BACKGROUND
oenococcus oeni, formerly leuconostoc oenos, is a member of the lactic acid bacteria  and is noted for its ability to perform malolactic fermentation  in wine, a de-acidification reaction in which malic acid is decarboxylated to lactic acid  <cit> .
mlf is particularly common in the production of red wines  where the decarboxylation reaction and associated by-products impart favourable sensory characteristics and decrease the likelihood of spoilage .
o. oeni is found on grapes or in the natural environment at very low levels, but can be commonly found in the hostile environment of wine, where it readily grows in low ph, presence of alcohol and scarcity of nutrients that inhibit the growth of other microbes .
in addition to spontaneously occurring mlf, purified strains of o. oeni are commonly added to wine as starter cultures to enable more reliable secondary fermentation .
specific strains are selected for this purpose based on production of desirable flavour compounds and/or resilience to stresses such as acidity, ethanol, sulfites and phenolic compounds.
understanding the genotypic attributes of this species is important for identifying these industrially-relevant phenotypes.
recent genomic sequencing efforts  have revealed that o. oeni has a compact genome of approximately  <dig>  mb which presumably has resulted from specialisation of this microbe in the relatively narrow ecological niche of wine  <cit> .
despite this streamlined genome, previous comparative genomic studies of o. oeni have shown substantial inter-strain genomic variation, with up to 10 % variation in protein coding genes between strains, including those participating in sugar utilisation and transport, exopolysaccharide biosynthesis and amino-acid biosynthesis  <cit> .
in contrast to the historical use of a single strain genome as the de facto “reference” for any one species, the ongoing reduction in the cost of whole-genome sequencing now allows for large numbers of representatives from within the same species to be sequenced.
intra-specific comparison of the variation in coding potential of these strains has led to the conceptualisation of the pan-genome – the full complement of genes for a species  <cit> .
very recently, the first bacterial consensus “pan-chromosome” of acinetobacter baumannii was assembled independent of any pre-assigned genome reference and identified both invariant  and variable  regions within the chromosome  <cit> .
this allowed for the order and orientation of core genes and flexible genomic regions to be established and led to the characterisation and comparison of clusters of functionally-related genes.
in this study, we have utilised this approach to assemble the first o. oeni pan-genome.
in conjunction with  <dig> previously described genome sequences, we sequenced the genomes of a further  <dig> strains from commercial and environmental sources.
by utilising this expanded set of strains, we have broadened the scope and scale of genomic comparisons and provided a genetic basis for phenotypic characterisations of this industrially-important microbe.
specifically, we report the pan-genome assembly and phylogenomic association of regions predicted to encode intra-specific variations in amino acid biosynthesis, sugar transport and utilisation and natural competence.
RESULTS
genome sequencing of o. oeni
genome sequences of  <dig> wine isolates and four cider isolates, predominantly originating from australia and france, but also from lebanon, usa, switzerland and england were used in this study,  <dig> of which have been previously characterised  <cit> .
on average, the additional  <dig> genome sequences were each assembled from  <dig>  illumina sequencing reads  into  <dig> contigs, forming a consensus sequence of  <dig> ,000 bp in size and with  <dig> predicted protein-coding sequences.
genetic diversity of o. oeni
independent of coding-region predictions, the genetic relatedness of the various strains were deduced from the patterns of single-nucleotide polymorphisms  from reference-based read mapping .
the resulting neighbour-joining dendrogram could be broadly split into two major genetic groups .
group a was in turn comprised of two subgroups; one genetically diverse and a second that contained a very large number of highly-related strains.
relative to group a, group b represents a highly-divergent clade comprised of genetically-distant strains.
consistent with previous reports  <cit> , three out of four of the cider isolates cluster closely together in this group.
however, while it has been suggested that this reflects domestication of o. oeni in a cider environment, the presence of numerous neighbouring wine-derived strains suggests that information from additional strains isolated from cider is required before any conclusions regarding the possibility of a cider-specific subset of o. oeni can be reached.fig.
<dig> neighbour-joining phylogeny based on whole-genome alignments of  <dig> o. oeni strains.
the strain id, region of isolation, date of isolation and the grape variety are as indicated.
the tree was characterised into two broad genetic groups
the variety, region and year of isolation did not appear to influence the clustering of strains into specific groups with the exception of the upper, closely-related group which is mostly comprised of australian isolates .
approximately 60 % of the known australian isolates, but only 15 % of the known non-australian isolates clustered into this genetic group.
strains isolated from france, switzerland and the usa were also found in this closely-related group, making assertions about the ancestral geographic origin of these strains difficult.
unfortunately, very little is known regarding the stage of fermentation these strains were isolated from.
strains in this group may represent a robust variety that is capable of out-competing other strains during fermentation.
sampling late in the fermentation would therefore result in over-representation of this phylotype.
another possibility is that strains in this group are well suited to australian winemaking conditions and the enrichment of australian isolates in this genetic group is actually an accurate representation of the broader australian population.
as the concept of regional identity is very important in the valuation of wine and can be influenced by the bacterial strains performing mlf, further investigation whether australian wines are typically dominated by this very closely-related subset of o. oeni population compared to other geographic regions represents a research direction worth consideration.
the o. oeni pan-genome
previous comparative genomic studies of much smaller cohorts of o. oeni strains revealed substantial genomic diversity between some isolates .
despite these efforts, the full extent of the pan-genome remains unclear.
the core- and pan-genome sizes of o. oeni were therefore determined for this large collection of strains using the pan-genome ortholog clustering tool, panoct  <cit> .
unlike pure sequence-based clustering tools, panoct differentiates paralogous and non-paralogous orfs using the conserved gene neighbourhood to separate duplicated gene families.
thus, singleton clusters can be formed from insertion sequence  elements that are in novel contexts, even though the is elements are identical  <cit> .
in this context, there were  <dig> core clusters  and  <dig> variable clusters assembled from the  <dig> strains .fig.
