Complete genome sequences of 11 Streptococcus pneumoniae strains isolated from acute infectious purpura fulminans, sepsis, and pneumonia.

The complete genome sequences of 11 Japanese Streptococcus pneumoniae isolates were determined by hybrid assembly of long and short reads, including two strains isolated from patients with acute infectious purpura fulminans, six strains from patients with sepsis, and three strains from patients with pneumonia.

Spore-forming properties and enhanced oxygen tolerance of butyrate-producing Anaerostipes spp.

OBJECTIVES: Butyrate producing bacteria are promising candidates for next-generation probiotics. However, they are extremely sensitive to oxygen, which is a significant obstacle to their inclusion in food matrices in a viable form. The present study characterized the spore-forming properties and stress tolerance of human gut butyrate-producing Anaerostipes spp. METHODS: Spore formation properties in six species of Anaerostipes spp. were studied by in vitro and in silico tests. RESULTS: Spores were observed from the cells of three species using microscopic analyses, while the remaining three did not form spores under the tested conditions. Spore-forming properties were confirmed by an ethanol treatment. The spores of Anaerostipes caccae were tolerant to oxygen and survived for 15 weeks under atmospheric conditions. Spores tolerated heat stress at 70 °C, but not at 80 °C. An in silico analysis of the conservation of potential sporulation signature genes revealed that the majority of human gut butyrate-producing bacteria were classified as potential spore formers. Comparative genomics revealed that three spore-forming Anaerostipes spp. specifically possessed the spore formation-related genes of bkdR, sodA, and splB, which may be key genes for different sporulation properties in Anaerostipes spp. CONCLUSIONS: The present study demonstrated the enhanced stress tolerance of butyrate producing Anaerostipes spp. for future probiotic application. Presence of specific gene(s) are possibly keys for sporulation in Anaerostipes spp.

16S rRNA gene sequence diversity in Faecalibacterium prausnitzii-complex taxa has marked impacts on quantitative analysis.

Faecalibacterium prausnitzii has been suggested as a biomarker of a healthy microbiota in human adults. Here, we report a taxonomic study of F. prausnitzii using genomic information and evaluation of the quantitative real-time PCR (qPCR) assay by focusing on specific primers to quantify its population. Average nucleotide identity values revealed that strains deposited as F. prausnitzii in a public database were separated into eight genomogroups with significant differences at the species level. A total of six of the 10 primer pairs used in the previous studies for qPCR of F. prausnitzii contained sequence mismatches to 16S rRNA gene sequences of the tested strains with markedly different levels by in silico analysis. In vitro primer evaluation by qPCR generally agreed with the in silico analysis, and markedly reduced amount of DNA was recorded by qPCR in combination with the primer pairs containing sequence mismatches. The present study demonstrated that a part of the accumulated knowledge on F. prausnitzii is maybe based on biased results.

Oligosaccharide Metabolism and Lipoteichoic Acid Production in Lactobacillus gasseri and Lactobacillus paragasseri.

Lactobacillus gasseri and Lactobacillus paragasseri are human commensal lactobacilli that are candidates for probiotic application. Knowledge of their oligosaccharide metabolic properties is valuable for synbiotic application. The present study characterized oligosaccharide metabolic systems and their impact on lipoteichoic acid (LTA) production in the two organisms, i.e., L. gasseri JCM 1131T and L. paragasseri JCM 11657. The two strains grew well in medium with glucose but poorly in medium with raffinose, and growth rates in medium with kestose differed between the strains. Oligosaccharide metabolism markedly influenced their LTA production, and apparent molecular size of LTA in electrophoresis recovered from cells cultured with glucose and kestose differed from that from cells cultured with raffinose in the strains. On the other hand, more than 15-fold more LTA was observed in the L. gasseri cells cultured with raffinose when compared with glucose or kestose after incubation for 15 h. Transcriptome analysis identified glycoside hydrolase family 32 enzyme as a potential kestose hydrolysis enzyme in the two strains. Transcriptomic levels of multiple genes in the dlt operon, involved in D-alanine substitution of LTA, were lower in cells cultured with raffinose than in those cultured with kestose or glucose. This suggested that the different sizes of LTA observed among the carbohydrates tested were partly due to different levels of alanylation of LTA. The present study indicates that available oligosaccharide has the impact on the LTA production of the industrially important lactobacilli, which might influence their probiotic properties.

