BACKGROUND
acetic acid bacteria  are a group of microorganisms that belong to the family of the acetobacteraceae of the alpha-proteobacteria  <cit> .
aab can be found on  fruits and flowers  <cit> , in fermented foods  <cit> , and as members of the drosophila gut  <cit> .
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  <cit>  as well as for fine chemical productions such as those of ascorbic acid and cellulose  <cit> .
furthermore, aab can occur as spoilage bacteria, as can be the case in beer, wine, and cider fermentations  <cit> .
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  and aldehyde dehydrogenase  enzymes  <cit> .
currently, aab include twelve genera, among which acetobacter, gluconobacter, and gluconacetobacter are the most studied ones  <cit> .
the genus acetobacter is one of the most interesting from a biotechnological point of view  <cit> , because of its ability to oxidize ethanol into acetate while tolerating high acetic acid concentrations in the environment  <cit> .
different species within the acetobacter genus are distinguished, among which acetobacter pasteurianus, acetobacter aceti and acetobacter pomorum are important in industrial vinegar production  <cit> , acetobacter cerevisiae is present in beer and on grapes  <cit> , and acetobacter tropicalis and acetobacter senegalensis are involved in the cocoa bean fermentation process  <cit> .
acetobacter species are able to oxidize acetate completely  and use ubiquinones of the q- <dig> type, the latter being in contrast with species of the aab genera gluconacetobacter and gluconobacter that contain mainly ubiquinones of the q- <dig> type  <cit> .
at present, a. pasteurianus ifo  <dig>  is the only member of the genus acetobacter of which the genome has been sequenced completely, including six plasmids  <cit> .
however, draft genomes are available for a. pasteurianus 3p <dig>   <cit> , a. pasteurianus nbrc  <dig>   <cit> , a. pasteurianus subsp.
pasteurianus lmg 1262t   <cit> , a. aceti nbrc  <dig>   <cit> , a. pomorum dm <dig>   <cit> , and a. tropicalis nbrc  <dig>   <cit> .
in general, acetobacter species possess relatively small genomes , including plasmids in particular cases  <cit> .
acetobacter pasteurianus strains are used for vinegar fermentations worldwide  <cit>  and also occur in beer as spoilers  <cit> .
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  <cit> .
spontaneous cocoa bean fermentation is characterized by a succession of microbial activities carried out by yeasts , involved in depectinization and ethanol formation; lactic acid bacteria , involved in citric acid and fructose conversion and lactic acid production; and aab , 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  <cit> .
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  <cit> .
furthermore, a. pasteurianus 386b is a thermotolerant strain with high resistance to ethanol and acetic acid  <cit> .
these functional properties make it an ideal starter culture strain for cocoa bean fermentations  <cit> .
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  <cit> .
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
<dig> pyrosequencing and sequence annotation
the 454-pyrosequencing run of the a. pasteurianus 386b genomic dna yielded  <dig>  reads with a total number of  <dig> ,894 bp that were assembled into  <dig> scaffolds, consisting of  <dig> large  contigs and  <dig> small  contigs.
computational analysis of the sequence assembly indicated the presence of seven plasmids.
the gaps in the chromosome and plasmids were closed by pcr assays followed by sequencing of the corresponding amplicons, resulting in a final assembly of the circular chromosome with a size of  <dig> ,679 bp and the seven plasmids ranging in size from  <dig>  bp to  <dig>  bp .
gene finding and annotation of the a. pasteurianus 386b genome with the gendb software resulted in  <dig>  and  <dig> protein-coding sequences  for the chromosome and plasmids, respectively.
furthermore, five ribosomal rna  operons were detected and  <dig> trna genes were predicted.
clustered regularly interspaced short palindromic repeats  were not found.
relevant features deduced from the genome sequence of a. pasteurianus 386b are summarized in table  <dig>
general architecture of the a. pasteurianus 386b genome
a plot of the calculated g/c skew  indicated a bidirectional replication mechanism of the chromosome , which was confirmed by the biased distribution of architecture imparting sequences  on the leading and lagging strands  <cit> , dividing the chromosome of a. pasteurianus 386b into two replichores of similar sizes .
this enabled the prediction of the origin of chromosomal replication , located near the dnaa-coding region , as well as a replication termination  region at position  <dig> , <dig> on the chromosomal map  <cit> .
the sequence of the 32-bp dif region was aligned with the consensus sequence of gamma-proteobacterial dif sites  <cit> .