<dig> the core- and pan-genomes of o. oeni.
a. distribution of protein cluster sizes generated from the comparison of  <dig> genomes.
b. calculation of core- and pan-genome sizes including exponential law models to fit the medians.
c. distribution of blast best-hits by genus for clusters with no o. oeni match in the ncbi non-redundant dataset.
the size of the circles represents the number of assigned hits
in order to determine if the genetic diversity of o. oeni had been sufficiently sampled, medians and exponential law regressions were calculated from  <dig> randomly sampled combinations of  <dig> strains .
the sequencing of approximately  <dig> strains was sufficient to estimate the final core-genome size of  <dig> genes.
the size of the pan-genome was predicted to continue to expand, albeit at a slowing rate, beyond the size calculated using  <dig> genomes , indicating that the o. oeni pan-genome is still “open”.
in addition, to ensure that potential bias derived from oversampling of closely-related strains, such as those in group a, did not confound the core- and pan-genome estimates,  <dig> of the very closely-related genomes in group a were excluded from the analysis.
the estimations of core- and pan-genome sizes were not substantially different when compared to analysis of the complete set of genomes, indicating a negligible bias in the original calculations .
using  <dig> iterations of  <dig> randomly sampled genomes, the median core-genome sizes were  <dig> and  <dig>  and median pan-genome sizes were  <dig> and  <dig> for the full set and partial set respectively.
further genome sequencing is therefore expected to be required to characterise the entire spectrum of genetic diversity in o. oeni, however additional variation is likely to be rare.
whilst prioritising future sequencing towards strains from winemaking locales not currently represented in this study may accelerate the discovery of additional genetic diversity, it is interesting to note that phylogenomic analysis of a small number of o. oeni strains from italy  and south america  <cit> , which were released after this analysis was initiated, indicate that these strains actually fall within the existing genomic clades and so may not provide a substantial amount of additional variation .
the o. oeni genome has previously been described to contain regions likely to have been horizontally-acquired from members of the lactobacillales  <cit> .
to infer the evolutionary relationship of o. oeni on a larger complement of strains, blast best hits were attributed to each cluster.
a total of  <dig> clusters  did not display o. oeni as a best match in the ncbi non-redundant dataset.
as could be expected, all of these clusters were found within the variable  genome and indicate new orfs that have previously not been identified in other annotated strains of o. oeni.
these non-o.
oeni clusters appear to originate from members of lactobacillales family, particularly the genera lactobacillus, oenococcus, leuconostoc and enterrococcus .
a comparatively small number of clusters appear to originate from outside lactobacillales, including members of the bacillales and bacteroidales families, and the phyla actinobacteria, bacteroidetes and proteobacteria .
pan-genome assembly
substantial genomic variation in exopolysaccharide and amino acid biosynthesis, and sugar transport and utilisation have been reported previously for o. oeni  <cit> .
amongst bacteria, these variations are often due to the insertion of mobile elements or variable regions described as flexible genomic islands , which usually contain highly conserved orfs from bacteriophage .
establishing a syntenic order of sequences was therefore critical for determining the orthology of genomic regions and to reflect important functional relationships between genes .
to capture this information, a consensus core-genome and fgi assemblies were computed for the o. oeni pan-genome as described by chan et al.
<cit>  .
this methodology links clusters together based on the consensus of the layout of orfs in individual de novo genome assemblies.
in this study, the psu- <dig> strain was used as a basal reference sequence to initially guide the arrangement of the clusters and this ultimately resulted in a core-genome assembly that closely resembles the arrangement of the psu- <dig> genome .
to help elucidate whether individual orfs in a cluster are likely to be truncated, the lengths of each peptide sequence were calculated as a percentage of the longest peptide sequence in the cluster .fig.
<dig> visualisation of the core-genome and fgi assemblies.
full versions of the annotated assemblies are available in additional file  <dig>  a. core-genome assembly of  <dig> clusters.
b. concatenated fgi assemblies of  <dig> clusters into  <dig> fgis
clusters at the beginning and end of the core-genome assembly  were linked, indicating assembly into a circular topology, as expected.
the core-genome assembly revealed several large regions, up to  <dig> orfs in size, which were completely absent in some clades of the genetic relatedness dendrogram .
in addition to generating an assembled consensus core-genome, fgis were also assembled.
the fgis were exclusively linear in topology and were located in specific clades of the relatedness dendrogram .
a total of  <dig> clusters were assembled into  <dig> fgis, the largest of which representing a bacteriophage insertion containing  <dig> orfs.
interestingly, several fgis were found to be unique to the closely related genetic group that consists mostly of australian isolates.
amino acid biosynthesis
o. oeni has previously been reported to exhibit a variety of amino acid auxotrophies, with many strains showing intra-specific genomic differences .
early genomic analysis of the psu- <dig> strain using the cog database  <cit>  suggested the capacity for biosynthesis of eight amino acids: alanine, aspartate, asparagine, cysteine, glutamine, lysine, methionine and threonine  <cit> .
in this expanded collection of strains and utilising kegg, rast and blast annotations, pathways leading to the biosynthesis of nine amino acids were observed in at least one strain .table  <dig> complete amino acid biosynthesis pathways in o. oeni
the presence or absence of the complete sets of enzymes for each of these pathways in each strain was compiled and correlated with the genetic relatedness dendrogram  and highlighted in a pathway overview .
the complete pathways to synthesise glutamine, glycine, serine, cysteine, proline, aspartate and threonine were found to be conserved across the majority of strains.