Niche-specific adaptation of Lactobacillus helveticus strains isolated from malt whisky and dairy fermentations.

Lactobacillus helveticus is a well characterized lactobacillus for dairy fermentations that is also found in malt whisky fermentations. The two environments contain considerable differences related to microbial growth, including the presence of different growth inhibitors and nutrients. The present study characterized L. helveticus strains originating from dairy fermentations (called milk strains hereafter) and malt whisky fermentations (called whisky strains hereafter) by in vitro phenotypic tests and comparative genomics. The whisky strains can tolerate ethanol more than the milk strains, whereas the milk strains can tolerate lysozyme and lactoferrin more than the whisky strains. Several plant-origin carbohydrates, including cellobiose, maltose, sucrose, fructooligosaccharide and salicin, were generally metabolized only by the whisky strains, whereas milk-derived carbohydrates, i.e. lactose and galactose, were metabolized only by the milk strains. Milk fermentation properties also distinguished the two groups. The general genomic characteristics, including genomic size, number of coding sequences and average nucleotide identity values, differentiated the two groups. The observed differences in carbohydrate metabolic properties between the two groups correlated with the presence of intact specific enzymes in glycoside hydrolase (GH) families GH1, GH4, GH13, GH32 and GH65. Several GHs in the milk strains were inactive due to the presence of stop codon(s) in genes encoding the GHs, and the inactivation patterns of the genes encoding specific enzymes assigned to GH1 in the milk strains suggested a possible diversification manner of L. helveticus strains. The present study has demonstrated how L. helveticus strains have adapted to their habitats.

Unique niche-specific adaptation of fructophilic lactic acid bacteria and proposal of three Apilactobacillus species as novel members of the group.

BACKGROUND: Fructophilic lactic acid bacteria (FLAB) found in D-fructose rich niches prefer D-fructose over D-glucose as a growth substrate. They need electron acceptors for growth on D-glucose. The organisms share carbohydrate metabolic properties. Fructobacillus spp., Apilactobacillus kunkeei, and Apilactobacillus apinorum are members of this unique group. Here we studied the fructophilic characteristics of recently described species Apilactobacillus micheneri, Apilactobacillus quenuiae, and Apilactobacillus timberlakei. RESULTS: The three species prefer D-fructose over D-glucose and only metabolize D-glucose in the presence of electron acceptors. The genomic characteristics of the three species, i.e. small genomes and thus a low number of coding DNA sequences, few genes involved in carbohydrate transport and metabolism, and partial deletion of adhE gene, are characteristic of FLAB. The three species thus are novel members of FLAB. Reduction of genes involved in carbohydrate transport and metabolism in accordance with reduction of genome size were the common characteristics of the family Lactobacillaceae, but FLAB markedly reduced the gene numbers more than other species in the family. Pan-genome analysis of genes involved in metabolism displayed a lack of specific carbohydrate metabolic pathways in FLAB, leading to a unique cluster separation. CONCLUSIONS: The present study expanded FLAB group. Fructose-rich environments have induced similar evolution in phylogenetically distant FLAB species. These are examples of convergent evolution of LAB.

Characterization of the microbiota and chemical properties of pork loins during dry aging.

