Comparative analysis of the Oenococcus oeni pan genome reveals genetic diversity in industrially-relevant pathways

ABSTRACT: BACKGROUND: Oenococcus oeni, a member of the lactic acid bacteria, is one of a limited number of microorganisms that not only survive, but actively proliferate in wine. It is is also unusual as, unlike the majority of bacteria present in wine, it is beneficial to wine quality rather than causing spoilage. These benefits are realised primarily through catalysing malolactic fermentation, but also through imparting other positive sensory properties. However, many of these industrially-important secondary attributes have been shown to be strain-dependent and their genetic basis it yet to be determined. RESULTS: In order to investigate the scale and scope of genetic variation in O. oeni, we have performed whole-genome sequencing on eleven strains of this bacterium, bringing the total number of strains for which genome sequences are available to fourteen. While any single strain of O. oeni was shown to contain around 1800 protein-coding genes, in-depth comparative annotation based on genomic synteny and protein orthology identified over 2800 orthologous open reading frames that comprise the pan genome of this species, and less than 1200 genes that make up the conserved genomic core present in all of the strains. The expansion of the pan genome relative to the coding potential of individual strains was shown to be due to the varied presence and location of multiple distinct bacteriophage sequences and also in various metabolic functions with potential impacts on the industrial performance of this species, including cell wall exopolysaccharide biosynthesis, sugar transport and utilisation and amino acid biosynthesis. CONCLUSIONS: By providing a large cohort of sequenced strains, this study provides a broad insight into the genetic variation present within O. oeni. This data is vital to understanding and harnessing the phenotypic variation present in this economically-important species.     

Oligopeptide assimilation and transport by Oenococcus oeni

Aims: Oenococcus oeni is a slow‐growing wine bacterium with a low growth yield. It thrives better on complex nitrogen sources than on free amino‐acid medium. We aimed to characterize the oligopeptide use of this micro‐organism. Methods and Results: Several peptides of two to eight amino‐acid residues were able to provide essential amino acids. The disappearance of various peptides from extracellular medium was assessed with whole cells. Initial rates of utilization varied with the peptide, and free amino acids were released into the medium. Conclusions: Oenococcus oeni was able to transport the oligopeptides with two to five amino‐acid residues tested and to hydrolyse them further. Significance and Impact of the Study: This study has clear implications for the relationship between wine nitrogen composition and the ability of O. oeni to cope with its environment.     

Intraspecific diversity of Oenococcus oeni isolated during red wine-making in Japan

Using molecular and chemotaxonomic techniques, we studied the intraspecific diversity of Oenococcus oeni, a lactic acid bacterium isolated during red wine-making in Japan. The results confirmed high values of DNA-DNA relatedness and strong similarity among 16S rDNA sequences of the isolates with the O. oeni-type strain. Pulsed-field gel electrophoresis (PFGE) by NotI identified four patterns among the strains. Three different patterns of lactate dehydrogenase mobility were seen and there was a strong correlation between PFGE pattern and mobility. The present results suggest that the different strains of O. oeni comprise one species, and that variations in the genomic profiles of the different strains of O. oeni, including Japanese isolates are well correlated.     

Intraspecific diversity of Oenococcus oeni strains determined by sequence analysis of target genes

Using molecular techniques and sequencing, we studied the intraspecific diversity of Oenococcus oeni, a lactic acid bacterium involved in red winemaking. A relationship between the phenotypic and genotypic characterization of 16 O. oeni strains isolated from wine with different levels of enological potential was shown. The study was based on the comparative genomic analysis by subtractive hybridization between two strains of O. oeni with opposite enological potential. The genomic sequences obtained from subtractive hybridization were amplified by polymerase chain reaction and sequenced for the 16 strains. A considerable diversity among strains of O. oeni was observed.     