the ability to synthesise aspartate from lactic and malic acids was predicted to be disrupted in certain phylogenomic clades due to the presence of a frameshift mutation in pyruvate orthophosphate dikinase , which is responsible for the conversion of pyruvate into phosphoenolpyruvate.
threonine biosynthesis deficiencies were also observed in specific clades.
loss of threonine biosynthesis capability exhibited intra-specific differences, as the deficient enzyme varied between strains and particularly in homoserine kinase  where two different truncated versions of the peptide sequence were observed.
the ability to synthesise leucine and arginine was predicted to exist in a small proportion of strains and was typically restricted to several small clades.
loss of a functional leucine biosynthesis pathway was attributed to mutations within 3-isopropylmalate dehydrogenase  and isoproylmalate isomerase .
similar to threonine biosynthesis, loss of the complete arginine biosynthesis pathway is attributed to several different mutations within genes from throughout the entire pathway.
the ability to synthesise a tenth amino acid, alanine from aspartate, remains a possibility in three strains  since the orf encoding an aspartate 4-decarboxylase  was found in fgis excluded from the assembly because they contained less than three orfs.
in addition to these biosynthesis pathways, several incomplete pathways were also observed .fig.
<dig> intra-specific differences in amino acid biosynthesis, sugar transport and utilisation and natural competence.
orfs which contained a contig break are shaded in a lighter colour.
a. intra-specific differences in amino acid biosynthesis.
each pathway requires multiple enzymes, as described by their kegg module numbers.
b. intra-specific differences in pts components.
each sugar-specific system requires multiple subunits .
c. intra-specific differences in the genes involved in five-carbon sugar utilisation, as described in fig.
<dig>  d. intra-specific differences in the genes encoding natural competence proteins
fig.
<dig> overview of amino acid biosynthesis pathways in o. oeni.
kegg, rast and blast annotations were used determine the presence of orfs associated with amino acid biosynthesis across  <dig> strains.
pathways containing the full set of required genes, mostly between two amino acids , are highlighted in blue and represented in fig.
4a.
orfs forming incomplete pathways are highlighted in green.
pathways to make nine different amino acids were observed
since amino acid concentrations are low in wine, amino acid biosynthesis capabilities are considered to be an important growth requirement.
early phenotypic studies predicted between five and thirteen amino acids to be essential for the growth of different strains of o. oeni .
furthermore at least two organic acids, malic and citric acid, were involved in the biosynthesis of aspartate-derived amino acids  <cit> .
a recent study which utilised a more sensitive methodology reported that two different o. oeni strains were auxotrophic for  <dig> and  <dig> amino acids, respectively  <cit> .
of the  <dig> essential amino acids found in one of these strains, only  <dig> were found to be essential in alternate strains from previous phenotypic studies, possibly reflecting substantial intra-specific variation.
of the  <dig> genomes analysed in this report,  <dig> to  <dig> amino acids were predicted to be unable to be converted from other amino acids or organic acids .
o. oeni and other lactic acid bacteria are often described as having exacting nutritional requirements.
commercially used started cultures are often selected based on their resilience to wine stress conditions such as ethanol concentration, ph and temperature.
despite this, the rate of mlf can be substantially affected by nutrient availability  <cit> , often resulting in sluggish or stuck fermentations.
comprehensive characterisation of amino acid auxotrophies can be useful for identifying essential nutritional requirements to help assess the suitability of wines or added nutrients for microbial growth and fermentability.
furthermore, it may also be used to assess the microbial stability of finished wines  <cit> .
phosphotransferase enzyme systems in fgis
the range of sugars that o. oeni is capable of utilising is strain dependent  <cit> .
previous studies have revealed intra-specific variation in the phosphotransferase system  enzyme ii sugar transporters  <cit> .
similar to the characterisations of amino acid biosynthesis, variation in pts enzyme ii components  were analysed in this expanded set of strains .
four phosphotransferases, containing all of the required subunits, were conserved in the majority of strains: mannose-specific ii, galactitol-specific ii, cellobiose-specific ii and beta-glucoside-specific ii.
two phosphotransferases were observed to correspond to specific clades: the fructose-specific ii and ascorbate-specific ii.
the full complement of subunits of the fructose-specific ii transporter was conferred by the presence of an fgi encoding fructose-specific iib and iic components.
this fgi was comparatively large with  <dig> orfs encoding various cell wall related proteins  and generally corresponded to the group a clade.
for the ascorbate-specific ii transporter, the majority of strains encoded the ascorbate-specific iia and iic subunits however only certain clade-specific strains encoded the ascorbate-specific iib subunit.
in addition to these differences, two highly strain-specific phosphotransferases were observed: sucrose-specific ii and lactose-specific ii.
the sucrose-specific iia and iibc subunits occurred in an fgi specific to the strain baa- <dig>  upon further investigation, the fgi encoding these subunits was predicted to also encode additional sucrose-related proteins including sucrose operon repressors and both a partial and complete sucrose-6-phosphate hydrolase.
this group of orfs would therefore be expected to allow for the perception, transport and metabolism of sucrose .
the orf encoding the lactose-specific iia component was predicted to be present only in the s <dig> strain whereas the iib and iic components were commonly found in other strains.
it is interesting to note that despite o. oeni existing in a relatively specific ecological niche, this bacterium retains diversity in the specific collection of pts systems encoded in each strain.
five-carbon sugar utilisation pathways in fgis
it has been previously reported that o. oeni exhibits strain-dependent sugar utilisation phenotypes, particularly with the five-carbon sugars arabinose, xylulose and xylose and the metabolic pathways for arabinose and xylulose utilisation have previously been shown to be strain-specific  <cit> .
by comparing this larger set of strains, it was possible to define the extent of the arabinose and xylulose utilisation pathways .
l-arabinose utilisation is encoded by a set of three enzymes   which were present in the core-genome assembly, indicating that they were present in at least 75 % of the strains, however the enzyme required for the hydrolysis of the arabinose polymer arabinan  was only found in a subset of strains predominantly found in group b of the genetic relatedness dendrogram .
three enzymes responsible for l-xylulose utilisation   were found to be encoded in adjacent positions within the same fgi  and generally appeared in a closely-related clade in group a of fig.