Dry aging (DA) allows for the storage of meat without packaging at 0 to 3°C for several weeks. It enhances the production of pleasant flavors, tenderness, and juiciness in meat. Due to the long storage period and roles of indigenous microbiota in the maturation of several meat products, the microbiota of DA meat is of interest in terms of microbial contributions and food hygiene but has not yet been characterized in detail. This study identified the microbiota of pork loins during DA using culturing and culture-independent meta-16S rRNA gene sequencing and elucidated its characteristics. The amounts of free amino acids and profiles of aroma-active compounds were also monitored by high-performance liquid chromatography and gas chromatography, respectively. The meta-16S rRNA gene sequencing revealed that Pseudomonas spp. generally dominated the microbiota throughout DA; however, the culturing analysis showed marked changes in the species composition during DA. Acinetobacter spp. were the second most dominant bacteria before DA in the culture-independent analysis but became a minor population during DA. The cell numbers of yeasts showed an increased tendency during DA, and Debaryomyces hansenii was the only microorganism isolated from all meat samples throughout DA. Well-known foodborne pathogens were not observed in two microbiota analyses. The amounts of free amino acids were increased by DA, and the number of aroma-active compounds and their flavor dilution values markedly changed during DA. Most microbial isolates showed positive reactions with proteolytic and lipolytic activities, suggesting their contribution to tenderness and aroma production in DA meats.

Characterization of fructooligosaccharide metabolism and fructooligosaccharide-degrading enzymes in human commensal butyrate producers.

Butyrate produced by gut microbiota has multiple beneficial effects on host health, and oligosaccharides derived from host diets and glycans originating from host mucus are major sources of its production. A significant reduction of butyrate-producing bacteria has been reported in patients with inflammatory bowel diseases and colorectal cancers. Although gut butyrate levels are important for host health, oligosaccharide metabolic properties in butyrate producers are poorly characterized. We studied the metabolic properties of fructooligosaccharides (FOSs) and other prebiotic oligosaccharides (i.e. raffinose and xylooligosaccharides; XOSs) in gut butyrate producers. 1-Kestose (kestose) and nystose, FOSs with degrees of polymerization of 3 and 4, respectively, were also included. Fourteen species of butyrate producers were divided into four groups based on their oligosaccharide metabolic properties, which are group A (two species) metabolizing all oligosaccharides tested, group F (four species) metabolizing FOSs but not raffinose and XOSs, group XR (four species) metabolizing XOSs and/or raffinose but not FOSs, and group N (four species) metabolizing none of the oligosaccharides tested. Species assigned to groups A and XR are rich glycoside hydrolase (GH) holders, whereas those in groups F and N are the opposite. In total, 17 enzymes assigned to GH32 were observed in nine of the 14 butyrate producers tested, and species that metabolized FOSs had at least one active GH32 enzyme. The GH32 enzymes were divided into four clusters by phylogenetic analysis. Heterologous gene expression analysis revealed that the GH32 enzymes in each cluster had similar FOS degradation properties within clusters, which may be linked to the conservation/substitution of amino acids to bind with substrates in GH32 enzymes. This study provides important knowledge to understand the impact of FOS supplementation on the activation of gut butyrate producers. Abbreviations: SCFA, short chain fatty acid; FOS, fructooligosaccharide; XOS, xylooligosaccharide; CAZy, Carbohydrate Active Enzymes; CBM, carbohydrate-binding module; PUL, polysaccharide utilization locus; S6PH sucrose-6-phosphate hydrolase.

Kunkecin A, a New Nisin Variant Bacteriocin Produced by the Fructophilic Lactic Acid Bacterium, Apilactobacillus kunkeei FF30-6 Isolated From Honey Bees.

Apilactobacillus kunkeei FF30-6 isolated from healthy honey bees synthesizes the bacteriocin, which exhibits antimicrobial activity against Melissococcus plutonius. The bacteriocin, kunkecin A, was purified through three-step chromatography, and mass spectrometry revealed that its relative molecular mass was 4218.3. Edman degradation of purified kunkecin A showed only the N-terminal residue, isoleucine. Hence, alkaline alkylation made the subsequent amino acid residues accessible to Edman degradation, and 30 cycles were sequenced with 11 unidentified residues. Whole genome sequencing of A. kunkeei FF30-6, followed by Sanger sequencing, revealed that the genes encoding the proteins involved in lantibiotic biosynthesis were within the plasmid, pKUNFF30-6. Most of the identified proteins exhibited significant sequence similarities to the biosynthetic proteins of nisin A and its variants, such as subtilin. However, the kunkecin A gene cluster lacked the genes corresponding to nisI, nisR, and nisK of the nisin A biosynthetic gene cluster. A comparison of the gene products of kukA and nisA (kunkecin A and nisin A structural genes, respectively) suggested that they had similar post-translational modifications. Furthermore, the structure of kunkecin A was proposed based on a comparison of the observed and calculated relative molecular masses of kunkecin A. The structural analysis revealed that kunkecin A and nisin A had a similar mono-sulfide linkage pattern. Purified kunkecin A exhibited a narrow antibacterial spectrum, but high antibacterial activity against M. plutonius. Kunkecin A is the first bacteriocin to be characterized in fructophilic lactic acid bacteria and is the first nisin-type lantibiotic found in the family Lactobacillaceae.