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 <cit> .
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
synteny analysis revealed a highly conserved order of orthologous genes between the genome sequences of a. pasteurianus 386b and a. pasteurianus ifo  <dig> .
the chromosomal synteny was interrupted due to a few transposases/integrases and the presence of genes related to a prophage .
this prophage genomic segment had a size of about  <dig>  kb and comprised  <dig> genes .
thirteen, six, and six of these genes had homologues in the genomes of a. pasteurianus ifo  <dig>  gluconacetobacter diazotrophicus pal <dig>  and gluconobacter oxydans 621h, respectively .
the prophage-related region did not contain virulence-associated genes or genes coding for known proteins.
however, the region contained an integrase , a phage terminase , and a portal protein , amongst other prophage-related proteins.
no head maturation protease, coat protein, or tail measure protein were retrieved, indicating that the prophage is defective  <cit> .
surprisingly, the genome sequence of a. pasteurianus 386b contained only  <dig> transposases , none of which was of the is1380-type, an insertion sequence abundant in multiple a. pasteurianus strains  <cit> , indicating that the 386b strain is genetically more stable than currently known strains of a. pasteurianus <cit> .
in contrast, the genome sequence of a. pasteurianus ifo  <dig> contains  <dig> transposons , which are involved in the truncation of  <dig> genes  <cit> .
as these genes, such as a d-galactonate transporter , an oxalyl-coa decarboxylase , a membrane-bound transporter , an acetyl-coa:propanoate coa-transferase , and an oxidoreductase  were not truncated in a. pasteurianus 386b , they may contribute to specific metabolic features of this strain.
for instance, the presence of a functional acetyl-coa:propanoate coa-transferase may have implications on metabolic pathways involving acetate, an important substrate in the cocoa bean fermentation.
a comparison of all seven plasmids of a. pasteurianus 386b with all six plasmids of a. pasteurianus ifo  <dig> by means of the act and edgar software tools demonstrated that both strains did not share the same plasmids, as only very few homologous regions were retrieved .
for instance, papa01- <dig> and papa01- <dig>  the largest plasmids of a. pasteurianus ifo  <dig>  shared only  <dig> and  <dig> cds with apa386b_1p, the largest plasmid of a. pasteurianus 386b, respectively .
furthermore, apa386b_1p contained  <dig> unique genes, not shared with any of the two largest plasmids of a. pasteurianus ifo  <dig>
comparative analysis of the five available genome sequences of strains of the species a. pasteurianus revealed that there were  <dig>  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  <dig> strain-specific genes, of which  <dig> had no known function, which may be related with niche adaptations.
the  <dig> unique genes with an assigned function  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  indicates a possible role of this strain in pectin breakdown, an important metabolic process in the beginning of cocoa bean fermentations  <cit> .