<dig> fig.
<dig> variations in five-carbon sugar utilisation in o. oeni.
the predicted pathways for the assimilation of xylose, arabinose and xylulose and the individual enzymatic steps with their corresponding ec numbers are indicated.
the grey shading represents the number of strains out of  <dig> which contain that enzyme.
the boxes are shaded relative to each other on a square-root scale.
ec  <dig> .
<dig>  was most common, present in  <dig> strains
in addition to these pathways, it was also possible to define an fgi that is predicted to encode for the ability to utilise d-xylose via the pentose phosphate pathway, the first time that this pathway has been described in o. oeni.
this fgi is predicted to encode the two enzymes required to interconvert xylose to xylulose-5p  , in addition to a xylose transcriptional regulator and a d-xylose proton-symporter  and was generally confined to a single specific phylogenomic clade .
it is interesting to note that these two fgis  correspond to different clades.
given that these genomic regions are not found in other clades, it is tempting to hypothesise that specialisation of o. oeni in an environment composed of residual five-carbon sugars like xylose and arabinose  has directed the acquisition of these regions in different instances throughout the course of evolution.
natural competence of o. oeni
many bacteria are naturally competent and able to actively transport environmental dna fragments across their cell envelope and into their cytoplasm .
competence represents an important mechanism to allow for horizontal gene transfer as well as providing access to nutrients.
uptake of extra-cellular dna in gram-positive bacteria, such as o. oeni, requires a suite of proteins which include dna receptors , transmembrane pores , transformation pili , atp-dependent translocases  and additional proteins encoded by the comg operon.
substantial intra-specific diversity with o. oeni was observed for these natural competence proteins .
interestingly, the highly diverse clade  retains full-length peptide sequences for proteins that appear truncated elsewhere on the tree.
for example, strains in group b mostly retained full-length versions of comea whereas other strains contained one of three different frameshift mutations in the gene encoding comea, all resulting in prematurely-encoded stop codons .
comea is a bitopic membrane protein often described as being obligatory for natural genetic transformations.
comea consists of a transmembrane n-terminal domain and a c-terminal domain outside the cytoplasm membrane  <cit> .
the c-terminal domain contains a helix-hairpin-helix dna-binding motif which is the structural basis for non-sequence-specific recognition of dna  <cit> .
two of the three frameshift mutations preclude the entire dna-binding motif from being encoded and this is anticipated to have an adverse effect on the ability of o. oeni to bind dna from the extracellular environment.
it is unknown whether the predicted orf downstream of a premature stop is transcribed in vivo , however the loss of a large n-terminal end would presumably affect the functionality of this protein.fig.
<dig> intra-specific variation in the gene encoding the comea transmembrane dna receptor.
a. five variants were found to map to specific branches of the genetic relatedness dendrogram.
strains which contained a contig break in the orf encoding comea are shaded in light blue and assigned to the likely variant category.
b. alignment of predicted comea peptide sequences showing full-length  and truncated  versions.
variants b, c and d contained frameshift mutations resulting in prematurely-encoded stop codons which resulted in an additional orf being predicted in silico .
the c-terminal dna-binding motif is highlighted in red and is not encoded by variants c and d. variant b contains a premature stop within the dna-binding domain and still corresponds with genetically-distant strains.
variant d represents a frameshift mutation unique to the baa- <dig> strain.
c. nucleotide sequence alignment highlighting single nucleotide deletions causing frameshift mutations and truncation of the comea peptide sequence
conceivably, retention of the functional versions of comea and other competence proteins has allowed for a protracted evolutionary divergence of group b, as evidenced by the higher inter-strain branch lengths in the phylogeny .
with the exception of comgc, all the genes encoding these proteins were found in the core-genome assembly.
presence of the truncated versions of these proteins in the core-genome may indicate that most modern-day o. oeni strains share a naturally competent ancestor but have lost this competence by processes such as genome decay.
since o. oeni has become specialised to a relatively stable, abundant, simplified and less competitive ecological niche, the ability for it to adapt to environmental conditions by up taking extra-cellular dna is presumably no longer essential for survival and may actually serve to disrupt its already specialised genome.
to this day, the ability to reproducibly transform o. oeni for research purposes remains a considerable challenge.
given the intra-specific variations in their dna uptake machinery, careful selection of strains which may be more amenable to transformation provides a sensible avenue for researchers to explore.
CONCLUSIONS
like other industrial species, phenotypic variation in o. oeni will have direct economic consequences through impacts on product quality and production efficiencies.
this study has conducted the largest pan-genome analysis of o. oeni to date and expanded upon previous comparative genomic approaches by providing a consensus pan-genome assembly.
the pan-genome assembly provides a powerful tool for researchers to compare protein-coding genes across a large number of strains with the added benefit of being able to infer likely functional relationships between genes in conserved syntenic regions.
the applicability of the pan-genome assembly was demonstrated in this study by substantially expanding upon previous observations of intra-specific variation in amino acid biosynthesis and sugar transport and utilisation as well as characterising previously unreported variability in natural competence.
compilation of this vast amount of genomic information can be used to inform research on the industrial implications by allowing for identification of strains with combinations of desirable genetic, and therefore phenotypic, characteristics.