PCR-based screening, isolation, and partial characterization of motile lactobacilli from various animal feces.

BACKGROUND: Most lactobacilli found in animal intestines are generally non-motile, but there are few exceptions. Our previous work showed that Lactobacillus agilis BKN88, which is a highly motile strain originating from a chicken, takes advantage of motility in gut colonization in murine models, and thus motile lactobacilli likely have unique ecological characteristics conferred by motility. However, the ecology and habitat of gut-derived motile lactobacilli are still rarely understood. In addition, the limited availability of motile Lactobacillus isolates is one of the major obstacles for further studies. To gain insight into the ecology and habitat of the motile lactobacilli, we established a routinely applicable detection method for motile lactobacilli using PCR and subsequent selective isolation in semi-solid MRS medium for the collection of additional motile lactobacilli from animal feces. RESULTS: We applied the PCR detection using motile lactobacilli-specific primers, based on the motor switch protein gene (fliG) of flagella, to 120 animal feces, followed by selective isolation performed using 45 animal feces. As a result, motile lactobacilli were detected in 44 animal feces. In the selective isolation, 29 isolates of L. agilis and 2 isolates of L. ruminis were obtained from 8 animal species. CONCLUSIONS: These results indicated that motile lactobacilli are distributed in different animal species. Moreover, phylogenetic analysis of the L. agilis isolates suggests co-evolution with the host, and adaptation to a particular environmental niche.

Impact of kestose supplementation on the healthy adult microbiota in in vitro fecal batch cultures.

Prebiotics are widely used to shape a balanced microbiota in humans and animals. 1-Kestose (kestose) is one of the major components in commercialized short-chain fructooligosaccharide and is a promising prebiotic for infants. We herein studied the impact of kestose on the healthy adult microbiota in an in vitro fecal batch culture model. Stool samples obtained from seven healthy adults were diluted, inoculated into broth supplemented with or without 0.5% (w/v) kestose (kestose group and control group, respectively), and cultured under anaerobic conditions. Microbiota in the groups and stool samples were analyzed using 16S rRNA gene sequencing. At the phylum level, the kestose group showed increases in Bacteroidetes, whereas the control group showed increases in Proteobacteria. At the species level, Bifidobacterium longum was the only species showing significantly higher levels in the kestose group than in the control group and stool samples. On the other hand, levels of Escherichia coli were significantly higher in the control group than in stool samples, while the levels were not significantly different between the kestose group and stool samples. Quantitative PCR assays also revealed significantly higher levels of B. longum and lower tendency of E. coli in the kestose group than in the control group. These results suggest that supplementation with kestose increased the levels of beneficial microorganism and prevented the growth of risk-associated microorganisms related to disease development. Further interventional studies are needed to understand the health benefits of kestose in adult humans.

Pseudofructophilic Leuconostoc citreum Strain F192-5, Isolated from Satsuma Mandarin Peel.