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  <dig>  <cit> ; a. tropicalis has been isolated from spontaneous cocoa bean fermentation processes as well  <cit> .
furthermore, a pcr assay indicated that this polygalacturonase gene was not widespread amongst a. pasteurianus strains isolated from spontaneous cocoa bean fermentations .
this suggests that expression of this gene might contribute to the capability of a. pasteurianus 386b to dominate cocoa bean fermentations.
phylogenetic analysis of the available genomes of the genus acetobacter showed that the different acetobacter pasteurianus strains were clustered together .
furthermore, a. pasteurianus 386b was most related to a. pasteurianus nbrc  <dig>  a thermotolerant strain  <cit> .
comparative analysis of a. pasteurianus 386b in relation to other members of the acetobacteraceae family revealed that this strain had approximately 20% shared genes with members of the genus acidiphilium .
furthermore,  <dig> % of the genes are in common with the human pathogen granulibacter bethesdensis cgdnih <dig>  whereas members of the genera gluconobacter and gluconacetobacter were more closely related to a. pasteurianus 386b, namely  <dig>  and  <dig> - <dig> % shared genes, respectively.
the finding that a. pasteurianus 386b had more genes in common with gluconacetobacter species than with g. oxydans 621h was not in accordance with the phylogenetic relationship based on their complete 16s rrna gene sequence  <cit> .
this indicates that gluconacetobacter species are more closely related to acetobacter species than gluconobacter species are.
number of shared protein-coding sequences between a. pasteurianus 386b and members of the acetobacteraceae family for which a complete genome sequence is available.
the percentage of shared genes depicts the number of shared genes between two members in relation to the total amount of genes of both members.
intracellular metabolism of sugars and sugar derivatives
acetobacter pasteurianus 386b possessed all genes encoding the enzymes of the embden-meyerhof-parnas  pathway, except for the phosphofructokinase-coding gene, indicating incomplete glycolysis.
the absence of this gene has been reported before in a. pasteurianus ifo  <dig>  g. diazotrophicus pal <dig>  and g. oxydans 621h  <cit> .
however, all genes encoding the enzymes of the pentose-phosphate pathway  were found , suggesting that glucose is degraded via the ppp, as described previously for a. pasteurianus ifo  <dig> and a. aceti nbrc  <dig>  <cit> .
glucose, an important substrate in the cocoa bean fermentation process, could be taken up by sugar permeases  or a sugar symporter .
fructose-6-phosphate could be formed from n-acetyl-glucosamine-6-phosphate as well as from mannitol via fructose by a polyol oxidoreductase .
fructose-6-phosphate could be further metabolized by the emp pathway .
furthermore, the gene coding for glycerol kinase  was found, allowing the formation of dihydroxyacetone -phosphate from glycerol via glycerol 3-phosphate, which could be further metabolized by the emp pathway.
dha-phosphate could be formed by both fad- and nad-dependent glycerol 3-phosphate dehydrogenases .
next to this, the a. pasteurianus 386b genome contained genes coding for a glycerol uptake facilitator protein , suggesting that this strain is able to take up glycerol from the environment to use it as an energy source, which might be present as a substrate during the cocoa bean fermentation process owing to yeast metabolism.
dha-phosphate may be channeled into the lower part of the emp pathway.
the a. pasteurianus 386b genome sequence provided further evidence that acetate is formed out of ethanol by soluble, nad+-dependent adh  and aldh  intracellularly, or out of lactate by means of a lactate dehydrogenase  and pyruvate decarboxylase , as proposed before  <cit> ; f. moens, t. lefeber, and l. de vuyst, .
acetate, formed intracelluarly or available extracellularly, could be further oxidized via acetyl-coa into carbon dioxide and water by a modified tricarboxylic acid  cycle, as explained below.
genes encoding enzymes of the glyoxylate pathway were not present.
during the course of all aforementioned reactions, nadh+h+ is produced by several dehydrogenases.
indeed, next to the annotated dehydrogenases, the a. pasteurianus 386b genome revealed several putative dehydrogenases/oxidoreductases with a currently unknown function .
these uncharacterized oxidoreductases included four aldehyde dehydrogenases,  <dig> short-chain dehydrogenases/reductases , a zinc-binding dehydrogenase, and an oxidoreductase containing a nad+-binding rossmann-fold domain.
intracellular dehydrogenase activity is indispensable for the intermediary metabolism of aab  <cit> .
