Investigation of the probiotic and metabolic potential of Fructobacillus tropaeoli and Apilactobacillus kunkeei from apiaries.

Honeybee products have been among important consumer products throughout history. Microbiota has attracted attention in recent years due to both their probiotic value and industrial potential. Fructophilic lactic acid bacteria (FLAB), whose field of study has been expanding rapidly in the last 20 years, are among the groups that can be isolated from the bee gut. This study aimed to isolate FLAB from the honeybees of two different geographic regions in Turkey and investigate their probiotic, metabolic and anti-quorum sensing (anti-QS) potential. Metabolic properties were investigated based on fructose toleration and acid and diacetyl production while the probiotic properties of the isolates were determined by examining pH, pepsin, pancreatin resistance, antimicrobial susceptibility, and antimicrobial activity. Anti-QS activities were also evaluated with the Chromobacterium violaceum biosensor strain. Two FLAB members were isolated and identified by the 16S rRNA analysis as Fructobacillus tropaeoli and Apilactobacillus kunkeei, which were found to be tolerant to high fructose, low pH, pepsin, pancreatin, and bile salt environments. Both isolates showed anti-QS activity against the C. violaceum biosensor strain and no diacetyl production. The daily supernatants of the isolates inhibited the growth of Enterococcus faecalis ATCC 29212 among the selected pathogens. The isolates were found resistant to kanamycin, streptomycin, erythromycin, and clindamycin. In the evaluation of the probiotic potential of these species, the negative effect of antibiotics and other chemicals to which honeybees are directly or indirectly exposed draws attention within the scope of the "One Health" approach.

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.

A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae.

The genus Lactobacillus comprises 261 species (at March 2020) that are extremely diverse at phenotypic, ecological and genotypic levels. This study evaluated the taxonomy of Lactobacillaceae and Leuconostocaceae on the basis of whole genome sequences. Parameters that were evaluated included core genome phylogeny, (conserved) pairwise average amino acid identity, clade-specific signature genes, physiological criteria and the ecology of the organisms. Based on this polyphasic approach, we propose reclassification of the genus Lactobacillus into 25 genera including the emended genus Lactobacillus, which includes host-adapted organisms that have been referred to as the Lactobacillus delbrueckii group, Paralactobacillus and 23 novel genera for which the names Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus and Lentilactobacillus are proposed. We also propose to emend the description of the family Lactobacillaceae to include all genera that were previously included in families Lactobacillaceae and Leuconostocaceae. The generic term 'lactobacilli' will remain useful to designate all organisms that were classified as Lactobacillaceae until 2020. This reclassification reflects the phylogenetic position of the micro-organisms, and groups lactobacilli into robust clades with shared ecological and metabolic properties, as exemplified for the emended genus Lactobacillus encompassing species adapted to vertebrates (such as Lactobacillus delbrueckii, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensensii, Lactobacillus johnsonii and Lactobacillus acidophilus) or invertebrates (such as Lactobacillus apis and Lactobacillus bombicola).

How fructophilic lactic acid bacteria may reduce the FODMAPs content in wheat-derived baked goods: a proof of concept.

BACKGROUND: FODMAPs (Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols) intake is associated with the onset of irritable bowel syndrome symptoms. FODMAPs in wheat-derived baked goods may be reduced via bioprocessing by endogenous enzymes and/or microbial fermentation. Because of the inherent enzyme activities, bread made by baker's yeast and sourdough may result in decreased levels of FODMAPs, whose values are, however, not enough low for people sensitive to FODMAPs. RESULTS: Our study investigated the complementary capability of targeted commercial enzymes and metabolically strictly fructophilic lactic acid bacteria (FLAB) to hydrolyze fructans and deplete fructose during wheat dough fermentation. FLAB strains displayed higher fructose consumption rate compared to conventional sourdough lactic acid bacteria. Fructose metabolism by FLAB was faster than glucose. The catabolism of mannitol with the goal of its reuse by FLAB was also investigated. Under sourdough conditions, higher fructans breakdown occurred in FLAB inoculated doughs compared to conventional sourdough bacteria. Preliminary trials allowed selecting Apilactobacillus kunkeei B23I and Fructobacillus fructosus MBIII5 as starter candidates, which were successfully applied in synergy with commercial invertase for low FODMAPs baking. CONCLUSIONS: Results of this study clearly demonstrated the potential of selected strictly FLAB to strongly reduce FODMAPs in wheat dough, especially under liquid-dough and high oxygenation conditions.