Fructophilic lactic acid bacteria (FLAB), composed of Fructobacillus spp., Lactobacillus kunkeei, and Lactobacillus apinorum, are unique in that they prefer d-fructose over d-glucose as a carbon source. Strain F192-5, isolated from the peel of a satsuma mandarin and identified as Leuconostoc citreum, grows well on d-fructose but poorly on d-glucose and produces mainly lactate and acetate, with trace amounts of ethanol, from the metabolism of d-glucose. These characteristics are identical to those of obligate FLAB. However, strain F192-5 ferments a greater variety of carbohydrates than known FLAB. Comparative analyses of the genomes of strain F192-5 and reference strains of L. citreum revealed no signs of specific gene reductions, especially genes involved in carbohydrate transport and metabolism, in the genome of F192-5. The bifunctional alcohol/acetaldehyde dehydrogenase gene (adhE) is conserved in strain F192-5 but is not transcribed. This is most likely due to a deletion in the promoter region upstream of the adhE gene. Strain F192-5 did, however, ferment d-glucose when transformed with a plasmid containing the allochthonous adhE gene. L. citreum F192-5 is an example of a pseudo-FLAB strain with a deficiency in d-glucose metabolism. This unique phenotypic characteristic appears to be strain specific within the species L. citreum This might be one of the strategies lactic acid bacteria use to adapt to diverse environmental conditions.IMPORTANCE Obligate fructophilic lactic acid bacteria (FLAB) lack the metabolic pathways used in the metabolism of most carbohydrates and differ from other lactic acid bacteria in that they prefer to ferment d-fructose instead of d-glucose. These characteristics are well conserved at the genus or species level. Leuconostoc citreum F192-5 shows similar growth characteristics. However, the strain is metabolically and genomically different from obligate FLAB. This is an example of a strain that evolved a pseudo-FLAB phenotype to adapt to a fructose-rich environment.

Introduction of bifunctional alcohol/acetaldehyde dehydrogenase gene (adhE) in Fructobacillus fructosus settled its fructophilic characteristics.

Fructophilic lactic acid bacteria (FLAB) are unique in the sense that they prefer D-fructose over D-glucose as main carbon source. If D-glucose is metabolised, electron acceptors are required and significant levels of acetate are produced. These bacteria are found in environments rich in D-fructose, such as flowers, fruits and the gastrointestinal tract of insects feeding on fructose-rich diets. Fructobacillus spp. are representatives of this unique group, and their fructophilic characteristics are well conserved. In this study, the bifunctional alcohol/acetaldehyde dehydrogenase gene (adhE) from Leuconostoc mesenteroides NRIC 1541T was cloned into a plasmid and transferred to Fructobacillus fructosus NRIC 1058T. Differences in biochemical characteristics between the parental strain (NRIC 1058T) and the transformants were compared. Strain 1-11, transformed with the adhE gene, did not show any fructophilic characteristics, and the strain grew well on D-glucose without external electron acceptors. Accumulation of acetic acid, which was originally seen in the parental strain, was replaced with ethanol in the transformed strain. Furthermore, in silico analyses revealed that strain NRIC 1058T lacked the sugar transporters/permeases and enzymes required for conversion of metabolic intermediates. This may be the reason for poor carbohydrate metabolic properties recorded for FLAB.

Lactobacillus paragasseri sp. nov., a sister taxon of Lactobacillus gasseri, based on whole-genome sequence analyses.

Three strains, JCM 5343T, JCM 5344 and JCM 1130, currently identified as Lactobacillus gasseri, were investigated using a polyphasic taxonomic approach. Although these strains shared high 16S rRNA gene sequence similarities with L. gasseri ATCC 33323T (99.9 %), they formed a clade clearly distinct from ATCC 33323T based on whole-genome relatedness. The average nucleotide identity and in silico DNA-DNA hybridization values of these three strains compared to L. gasseri ATCC 33323T were 93.4-93.7 and 53.1-54.1 %, respectively, and both were less than the widely accepted threshold to distinguish two species (95 and 70 %, respectively). The three strains were Gram-stain positive, non-motile, non-spore-forming, catalase-negative and rod-shaped bacteria. They grew at 25-45 °C and in the presence of 2.0 % (w/v) NaCl. The major fatty acids of the three strains were C16 : 0 and C18 : 1 ω9c. Based on phylogenetic analyses of the phenylalanyl-tRNA synthase alpha subunit and RNA polymerase alpha subunit genes, and on phenotypic and chemotaxonomic characteristics, strains JCM 5343T, JCM 5344 and JCM 1130 represent a novel species distinct from L. gasseri, for which we propose the name Lactobacillusparagasseri sp. nov. In addition, a large portion of genomes currently labelled as L. gasseri in the public sequence database should be reclassified as L. paragasseri based on whole-genome relatedness.