 oenococcus oeni, formerly leuconostoc oenos, is a member of the lactic acid bacteria and is noted for its ability to perform malolactic fermentation in wine, a de-acidification reaction in which malic acid is decarboxylated to lactic acid. mlf is particularly common in the production of red wines where the decarboxylation reaction and associated by-products impart favourable sensory characteristics and decrease the likelihood of spoilage. o. oeni is found on grapes or in the natural environment at very low levels, but can be commonly found in the hostile environment of wine, where it readily grows in low ph, presence of alcohol and scarcity of nutrients that inhibit the growth of other microbes. in addition to spontaneously occurring mlf, purified strains of o. oeni are commonly added to wine as starter cultures to enable more reliable secondary fermentation. specific strains are selected for this purpose based on production of desirable flavour compounds and/or resilience to stresses such as acidity, ethanol, sulfites and phenolic compounds. understanding the genotypic attributes of this species is important for identifying these industrially-relevant phenotypes. recent genomic sequencing efforts have revealed that o. oeni has a compact genome of approximately  mb which presumably has resulted from specialisation of this microbe in the relatively narrow ecological niche of wine. despite this streamlined genome, previous comparative genomic studies of o. oeni have shown substantial inter-strain genomic variation, with up to 10 % variation in protein coding genes between strains, including those participating in sugar utilisation and transport, exopolysaccharide biosynthesis and amino-acid biosynthesis. in contrast to the historical use of a single strain genome as the de facto “reference” for any one species, the ongoing reduction in the cost of whole-genome sequencing now allows for large numbers of representatives from within the same species to be sequenced. intra-specific comparison of the variation in coding potential of these strains has led to the conceptualisation of the pan-genome – the full complement of genes for a species. very recently, the first bacterial consensus “pan-chromosome” of acinetobacter baumannii was assembled independent of any pre-assigned genome reference and identified both invariant and variable regions within the chromosome. this allowed for the order and orientation of core genes and flexible genomic regions to be established and led to the characterisation and comparison of clusters of functionally-related genes. in this study, we have utilised this approach to assemble the first o. oeni pan-genome. in conjunction with previously described genome sequences, we sequenced the genomes of a further strains from commercial and environmental sources. by utilising this expanded set of strains, we have broadened the scope and scale of genomic comparisons and provided a genetic basis for phenotypic characterisations of this industrially-important microbe. specifically, we report the pan-genome assembly and phylogenomic association of regions predicted to encode intra-specific variations in amino acid biosynthesis, sugar transport and utilisation and natural competence.  genome sequencing of o. oeni genome sequences of wine isolates and four cider isolates, predominantly originating from australia and france, but also from lebanon, usa, switzerland and england were used in this study, of which have been previously characterised. on average, the additional genome sequences were each assembled from illumina sequencing reads into contigs, forming a consensus sequence of,000 bp in size and with predicted protein-coding sequences. genetic diversity of o. oeni independent of coding-region predictions, the genetic relatedness of the various strains were deduced from the patterns of single-nucleotide polymorphisms from reference-based read mapping. the resulting neighbour-joining dendrogram could be broadly split into two major genetic groups. group a was in turn comprised of two subgroups; one genetically diverse and a second that contained a very large number of highly-related strains. relative to group a, group b represents a highly-divergent clade comprised of genetically-distant strains. consistent with previous reports, three out of four of the cider isolates cluster closely together in this group. however, while it has been suggested that this reflects domestication of o. oeni in a cider environment, the presence of numerous neighbouring wine-derived strains suggests that information from additional strains isolated from cider is required before any conclusions regarding the possibility of a cider-specific subset of o. oeni can be reached.fig. neighbour-joining phylogeny based on whole-genome alignments of o. oeni strains. the strain id, region of isolation, date of isolation and the grape variety are as indicated. the tree was characterised into two broad genetic groups the variety, region and year of isolation did not appear to influence the clustering of strains into specific groups with the exception of the upper, closely-related group which is mostly comprised of australian isolates. approximately 60 % of the known australian isolates, but only 15 % of the known non-australian isolates clustered into this genetic group. strains isolated from france, switzerland and the usa were also found in this closely-related group, making assertions about the ancestral geographic origin of these strains difficult. unfortunately, very little is known regarding the stage of fermentation these strains were isolated from. strains in this group may represent a robust variety that is capable of out-competing other strains during fermentation. sampling late in the fermentation would therefore result in over-representation of this phylotype. another possibility is that strains in this group are well suited to australian winemaking conditions and the enrichment of australian isolates in this genetic group is actually an accurate representation of the broader australian population. as the concept of regional identity is very important in the valuation of wine and can be influenced by the bacterial strains performing mlf, further investigation whether australian wines are typically dominated by this very closely-related subset of o. oeni population compared to other geographic regions represents a research direction worth consideration. the o. oeni pan-genome previous comparative genomic studies of much smaller cohorts of o. oeni strains revealed substantial genomic diversity between some isolates. despite these efforts, the full extent of the pan-genome remains unclear. the core- and pan-genome sizes of o. oeni were therefore determined for this large collection of strains using the pan-genome ortholog clustering tool, panoct. unlike pure sequence-based clustering tools, panoct differentiates paralogous and non-paralogous orfs using the conserved gene neighbourhood to separate duplicated gene families. thus, singleton clusters can be formed from insertion sequence elements that are in novel contexts, even though the is elements are identical. in this context, there were core clusters and variable clusters assembled from the strains.fig. the core- and pan-genomes of o. oeni. a. distribution of protein cluster sizes generated from the comparison of genomes. b. calculation of core- and pan-genome sizes