Characterization of the genome composition of Bartonella koehlerae by microarray comparative genomic hybridization profiling

Bartonella henselae is present in a wide range of wild and domestic feline hosts and causes cat-scratch disease and bacillary angiomatosis in humans. We have estimated here the gene content of Bartonella koehlerae, a novel species isolated from cats that was recently identified as an agent of human endocarditis. The investigation was accomplished by comparative genomic hybridization (CGH) to a microarray constructed from the sequenced 1.93-Mb genome of B. henselae. Control hybridizations of labeled DNA from the human pathogen Bartonella quintana with a reduced genome of 1.58 Mb were performed to evaluate the accuracy of the array for genes with known levels of sequence divergence. Genome size estimates of B. koehlerae by pulsed-field gel electrophoresis matched that calculated by the CGH, indicating a genome of 1.7 to 1.8 Mb with few unique genes. As in B. quintana, sequences in the prophage and the genomic islands were reported absent in B. koehlerae. In addition, sequence variability was recorded in the chromosome II-like region, where B. koehlerae showed an intermediate retention pattern of both coding and noncoding sequences. Although most of the genes missing in B. koehlerae are also absent from B. quintana, its phylogenetic placement near B. henselae suggests independent deletion events, indicating that host specificity is not solely attributed to genes in the genomic islands. Rather, the results underscore the instability of the genomic islands even within bacterial populations adapted to the same host-vector system, as in the case of B. henselae and B. koehlerae.     

Extensive genomic diversity in pathogenic Escherichia coli and Shigella Strains revealed by comparative genomic hybridization microarray

Escherichia coli, including the closely related genus Shigella, is a highly diverse species in terms of genome structure. Comparative genomic hybridization (CGH) microarray analysis was used to compare the gene content of E. coli K-12 with the gene contents of pathogenic strains. Missing genes in a pathogen were detected on a microarray slide spotted with 4,071 open reading frames (ORFs) of W3110, a commonly used wild-type K-12 strain. For 22 strains subjected to the CGH microarray analyses 1,424 ORFs were found to be absent in at least one strain. The common backbone of the E. coli genome was estimated to contain about 2,800 ORFs. The mosaic distribution of absent regions indicated that the genomes of pathogenic strains were highly diversified because of insertions and deletions. Prophages, cell envelope genes, transporter genes, and regulator genes in the K-12 genome often were not present in pathogens. The gene contents of the strains tested were recognized as a matrix for a neighbor-joining analysis. The phylogenic tree obtained was consistent with the results of previous studies. However, unique relationships between enteroinvasive strains and Shigella, uropathogenic, and some enteropathogenic strains were suggested by the results of this study. The data demonstrated that the CGH microarray technique is useful not only for genomic comparisons but also for phylogenic analysis of E. coli at the strain level.     

Functional divergence in the genus Oenococcus as predicted by genome sequencing of the newly-described species, Oenococcus kitaharae

Oenococcus kitaharae is only the second member of the genus Oenococcus to be identified and is the closest relative of the industrially important wine bacterium Oenococcus oeni. To provide insight into this new species, the genome of the type strain of O. kitaharae, DSM 17330, was sequenced. Comparison of the sequenced genomes of both species show that the genome of O. kitaharae DSM 17330 contains many genes with predicted functions in cellular defence (bacteriocins, antimicrobials, restriction-modification systems and a CRISPR locus) which are lacking in O. oeni. The two genomes also appear to differentially encode several metabolic pathways associated with amino acid biosynthesis and carbohydrate utilization and which have direct phenotypic consequences. This would indicate that the two species have evolved different survival techniques to suit their particular environmental niches. O. oeni has adapted to survive in the harsh, but predictable, environment of wine that provides very few competitive species. However O. kitaharae appears to have adapted to a growth environment in which biological competition provides a significant selective pressure by accumulating biological defence molecules, such as bacteriocins and restriction-modification systems, throughout its genome.     

Intraspecific genetic diversity of Oenococcus oeni as derived from DNA fingerprinting and sequence analyses.