Fructophilic Lactic Acid Bacteria, a Unique Group of Fructose-Fermenting Microbes.

Fructophilic lactic acid bacteria (FLAB) are a recently discovered group, consisting of a few Fructobacillus and Lactobacillus species. Because of their unique characteristics, including poor growth on glucose and preference of oxygen, they are regarded as "unconventional" lactic acid bacteria (LAB). Their unusual growth characteristics are due to an incomplete gene encoding a bifunctional alcohol/acetaldehyde dehydrogenase (adhE). This results in the imbalance of NAD/NADH and the requirement of additional electron acceptors to metabolize glucose. Oxygen, fructose, and pyruvate are used as electron acceptors. FLAB have significantly fewer genes for carbohydrate metabolism than other LAB, especially due to the lack of complete phosphotransferase system (PTS) transporters. They have been isolated from fructose-rich environments, including flowers, fruits, fermented fruits, and the guts of insects that feed on plants rich in fructose, and are separated into two groups on the basis of their habitats. One group is associated with flowers, grapes, wines, and insects, and the second group is associated with ripe fruits and fruit fermentations. Species associated with insects may play a role in the health of their host and are regarded as suitable vectors for paratransgenesis in honey bees. Besides their impact on insect health, FLAB may be promising candidates for the promotion of human health. Further studies are required to explore their beneficial properties in animals and humans and their applications in the food industry.

The ability of human intestinal anaerobes to metabolize different oligosaccharides: Novel means for microbiota modulation?

Prebiotic oligosaccharides are known to have significant impacts on gut microbiota and are thus widely used to program healthy microbiota composition and activity from infants to the elderly. Bifidobacteria and lactobacilli are among the major target microorganisms of oligosaccharides, but the metabolic properties of oligosaccharides in other predominant gut microbes have not been well characterized. In the present study, we demonstrated the metabolic properties of six oligosaccharides in 31 key gut anaerobes. Bifidobacteria readily metabolized fructooligosaccharide (FOSs) with degree of polymerization (DP) 3, i.e. 1-kestose, but several strains used did not actively metabolize FOSs with DP4 and DP5, i.e. nystose and fructosylnystose. Akkermansia muciniphila, a potential new probiotic against obesity, did not show significant growth with any of the oligosaccharides tested. The butyrate producer Anaerostipes caccae grew well on 1-kestose but poorly on FOS mixtures, whereas it contained 1-kestose at 30%. Bacteroides-Parabacteroides group species were separated into two groups based on oligosaccharide metabolic properties. One group metabolized well most of the oligosaccharides tested, but the others metabolized only 1 or 2 selected oligosaccharides. Oligosaccharide profiles after culturing revealed that Bifidobacterium spp. preferentially metabolized shorter oligosaccharides (DP3) in the mixtures, whereas Bacteroides-Parabacteroides spp. did not show oligosaccharide selectivity for metabolism or rather preferred longer oligosaccharides (>DP4). The fermentation profiles indicated specific links between the microbial end-products and specific gut microbes. Available carbohydrates had a significant impact on the accumulation of amino acid-derived bacterial metabolites (i.e. phenol, p-cresol, indole and skatole) and short chain fatty acids. The results assist in predicting the impact of oligosaccharides in human intervention and gut microbiota modulation.

In vitro and in silico characterisation of Lactobacillus paraplantarum D2-1, a starter culture for soymilk fermentation.

Soymilk contains several functional nutrients and is thus a promising ingredient for production of functional foods. The present research aimed to study starter properties, functional characteristics and safety of Lactobacillus paraplantarum D2-1, a promising starter culture for soymilk fermentation. Strain D2-1 actively fermented soymilk within 24 h but had weak activity of additional acid production after 7 d. Succinate and acetoin, which could be linked to flavour and taste, were accumulated in fermented soymilk. In vitro study revealed that the organism has several beneficial properties, including high survival ability in artificial gastric juice, high abilities of mucus adhesion and biofilm formation and production of γ-aminobutyric acid and conjugated linoleic acid, without any significant risks for consumption. Genome sequencing supported the desirable metabolic properties of the strain. These results indicate that L. paraplantarum D2-1 is a suitable starter for soymilk fermentation and is a promising probiotic candidate that can be used safely.