Major role of lactate dehydrogenase D-LDH1 for the synthesis of lactic acid in Fructobacillus tropaeoli CRL 2034.

The enzymes D- and L-lactate dehydrogenase are involved in the reduction of pyruvate to D(+)- and L(-)-lactate, respectively. The fig-origin strain Fructobacillus tropaeoli CRL 2034 produces D- and L-lactic acids in a 9:1 ratio. In this work, two D-ldh (ldh1 and ldh2) and one L-ldh (ldh3) genes were found in the CRL 2034 genome. ldh1 and ldh2 are homologous (79% identity) and organized as contiguous operons, each gene containing 996 base pair (bp) and encoding for a 331-amino acid (aa) protein (74% identity). In contrast, ldh3 is a 927-bp gene coding for a 308-aa protein. The identity between ldh1/ldh2 and ldh3 was lower than 48%. To elucidate the role of these genes in the synthesis of lactic acid by the Fructobacillus strain, plasmid insertion mutants in each gene were generated and characterized. The growth kinetic parameters were affected only in CRL2034 ldh1::pRV300 cells, this mutant showing the lowest total lactic acid production (4.50 ± 0.15 versus 6.36 ± 0.67 g/L of wild-type strain), with a D/L ratio of 7.1:2.9. These results showed that the ldh1 gene is primarily responsible for lactic acid production by the studied strain. A comparative analysis among strains of the five Fructobacillus species revealed that the identity of D-LDH proteins was higher than 70%, while the identity of L-LDH was over 60%. Finally, phylogenetic analysis of D- and L-LDHs revealed that only D-LDH phylogeny was consistent to the phylogenetic evolution among Fructobacillus and evolutionarily related genera. Key Points •F. tropaeoli CRL 2034 harbors three ldh genes in its genome. •ldh1 and ldh2 encode D-lactate dehydrogenase; ldh3 encodes L-lactate dehydrogenase. •Gene ldh1 plays the major role in lactic acid production by strain CRL 2034. •Fructobacillus D-LDH phylogeny was consistent to phylogenetic evolution.

Exploring the Genome of Fructobacillus tropaeoli CRL 2034, a Fig-Origin Strain that Produces High Levels of Mannitol from Fructose.

We report the draft genome sequence of Fructobacillus tropaeoli CRL 2034, a strain isolated from ripe fig in Tucumán province, Argentina. The interest in studying the genome of this fructophilic lactic acid bacterium strain was motivated by its ability to produce high levels of mannitol from fructose. This polyol has multiple industrial applications; however, it is mainly used as low calorie sugar in the food industry. The assembled genome of this strain consists of a 1.66-Mbp circular chromosome with 1465 coding sequences and a G+C content of 44.6%. The analysis of this genome supports the one step reaction of fructose reduction to mannitol by the mannitol 2-dehydrogenase enzyme, which together with a fructose permease, were identified as involved in mannitol synthesis. In addition, a phylogenetic analysis was performed including other Leuconostocaceae members to which the Fructobacillus genus belongs to; according to the 16S rRNA gene sequences, the strain CRL 2034 was located in the Fructobacillus clade. The present genome sequence could be useful to further elucidate regulatory processes of mannitol and other bioactive metabolites and to highlight the biotechnological potential of this fruit-origin Fructobacillus strain.

Functional characterization and in vitro screening of Fructobacillus fructosus MCC 3996 isolated from Butea monosperma flower for probiotic potential.