including exponential law models to fit the medians. c. distribution of blast best-hits by genus for clusters with no o. oeni match in the ncbi non-redundant dataset. the size of the circles represents the number of assigned hits in order to determine if the genetic diversity of o. oeni had been sufficiently sampled, medians and exponential law regressions were calculated from randomly sampled combinations of strains. the sequencing of approximately strains was sufficient to estimate the final core-genome size of genes. the size of the pan-genome was predicted to continue to expand, albeit at a slowing rate, beyond the size calculated using genomes, indicating that the o. oeni pan-genome is still “open”. in addition, to ensure that potential bias derived from oversampling of closely-related strains, such as those in group a, did not confound the core- and pan-genome estimates, of the very closely-related genomes in group a were excluded from the analysis. the estimations of core- and pan-genome sizes were not substantially different when compared to analysis of the complete set of genomes, indicating a negligible bias in the original calculations. using iterations of randomly sampled genomes, the median core-genome sizes were and and median pan-genome sizes were and for the full set and partial set respectively. further genome sequencing is therefore expected to be required to characterise the entire spectrum of genetic diversity in o. oeni, however additional variation is likely to be rare. whilst prioritising future sequencing towards strains from winemaking locales not currently represented in this study may accelerate the discovery of additional genetic diversity, it is interesting to note that phylogenomic analysis of a small number of o. oeni strains from italy and south america, which were released after this analysis was initiated, indicate that these strains actually fall within the existing genomic clades and so may not provide a substantial amount of additional variation. the o. oeni genome has previously been described to contain regions likely to have been horizontally-acquired from members of the lactobacillales. to infer the evolutionary relationship of o. oeni on a larger complement of strains, blast best hits were attributed to each cluster. a total of clusters did not display o. oeni as a best match in the ncbi non-redundant dataset. as could be expected, all of these clusters were found within the variable genome and indicate new orfs that have previously not been identified in other annotated strains of o. oeni. these non-o. oeni clusters appear to originate from members of lactobacillales family, particularly the genera lactobacillus, oenococcus, leuconostoc and enterrococcus. a comparatively small number of clusters appear to originate from outside lactobacillales, including members of the bacillales and bacteroidales families, and the phyla actinobacteria, bacteroidetes and proteobacteria. pan-genome assembly substantial genomic variation in exopolysaccharide and amino acid biosynthesis, and sugar transport and utilisation have been reported previously for o. oeni. amongst bacteria, these variations are often due to the insertion of mobile elements or variable regions described as flexible genomic islands, which usually contain highly conserved orfs from bacteriophage. establishing a syntenic order of sequences was therefore critical for determining the orthology of genomic regions and to reflect important functional relationships between genes. to capture this information, a consensus core-genome and fgi assemblies were computed for the o. oeni pan-genome as described by chan et al.. this methodology links clusters together based on the consensus of the layout of orfs in individual de novo genome assemblies. in this study, the psu- strain was used as a basal reference sequence to initially guide the arrangement of the clusters and this ultimately resulted in a core-genome assembly that closely resembles the arrangement of the psu- genome. to help elucidate whether individual orfs in a cluster are likely to be truncated, the lengths of each peptide sequence were calculated as a percentage of the longest peptide sequence in the cluster.fig. visualisation of the core-genome and fgi assemblies. full versions of the annotated assemblies are available in additional file a. core-genome assembly of clusters. b. concatenated fgi assemblies of clusters into fgis clusters at the beginning and end of the core-genome assembly were linked, indicating assembly into a circular topology, as expected. the core-genome assembly revealed several large regions, up to orfs in size, which were completely absent in some clades of the genetic relatedness dendrogram. in addition to generating an assembled consensus core-genome, fgis were also assembled. the fgis were exclusively linear in topology and were located in specific clades of the relatedness dendrogram. a total of clusters were assembled into fgis, the largest of which representing a bacteriophage insertion containing orfs. interestingly, several fgis were found to be unique to the closely related genetic group that consists mostly of australian isolates. amino acid biosynthesis o. oeni has previously been reported to exhibit a variety of amino acid auxotrophies, with many strains showing intra-specific genomic differences. early genomic analysis of the psu- strain using the cog database suggested the capacity for biosynthesis of eight amino acids: alanine, aspartate, asparagine, cysteine, glutamine, lysine, methionine and threonine. in this expanded collection of strains and utilising kegg, rast and blast annotations, pathways leading to the biosynthesis of nine amino acids were observed in at least one strain.table complete amino acid biosynthesis pathways in o. oeni the presence or absence of the complete sets of enzymes for each of these pathways in each strain was compiled and correlated with the genetic relatedness dendrogram and highlighted in a pathway overview. the complete pathways to synthesise glutamine, glycine, serine, cysteine, proline, aspartate and threonine were found to be conserved across the majority of strains. the ability to synthesise aspartate from lactic and malic acids was predicted to be disrupted in certain phylogenomic clades due to the presence of a frameshift mutation in pyruvate orthophosphate dikinase, which is responsible for the conversion of pyruvate into phosphoenolpyruvate. threonine biosynthesis deficiencies were also observed in specific clades. loss of threonine biosynthesis capability exhibited intra-specific differences, as the deficient enzyme varied between strains and particularly in homoserine kinase where two different truncated versions of the peptide sequence were observed. the ability to synthesise leucine and arginine was predicted to exist in a small proportion of strains and was typically restricted to several small clades. loss of a functional leucine biosynthesis pathway was attributed to mutations within 3-isopropylmalate dehydrogenase and isoproylmalate isomerase. similar to threonine biosynthesis, loss of the complete arginine biosynthesis pathway is attributed to several different mutations within genes from throughout the entire pathway. the ability to synthesise a tenth amino acid, alanine from aspartate, remains a possibility in three strains since the orf encoding an aspartate 4-decarboxylase