Lactobacillus apinorum belongs to the fructophilic lactic acid bacteria.

Potential fructophilic characteristics of Lactobacillus apinorum, originally isolated from the guts of honeybees (Apis mellifera), were studied in the present study. The species showed typical fructophilic growth characteristics, i.e., active growth on d-fructose, poor growth on d-glucose, and accelerated growth on d-glucose in the presence of electron acceptors. Biochemical characteristics strongly supported classification of the species into fructophilic lactic acid bacteria (FLAB). Furthermore, genetic analyses suggested that the species underwent extensive gene reduction, similar to that recorded for Lactobacillus kunkeei and other FLAB. These data clearly indicated that L. apinorum is the second fructophilic species within the genus Lactobacillus.

Genomic characterization of a fructophilic bee symbiont Lactobacillus kunkeei reveals its niche-specific adaptation.

Lactobacillus kunkeei is classified as a sole obligate fructophilic lactic acid bacterium that is found in fructose-rich niches, including the guts of honeybees. The species is differentiated from other lactobacilli based on its poor growth with glucose, enhanced growth in the presence of oxygen and other electron acceptors, and production of high concentrations of acetate from the metabolism of glucose. These characteristics are similar to phylogenetically distant Fructobacillus spp. In the present study, the genomic structure of L. kunkeei was characterized by using 16 different strains, and it had significantly less genes and smaller genomes when compared with other lactobacilli. Functional gene classification revealed that L. kunkeei had lost genes specifically involved in carbohydrate transport and metabolism. The species also lacked most of the genes for respiration, although growth was enhanced in the presence of oxygen. The adhE gene of L. kunkeei, encoding a bifunctional alcohol dehydrogenase (ADH)/aldehyde dehydrogenase (ALDH) protein, lacked the part encoding the ADH domain, which is reported here for the first time in lactic acid bacteria. The deletion resulted in the lack of ADH activity, implying a requirement for electron acceptors in glucose assimilation. These results clearly indicated that L. kunkeei had undergone a specific reductive evolution in order to adapt to fructose-rich environments. The reduction characteristics were similar to those of Fructobacillus spp., but distinct from other lactobacilli with small genomes, such as Lactobacillus gasseri and Lactobacillus vaginalis. Fructose-richness thus induced an environment-specific gene reduction in phylogenetically distant microorganisms.

Comparative genomics of Fructobacillus spp. and Leuconostoc spp. reveals niche-specific evolution of Fructobacillus spp.

BACKGROUND: Fructobacillus spp. in fructose-rich niches belong to the family Leuconostocaceae. They were originally classified as Leuconostoc spp., but were later grouped into a novel genus, Fructobacillus, based on their phylogenetic position, morphology and specific biochemical characteristics. The unique characters, so called fructophilic characteristics, had not been reported in the group of lactic acid bacteria, suggesting unique evolution at the genome level. Here we studied four draft genome sequences of Fructobacillus spp. and compared their metabolic properties against those of Leuconostoc spp. RESULTS: Fructobacillus species possess significantly less protein coding sequences in their small genomes. The number of genes was significantly smaller in carbohydrate transport and metabolism. Several other metabolic pathways, including TCA cycle, ubiquinone and other terpenoid-quinone biosynthesis and phosphotransferase systems, were characterized as discriminative pathways between the two genera. The adhE gene for bifunctional acetaldehyde/alcohol dehydrogenase, and genes for subunits of the pyruvate dehydrogenase complex were absent in Fructobacillus spp. The two genera also show different levels of GC contents, which are mainly due to the different GC contents at the third codon position. CONCLUSION: The present genome characteristics in Fructobacillus spp. suggest reductive evolution that took place to adapt to specific niches.