved in the oxidation of a broad range of substrates, such as carbohydrates and polyols . Genome analysis revealed that ubiquinol, generated by the aforementioned membrane-bound dehydrogenases, could be reoxidized by a cytochrome bo3-type ubiquinol oxidase (APA386B_1578 - APA386B_1581) and a cytochrome bd-type ubiquinol oxidase (cyanide-insensitive terminal oxidase), whereby the encoding genes of the latter were present twice in the genome sequence of A. pasteurianus 386B (cydAB; APA386B_472 - APA386B_473 and APA386B_1010 - APA386B_1011). Both terminal oxidases reduce oxygen to water when reoxidizing ubiquinol into ubiquinone (Figure 5B).
The genes coding for a proton-translocating nicotinamide nucleotide transhydrogenase were present in the genome of A. pasteurianus 386B (pnt; APA386B_1508 - APA386B_1510; Figure 5C). This enzyme might oxidize NADPH+H+ derived from the intermediary metabolism, thereby translocating protons across the cytoplasmic membrane. The NADH+H+ derived from transhydrogenase activity might subsequently be reoxidized by complex I of the respiratory chain, whereas the NADP+ derived from the transhydrogenase can be reduced by the NADP+-dependent ALDH . Indeed, the genes coding for a complete proton-translocating respiratory chain complex I were retrieved (nuoA-nuoN; APA386B_556 - APA386B_567; Figure 5C). This complex I is absent in G. oxydans, which may be related to its lower growth yields . Furthermore, a membrane-bound succinate dehydrogenase/fumarate reductase was found (sdhABCD; APA386B_1513 - APA386B_1516; Figure 5C), which is not only part of the respiratory chain, but also plays an important role in the TCA cycle, thereby enabling overoxidation of acetic acid . Genes encoding both a bc 1 complex (ubiquinol:cytochrome c oxidoreductase; petABC; APA386B_775 - APA386B_777) as well as cytochrome c (cytC; APA386B_367 and APA386B_906) were identified in the genome of A. pasteurianus 386B. Furthermore, genes for the transport of heme c and the maturation of cytochrome c were present (ccmFGHI; APA386B_2396 - APA386B_2400) . However, it is unknown if this strain is able to reoxidize the reduced form of cytochrome c, as cytochrome c oxidase (complex IV) is probably inactive, because only subunit I (APA386B_609) was present in the genome.
In silico analysis of mechanisms involved in acid tolerance
A first strategy of A. pasteurianus 386B to tolerate high levels of acetic acid may consist of a cytosolic acetate-assimilating detoxification pathway. This involves a conversion of acetate to acetyl-CoA, which is performed either by acetyl-CoA synthetase (acn; APA386B_2214 and APA386B_1843; Figure 4, reaction 36) or by acetate kinase (ackA; APA386B_335; Figure 4, reaction 37) and phosphate acetyltransferase (pta; APA386B_336; Figure 4, reaction 38). Both pathways were present in the A. pasteurianus 386B genome and are known to be upregulated when citrate oxidation takes place . This suggests that the presence of two copies of the acn gene in this strain provides an advantage for efficient acetate assimilation. Alternatively, acetate can be converted into acetyl-CoA via a modified TCA cycle . Indeed, all genes encoding the enzymes of the TCA cycle were retrieved, except for succinyl-CoA synthetase. This function is bypassed by succinyl-CoA: acetate CoA transferase (SCACT, aarC; APA386B_2589; Figure 4, reaction 50). Similarly, the gene for malate dehydrogenase was not found, but oxidation of malate into oxaloacetate can be catalyzed by malate:quinone oxidoreductase (mqo; APA386B_2675; Figure 4, reaction 48) . A second mechanism in acid tolerance probably involves the presence of an acetic acid resistance ABC transporter (aatA; APA386B_103), an efflux pump in the cytoplasmic membrane capable of exporting acetic acid . Thirdly, A. pasteurianus 386B contained the gene cluster involved in pellicle polysaccharide formation (polABCDE; APA386B_1394 - APA386B_1398), preventing the diffusion of acetic acid into the cytoplasm [46, 61, 62]. Fourthly, the genes coding for urease (ureDABCEFG; APA386B_1179 - APA386B_1184), an urea transporter (urtABCDE; APA386B_1640 - APA386B_1644), an allophanate hydrolase (APA386B_936 - APA386B_937), and an urea carboxylase (APA386B_218) were present, indicating the ability to transport urea and convert it into ammonia, which may contribute to the survival of A. pasteurianus 386B in acidic environments, such as the cocoa pulp-bean mass (pH 3.5 – 4.5). The human pathogenic Gr. bethesdensis CGDNIH1 contains this mechanism too, although it may not be widespread among AAB strains, as it is absent in G. oxydans 621H and Ga. diazotrophicus Pal5 [25, 26, 63]. Lastly, genome analysis of A. pasteurianus 386B revealed the presence of genes coding for cytoplasmic components that are adapted to intracellular acidification. This is the case for N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) mutase (purE; APA386B_2565), a protein involved in purine biosynthesis. Indeed, N5-CAIR mutase of A. pasteurianus 386B is 99% identical to its orthologue in A. aceti 1023, the latter strain being adapted to an acid cytosol . Similarly, alanine racemase (alr; APA386B_1310), a protein involved in peptidoglycan biosynthesis, is 92% identical to the A. aceti 1023 orthologue, a protein known to function at low pH . In addition, the sequence similarity of both N5-CAIR mutase and alanine racemase between A. pasteurianus 386B and A. aceti 1023 was higher than between A. pasteurianus 386B and any other sequenced AAB strain (data not shown), indicating that the presence of acid-stable proteins is not widespread.