The intraspecific genetic diversity of Oenococcus oeni, the key organism in the malolactic fermentation of wine, has been evaluated by random amplified polymorphic DNA (RAPD), ribotyping, small-plasmid content, and sequencing of RAPD markers with widespread distribution among the strains. Collection strains representing the diversity of this species have been studied together with some new isolates, many of which were obtained from wines produced by spontaneous malolactic fermentation. The RAPD profiles were strain specific and discerned two main groups of strains coincident with clusters obtained by macrorestriction typing in a previous work. Ribotyping and the conservation of RAPD markers indicates that O. oeni is a relatively homogeneous species. Furthermore, identical DNA sequences of some RAPD markers among strains representative of the most divergent RAPD clusters indicates that O. oeni is indeed a phylogenetically tight group, probably corresponding to a single clone, or clonal line of descent, specialized to grow in the wine environment and universally spread.     

Allelic diversity and population structure in Oenococcus oeni as determined from sequence analysis of housekeeping genes

Oenococcus oeni is the organism of choice for promoting malolactic fermentation in wine. The population biology of O. oeni is poorly understood and remains unclear. For a better understanding of the mode of genetic variation within this species, we investigated by using multilocus sequence typing (MLST) with the gyrB, pgm, ddl, recP, and mleA genes the genetic diversity and genetic relationships among 18 O. oeni strains isolated in various years from wines of the United States, France, Germany, Spain, and Italy. These strains have also been characterized by ribotyping and restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S-23S rRNA gene intergenic spacer region (ISR). Ribotyping grouped the strains into two groups; however, the RFLP analysis of the ISRs showed no differences in the strains analyzed. In contrast, MLST in oenococci had a good discriminatory ability, and we have found a higher genetic diversity than indicated by ribotyping analysis. All sequence types were represented by a single strain, and all the strains could be distinguished from each other because they had unique combinations of alleles. Strains assumed to be identical showed the same sequence type. Phylogenetic analyses indicated a panmictic population structure in O. oeni. Sequences were analyzed for evidence of recombination by split decomposition analysis and analysis of clustered polymorphisms. All results indicated that recombination plays a major role in creating the genetic heterogeneity of O. oeni. A low standardized index of association value indicated that the O. oeni genes analyzed are close to linkage equilibrium. This study constitutes the first step in the development of an MLST method for O. oeni and the first example of the application of MLST to a nonpathogenic food production bacteria.     

Novel multi-nucleotide polymorphisms in the human genome characterized by whole genome and exome sequencing

Genomic sequence comparisons between individuals are usually restricted to the analysis of single nucleotide polymorphisms (SNPs). While the interrogation of SNPs is efficient, they are not the only form of divergence between genomes. In this report, we expand the scope of polymorphism detection by investigating the occurrence of double nucleotide polymorphisms (DNPs) and triple nucleotide polymorphisms (TNPs), in which two or three consecutive nucleotides are altered compared to the reference sequence. We have found such DNPs and TNPs throughout two complete genomes and eight exomes. Within exons, these novel polymorphisms are over-represented amongst protein-altering variants; nearly all DNPs and TNPs result in a change in amino acid sequence and, in some cases, two adjacent amino acids are changed. DNPs and TNPs represent a potentially important new source of genetic variation which may underlie human disease and they should be included in future medical genetics studies. As a confirmation of the damaging nature of xNPs, we have identified changes in the exome of a glioblastoma cell line that are important in glioblastoma pathogenesis. We have found a TNP causing a single amino acid change in LAMC2 and a TNP causing a truncation of HUWE1.     