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  and glutamine synthetase , could be taken up by a specific ammonia transporter .
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  <cit> .
the pathway consisted of trehalose-6-phosphate synthase , trehalose-6-phosphate phosphatase , and trehalase .
in addition, genes coding for the mechanosensitive channels mscl  and mscs  were present in the genome sequence of a. pasteurianus 386b, which generally play an important role in osmotolerance  <cit> .
membrane-bound dehydrogenases and respiratory chain
acetobacter pasteurianus 386b possessed several membrane-bound dehydrogenases that channel electrons into the respiratory chain .
a first group of dehydrogenases depend on the cofactor pyrroloquinoline quinone , among which pqq-dependent alcohol dehydrogenase  and pqq-dependent glucose dehydrogenase, allowing the conversion of ethanol into acetaldehyde and glucose into gluconate, respectively, both substrates being available during cocoa bean fermentations.
the genes coding for the three subunits of pqq-adh were not clustered together in the genome, as the gene encoding the smallest subunit  was separated from the other two genes .
this gene organization in a. pasteurianus has been suggested before  <cit> .
further, the genome of a. pasteurianus 386b contained two uncharacterized, membrane-bound, pqq-dependent oxidoreductases with five transmembrane helices .
proteins for the synthesis of the cofactor pqq are encoded by the pqqabcde operon .
in contrast to other aab, the genome of a. pasteurianus 386b did not possess the major polyol dehydrogenase , an enzyme of industrial importance  <cit> .
indeed, sldab is for instance able to oxidize d-sorbitol into l-sorbose , gluconate into 5-ketogluconate , and glycerol into dihydroxyacetone   <cit> .
a second group of membrane-bound dehydrogenases contains flavines as cofactor.
the genes coding for flavine adenine dinucleotide -dependent sorbitol dehydrogenase  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  <cit> .
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 .
in addition, a. pasteurianus 386b possessed six membrane-bound oxidoreductases with unknown function .
these oxidoreductases are also present in the genomes of a. pasteurianus ifo  <dig>  ga. diazotrophicus pal <dig>  and g. oxydans 621h , and could be involved in the oxidation of a broad range of substrates, such as carbohydrates and polyols  <cit> .
genome analysis revealed that ubiquinol, generated by the aforementioned membrane-bound dehydrogenases, could be reoxidized by a cytochrome bo3-type ubiquinol oxidase  and a cytochrome bd-type ubiquinol oxidase , whereby the encoding genes of the latter were present twice in the genome sequence of a. pasteurianus 386b .
both terminal oxidases reduce oxygen to water when reoxidizing ubiquinol into ubiquinone .
the genes coding for a proton-translocating nicotinamide nucleotide transhydrogenase were present in the genome of a. pasteurianus 386b .
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  <cit> .
indeed, the genes coding for a complete proton-translocating respiratory chain complex i were retrieved .
this complex i is absent in g. oxydans, which may be related to its lower growth yields  <cit> .
furthermore, a membrane-bound succinate dehydrogenase/fumarate reductase was found , 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  <cit> .
genes encoding both a bc <dig> complex  as well as cytochrome c  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   <cit> .
however, it is unknown if this strain is able to reoxidize the reduced form of cytochrome c, as cytochrome c oxidase  is probably inactive, because only subunit i  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  or by acetate kinase  and phosphate acetyltransferase .
both pathways were present in the a. pasteurianus 386b genome and are known to be upregulated when citrate oxidation takes place  <cit> .
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  <cit> .