In silico analysis of mechanisms involved in thermotolerance
As described above, genome-wide phylogenetic analysis of A. pasteurianus 386B revealed that this strain is most related to the thermotolerant strain A. pasteurianus NBRC 101655. In addition, adaptive mutation resulted in 14 validated mutations, involved in improved thermotolerance of this strain . Five of these regions were also modified in A. pasteurianus 386B and not in A. pasteurianus NBRC 101655 (Additional file 7). For example, one of the two genes coding for cytochrome c (APA386B_906), contained three synonymous mutations and one nonsynonymous mutation. Furthermore, the genes necessary for growth at high temperatures (42°C) of the thermotolerant strain A. tropicalis NBRC 101654 have been identified recently . Although this strain belongs to a different species than A. pasteurianus 386B, all genes of A. tropicalis NBRC 101654 necessary for growth at high temperatures were found in the genome sequence of A. pasteurianus 386B as well (Additional file 7). This indicates that the latter strain, when thriving in the high-temperature cocoa pulp-bean mass, may use the same mechanisms towards heat stress as A. tropicalis NBRC 101654.
The complete genome sequence of A. pasteurianus 386B, a strain originating from a spontaneous cocoa bean heap fermentation in Ghana, was determined, annotated, and described in this study. The global overview of all genes and pathways obtained provided comprehensive insights into the metabolic features regarding important substrates (such as ethanol, glucose, acetate, lactate, and glycerol) and stresses (such as acidic and heat stress) during the cocoa bean fermentation process.
Comparative genome analysis provided information regarding niche adaptations of this strain. For example, the presence of a gene coding for an endopolygalacturonase was discovered. This enzyme is involved in the breakdown of pectin, a compound responsible for the viscosity of the cocoa pulp-bean mass. Although depectinization is mainly important in the beginning (anaerobic yeast activity phase) of the cocoa bean fermentation process, the activity of the pectinolytic enzymes allows air to enter the cocoa pulp-bean mass, which promotes the growth of obligate aerobic AAB. Therefore, the presence of this gene could be an important prerequisite for survival and performance of AAB during cocoa bean fermentations. Furthermore, the comparative genome analysis revealed that the genome of A. pasteurianus 386B contained a low number of transposases, resulting in the absence of truncated genes, which might be important for expression under cocoa bean fermentation conditions.
Genome analysis unraveled various mechanisms of A. pasteurianus 386B to tolerate stress conditions occurring in a cocoa bean fermentation ecosystem. As also active prophages were absent in the genome sequence, these findings indicate that A. pasteurianus 386B is genetically more stable compared with other fully characterized AAB, contributing to the prerequisites of a starter culture strain.
All these findings support that this strain is a suitable functional starter culture for controlled cocoa bean fermentation processes. In addition, the results presented in this study will enable analysis of the transcriptome of A. pasteurianus 386B, which will provide insight into its metabolic activity. Finally, the characteristics of A. pasteurianus 386B revealed in this study are essential to generate further insights into the functional role of AAB in general, and A. pasteurianus in particular, during the cocoa bean fermentation process, which is of great importance to select an appropriate starter culture for homogeneous, fast, and successfully controlled fermentation processes.
Bacterial strain and growth conditions
Acetobacter pasteurianus 386B was originally isolated from a spontaneous cocoa bean heap fermentation carried out in Ghana . The strain was stored at −80°C in mannitol-yeast extract-peptone (MYP) medium [2.5% D-mannitol, 0.5% yeast extract, and 0.3% bacteriological peptone (Oxoid, Basingstoke, Hampshire, United Kingdom), w/v], supplemented with 25% (v/v) glycerol as a cryoprotectant. To obtain cell pellets, A. pasteurianus 386B was propagated in MYP medium twice (aerobic incubation at 30°C overnight), followed by centrifugation (21,036 × g, 15 min, 4°C) of 2-ml cultures.
DNA extraction and 454 pyrosequencing
Total genomic DNA was extracted from cell pellets using the High Pure PCR Template Preparation Kit (Roche Applied Science, Mannheim, Germany), followed by RNase treatment and purification using the High Pure PCR Product Purification Kit (Roche Applied Science), always according to the manufacturer’s instructions. To confirm the identity of the bacterial strain grown, a 16S rRNA gene-specific region was amplified based on the genomic DNA extracted, as described previously . Amplicons were purified using the Wizard SV Gel and PCR Clean up system (Promega, Madison, WI, USA) and sequenced at a commercial facility using Sanger sequencing (VIB Genetic Service Facility, Antwerp, Belgium). 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).
For genome sequencing, a total amount of 5 μg of genomic DNA was used for the construction of an 8-kb paired-end library with the GS FLX Titanium Library Paired-End Adaptors Kit and the GS FLX Titanium Rapid Library Prep Kit (Roche Applied Science) according to the manufacturer’s instructions. 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). The final emulsion PCR was performed using the GS FLX Titanium LV emPCR kit (Lib-L; Roche Applied Science). Pyrosequencing was performed on a Genome Sequencer GS FLX instrument using Titanium chemistry (Roche Applied Science) with the sample occupying one region of a four-region gasket. Library preparation and pyrosequencing were performed by the VIB Nucleomics Core Facility (Leuven, Belgium). Reads were assembled using the GS De Novo Assembler version 2.5.3 with default parameters.