The fructophilic bacterium Fructobacillus fructosus MCC 3996 described in the present investigation was isolated from the nectar of Butea monosperma flower and evaluated in vitro for the manifestation of probiotic features. The strain utilizes fructose faster than glucose and is capable to grow in the range of 1-35% fructose concentration (optimum 5% w/v) and thus denotes its fructophilic nature. In vitro assessments of the strain have examined for the endurance in acidic environment/gastric juice, the better auto-aggregation ability even in the presence of hydrolytic enzymes, co-aggregation with pathogenic bacteria, hydrophobicity properties and no haemolytic activity to elucidate its feasible probiotic use. The significant antagonistic activity against several detrimental bacteria, despite lacking the bacteriocin secretion, is an astonishing feature. Owing to the indigenous origin of the isolate, it could be used as a probiotic, starter culture, and/or the active ingredient of food formulation may contribute to improve the desirable fermentation, long-term storage and nutritional benefits of foods especially rich in fructose. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided in vitro evidence that Fructobacillus fructosus MCC 3996 have endurance in acidic gastric juice, better co-aggregation, auto-aggregation properties, splendid antagonistic activities against several bacteria involved in food spoilage/human infections, pertinent antibiotic susceptibility profile and no haemolytic activity. Also, F. fructosus have the capability to survive in the appreciable amount of fructose, and this advocates that the strain could be used as starter culture and/or the active ingredient of fructose-rich foods. The current in vitro study provided a strong basis for further in vivo research to identify the health beneficial characteristics of F. fructosus and its potential could be effectively utilized as health-boosting ingredient in food and pharmaceutical industries.

Contribution of Leuconostocaceae to CO2-mediated bloater defect in cucumber fermentation.

Fermented cucumber bloater defect, caused by the accumulation of microbiologically produced carbon dioxide (CO2), creates significant economic losses for the pickling industry. The ability of Leuconostocaceae, indigenous to cucumber, to grow and produce CO2 during a fermentation and cause bloater defect was evaluated. Leuconostocaceae grew and produced over 40% CO2 in cucumber juice medium, used as a model for cucumber fermentation. The inoculation of Leuconostocaceae to 5 Log CFU/g in cucumber fermentations brined with 25 mM calcium chloride and 6 mM potassium sorbate resulted in no significant differences in bloater defect, colony counts from MRS and VRBG agar plates or the fermentation biochemistry; suggesting an inability of the inoculated bacterial species to prevail in the bioconversion. Acidified cucumbers were subjected to a fermentation inoculated with a Leuconostoc lactis starter culture after raising the pH to 5.9 ± 0.4. CO2 was produced in the acidified cucumber fermentations to 13.6 ± 3.5% yielding a bloater index of 21.3 ± 6.4; while 8.6 ± 0.8% CO2 and a bloater index of 5.2 ± 5.9 were observed in the non-inoculated control jars. Together the data collected demonstrate that Leuconostocaceae can produce enough CO2 to contribute to bloater defect, if not outcompeted by the leading lactic acid bacteria in a cucumber fermentation.

Evaluation of lactic acid bacteria strains isolated from fructose-rich environments for their mannitol-production and milk-gelation abilities.