Allelic genome structural variations in maize detected by array comparative genome hybridization

DNA polymorphisms such as insertion/deletions and duplications affecting genome segments larger than 1 kb are known as copy-number variations (CNVs) or structural variations (SVs). They have been recently studied in animals and humans by using array-comparative genome hybridization (aCGH), and have been associated with several human diseases. Their presence and phenotypic effects in plants have not been investigated on a genomic scale, although individual structural variations affecting traits have been described. We used aCGH to investigate the presence of CNVs in maize by comparing the genome of 13 maize inbred lines to B73. Analysis of hybridization signal ratios of 60,472 60-mer oligonucleotide probes between inbreds in relation to their location in the reference genome (B73) allowed us to identify clusters of probes that deviated from the ratio expected for equal copy-numbers. We found CNVs distributed along the maize genome in all chromosome arms. They occur with appreciable frequency in different germplasm subgroups, suggesting ancient origin. Validation of several CNV regions showed both insertion/deletions and copy-number differences. The nature of CNVs detected suggests CNVs might have a considerable impact on plant phenotypes, including disease response and heterosis.     

Role of secondary transporters and phosphotransferase systems in glucose transport by Oenococcus oeni

Glucose uptake by the heterofermentative lactic acid bacterium Oenococcus oeni B1 was studied at the physiological and gene expression levels. Glucose- or fructose-grown bacteria catalyzed uptake of [(14)C]glucose over a pH range from pH 4 to 9, with maxima at pHs 5.5 and 7. Uptake occurred in two-step kinetics in a high- and low-affinity reaction. The high-affinity uptake followed Michaelis-Menten kinetics and required energization. It accumulated the radioactivity of glucose by a factor of 55 within the bacteria. A large portion (about 80%) of the uptake of glucose was inhibited by protonophores and ionophores. Uptake of the glucose at neutral pH was not sensitive to degradation of the proton potential, Δp. Expression of the genes OEOE_0819 and OEOE_1574 (here referred to as 0819 and 1574), coding for secondary transporters, was induced by glucose as identified by quantitative real-time (RT)-PCR. The genes 1574 and 0819 were able to complement growth of a Bacillus subtilis hexose transport-deficient mutant on glucose but not on fructose. The genes 1574 and 0819 therefore encode secondary transporters for glucose, and the transports are presumably Δp dependent. O. oeni codes, in addition, for a phosphotransferase transport system (PTS) (gene OEOE_0464 [0464] for the permease) with similarity to the fructose- and mannose-specific PTS of lactic acid bacteria. Quantitative RT-PCR showed induction of the gene 0464 by glucose and by fructose. The data suggest that the PTS is responsible for Δp-independent hexose transport at neutral pH and for the residual Δp-independent transport of hexoses at acidic pH.     

Genotypic analysis of multiple loci in somatic cells by whole genome amplification.

To screen multiple loci in small purified samples of diploid and aneuploid cells a PCR-based technique of whole genome amplification was adapted to the study of somatic lesions. DNA samples from different numbers of flow-sorted diploid and aneuploid cells from biopsies were amplified with a degenerate 15mer primer. Aliquots of these reactions were then used in locus-specific reactions using a single round of PCR cycles with individual sets of primers representing polymorphic markers for different regions. As a result, polymorphic markers for different chromosomal regions, including VNTRs and dinucleotide repeats, can be used to perform up to 30 locus-specific PCR assays with a single sample obtained from fewer than 1000 cells.     

Yersinia genome diversity disclosed by Yersinia pestis genome-wide DNA microarray

The genus Yersinia includes 11 species, 3 of which (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica) are pathogenic for humans. The remaining 8 species (Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. bercovieri, Y. mollaretii, Y. rohdei, Y. ruckeri, and Y. aldovae) are merely opportunistic pathogens found mostly in the environment. In this work, the genomic differences among Yersinia were determined using a Y. pestis-specific DNA microarray. The results revealed 292 chromosomal genes that were shared by all Yersinia species tested, constituting the conserved gene pool of the genus Yersinia. Hierarchical clustering analysis of the microarray data revealed the genetic relationships among all 11 species in this genus. The microarray analysis in conjunction with PCR screening greatly reduced the number of chromosomal genes (32) specific for Y. pestis to 16 genes and uncovered a high level of genomic plasticity within Y. pseudotuberculosis, indicating that its different serotypes have undergone an extensively parallel loss or acquisition of genetic content, which is likely to be important for its adaptation to changes in environmental niches.     