PCR-based gap closure
To close remaining gaps in the assembled genome sequence, PCR primers were designed based on contig ends using the Consed program  and synthesized in a commercial facility (Integrated DNA Technologies, Leuven, Belgium). PCR assays were performed using a DNA T3000 thermocycler (Biometra, Goettingen, Germany), containing 50 ng of genomic DNA, 100 μM of each dNTP (Sigma-Aldrich, St. Louis, MO, USA), 5 pmole of each primer, 5 μL of 10 × PCR reaction buffer (Fermentas, St. Leon-Rot, Germany), 1.875 U of Pfu DNA Polymerase (Fermentas), and sterile ultrapure water in a final volume of 50 μL. Following amplification, PCR product sizes were verified using a 1.0-% (w/v) agarose gel and the remaining reaction mixture was purified using the Wizard SV Gel and PCR Clean up system (Promega). Amplicons were sequenced in a commercial facility using Sanger sequencing technology (Macrogen Europe, Amsterdam, The Netherlands). All DNA sequences obtained were uploaded into the Consed program, manually inspected, and integrated into the genome assembly to generate the complete genome sequence of A. pasteurianus 386B. To facilitate gap closure and assembly validation, contigs were mapped to the A. pasteurianus IFO 3283 genome by means of the r2cat tool [20, 69].
Genome analysis and annotation
Automatic gene prediction and annotation of the assembled genome sequence were carried out using a local installation of the bacterial genome annotation system GenDB v2.2 . A combined gene prediction strategy was applied by using GLIMMER 2.1 and the CRITICA program suite [71, 72]. Putative ribosomal binding sites and tRNA genes were identified with the RBSfinder tool  and tRNAscan-SE . The deduced proteins were functionally characterized by REGANOR  using automated searches in public databases, including SWISS-PROT and TrEMBL , Pfam , KEGG , and TIGRFAM . Additionally, SignalP (detection of signal peptides) , helix-turn-helix (identification of helix-turn-helix DNA binding motifs) , and TMHMM (detection of transmembrane regions)  were applied. Each gene was functionally classified by assigning a Cluster of Orthologous groups (COG) number  and a Gene Ontology (GO) number . The automated gene prediction and annotation was followed by manual curation of the data. To correct for over-annotation, short CDS without functional annotation, with low confidence scores inferred by the GenDB platform, and with overlaps with other CDS were eliminated from the final annotation. A genome plot of A. pasteurianus 386B was generated with the DNAPlotter tool . The origin of chromosomal replication of A. pasteurianus 386B was predicted with the Ori-Finder tool . CRISPRs were searched for with the CRISPRFinder tool .
Phylogenetic analysis and comparative genomics
A phylogenetic analysis was performed using complete and draft genome sequences of members of the family Acetobacteraceae. Therefore, the annotated genome sequences of the finished genomes of A. pasteurianus IFO 3283 (including plasmids), Acidiphilium multivorum AIU301, Acidiphilium cryptum JF-5, Ga. diazotrophicus Pal5, Ga. medellinensis NBRC 3288 (formerly Gluconacetobacter xylinus NBRC 3288), and G. oxydans 621H were used [25–27, 63, 87]. Furthermore, the draft genome sequences (contigs and scaffolds) of A. pasteurianus subsp. pasteurianus LMG 1262T, A. pasteurianus NBRC 101655, A. pomorum DM001, A. tropicalis NBRC 101654, and A. aceti NBRC 14818 were included [2, 5, 20, 22, 23]. As no annotation of the draft genome sequence of A. pasteurianus 3P3  was available, the draft genome was annotated using the GenDB platform as described above. The manually curated genome sequence of A. pasteurianus 386B, together with the plasmids identified, was incorporated as well. Comparative analysis of these genome sequences, including synteny analyses, identification and classification of orthologous genes, and phylogenetic analysis was accomplished by the EDGAR software framework using default parameters . In addition, the Artemis Comparison Tool (ACT) was applied to identify similarity between the different plasmids of A. pasteurianus 386B and A. pasteurianus IFO 3283 , using the BLASTN algorithm with default parameters .
The annotated sequences of the A. pasteurianus chromosome and plasmids were deposited in the DDBJ/EMBL/GenBank database (Sequencing Project PRJEB1172). The accession numbers are listed in Table 1.
Acetic acid bacteria
Artemis comparison tool
Architecture imparting sequences
Cluster of orthologous groups
Cocoa pulp simulation medium
Clustered regularly interspaced short palindromic repeat
Flavine adenine dinucleotide
Lactic acid bacteria
Succinyl-CoA:acetate CoA transferase
We thank Dipl.-Inform. Jochen Blom and Dr. Alexander Goesmann [Bioinformatics Resource Facility, Center for Biotechnology (CeBiTec), Bielefeld University, Germany] for performing the EDGAR computational analysis and assistance with the installation of the GenDB platform, respectively. This research was financed by the Research Council of the Vrije Universiteit Brussel and the Research Foundation Flanders (FWO; project number 1.5.108.09N). KI is 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.