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 .
similarly, the gene for malate dehydrogenase was not found, but oxidation of malate into oxaloacetate can be catalyzed by malate:quinone oxidoreductase   <cit> .
a second mechanism in acid tolerance probably involves the presence of an acetic acid resistance abc transporter , an efflux pump in the cytoplasmic membrane capable of exporting acetic acid  <cit> .
thirdly, a. pasteurianus 386b contained the gene cluster involved in pellicle polysaccharide formation , preventing the diffusion of acetic acid into the cytoplasm  <cit> .
fourthly, the genes coding for urease , an urea transporter , an allophanate hydrolase , and an urea carboxylase  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 .
the human pathogenic gr.
bethesdensis cgdnih <dig> contains this mechanism too, although it may not be widespread among aab strains, as it is absent in g. oxydans 621h and ga. diazotrophicus pal <dig>  <cit> .
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  mutase , a protein involved in purine biosynthesis.
indeed, n5-cair mutase of a. pasteurianus 386b is 99% identical to its orthologue in a. aceti  <dig>  the latter strain being adapted to an acid cytosol  <cit> .
similarly, alanine racemase , a protein involved in peptidoglycan biosynthesis, is 92% identical to the a. aceti  <dig> orthologue, a protein known to function at low ph  <cit> .
in addition, the sequence similarity of both n5-cair mutase and alanine racemase between a. pasteurianus 386b and a. aceti  <dig> was higher than between a. pasteurianus 386b and any other sequenced aab strain , 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  <dig>  in addition, adaptive mutation resulted in  <dig> validated mutations, involved in improved thermotolerance of this strain  <cit> .
five of these regions were also modified in a. pasteurianus 386b and not in a. pasteurianus nbrc  <dig> .
for example, one of the two genes coding for cytochrome c , contained three synonymous mutations and one nonsynonymous mutation.
furthermore, the genes necessary for growth at high temperatures  of the thermotolerant strain a. tropicalis nbrc  <dig> have been identified recently  <cit> .
although this strain belongs to a different species than a. pasteurianus 386b, all genes of a. tropicalis nbrc  <dig> necessary for growth at high temperatures were found in the genome sequence of a. pasteurianus 386b as well .
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  <dig>
CONCLUSIONS
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  and stresses  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  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.

 acetic acid bacteria are a group of microorganisms that belong to the family of the acetobacteraceae of the alpha-proteobacteria. aab can be found on fruits and flowers, in fermented foods, 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 as well as for fine chemical productions such as those of ascorbic acid and cellulose. furthermore, aab can occur as spoilage bacteria, as can be the case in beer, wine, and cider fermentations. 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 and aldehyde dehydrogenase enzymes. currently, aab include twelve genera, among which acetobacter, gluconobacter, and gluconacetobacter are the most studied ones. the genus acetobacter is one of the most interesting from a biotechnological point of view, 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, acetobacter cerevisiae is present in beer and on grapes, and acetobacter tropicalis and acetobacter senegalensis are involved in the cocoa bean fermentation process. acetobacter species are able to oxidize acetate completely and use ubiquinones of the q- type, the latter being in contrast with species of the aab genera gluconacetobacter and gluconobacter that contain mainly ubiquinones of the q- type. at present, a. pasteurianus ifo 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 3p, a. pasteurianus nbrc, a. pasteurianus subsp. pasteurianus lmg 1262t, a. aceti nbrc, a. pomorum dm, and a. tropicalis nbrc. in general, acetobacter species possess relatively small genomes, including plasmids in particular cases. acetobacter pasteurianus strains are used for vinegar fermentations worldwide 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. spontaneous cocoa bean fermentation is characterized by a succession of microbial activities carried out by yeasts, involved in depectinization and ethanol formation; lactic acid bacteria, involved in citric acid and fructose conversion and lactic acid production; and aab, 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. 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. furthermore, a. pasteurianus 386b is a thermotolerant strain with high resistance to ethanol and acetic acid. 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. 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.  