was found in fgis excluded from the assembly because they contained less than three orfs. in addition to these biosynthesis pathways, several incomplete pathways were also observed.fig. intra-specific differences in amino acid biosynthesis, sugar transport and utilisation and natural competence. orfs which contained a contig break are shaded in a lighter colour. a. intra-specific differences in amino acid biosynthesis. each pathway requires multiple enzymes, as described by their kegg module numbers. b. intra-specific differences in pts components. each sugar-specific system requires multiple subunits. c. intra-specific differences in the genes involved in five-carbon sugar utilisation, as described in fig.  d. intra-specific differences in the genes encoding natural competence proteins fig. overview of amino acid biosynthesis pathways in o. oeni. kegg, rast and blast annotations were used determine the presence of orfs associated with amino acid biosynthesis across strains. pathways containing the full set of required genes, mostly between two amino acids, are highlighted in blue and represented in fig. 4a. orfs forming incomplete pathways are highlighted in green. pathways to make nine different amino acids were observed since amino acid concentrations are low in wine, amino acid biosynthesis capabilities are considered to be an important growth requirement. early phenotypic studies predicted between five and thirteen amino acids to be essential for the growth of different strains of o. oeni. furthermore at least two organic acids, malic and citric acid, were involved in the biosynthesis of aspartate-derived amino acids. a recent study which utilised a more sensitive methodology reported that two different o. oeni strains were auxotrophic for and amino acids, respectively. of the essential amino acids found in one of these strains, only were found to be essential in alternate strains from previous phenotypic studies, possibly reflecting substantial intra-specific variation. of the genomes analysed in this report, to amino acids were predicted to be unable to be converted from other amino acids or organic acids. o. oeni and other lactic acid bacteria are often described as having exacting nutritional requirements. commercially used started cultures are often selected based on their resilience to wine stress conditions such as ethanol concentration, ph and temperature. despite this, the rate of mlf can be substantially affected by nutrient availability, often resulting in sluggish or stuck fermentations. comprehensive characterisation of amino acid auxotrophies can be useful for identifying essential nutritional requirements to help assess the suitability of wines or added nutrients for microbial growth and fermentability. furthermore, it may also be used to assess the microbial stability of finished wines. phosphotransferase enzyme systems in fgis the range of sugars that o. oeni is capable of utilising is strain dependent. previous studies have revealed intra-specific variation in the phosphotransferase system enzyme ii sugar transporters. similar to the characterisations of amino acid biosynthesis, variation in pts enzyme ii components were analysed in this expanded set of strains. four phosphotransferases, containing all of the required subunits, were conserved in the majority of strains: mannose-specific ii, galactitol-specific ii, cellobiose-specific ii and beta-glucoside-specific ii. two phosphotransferases were observed to correspond to specific clades: the fructose-specific ii and ascorbate-specific ii. the full complement of subunits of the fructose-specific ii transporter was conferred by the presence of an fgi encoding fructose-specific iib and iic components. this fgi was comparatively large with orfs encoding various cell wall related proteins and generally corresponded to the group a clade. for the ascorbate-specific ii transporter, the majority of strains encoded the ascorbate-specific iia and iic subunits however only certain clade-specific strains encoded the ascorbate-specific iib subunit. in addition to these differences, two highly strain-specific phosphotransferases were observed: sucrose-specific ii and lactose-specific ii. the sucrose-specific iia and iibc subunits occurred in an fgi specific to the strain baa- upon further investigation, the fgi encoding these subunits was predicted to also encode additional sucrose-related proteins including sucrose operon repressors and both a partial and complete sucrose-6-phosphate hydrolase. this group of orfs would therefore be expected to allow for the perception, transport and metabolism of sucrose. the orf encoding the lactose-specific iia component was predicted to be present only in the s strain whereas the iib and iic components were commonly found in other strains. it is interesting to note that despite o. oeni existing in a relatively specific ecological niche, this bacterium retains diversity in the specific collection of pts systems encoded in each strain. five-carbon sugar utilisation pathways in fgis it has been previously reported that o. oeni exhibits strain-dependent sugar utilisation phenotypes, particularly with the five-carbon sugars arabinose, xylulose and xylose and the metabolic pathways for arabinose and xylulose utilisation have previously been shown to be strain-specific. by comparing this larger set of strains, it was possible to define the extent of the arabinose and xylulose utilisation pathways. l-arabinose utilisation is encoded by a set of three enzymes which were present in the core-genome assembly, indicating that they were present in at least 75 % of the strains, however the enzyme required for the hydrolysis of the arabinose polymer arabinan was only found in a subset of strains predominantly found in group b of the genetic relatedness dendrogram. three enzymes responsible for l-xylulose utilisation were found to be encoded in adjacent positions within the same fgi and generally appeared in a closely-related clade in group a of fig.  fig. variations in five-carbon sugar utilisation in o. oeni. the predicted pathways for the assimilation of xylose, arabinose and xylulose and the individual enzymatic steps with their corresponding ec numbers are indicated. the grey shading represents the number of strains out of which contain that enzyme. the boxes are shaded relative to each other on a square-root scale. ec. was most common, present in strains in addition to these pathways, it was also possible to define an fgi that is predicted to encode for the ability to utilise d-xylose via the pentose phosphate pathway, the first time that this pathway has been described in o. oeni. this fgi is predicted to encode the two enzymes required to interconvert xylose to xylulose-5p, in addition to a xylose transcriptional regulator and a d-xylose proton-symporter and was generally confined to a single specific phylogenomic clade. it is interesting to note that these two fgis correspond to different clades. given that these genomic regions are not found in other clades, it is tempting to hypothesise that specialisation of o. oeni in an environment composed of residual five-carbon sugars like xylose and arabinose has directed the acquisition of these regions in different instances throughout the course of evolution. natural competence of o. oeni many bacteria are naturally competent and able to actively transport environmental dna