- Kersters K, Lisdiyanti P, Komagata K, Swings J: The family Acetobacteraceae: the genera Acetobacter, Acidomonas, Asaia, Gluconacetobacter, Gluconobacter, and Kozakia. The Prokaryotes. Edited by: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E. 2006, New York: Springer, 163-200.View Article
- Matsutani M, Hirakawa H, Nishikura M, Soemphol W, Ali IAI, Yakushi T, Matsushita K: Increased number of arginine-based salt bridges contributes to the thermotolerance of thermotolerant acetic acid bacteria, Acetobacter tropicalis SKU1100. Biochem Biophys Res Commun. 2011, 409: 120-124. 10.1016/j.bbrc.2011.04.126.View ArticlePubMed
- Sengun IY, Karabiyikli S: Importance of acetic acid bacteria in food industry. Food Control. 2011, 22: 647-656. 10.1016/j.foodcont.2010.11.008.View Article
- Soemphol W, Deeraksa A, Matsutani M, Yakushi T, Toyama H, Adachi O, Yamada M, Matsushita K: Global analysis of the genes involved in the thermotolerance mechanism of thermotolerant Acetobacter tropicalis SKU1100. Biosci Biotechnol Biochem. 2011, 75: 1921-1928. 10.1271/bbb.110310.View ArticlePubMed
- Shin SC, Kim S-H, You H, Kim B, Kim AC, Lee K-A, Yoon J-H, Ryu J-H, Lee W-J: Drosophila microbiome modulates host developmental and metabolic homeostasis via insulin signaling. Science. 2011, 334: 670-674. 10.1126/science.1212782.View ArticlePubMed
- De Vuyst L, Lefeber T, Papalexandratou Z, Camu N: The functional role of lactic acid bacteria in cocoa bean fermentation. Biotechnology of Lactic Acid Bacteria: Novel Applications. Edited by: Mozzi F, Raya RR, Vignolo GM. 2010, Oxford, UK: Wiley-Blackwel, 301-325.View Article
- Adachi O, Moonmangmee D, Toyama H, Yamada M, Shinagawa E, Matsushita K: New developments in oxidative fermentation. Appl Microbiol Biotechnol. 2003, 60: 643-653.View ArticlePubMed
- Raspor P, Goranovic D: Biotechnological applications of acetic acid bacteria. Crit Rev Biotechnol. 2008, 28: 101-124. 10.1080/07388550802046749.View ArticlePubMed
- Bremus C, Herrmann U, Bringer-Meyer S, Sahm H: The use of microorganisms in L-ascorbic acid production. J Biotechnol. 2006, 124: 196-205. 10.1016/j.jbiotec.2006.01.010.View ArticlePubMed
- Römling U: Molecular biology of cellulose production in bacteria. Res Microbiol. 2002, 153: 205-212. 10.1016/S0923-2508(02)01316-5.View ArticlePubMed
- Matsushita K, Toyama H, Adachi O: Respiratory chains and bioenergetics of acetic acid bacteria. Adv Microb Physiol. 1994, 36: 247-301.View ArticlePubMed
- González Á, Mas A: Differentiation of acetic acid bacteria based on sequence analysis of 16S-23S rRNA gene internal transcribed spacer sequences. Int J Food Microbiol. 2011, 147: 217-222. 10.1016/j.ijfoodmicro.2011.04.005.View ArticlePubMed
- Matsutani M, Hirakawa H, Yakushi T, Matsushita K: Genome-wide phylogenetic analysis of Gluconobacter, Acetobacter, and Gluconacetobacter. FEMS Microbiol Lett. 2010, 315: 122-128.View ArticlePubMed
- Zahoor T, Siddique F, Farooq U: Isolation and characterization of vinegar culture (Acetobacter aceti) from indigenous sources. Br Food J. 2006, 108: 429-439. 10.1108/00070700610668405.View Article
- Sokollek SJ, Hertel C, Hammes WP: Description of Acetobacter oboediens sp. nov. and Acetobacter pomorum sp. nov., two new species isolated from industrial vinegar fermentations. Int J Syst Bacteriol. 1998, 48: 935-940. 10.1099/00207713-48-3-935.View ArticlePubMed
- Cleenwerck I, Vandemeulebroecke K, Janssens D, Swings J: Re-examination of the genus Acetobacter, with descriptions of Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov. Int J Syst Evol Microbiol. 2002, 52: 1551-1558. 10.1099/ijs.0.02064-0.PubMed
- Prieto C, Jara C, Mas A, Romero J: Application of molecular methods for analysing the distribution and diversity of acetic acid bacteria in Chilean vineyards. Int J Food Microbiol. 2007, 115: 348-355. 10.1016/j.ijfoodmicro.2006.12.017.View ArticlePubMed
- Camu N, De Winter T, Verbrugghe K, Cleenwerck I, Vandamme P, Takrama JS, Vancanneyt M, De Vuyst L: Dynamics and biodiversity of populations of lactic acid bacteria and acetic acid bacteria involved in spontaneous heap fermentation of cocoa beans in Ghana. Appl Environ Microbiol. 2007, 73: 1809-1824. 10.1128/AEM.02189-06.PubMed CentralView ArticlePubMed
