The whole-genome sequence of Bombella intestini LMG 28161T, an endosymbiotic acetic acid bacterium (AAB) occurring in bumble bees, was determined to investigate the molecular mechanisms underlying its metabolic capabilities. The draft genome sequence of B. intestini LMG 28161T was 2.02 Mb. Metabolic carbohydrate pathways were in agreement with the metabolite analyses of fermentation experiments and revealed its oxidative capacity towards sucrose, D-glucose, D-fructose and D-mannitol, but not ethanol and glycerol. The results of the fermentation experiments also demonstrated that the lack of effective aeration in small-scale carbohydrate consumption experiments may be responsible for the lack of reproducibility of such results in taxonomic studies of AAB. Finally, compared to the genome sequences of its nearest phylogenetic neighbor and of three other insect associated AAB strains, the B. intestini LMG 28161T genome lost 69 orthologs and included 89 unique genes. Although many of the latter were hypothetical they also included several type IV secretion system proteins, amino acid transporter/permeases and membrane proteins which might play a role in the interaction with the bumble bee host.
Citation: Li L, Illeghems K, Van Kerrebroeck S, Borremans W, Cleenwerck I, Smagghe G, et al. (2016) Whole-Genome Sequence Analysis of Bombella intestini LMG 28161T, a Novel Acetic Acid Bacterium Isolated from the Crop of a Red-Tailed Bumble Bee, Bombus lapidarius. PLoS ONE 11(11): e0165611. https://doi.org/10.1371/journal.pone.0165611
Editor: Daniele Daffonchio, University of Milan, ITALY
Received: April 19, 2016; Accepted: October 15, 2016; Published: November 16, 2016
Copyright: © 2016 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All genome sequence files are available from the NCBI database (BioProject no. PRJNA235371).
Funding: The BCCM/LMG collection is supported by the Federal Public Planning Service—Science Policy, Belgium (contract no. C4/00/BCCM). The authors acknowledge the financial support of the Research Foundation-Flanders (FWO-Vlaanderen; http://www.fwo.be; grant no. G.0.910.10.N.10), the Research Council of Ghent University and the Vrije Universiteit Brussel (http://www.vub.ac.be/en/research; SRP7, IRP2 and IOF342 projects) and the Hercules Foundation (grant number UABR09004). L.L. acknowledges the Chinese Scholarship Council (http://www.csc.edu.cn; file no. 2011533075) and Ghent University Co-Funding (http://www.ugent.be/nl/onderzoek/financiering/; grant no. 01SC1111).
Competing interests: The authors have declared that no competing interests exist.
Acetic acid bacteria (AAB) are best known for their production of acetic acid from ethanol during vinegar and cocoa bean fermentation [1–3]. Some AAB are also of interest to the industry because of their capacity to produce cellulose or other chemicals, such as L-sorbose involved in the synthesis of vitamin C . Furthermore, AAB occur as plant growth-promoting bacteria , insect endosymbionts , and as spoilers of many kinds of beverages such as wine and beer . AAB are classified in the family Acetobacteraceae within the Alphaproteobacteria. Recent studies of the symbiotic relationship between AAB and several insect hosts have revealed that this symbiosis relies on sugar-based diets such as nectar, fruit sugar, or phloem sap [6, 8]. During a study of the bumble bee and honey bee gut microbiota, an Acetobacteraceae operational taxonomic unit, referred to as Alpha-2.2, was repeatedly detected in the digestive tract of honey bees (Apis spp.) and bumble bees (Bombus spp.) [9–13]. These bacteria were categorized as one of the core bacteria in Bombus bimaculatus  and its presence in wild bumble bees (Bombus) was positively associated with Crithidia infection . The Alpha-2.2 strain LMG 28161T was recently isolated from the crop of a red-tailed bumble bee, Bombus lapidarius; it showed 97–99% pairwise 16S rRNA gene sequence identity to Alpha-2.2 sequences and was formally classified into a novel genus as Bombella intestini . Another Alpha-2.2 isolate, A29, was recently described as “Parasaccharibacter apium” and showed 98.9% 16S rRNA sequence similarity with B. intestini LMG 28161T; it was proven to be helpful in improving honey bee resistance to Nosema infections . B. intestini LMG 28161T shows distinctive phenotypic features from other AAB, such as Gluconobacter and Acetobacter . In the present study, the genomic characteristics of B. intestini LMG 28161T were examined through a whole-genome sequencing approach and its capability to oxidize the main components of nectar and honey, i.e. sucrose, glucose and fructose  and D-mannitol, a six-carbon sugar alcohol that is widely distributed in plants, were explored  through cultivation experiments under both aerobic and micro-aerobic conditions. Genomes of three insect associated AAB strains, i. e. Asaia platycodi SF2.1, Commensalibacter intestini A911, Saccharibacter sp. AM169 and of its nearest phylogenetic neighbor, Saccharibacter floricola DSM 15669T (an organism isolated from the pollen of Japanese flowers) were used in an ortholog analysis to explore the genetic symbiotic traits in B. intestini.