pyrosequencing and sequence annotation the 454-pyrosequencing run of the a. pasteurianus 386b genomic dna yielded reads with a total number of,894 bp that were assembled into scaffolds, consisting of large contigs and small contigs. computational analysis of the sequence assembly indicated the presence of seven plasmids. the gaps in the chromosome and plasmids were closed by pcr assays followed by sequencing of the corresponding amplicons, resulting in a final assembly of the circular chromosome with a size of,679 bp and the seven plasmids ranging in size from  bp to  bp. gene finding and annotation of the a. pasteurianus 386b genome with the gendb software resulted in and protein-coding sequences for the chromosome and plasmids, respectively. furthermore, five ribosomal rna operons were detected and trna genes were predicted. clustered regularly interspaced short palindromic repeats were not found. relevant features deduced from the genome sequence of a. pasteurianus 386b are summarized in table  general architecture of the a. pasteurianus 386b genome a plot of the calculated g/c skew indicated a bidirectional replication mechanism of the chromosome, which was confirmed by the biased distribution of architecture imparting sequences on the leading and lagging strands, dividing the chromosome of a. pasteurianus 386b into two replichores of similar sizes. this enabled the prediction of the origin of chromosomal replication, located near the dnaa-coding region, as well as a replication termination region at position, 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 synteny analysis revealed a highly conserved order of orthologous genes between the genome sequences of a. pasteurianus 386b and a. pasteurianus ifo. the chromosomal synteny was interrupted due to a few transposases/integrases and the presence of genes related to a prophage. this prophage genomic segment had a size of about  kb and comprised genes. thirteen, six, and six of these genes had homologues in the genomes of a. pasteurianus ifo gluconacetobacter diazotrophicus pal and gluconobacter oxydans 621h, respectively. the prophage-related region did not contain virulence-associated genes or genes coding for known proteins. however, the region contained an integrase, a phage terminase, and a portal protein, amongst other prophage-related proteins. no head maturation protease, coat protein, or tail measure protein were retrieved, indicating that the prophage is defective. surprisingly, the genome sequence of a. pasteurianus 386b contained only transposases, none of which was of the is1380-type, an insertion sequence abundant in multiple a. pasteurianus strains, indicating that the 386b strain is genetically more stable than currently known strains of a. pasteurianus. in contrast, the genome sequence of a. pasteurianus ifo contains transposons, which are involved in the truncation of genes. as these genes, such as a d-galactonate transporter, an oxalyl-coa decarboxylase, a membrane-bound transporter, an acetyl-coa:propanoate coa-transferase, and an oxidoreductase were not truncated in a. pasteurianus 386b, they may contribute to specific metabolic features of this strain. for instance, the presence of a functional acetyl-coa:propanoate coa-transferase may have implications on metabolic pathways involving acetate, an important substrate in the cocoa bean fermentation. a comparison of all seven plasmids of a. pasteurianus 386b with all six plasmids of a. pasteurianus ifo by means of the act and edgar software tools demonstrated that both strains did not share the same plasmids, as only very few homologous regions were retrieved. for instance, papa01- and papa01- the largest plasmids of a. pasteurianus ifo shared only and cds with apa386b_1p, the largest plasmid of a. pasteurianus 386b, respectively. furthermore, apa386b_1p contained unique genes, not shared with any of the two largest plasmids of a. pasteurianus ifo comparative analysis of the five available genome sequences of strains of the species a. pasteurianus revealed that there were 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 strain-specific genes, of which had no known function, which may be related with niche adaptations. the unique genes with an assigned function 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 indicates a possible role of this strain in pectin breakdown, an important metabolic process in the beginning of cocoa bean fermentations. 