- Yamada Y, Hoshino K-i, Ishikawa T: The phylogeny of acetic acid bacteria based on the partial sequences of 16S ribosomal RNA: the elevation of the subgenus Gluconoacetobacter to the generic level. Biosci Biotechnol Biochem. 1997, 61: 1244-1251. 10.1271/bbb.61.1244.View ArticlePubMed
- Azuma Y, Hosoyama A, Matsutani M, Furuya N, Horikawa H, Harada T, Hirakawa H, Kuhara S, Matsushita K, Fujita N, Shirai M: Whole-genome analyses reveal genetic instability of Acetobacter pasteurianus. Nucleic Acids Res. 2009, 37: 5768-5783. 10.1093/nar/gkp612.PubMed CentralView ArticlePubMed
- Andrés-Barrao C, Falquet L, Calderon-Copete SP, Descombes P, Ortega Pérez R, Barja F: Genome sequences of the high-acetic acid-resistant bacteria Gluconacetobacter europaeus LMG 18890T and G. europaeus LMG 18494 (reference strains), G. europaeus 5P3, and Gluconacetobacter oboediens 174Bp2 (isolated from vinegar). J Bacteriol. 2011, 193: 2670-2671. 10.1128/JB.00229-11.PubMed CentralView ArticlePubMed
- Matsutani M, Hirakawa H, Saichana N, Soemphol W, Yakushi T, Matsushita K: Genome-wide phylogenetic analysis of differences in thermotolerance among closely related Acetobacter pasteurianus strains. Microbiology. 2012, 158: 229-239. 10.1099/mic.0.052134-0.View ArticlePubMed
- Sakurai K, Arai H, Ishii M, Igarashi Y: Transcriptome response to different carbon sources in Acetobacter aceti. Microbiology. 2010, 157: 899-910.View ArticlePubMed
- Lisdiyanti P, Katsura K, Potacharoen W, Navarro RR, Yamada Y, Uchimura T, Komagata K: Diversity of acetic acid bacteria in Indonesia, Thailand, and the Philippines. Microbiol Cult Coll. 2003, 19: 91-98.
- Prust C, Hoffmeister M, Liesegang H, Wiezer A, Fricke WF, Ehrenreich A, Gottschalk G, Deppenmeier U: Complete genome sequence of the acetic acid bacterium Gluconobacter oxydans. Nat Biotechnol. 2005, 23: 195-200. 10.1038/nbt1062.View ArticlePubMed
- Bertalan M, Albano R, de Padua V, Rouws L, Rojas C, Hemerly A, Teixeira K, Schwab S, Araujo J, Oliveira A, Franca L, Magalhaes V, Alqueres S, Cardoso A, Almeida W, Loureiro M, Nogueira E, Cidade D, Oliveira D, Simao T, Macedo J, Valadao A, Dreschsel M, Freitas F, Vidal M, Guedes H, Rodrigues E, Meneses C, Brioso P, Pozzer L: Complete genome sequence of the sugarcane nitrogen-fixing endophyte Gluconacetobacter diazotrophicus Pal5. BMC Genomics. 2009, 10: 450-10.1186/1471-2164-10-450.PubMed CentralView ArticlePubMed
- Ogino H, Azuma Y, Hosoyama A, Nakazawa H, Matsutani M, Hasegawa A, Otsuyama K-i, Matsushita K, Fujita N, Shirai M: Complete genome sequence of NBRC 3288, a unique cellulose-nonproducing strain of Gluconacetobacter xylinus isolated from vinegar. J Bacteriol. 2011, 193: 6997-6998. 10.1128/JB.06158-11.PubMed CentralView ArticlePubMed
- Vegas C, Mateo E, González Á, Jara C, Guillamón JM, Poblet M, Torija MJ, Mas A: Population dynamics of acetic acid bacteria during traditional wine vinegar production. Int J Food Microbiol. 2010, 138: 130-136. 10.1016/j.ijfoodmicro.2010.01.006.View ArticlePubMed
- Gullo M, Caggia C, De Vero L, Giudici P: Characterization of acetic acid bacteria in ‘traditional balsamic vinegar’. Int J Food Microbiol. 2006, 106: 209-212. 10.1016/j.ijfoodmicro.2005.06.024.View ArticlePubMed
- Haruta S, Ueno S, Egawa I, Hashiguchi K, Fujii A, Nagano M, Ishii M, Igarashi Y: Succession of bacterial and fungal communities during a traditional pot fermentation of rice vinegar assessed by PCR-mediated denaturing gradient gel electrophoresis. Int J Food Microbiol. 2006, 109: 79-87. 10.1016/j.ijfoodmicro.2006.01.015.View ArticlePubMed
- Papalexandratou Z, Vrancken G, De Bruyne K, Vandamme P, De Vuyst L: Spontaneous organic cocoa bean box fermentations in Brazil are characterized by a restricted species diversity of lactic acid bacteria and acetic acid bacteria. Food Microbiol. 2011, 28: 1326-1338. 10.1016/j.fm.2011.06.003.View ArticlePubMed
- Nielsen DS, Teniola OD, Ban-Koffi L, Owusu M, Andersson TS, Holzapfel WH: The microbiology of Ghanaian cocoa fermentations analysed using culture-dependent and culture-independent methods. Int J Food Microbiol. 2007, 114: 168-186. 10.1016/j.ijfoodmicro.2006.09.010.View ArticlePubMed
- Illeghems K, De Vuyst L, Papalexandratou Z, Weckx S: Phylogenetic analysis of a spontaneous cocoa bean fermentation metagenome reveals new insights into its bacterial and fungal community diversity. PLoS One. 2012, 7: e38040-10.1371/journal.pone.0038040.PubMed CentralView ArticlePubMed