Results and Discussion
General genome features
The genome sequencing of B. intestini strain LMG 28161T, an endosymbiotic acetic acid bacterium occurring in bumble bees, yielded more than 6 million reads of 2 x 100 bp with a genome coverage of 299.0 x. All reads were assembled into 12 contigs of 1,402 to 670,914 nucleotides. Automated gene prediction and annotation of the assembled genome sequences resulted after manual curation in a draft genome of 2.02 Mb with an average G + C content of 54.9%. The latter value was identical to the DNA G+C content determined through an enzymatic degradation method  and separation of the nucleoside mixture through high-performance liquid chromatography . The general genome features are summarized in Table 1. The genome size of B. intestini is most similar to that of Saccharibacter sp. AM169 (1.9 Mb) which was isolated from the honey bee Apis mellifera ; however, it is smaller than that of most other AAB that have been determined (2.7–3.9 Mb) [2, 18–20]. The reduced genome size may be indicative for gene loss, which can cause reduced functional capabilities, a typical feature of niche-specific microorganisms, such as bacterial endosymbionts and may suggest that B. intestini has adapted to the bumble bee digestive tract [13, 21–22].
No plasmids were found during the assembly. The final annotation resulted in 1574 coding sequences (CDSs) and 54 RNA sequences, including three rRNA genes (5S, 16S and 23S), 50 tRNA genes, and one non-coding RNA. Three clustered regularly interspaced short palindromic repeats (CRISPRs) were found on contig 1, contig 2, and contig 5. CRISPR-associated CDSs were found on contig 1 (AL01_08840, AL01_08855) and contig 5 (AL01_03255, AL01_03260, AL01_03265). The three CRISPR arrays are 233 bp, 1736 bp and 1736 bp in length, with 3, 28 and 28 spacers, respectively. The draft genome was submitted to NCBI under the BioProject PRJNA235371.
Metabolic pathways of carbohydrates
Based on the annotated draft genome, central metabolic pathways including the Embden-Meyerhof-Parnas (EMP) pathway, the pentose-phosphate pathway (PPP), the pyruvate pathway, and the tricarboxylic acid (TCA) cycle were reconstructed (Fig 1).
Dashed arrows represent missing steps in the pathways due to the absence of corresponding genes. Genes encoding enzymes that catalyze the reaction between pyruvate and methylglyoxal in pyruvate metabolism and those that catalyze the conversion of succinyl-CoA to succinate, succinate to fumarate and malate to oxaloacetate in the TCA cycle were not found in the draft genome. 1, glucokinase (AL01_01675); 2, glucose-6-phosphate isomerase (AL01_06115); 3, aldose epimerase (AL01_04275); 4, fructose 1, 6-bisphosphatase (AL01_08695); 5, fructose-bisphosphate aldolase (AL01_06890); 6, glyceraldehyde-3-phosphate dehydrogenase (AL01_03750); 7, phosphoglycerate kinase (AL01_03755); 8, phosphoglycerate mutase (AL01_07435, AL01_07655); 9, enolase (AL01_00860); 10, pyruvate kinase (AL01_00625); 11, malate dehydrogenase (AL01_05845); 12, pyruvate dehydrogenase (AL01_00915, AL01_00920, AL01_03860); 13, pyruvate dehydrogenase E2 (AL01_00925); 14, dihydrolipoamide dehydrogenase (AL01_00930); 15, lactate dehydrogenase (AL01_06935); 16, hydroxyacylglutathione hydrolase (AL01_04950); 17, lactoylgluthathione lyase (AL01_00090); 18, pyruvate decarboxylase (AL01_08375); 19, alcohol dehydrogenase (AL01_01980, AL01_07015); 20, citrate synthase (AL01_06255); 21, aconitate hydratase 1 (AL01_06260); 22, NADP+-dependent isocitrate dehydrogenase (AL01_06250); 23, 2-oxoglutarate dehydrogenase E1 (AL01_08340); 24, dihydrolipoyllysine succinyltransferase (AL01_07740); 25, dihydrolipoamide dehydrogenase (AL01_00930); 26, fumarate hydratase (AL01_05840); 27, PQQ-dependent glucose dehydrogenase (AL01_09305); 28, gluconolactonase (AL01_06230); 29, lactate dehydrogenase (AL01_06935); 30, phosphogluconate dehydrogenase (AL01_06120); 31, transketolase (AL01_06110); 32, transaldolase (AL01_06115); 33, gluconate 2-dehydrogenase (AL01_07015); 34, 6-phosphogluconate dehydrogenase (AL01_06120); 35, ribose-5-phosphate isomerase (AL01_06135); 36, glucose-6-phosphate dehydrogenase (AL01_02790); 37, 6-phosphogluconolactonase (AL01_06130); 38, ribulose-phosphate 3-epimerase (AL01_09060); 39, polyol:NADP oxidoreductase (AL01_07080); 40, phosphomannomutase (AL_0102400); 41, mannose-1-phosphate guanyltransferase (AL01_07360); 42, carbohydrate kinase (AL01_03675); 43, mannose06-phosphate isomerase (AL01_00140); 44, 2-dehydro-3-deoxyphosphogluconate aldolase (AL01_04330).