fragments across their cell envelope and into their cytoplasm. competence represents an important mechanism to allow for horizontal gene transfer as well as providing access to nutrients. uptake of extra-cellular dna in gram-positive bacteria, such as o. oeni, requires a suite of proteins which include dna receptors, transmembrane pores, transformation pili, atp-dependent translocases and additional proteins encoded by the comg operon. substantial intra-specific diversity with o. oeni was observed for these natural competence proteins. interestingly, the highly diverse clade retains full-length peptide sequences for proteins that appear truncated elsewhere on the tree. for example, strains in group b mostly retained full-length versions of comea whereas other strains contained one of three different frameshift mutations in the gene encoding comea, all resulting in prematurely-encoded stop codons. comea is a bitopic membrane protein often described as being obligatory for natural genetic transformations. comea consists of a transmembrane n-terminal domain and a c-terminal domain outside the cytoplasm membrane. the c-terminal domain contains a helix-hairpin-helix dna-binding motif which is the structural basis for non-sequence-specific recognition of dna. two of the three frameshift mutations preclude the entire dna-binding motif from being encoded and this is anticipated to have an adverse effect on the ability of o. oeni to bind dna from the extracellular environment. it is unknown whether the predicted orf downstream of a premature stop is transcribed in vivo, however the loss of a large n-terminal end would presumably affect the functionality of this protein.fig. intra-specific variation in the gene encoding the comea transmembrane dna receptor. a. five variants were found to map to specific branches of the genetic relatedness dendrogram. strains which contained a contig break in the orf encoding comea are shaded in light blue and assigned to the likely variant category. b. alignment of predicted comea peptide sequences showing full-length and truncated versions. variants b, c and d contained frameshift mutations resulting in prematurely-encoded stop codons which resulted in an additional orf being predicted in silico. the c-terminal dna-binding motif is highlighted in red and is not encoded by variants c and d. variant b contains a premature stop within the dna-binding domain and still corresponds with genetically-distant strains. variant d represents a frameshift mutation unique to the baa- strain. c. nucleotide sequence alignment highlighting single nucleotide deletions causing frameshift mutations and truncation of the comea peptide sequence conceivably, retention of the functional versions of comea and other competence proteins has allowed for a protracted evolutionary divergence of group b, as evidenced by the higher inter-strain branch lengths in the phylogeny. with the exception of comgc, all the genes encoding these proteins were found in the core-genome assembly. presence of the truncated versions of these proteins in the core-genome may indicate that most modern-day o. oeni strains share a naturally competent ancestor but have lost this competence by processes such as genome decay. since o. oeni has become specialised to a relatively stable, abundant, simplified and less competitive ecological niche, the ability for it to adapt to environmental conditions by up taking extra-cellular dna is presumably no longer essential for survival and may actually serve to disrupt its already specialised genome. to this day, the ability to reproducibly transform o. oeni for research purposes remains a considerable challenge. given the intra-specific variations in their dna uptake machinery, careful selection of strains which may be more amenable to transformation provides a sensible avenue for researchers to explore.  like other industrial species, phenotypic variation in o. oeni will have direct economic consequences through impacts on product quality and production efficiencies. this study has conducted the largest pan-genome analysis of o. oeni to date and expanded upon previous comparative genomic approaches by providing a consensus pan-genome assembly. the pan-genome assembly provides a powerful tool for researchers to compare protein-coding genes across a large number of strains with the added benefit of being able to infer likely functional relationships between genes in conserved syntenic regions. the applicability of the pan-genome assembly was demonstrated in this study by substantially expanding upon previous observations of intra-specific variation in amino acid biosynthesis and sugar transport and utilisation as well as characterising previously unreported variability in natural competence. compilation of this vast amount of genomic information can be used to inform research on the industrial implications by allowing for identification of strains with combinations of desirable genetic, and therefore phenotypic, characteristics. 
by assembling a consensus pan-genome from a large number of strains, this study provides a tool for researchers to readily compare protein-coding genes across strains and infer functional relationships between genes in conserved syntenic regions. whole genome comparison was performed on strains of o. oeni; from this rich source of genomic information consensus pan-genome assemblies of the invariant and variable regions of this organism were established. oenococcus oeni is a lactic acid bacterium that is specialised for growth in the ecological niche of wine, where it is noted for its ability to perform the secondary, malolactic fermentation that is often required for many types of wine. genetic variation in amino acid biosynthesis and sugar transport and utilisation was found to be common between strains. furthermore, we characterised previously-unreported intra-specific genetic variations in the natural competence of this microbe.
 oenococcus oeni is a lactic acid bacterium that is specialised for growth in the ecological niche of wine, where it is noted for its ability to perform the secondary, malolactic fermentation that is often required for many types of wine. expanding the understanding of strain-dependent genetic variations in its small and streamlined genome is important for realising its full potential in industrial fermentation processes.  whole genome comparison was performed on strains of o. oeni; from this rich source of genomic information consensus pan-genome assemblies of the invariant and variable regions of this organism were established. genetic variation in amino acid biosynthesis and sugar transport and utilisation was found to be common between strains. furthermore, we characterised previously-unreported intra-specific genetic variations in the natural competence of this microbe.  by assembling a consensus pan-genome from a large number of strains, this study provides a tool for researchers to readily compare protein-coding genes across strains and infer functional relationships between genes in conserved syntenic regions. this establishes a foundation for further genetic, and thus phenotypic, research of this industrially-important species. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords comparative genomicsoenococcusindustrial microbiologypan-genomeassemblyamino acidphosphotransferasecompetenceorthologissue-copyright-statement© the author 2016 