- Schwan RF, Wheals AE: The microbiology of cocoa fermentation and its role in chocolate quality. Crit Rev Food Sci Nutr. 2004, 44: 205-221. 10.1080/10408690490464104.View ArticlePubMed
- Lefeber T, Janssens M, Moens F, Gobert W, De Vuyst L: Interesting starter culture strains for controlled cocoa bean fermentation revealed by simulated cocoa pulp fermentations of cocoa-specific lactic acid bacteria. Appl Environ Microbiol. 2011, 77: 6694-6698. 10.1128/AEM.00594-11.PubMed CentralView ArticlePubMed
- Camu N, De Winter T, Addo SK, Takrama JS, Bernaert H, De Vuyst L: Fermentation of cocoa beans: influence of microbial activities and polyphenol concentrations on the flavour of chocolate. J Sci Food Agric. 2008, 88: 2288-2297. 10.1002/jsfa.3349.View Article
- Lefeber T, Janssens M, Camu N, De Vuyst L: Kinetic analysis of strains of lactic acid bacteria and acetic acid bacteria in cocoa pulp simulation media toward development of a starter culture for cocoa bean fermentation. Appl Environ Microbiol. 2010, 76: 7708-7716. 10.1128/AEM.01206-10.PubMed CentralView ArticlePubMed
- Lefeber T, Papalexandratou Z, Gobert W, Camu N, De Vuyst L: On-farm implementation of a starter culture for improved cocoa bean fermentation and its influence on the flavour of chocolates produced thereof. Food Microbiol. 2012, 30: 379-392. 10.1016/j.fm.2011.12.021.View ArticlePubMed
- Hendrickson H, Lawrence JG: Selection for chromosome architecture in bacteria. J Mol Evol. 2006, 62: 615-629. 10.1007/s00239-005-0192-2.View ArticlePubMed
- Gao F, Zhang C-T: Ori-finder: a web-based system for finding oriC s in unannotated bacterial genomes. BMC Bioinforma. 2008, 9: 79-10.1186/1471-2105-9-79.View Article
- Hendrickson H, Lawrence JG: Mutational bias suggests that replication termination occurs near the dif site, not at ter sites. Mol Microbiol. 2007, 64: 42-56. 10.1111/j.1365-2958.2007.05596.x.View ArticlePubMed
- Canchaya C, Proux C, Fournous G, Bruttin A, Brüssow H: Prophage genomics. Microbiol Mol Biol Rev. 2003, 67: 238-276. 10.1128/MMBR.67.2.238-276.2003.PubMed CentralView ArticlePubMed
- Takemura H, Horinouchi S, Beppu T: Novel insertion sequence IS1380 from Acetobacter pasteurianus is involved in loss of ethanol-oxidizing ability. J Bacteriol. 1991, 173: 7070-7076.PubMed CentralPubMed
- Ardhana MM, Fleet GH: The microbial ecology of cocoa bean fermentations in Indonesia. Int J Food Microbiol. 2003, 86: 87-99. 10.1016/S0168-1605(03)00081-3.View ArticlePubMed
- Schwan RF, Rose AH, Board RG: Microbial fermentation of cocoa beans, with emphasis on enzymatic degradation of the pulp. J Appl Bacteriol Symposium Supplement. 1995, 79: 96S-107S.
- Kanchanarach W, Theeragool G, Inoue T, Yakushi T, Adachi O, Matsushita K: Acetic acid fermentation of Acetobacter pasteurianus: relationship between acetic acid resistance and pellicle polysaccharide formation. Biosci Biotechnol Biochem. 2010, 74: 1591-1597. 10.1271/bbb.100183.View ArticlePubMed
- Kanchanarach W, Theeragool G, Yakushi T, Toyama H, Adachi O, Matsushita K: Characterization of thermotolerant Acetobacter pasteurianus strains and their quinoprotein alcohol dehydrogenases. Appl Microbiol Biotechnol. 2010, 85: 741-751. 10.1007/s00253-009-2203-5.View ArticlePubMed
- Cleenwerck I, De Vos P: Polyphasic taxonomy of acetic acid bacteria: an overview of the currently applied methodology. Int J Food Microbiol. 2008, 125: 2-14. 10.1016/j.ijfoodmicro.2007.04.017.View ArticlePubMed
- Chandra Raj K, Ingram L, Maupin-Furlow J: Pyruvate decarboxylase: a key enzyme for the oxidative metabolism of lactic acid by Acetobacter pasteurianus. Arch Microbiol. 2001, 176: 443-451. 10.1007/s002030100348.View ArticlePubMed
- Argüelles JC: Physiological roles of trehalose in bacteria and yeasts: a comparative analysis. Arch Microbiol. 2000, 174: 217-224. 10.1007/s002030000192.View ArticlePubMed
- Booth IR, Edwards MD, Black S, Schumann U, Miller S: Mechanosensitive channels in bacteria: signs of closure?. Nat Rev Microbiol. 2007, 5: 431-440. 10.1038/nrmicro1659.View ArticlePubMed
- Kondo K, Beppu T, Horinouchi S: Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus. J Bacteriol. 1995, 177: 5048-5055.PubMed CentralPubMed
- Gupta A, Singh VK, Qazi GN, Kumar A: Gluconobacter oxydans: its biotechnological applications. J Mol Microbiol Biotechnol. 2001, 3: 445-456.PubMed