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; 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. this suggests that expression of this gene might contribute to the capability of a. pasteurianus 386b to dominate cocoa bean fermentations. phylogenetic analysis of the available genomes of the genus acetobacter showed that the different acetobacter pasteurianus strains were clustered together. furthermore, a. pasteurianus 386b was most related to a. pasteurianus nbrc a thermotolerant strain. comparative analysis of a. pasteurianus 386b in relation to other members of the acetobacteraceae family revealed that this strain had approximately 20% shared genes with members of the genus acidiphilium. furthermore, % of the genes are in common with the human pathogen granulibacter bethesdensis cgdnih whereas members of the genera gluconobacter and gluconacetobacter were more closely related to a. pasteurianus 386b, namely and - % shared genes, respectively. the finding that a. pasteurianus 386b had more genes in common with gluconacetobacter species than with g. oxydans 621h was not in accordance with the phylogenetic relationship based on their complete 16s rrna gene sequence. this indicates that gluconacetobacter species are more closely related to acetobacter species than gluconobacter species are. number of shared protein-coding sequences between a. pasteurianus 386b and members of the acetobacteraceae family for which a complete genome sequence is available. the percentage of shared genes depicts the number of shared genes between two members in relation to the total amount of genes of both members. intracellular metabolism of sugars and sugar derivatives acetobacter pasteurianus 386b possessed all genes encoding the enzymes of the embden-meyerhof-parnas pathway, except for the phosphofructokinase-coding gene, indicating incomplete glycolysis. the absence of this gene has been reported before in a. pasteurianus ifo g. diazotrophicus pal and g. oxydans 621h. however, all genes encoding the enzymes of the pentose-phosphate pathway were found, suggesting that glucose is degraded via the ppp, as described previously for a. pasteurianus ifo and a. aceti nbrc. glucose, an important substrate in the cocoa bean fermentation process, could be taken up by sugar permeases or a sugar symporter. fructose-6-phosphate could be formed from n-acetyl-glucosamine-6-phosphate as well as from mannitol via fructose by a polyol oxidoreductase. fructose-6-phosphate could be further metabolized by the emp pathway. furthermore, the gene coding for glycerol kinase was found, allowing the formation of dihydroxyacetone -phosphate from glycerol via glycerol 3-phosphate, which could be further metabolized by the emp pathway. dha-phosphate could be formed by both fad- and nad-dependent glycerol 3-phosphate dehydrogenases. next to this, the a. pasteurianus 386b genome contained genes coding for a glycerol uptake facilitator protein, suggesting that this strain is able to take up glycerol from the environment to use it as an energy source, which might be present as a substrate during the cocoa bean fermentation process owing to yeast metabolism. dha-phosphate may be channeled into the lower part of the emp pathway. the a. pasteurianus 386b genome sequence provided further evidence that acetate is formed out of ethanol by soluble, nad+-dependent adh and aldh intracellularly, or out of lactate by means of a lactate dehydrogenase and pyruvate decarboxylase, as proposed before; f. moens, t. lefeber, and l. de vuyst,. acetate, formed intracelluarly or available extracellularly, could be further oxidized via acetyl-coa into carbon dioxide and water by a modified tricarboxylic acid cycle, as explained below. genes encoding enzymes of the glyoxylate pathway were not present. during the course of all aforementioned reactions, nadh+h+ is produced by several dehydrogenases. indeed, next to the annotated dehydrogenases, the a. pasteurianus 386b genome revealed several putative dehydrogenases/oxidoreductases with a currently unknown function. these uncharacterized oxidoreductases included four aldehyde dehydrogenases, short-chain dehydrogenases/reductases, a zinc-binding dehydrogenase, and an oxidoreductase containing a nad+-binding rossmann-fold domain. intracellular dehydrogenase activity is indispensable for the intermediary metabolism of aab. 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 and glutamine synthetase, could be taken up by a specific ammonia transporter. 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, trehalose-6-phosphate phosphatase, and trehalase. in addition, genes coding for the mechanosensitive channels mscl and mscs were present in the genome sequence of a. pasteurianus 386b, which generally play an important role in osmotolerance. membrane-bound dehydrogenases and respiratory chain acetobacter pasteurianus 386b possessed several membrane-bound dehydrogenases that channel electrons into the respiratory chain. a first group of dehydrogenases depend on the cofactor pyrroloquinoline quinone, among which pqq-dependent alcohol dehydrogenase and pqq-dependent glucose dehydrogenase, allowing the conversion of ethanol into acetaldehyde and glucose into gluconate, respectively, both substrates being available during cocoa bean fermentations. the genes coding for the three subunits of pqq-adh were not clustered together in the genome, as the gene encoding the smallest subunit was