All genes encoding the enzymes of the EMP pathway were identified, except for the phosphofructokinase-coding gene, suggesting incomplete glycolysis. The absence of this gene in AAB has been reported before for Gluconobacter oxydans 621H, Acetobacter pasteurianus IFO 3283, and Gluconacetobacter diazotrophicus Pal5T [2, 18–20].
All genes encoding the enzymes of the PPP were identified, enabling degradation of hexoses such as glucose and fructose via this pathway. Uptake of hexoses appeared possible through a sugar transporter (AL01_05795 and AL01_06590) or a D-galactose transporter encoded by galP (AL01_03445, AL01_03450 and AL01_02185), which both belong to the major facilitator superfamily (MFS) [19, 23]. Phenotypic tests, using a method described previously , revealed that B. intestini LMG 28161T was capable to produce acid from several carbohydrates, including sucrose, D-glucose, D-fructose, D-galactose, D-mannitol, and D-mannose . A polyol oxidoreductase (AL01_07080) enabling the conversion of D-mannitol into D-fructose was also found, as well as genes encoding enzymes that catalyze D-mannose utilization (Fig 1). This supported the previous observation that B. intestini LMG LMG 28161T is able to produce acid from D-mannitol and D-mannose .
D-gluconate could be oxidized into 2-keto-D-gluconate by a membrane-bound gluconate 2-dehydrogenase (AL01_07015) (Fig 2A). A gene encoding gluconate-5-dehydrogenase was not found. This was in accordance with the previous observation that this microorganism can produce 2-keto-D-gluconate but not 5-keto-D-gluconate . A general alcohol dehydrogenase-coding gene (AL01_01980) was found, but no gene encoding an enzyme for the oxidation of acetaldehyde into acetate. This supported the phenotypic inability of this strain to produce acetate from ethanol . Furthermore, a gene encoding glycerol kinase was not detected, suggesting that glycerol could not be transferred into the cell and further utilized. This explained why this microorganism can not grow on or produce acid from glycerol .
A) Membrane bound dehydrogenase and B) respiratory chain of Bombella intestini LMG 28161T. a, membrane-bound glucose dehydrogenase (AL01_09305); b, gluconate 2-dehydrogenase (AL01_07015); c, membrane-bound lactate dehydrogenase (AL01_06935); d, electron transfer flavoprotein-ubiquinone oxidoreductase (AL01_08300); e, type II NADH dehydrogenase (AL01_05990); f, cytochrome bo3 ubiquinol oxidase (AL01_00470, AL01_00475, AL01_00480 and AL01_00485); g, ubiquinol-cytochrome c reductase (bc1 complex) (AL01_05885, AL01_08145, AL01_08150); h, Cyt C, cytochrome c (AL01_05875).
B. intestini LMG 28161T appeared to possess an incomplete TCA cycle. Genes coding for enzymes converting succinyl-CoA into succinate, succinate into fumarate, and malate into oxaloacetate were not identified. However, L-asparagine permease (AL01_09015) and L-aspartate oxidase (AL01_04960) were identified. The former could enable the microorganism to take up L-asparagine from the environment, which could then be hydrolyzed to L-aspartate. L-aspartate oxidase is a flavoprotein that acts on the CH-NH2 group of donors with oxygen as electron acceptor . Oxygen can be replaced by fumarate as electron acceptor, yielding succinate . The ability of the enzyme to use both oxygen and fumarate in cofactor re-oxidation enables it to function under both aerobic and anaerobic conditions . L-aspartate could be converted by aspartate aminotransferase (AL01_03035) into oxaloacetate to join the TCA cycle. As for fumarate, it could also be derived from L-aspartate via two different two-step reactions with adenylosuccinate or L-argininosuccinate as intermediates, catalyzed by adenylosuccinate synthetase (AL01_06765), adenylosuccinate lyase (AL01_00960), argininosuccinate synthase (AL01_09265) and argininosuccinate lyase (AL01_02240). Although the three above-mentioned substrates of the TCA cycle could be generated by other reactions, the amount of energy generated through the TCA cycle may be rather limited, as in a complete TCA cycle the three enzymatic reactions catalyzed by these three missing enzymes are accompanied by the generation of GTP, FADH2 or NADH .