- Shinagawa E, Matsushita K, Adachi O, Ameyama M: Purification and characterization of D-sorbitol dehydrogenase from membrane of Gluconobacter suboxydans var. α. Agric Biol Chem. 1982, 46: 135-141. 10.1271/bbb1961.46.135.View Article
- Garcia-Armisen T, Vercammen K, Rimaux T, Vrancken G, De Vuyst L, Cornelis P: Identification of a five-oxidoreductase-gene cluster from Acetobacter pasteurianus conferring ethanol-dependent acidification in Escherichia coli. Microbiology Open. 2012, 1: 25-32. 10.1002/mbo3.4.PubMed CentralView ArticlePubMed
- Greenfield S, Claus GW: Nonfunctional tricarboxylic acid cycle and the mechanism of glutamate biosynthesis in Acetobacter suboxydans. J Bacteriol. 1972, 112: 1295-1301.PubMed CentralPubMed
- Sanders C, Turkarslan S, Lee D-W, Onder O, Kranz RG, Daldal F: The cytochrome c maturation components CcmF, CcmH, and CcmI form a membrane-integral multisubunit heme ligation complex. J Biol Chem. 2008, 283: 29715-29722. 10.1074/jbc.M805413200.PubMed CentralView ArticlePubMed
- Saeki A, Matsushita K, Takeno S, Taniguchi M, Toyama H, Theeragool G, Lotong N, Adachi O: Enzymes responsible for acetate oxidation by acetic acid bacteria. Biosci Biotechnol Biochem. 1999, 63: 2102-2109. 10.1271/bbb.63.2102.View Article
- Mullins EA, Francois JA, Kappock TJ: A specialized citric acid cycle requiring succinyl-coenzyme A (CoA):acetate CoA-transferase (AarC) confers acetic acid resistance on the acidophile Acetobacter aceti. J Bacteriol. 2008, 190: 4933-4940. 10.1128/JB.00405-08.PubMed CentralView ArticlePubMed
- Nakano S, Fukaya M, Horinouchi S: Putative ABC transporter responsible for acetic acid resistance in Acetobacter aceti. Appl Environ Microbiol. 2006, 72: 497-505. 10.1128/AEM.72.1.497-505.2006.PubMed CentralView ArticlePubMed
- Deeraksa A, Moonmangmee S, Toyama H, Yamada M, Adachi O, Matsushita K: Characterization and spontaneous mutation of a novel gene, polE, involved in pellicle formation in Acetobacter tropicalis SKU1100. Microbiology. 2005, 151: 4111-4120. 10.1099/mic.0.28350-0.View ArticlePubMed
- Ali IAI, Akakabe Y, Moonmangmee S, Deeraksa A, Matsutani M, Yakushi T, Yamada M, Matsushita K: Structural characterization of pellicle polysaccharides of Acetobacter tropicalis SKU1100 wild type and mutant strains. Carbohydr Polym. 2011, 86: 1000-1006. 10.1016/j.carbpol.2011.05.055.View Article
- Greenberg DE, Porcella SF, Zelazny AM, Virtaneva K, Sturdevant DE, Kupko JJ, Barbian KD, Babar A, Dorward DW, Holland SM: Genome sequence analysis of the emerging human pathogenic acetic acid bacterium Granulibacter bethesdensis. J Bacteriol. 2007, 189: 8727-8736. 10.1128/JB.00793-07.PubMed CentralView ArticlePubMed
- Constantine CZ, Starks CM, Mill CP, Ransome AE, Karpowicz SJ, Francois JA, Goodman RA, Kappock TJ: Biochemical and structural studies of N5-carboxyaminoimidazole ribonucleotide mutase from the acidophilic bacterium Acetobacter aceti. Biochemistry. 2006, 45: 8193-8208. 10.1021/bi060465n.View ArticlePubMed
- Francois JA, Kappock TJ: Alanine racemase from the acidophile Acetobacter aceti. Protein Expr Purif. 2007, 51: 39-48. 10.1016/j.pep.2006.05.016.View ArticlePubMed
- Matsutani M, Nishikura M, Saichana N, Hatano T, Masud-Tippayasak U, Theergool G, Yakushi T, Matsushita K: Adaptive mutation of Acetobacter pasteurianus SKU1108 enhances acetic acid fermentation ability at high temperature. J Biotechnol. 2013, 165: 109-119. 10.1016/j.jbiotec.2013.03.006.View ArticlePubMed
- Ravyts F, Barbuti S, Frustoli MA, Parolari G, Saccani G, de Vuyst L, Leroy F: Competitiveness and antibacterial potential of bacteriocin-producing starter cultures in different types of fermented sausages. J Food Prot. 2008, 71: 1817-1827.PubMed
- Gordon D, Abajian C, Green P: Consed: a graphical tool for sequence finishing. Genome Res. 1998, 8: 195-202. 10.1101/gr.8.3.195.View ArticlePubMed
- Husemann P, Stoye J: R2cat: synteny plots and comparative assembly. Bioinformatics. 2010, 26: 570-571. 10.1093/bioinformatics/btp690.PubMed CentralView ArticlePubMed
- Meyer F, Goesmann A, McHardy AC, Bartels D, Bekel T, Clausen J, Kalinowski J, Linke B, Rupp O, Giegerich R, Puhler A: GenDB - an open source genome annotation system for prokaryote genomes. Nucleic Acids Res. 2003, 31: 2187-2195. 10.1093/nar/gkg312.PubMed CentralView ArticlePubMed