separated from the other two genes. this gene organization in a. pasteurianus has been suggested before. further, the genome of a. pasteurianus 386b contained two uncharacterized, membrane-bound, pqq-dependent oxidoreductases with five transmembrane helices. proteins for the synthesis of the cofactor pqq are encoded by the pqqabcde operon. in contrast to other aab, the genome of a. pasteurianus 386b did not possess the major polyol dehydrogenase, an enzyme of industrial importance. indeed, sldab is for instance able to oxidize d-sorbitol into l-sorbose, gluconate into 5-ketogluconate, and glycerol into dihydroxyacetone. a second group of membrane-bound dehydrogenases contains flavines as cofactor. the genes coding for flavine adenine dinucleotide -dependent sorbitol dehydrogenase 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. in addition, a. pasteurianus 386b possessed six membrane-bound oxidoreductases with unknown function. these oxidoreductases are also present in the genomes of a. pasteurianus ifo ga. diazotrophicus pal and g. oxydans 621h, 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 and a cytochrome bd-type ubiquinol oxidase, whereby the encoding genes of the latter were present twice in the genome sequence of a. pasteurianus 386b. both terminal oxidases reduce oxygen to water when reoxidizing ubiquinol into ubiquinone. the genes coding for a proton-translocating nicotinamide nucleotide transhydrogenase were present in the genome of a. pasteurianus 386b. 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. 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, 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 complex as well as cytochrome c 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. however, it is unknown if this strain is able to reoxidize the reduced form of cytochrome c, as cytochrome c oxidase is probably inactive, because only subunit i 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 or by acetate kinase and phosphate acetyltransferase. 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. similarly, the gene for malate dehydrogenase was not found, but oxidation of malate into oxaloacetate can be catalyzed by malate:quinone oxidoreductase. a second mechanism in acid tolerance probably involves the presence of an acetic acid resistance abc transporter, 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, preventing the diffusion of acetic acid into the cytoplasm. fourthly, the genes coding for urease, an urea transporter, an allophanate hydrolase, and an urea carboxylase 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. the human pathogenic gr. bethesdensis cgdnih contains this mechanism too, although it may not be widespread among aab strains, as it is absent in g. oxydans 621h and ga. diazotrophicus pal. 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 mutase, a protein involved in purine biosynthesis. indeed, n5-cair mutase of a. pasteurianus 386b is 99% identical to its orthologue in a. aceti the latter strain being adapted to an acid cytosol. similarly, alanine racemase, a protein involved in peptidoglycan biosynthesis, is 92% identical to the a. aceti 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 was higher than between a. pasteurianus 386b and any other sequenced aab strain, 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 in addition, adaptive mutation resulted in 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. for example, one of the two genes coding for cytochrome c, contained three synonymous mutations and one nonsynonymous mutation. furthermore, the genes necessary for growth at high temperatures of the thermotolerant strain a. tropicalis nbrc have been identified recently. although this strain belongs to a different species than a. pasteurianus 386b, all genes of a. tropicalis nbrc necessary for growth at high temperatures were found in the genome sequence of a. pasteurianus 386b as well. 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  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 and stresses 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 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. 
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. 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. however, the molecular mechanisms underlying its metabolic capabilities and niche adaptations are unknown. it is able to dominate the fermentation process, thereby resisting high acetic acid concentrations and temperatures. 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. the genome sequence of a. pasteurianus 386b is composed of a -mb chromosome and seven plasmids. the annotation of 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.
 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 -mb chromosome and seven plasmids. the annotation of 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. acetobacter pasteurianuscocoa bean fermentation pyrosequencing 