Mannitol is a sugar alcohol, or polyol, widely used in the food industry because of its low-calorie properties. Industrial production of mannitol is difficult and expensive. However, certain bacterial species are known to produce mannitol naturally, including certain lactic acid bacteria and fructophilic lactic acid bacteria (LAB). In this study, bacterial strains isolated from fructose-rich sources, including flowers, leaves, and honey, were identified by 16S rRNA sequence analysis as Leuconostoc, Fructobacillus, Lactococcus, and Lactobacillus species and 4 non-LAB species. DNA profiles generated by pulsed-field gel electrophoresis discriminated 32 strains of Leuconostoc mesenteroides and 6 Fructobacillus strains. Out of 41 LAB strains isolated, 32 were shown to harbor the mdh gene, which encodes the mannitol dehydrogenase enzyme, and several showed remarkable fructose tolerance even at 50% fructose concentrations, indicating their fructophilic nature. Several of the strains isolated, including Leuconostoc mesenteroides strains DPC 7232 and DPC 7261, Fructobacillus fructosus DPC 7237, and Fructobacillus fructosus DPC 7238, produced higher mannitol concentrations than did the positive control strain Limosilactobacillus reuteri DSM 20016 during an enzymatic screening assay. Mannitol concentrations were also examined via HPLC in 1% fructose de Man, Rogosa, and Sharpe medium (FMRS) or 1% fructose milk (FM). Among the strains, Fructobacillus fructosus DPC 7238 displayed high fructose utilization (9.27 g/L), high mannitol yield (0.99 g of mannitol/g of fructose), and greatest volumetric productivities (0.46 g/L per h) in FMRS. However, Leuconostoc mesenteroides DPC 7261 demonstrated the highest fructose utilization (8.99 g/L), mannitol yield (0.72 g of mannitol/g of fructose), and volumetric productivities (0.04 g/L per h) in FM. Storage modulus G' (>0.1 Pa) indicated a shorter gelation time for Limosilactobacillus reuteri DSM 20016 (8.73 h), followed by F. fructosus DPC 7238 (11.57 h) and L. mesenteroides DPC 7261 (14.52 h). Our results show that fructose-rich niches can be considered important sources of fructophilic LAB strains, with the potential to be used as starter cultures or adjunct cultures for the manufacture of mannitol-enriched fermented dairy products and beverages.

Dynamics and species diversity of lactic acid bacteria involved in the spontaneous fermentation of various palm tree saps during palm wine tapping in Côte d'Ivoire.

To document and speed up research on the usefulness and selection of potential health-promoting bacterial starter cultures from unexplored fermented saps of various palm species in Côte d'Ivoire, benchmark tapping processes were successfully developed and implemented at field level. Therefore, spontaneously fermented saps of three palm species (Elaeis guineensis, Raphia hookeri, Borassus aethiopum) were collected throughout tapping process and lactic acid bacteria (LAB) diversity and dynamics were studied through a multiphasic approach. Overall microbiological analysis revealed a LAB species diversity throughout tapping process. LAB isolates belonged to two main (GTG)5-PCR clusters, namely Fructobacillus durionis (40.33%) and Leuconostoc mesenteroides (45.66%), with Leuconostoc pseudomesenteroides, Lactobacillus paracasei, Lactobacillus fermentum Weissella cibaria, Enterococcus casseliflavus and Lactococcus lactis occurring occasionally. LAB diversity was higher in fermented saps from E. guineensis (8 species) than those of R. hookeri (5 species) and B. aethiopum (3 species). Dynamic study revealed that F. durionis and L. mesenteroides dominated the fermentations from the beginning until the end of tapping process in all palm wine types. But the earlier stages of the process were also populated by some species like W. cibaria, L. pseudomesenteroides and L. fermentum, which population decreased or disappeared after some days. Also, species of Enterococcus and Lactococcus genera were sporadically detected uniquely in sap from E. guineensis. This study is the first to investigate extensively the LAB diversity and dynamics throughout palm trees tapping process in Côte d'Ivoire and is relevant for future selection of health promoting bacteria.

Evaluation of efficient carbon, nitrogen sources, micro and macro nutrients for dextran production by Weissella cibaria CMGDEX3 by utilizing a modified multifactorial Placket-Burman statistical design.

In the present study previously isolated Weissella cibaria CMG DEX3 capable of producing high molecular weight, water soluble dextran (Ahmed et al., 2012) is characterized for most efficient less expensive carbon, nitrogen sources, micro and macro nutrients by utilizing a multifactorial Placket-Burman statistical design for optimization of dextran production. A twelve run Plackett-Burman experimental model with slight modification was utilized to evaluate the impact of ten diverse nutrients on the production of dextran by the bacterial isolate Weissella cibaria CMG DEX3.

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.

Enhanced mannitol biosynthesis by the fruit origin strain Fructobacillus tropaeoli CRL 2034.