- Delcher AL, Harmon D, Kasif S, White O, Salzberg SL: Improved microbial gene identification with GLIMMER. Nucleic Acids Res. 1999, 27: 4636-4641. 10.1093/nar/27.23.4636.PubMed CentralView ArticlePubMed
- Badger JH, Olsen GJ: CRITICA: coding region identification tool invoking comparative analysis. Mol Biol Evol. 1999, 16: 512-524. 10.1093/oxfordjournals.molbev.a026133.View ArticlePubMed
- Suzek BE, Ermolaeva MD, Schreiber M, Salzberg SL: A probabilistic method for identifying start codons in bacterial genomes. Bioinformatics. 2001, 17: 1123-1130. 10.1093/bioinformatics/17.12.1123.View ArticlePubMed
- Lowe TM, Eddy SR: tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997, 25: 955-964.PubMed CentralView ArticlePubMed
- Linke B, McHardy AC, Neuweger H, Krause L, Meyer F: REGANOR: A gene prediction server for prokaryotic genomes and a database of high quality gene predictions for prokaryotes. Appl Bioinformatics. 2006, 5: 193-198. 10.2165/00822942-200605030-00008.View ArticlePubMed
- Boeckmann B, Bairoch A, Apweiler R, Blatter MC, Estreicher A, Gasteiger E, Martin MJ, Michoud K, O’Donovan C, Phan I, Pilbout S, Schneider M: The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003. Nucleic Acids Res. 2003, 31: 365-370. 10.1093/nar/gkg095.PubMed CentralView ArticlePubMed
- Bateman A, Coin L, Durbin R, Finn RD, Hollich V, Griffiths-Jones S, Khanna A, Marshall M, Moxon S, Sonnhammer ELL, Studholme DJ, Yeats C, Eddy SR: The Pfam protein families database. Nucleic Acids Res. 2004, 32: D138-D141. 10.1093/nar/gkh121.PubMed CentralView ArticlePubMed
- Kanehisa M, Goto S: KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000, 28: 27-30. 10.1093/nar/28.1.27.PubMed CentralView ArticlePubMed
- Haft DH, Selengut JD, White O: The TIGRFAMs database of protein families. Nucleic Acids Res. 2003, 31: 371-373. 10.1093/nar/gkg128.PubMed CentralView ArticlePubMed
- Bendtsen JD, Nielsen H, von Heijne G, Brunak S: Improved prediction of signal peptides: signalP 3.0. J Mol Biol. 2004, 340: 783-795. 10.1016/j.jmb.2004.05.028.View ArticlePubMed
- Dodd IB, Egan JB: Improved detection of helix-turn-helix DNA-binding motif in protein sequences. Nucleic Acids Res. 1990, 18: 5019-5026. 10.1093/nar/18.17.5019.PubMed CentralView ArticlePubMed
- Krogh A, Larsson B, von Heijne G, Sonnhammer ELL: Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001, 305: 567-580. 10.1006/jmbi.2000.4315.View ArticlePubMed
- Tatusov RL, Natale DA, Garkavtsev IV, Tatusova TA, Shankavaram UT, Rao BS, Kiryutin B, Galperin MY, Fedorova ND, Koonin EV: The COG database: new developments in phylogenetic classification of proteins from complete genomes. Nucleic Acids Res. 2001, 29: 22-28. 10.1093/nar/29.1.22.PubMed CentralView ArticlePubMed
- Harris MA, Clark J, Ireland A, Lomax J, Ashburner M, Foulger R, Eilbeck K, Lewis S, Marshall B, Mungall C, Richter J, Rubin GM, Blake JA, Bult C, Dolan M, Drabkin H, Eppig JT, Hill DP, Ni L, Ringwald M, Balakrishnan R, Cherry JM, Christie KR, Costanzo MC, Dwight SS, Engel S, Fisk DG, Hirschman JE, Hong EL, Nash RS: The Gene Ontology (GO) database and informatics resource. Nucleic Acids Res. 2004, 32: D258-D261. 10.1093/nar/gkh036.View ArticlePubMed
- Carver T, Thomson N, Bleasby A, Berriman M, Parkhill J: DNAPlotter: circular and linear interactive genome visualization. Bioinformatics. 2009, 25: 119-120. 10.1093/bioinformatics/btn578.PubMed CentralView ArticlePubMed
- Grissa I, Vergnaud G, Pourcel C: CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 2007, 35: W52-W57. 10.1093/nar/gkm360.PubMed CentralView ArticlePubMed
- Castro C, Cleenwerck I, Trček J, Zuluaga R, De Vos P, Caro G, Aguirre R, Putaux J-L, Gañán P: Gluconacetobacter medellinensis sp. nov., cellulose- and non-cellulose producing acetic acid bacteria isolated from vinegar. Int J Syst Evol Microbiol. 2012, 63: 1119-1125.View ArticlePubMed
- Blom J, Albaum S, Doppmeier D, Pühler A, Vorhölter F-J, Zakrzewski M, Goesmann A: EDGAR: a software framework for the comparative analysis of prokaryotic genomes. BMC Bioinforma. 2009, 10: 1-14.View Article
- Carver TJ, Rutherford KM, Berriman M, Rajandream MA, Barrell BG, Parkhill J: ACT: the Artemis Comparison Tool. Bioinformatics. 2005, 21: 3422-3423. 10.1093/bioinformatics/bti553.View ArticlePubMed
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215: 403-410.View ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.