Control of Flavor Development in Wine during and after Malolactic Fermentation by Oenococcus oeni

During malolactic fermentation in wine by Oenococcus oeni, the degradation of citric acid was delayed compared to the degradation of malic acid. The maximum concentration of diacetyl, an intermediary compound in the citric acid metabolism with a buttery or nutty flavor, coincided with the exhaustion of malic acid in the wine. The maximum concentration of diacetyl obtained during malolactic fermentation was strongly dependent on the oxygen concentration and the redox potential of the wine and, to a lesser extent, on the initial citric acid concentration. The final diacetyl concentration in the wine was also dependent on the concentration of SO₂. Diacetyl combines rather strongly with SO₂ (K_f = 7.2 × 10³ M⁻¹ in 0.1 M malate buffer [pH 3.5] at 30°C). The reaction is exothermic and reversible. If the concentration of SO₂ decreases during storage of the wine, the diacetyl concentration increases again.     

Molecular and biochemical diversity of Oenococcus oeni strains isolated during spontaneous malolactic fermentation of Malvasia Nera wine

The diversity of indigenous Oenococcus oeni strains was investigated by molecular and biochemical characterization of isolates from Malvasia Nera wine, an economically important red wine of the Salento Region (Apulia, Italy), during spontaneous malolactic fermentation (MLF). A total of 82 isolates of this species, identified by species-specific PCR and 16S rDNA sequence analysis, was molecularly characterized by the amplified fragment length polymorphism (AFLP) technique. Three main groups resulted from cluster analysis and showed intraspecific homology higher than 50%, and a total of seven subgroups, with similarity values ranged from 80% to 98%, were obtained within these groups. Enzymatic activities, such as esterase, β-glucosidase, protease, and the consumption rate of l-malic acid, citric acid, acetaldehyde and arginine were assessed in the representative strains, according to AFLP analysis. The results showed different enzymatic activities and consumption rates of the tested metabolites among the strains. No correlation between molecular and biochemical data was observed. The evidence of biochemical variability observed among Malvasia Nera strains demonstrated that the wine aroma can be modulated depending on the strains involved in MLF. Hence, the heterogeneity existing within natural O. oeni populations represents an interesting ecological source that can be useful for technological purposes.     

Anti-Prelog Reduction of Prochiral Carbonyl Compounds by Oenococcus oeni in a Biphasic System

An aqueous-organic biphasic system was established and used with whole cells of Oenococcus oeni to reduce 2-octanone to (R)-2-octanol. The conversion reached 99% when the Tris/borate buffer was increased from 50 mM to 300 mM in the aqueous phase. In addition, the conversion increased as the log P value of the organic solvent changed from 0.5 to 6.6. Under optimized conditions, the conversion of (R)-2-octanol reached 99% from 0.5 M 2-octanone with an optical purity of 99% e.e. The biphasic system allows the anti-Prelog reduction of aliphatic and aromatic ketones to furnish (R)-configurated alcohols in high optical purity as well.     

Leptospire genomic diversity revealed by microarray-based comparative genomic hybridization

Comparative genomic hybridization was used to compare genetic diversity of five strains of Leptospira (Leptospira interrogans serovars Bratislava, Canicola, and Hebdomadis and Leptospira kirschneri serovars Cynopteri and Grippotyphosa). The array was designed based on two available sequenced Leptospira reference genomes, those of L. interrogans serovar Copenhageni and L. interrogans serovar Lai. A comparison of genetic contents showed that L. interrogans serovar Bratislava was closest to the reference genomes while L. kirschneri serovar Grippotyphosa had the least similarity to the reference genomes. Cluster analysis indicated that L. interrogans serovars Bratislava and Hebdomadis clustered together first, followed by L. interrogans serovar Canicola, before the two L. kirschneri strains. Confirmed/potential virulence factors identified in previous research were also detected in the tested strains.