Mannitol is a natural low-calorie sugar alcohol produced by certain (micro)organisms applicable in foods for diabetics due to its zero glycemic index. In this work, we evaluated mannitol production and yield by the fruit origin strain Fructobacillus tropaeoli CRL 2034 using response surface methodology with central composite design (CCD) as optimization strategy. The effect of the total saccharide (glucose + fructose, 1:2) content (TSC) in the medium (75, 100, 150, 200, and 225 g/l) and stirring (S; 50, 100, 200, 300 and 350 rpm) on mannitol production and yield by this strain was evaluated by using a 22 full-factorial CCD with 4 axial points (α = 1.5) and four replications of the center point, leading to 12 random experimental runs. Fermentations were carried out at 30 °C and pH 5.0 for 24 h. Minitab-15 software was used for experimental design and data analyses. The multiple response prediction analysis established 165 g/l of TSC and 200 rpm of S as optimal culture conditions to reach 85.03 g/l [95% CI (78.68, 91.39)] of mannitol and a yield of 82.02% [95% CI (71.98, 92.06)]. Finally, a validation experiment was conducted at the predicted optimum levels. The results obtained were 81.91 g/l of mannitol with a yield of 77.47% in outstanding agreement with the expected values. The mannitol 2-dehydrogenase enzyme activity was determined with 4.6-4.9 U/mg as the highest value found. To conclude, F. tropaeoli CRL 2034 produced high amounts of high-quality mannitol from fructose, being an excellent candidate for this polyol production.

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.

Three-phase succession of autochthonous lactic acid bacteria to reach a stable ecosystem within 7 days of natural bamboo shoot fermentation as revealed by different molecular approaches.

Microbial community structure and population dynamics during spontaneous bamboo shoot fermentation for production of 'soidon' (indigenous fermented food) in North-east India were studied using cultivation-dependent and cultivation-independent molecular approaches. Cultivation-dependent analyses (PCR-amplified ribosomal DNA restriction analysis and rRNA gene sequencing) and cultivation-independent analyses (PCR-DGGE, qPCR and Illumina amplicon sequencing) were conducted on the time series samples collected from three independent indigenous soidon fermentation batches. The current findings revealed three-phase succession of autochthonous lactic acid bacteria to attain a stable ecosystem within 7 days natural fermentation of bamboo shoots. Weissella spp. (Weissella cibaria, uncultured Weissella ghanensis) and Lactococcus lactis subsp. cremoris predominated the early phase (1-2 days) which was joined by Leuconostoc citreum during the mid-phase (3 days), while Lactobacillus brevis and Lactobacillus plantarum emerged and became dominant in the late phase (5-7 days) with concurrent disappearance of W. cibaria and L. lactis subsp. cremoris. Lactococcus lactis subsp. lactis and uncultured Lactobacillus acetotolerans were predominantly present throughout the fermentation with no visible dynamics. The above identified dominant bacterial species along with their dynamics can be effectively utilized for designing a starter culture for industrialization of soidon production. Our results showed that a more realistic view on the microbial ecology of soidon fermentation could be obtained by cultivation-dependent studies complemented with cultivation-independent molecular approaches. Moreover, the critical issues to be considered for reducing methodological biases while studying the microbial ecology of traditional food fermentation were also highlighted with this soidon fermentation model.

Paratransgenesis feasibility in the honeybee (Apis mellifera) using Fructobacillus fructosus commensal.

AIMS: To establish the molecular tools for honeybee paratransgenesis. METHODS AND RESULTS: Commensal bacteria were isolated from two honeybees. Based on 16S ribosomal RNA sequence analysis, some isolates were identified as Fructobacillus fructosus, Lactobacillus kunkeei, Gilliamella apicola, Acinetobacter spp, Arthrobacter spp and Pseudomonas spp. Rolling circle and theta replicons were successfully introduced into F. fructosus and Lact. kunkeei. Green fluorescent protein was expressed into both species. The 7·3 Kb Lactococcus lactis subsp. cremoris MG1363 operon encoding a cluster of five genes involved in the metabolism of galactose via the Leloir pathway was functionally expressed into a non-galactose-fermenting strain of F. fructosus enabling it to grow on galactose as a sole carbon source. CONCLUSIONS: Fructophilic lactic acid bacteria, F. fructosus and Lact. kunkeei, are amenable to extensive genetic manipulations. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study demonstrating the feasibility of genetically engineering honeybee commensals, thus establishing the tools necessary for honeybee paratransgenesis.

Fructophilic characteristics of Fructobacillus spp. may be due to the absence of an alcohol/acetaldehyde dehydrogenase gene (adhE).

Fructophilic strains of Leuconostoc spp. have recently been reclassified to a new genus, i.e., Fructobacillus. Members of the genus are differentiated from Leuconostoc spp. by their preference for fructose on growth, requirement of an electron acceptor for glucose metabolism, and the inability to produce ethanol from the fermentation of glucose. In the present study, enzyme activities and genes involved in ethanol production were studied, since this is the key pathway for NAD(+)/NADH cycling in heterofermentative lactic acid bacteria. Fructobacillus spp. has a weak alcohol dehydrogenase activity and has no acetaldehyde dehydrogenase activity, whereas both enzymes are active in Leuconostoc mesenteroides. The bifunctional alcohol/acetaldehyde dehydrogenase gene, adhE, was described in Leuconostoc spp., but not in Fructobacillus spp. These results suggested that, due to the deficiency of the adhE gene, the normal pathway for ethanol production is absent in Fructobacillus spp. This leads to a shortage of NAD(+), and the requirement for an electron acceptor in glucose metabolism. Fructophilic characteristics, as observed for Fructobacillus spp., are thus due to the absence of the adhE gene, and a phenotype that most likely evolved as a result of regressive evolution.

Characterization of strains of Weissella fabalis sp. nov. and Fructobacillus tropaeoli from spontaneous cocoa bean fermentations.

Six facultatively anaerobic, non-motile lactic acid bacteria were isolated from spontaneous cocoa bean fermentations carried out in Brazil, Ecuador and Malaysia. Phylogenetic analysis revealed that one of these strains, designated M75(T), isolated from a Brazilian cocoa bean fermentation, had the highest 16S rRNA gene sequence similarity towards Weissella fabaria LMG 24289(T) (97.7%), W. ghanensis LMG 24286(T) (93.3%) and W. beninensis LMG 25373(T) (93.4%). The remaining lactic acid bacteria isolates, represented by strain M622, showed the highest 16S rRNA gene sequence similarity towards the type strain of Fructobacillus tropaeoli (99.9%), a recently described species isolated from a flower in South Africa. pheS gene sequence analysis indicated that the former strain represented a novel species, whereas pheS, rpoA and atpA gene sequence analysis indicated that the remaining five strains belonged to F. tropaeoli; these results were confirmed by DNA-DNA hybridization experiments towards their respective nearest phylogenetic neighbours. Additionally, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry proved successful for the identification of species of the genera Weissella and Fructobacillus and for the recognition of the novel species. We propose to classify strain M75(T) ( = LMG 26217(T)  = CCUG 61472(T)) as the type strain of the novel species Weissella fabalis sp. nov.

Genomic diversity in Fructobacillus spp. isolated from fructose-rich niches.

The Fructobacillus genus is a group of obligately fructophilic lactic acid bacteria (FLAB) that requires the use of fructose or another electron acceptor for their growth. In this work, we performed a comparative genomic analysis within the genus Fructobacillus by using 24 available genomes to evaluate genomic and metabolic differences among these organisms. In the genome of these strains, which varies between 1.15- and 1.75-Mbp, nineteen intact prophage regions, and seven complete CRISPR-Cas type II systems were found. Phylogenetic analyses located the studied genomes in two different clades. A pangenome analysis and a functional classification of their genes revealed that genomes of the first clade presented fewer genes involved in the synthesis of amino acids and other nitrogen compounds. Moreover, the presence of genes strictly related to the use of fructose and electron acceptors was variable within the genus, although these variations were not always related to the phylogeny.