Membrane-bound dehydrogenases and respiratory chain
Compared to other genome-sequenced AAB strains, B. intestini LMG 28161T did not possess many membrane-bound dehydrogenases, as only three were found (Fig 2A), namely a cofactor pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (AL01_09305) allowing the conversion of glucose into gluconate, a flavine adenine dinucleotide (FAD)-dependent gluconate 2-dehydrogenase (AL01_07015) allowing the conversion of gluconate into 2-keto-gluconate, and a FAD-dependent D-lactate oxidase (AL01_06935) allowing the conversion of D-lactate into pyruvate. A pqqBCDE operon (AL01_07315, AL01_07320, AL01_07325 and AL01_07330) encoding proteins for the biosynthesis of the cofactor PQQ was found. In the genomes of G. oxydans 621H and A. pasteurianus 386B, a pqqABCDE operon responsible for PPQ biosynthesis is present . It has been shown previously that a pqqA mutant of G. oxydans 621H is unable to grow on D-mannitol, D-glucose, or glycerol as the sole energy source . Bombella intestini LMG 28161T could grow on D-glucose and D-mannitol as the sole energy source (see below), which indicates that pqqA was not vital for this strain.
Genes encoding ubiquinol-cytochrome c reductase (bc1 complex) (AL01_05885, AL01_08145, AL01_08150) and cytochrome c (AL01_05875) were found in the genome, but not cytochrome c oxidase-encoding genes. Genes encoding a type II NADH dehydrogenase (AL01_05990) and a NAD(P)H:ubiquinone oxidoreductase (AL01_07780) were present in the genome. Both these enzymes catalyze electron transfer from NADH to ubiquinone . A flavoprotein-ubiquinone oxidoreductase (AL01_08300) could catalyze electron transfer from flavoprotein to ubiquinone. The reduced product, ubiquinol, could diffuse within the membrane and be re-oxidized by cytochrome bo3 ubiquinole oxidase (AL01_00470, AL01_00475, AL01_00480 and AL01_00485) (Fig 2B). A previous study has suggested that AAB acquired ubiquinol oxidase from β/γ-Proteobacteria via horizontal gene transfer and created afterwards a truncated respiratory chain in which electron transfer to oxygen occurs via ubiquinol oxidase directly, accepting electrons from ubiquinol . The truncated respiratory chain would generate less energy, but allow rapid oxidations, which would be beneficial for AAB . Cytochrome bo3 oxidase has been detected in other AAB genomes and shows a high affinity for oxygen, possibly allowing their survival in environments with low oxygen availability, such as the insect gut .
Amino acid metabolism and nitrogen metabolism
Pathways for all proteinogenic amino acids except alanine were present. L-alanine could not be converted from L-aspartate due to the absence of aspartate 4-decarboxylase and might be taken from the environment via an amino acid transporter or permease. Glutamate can be converted to ornithine and enter the urea cycle, where ornithine as well as aspartate are eventually converted to fumarate and enter the citrate cycle. Nitrogen fixation pathways were absent; ammonia can be incorporated into glycine by glycine synthase or to cyclic amidines by NAD synthase.
Bumble bee endosymbionts-related features
An ortholog analysis of B. intestini LMG 28161T, A. platycodi SF2.1, C. intestini A911, Saccharibacter sp. AM169 and S. floricola DSM 15669T carried out in OrthoMCL resulted in 1397 ortholog groups, including 894 core orthologs. In total 69 orthologs were shared among the four reference genomes but not present in B. intestini LMG 28161T, including cytochrome d ubiquinol oxidase subunit I, II, 39 functional genes and 28 hypothetical genes (S1 Table). Compared to the above mentioned reference strains, B. intestini LMG 28161T possessed 86 unique genes, of which 63 were hypothetical protein coding sequences and 23 were functional genes (S2 Table). Among the 23 functional genes, genes encoding for five type IV secretion system proteins, three amino acid transporter/permeases and three membrane proteins were unique to B. intestini. ABC transporters and type IV secretion systems have been reported to be involved in the cross talk between endosymbionts and their insect hosts [17, 21, 27–28]. Multiple CDSs associated to ABC transporters were identified (Table 2). A signal recognition particle (SRP) complex (AL01_03075 and AL01_06750), which recognizes and targets specific proteins on the plasma membrane, was also present. The restriction modification system to degrade foreign DNA, which has been found in the genomes of two additional Bombus endosymbionts, Gilliamella apicola and Snodgrasella alvi , was not detected in the genome of B. intestini LMG 28161T; yet, CRISPR elements were present and may be used in the defense against bacteriophages.
During a monoculture fermentation experiment at 10-L scale, B. intestini LMG 28161T oxidized D-glucose from the beginning of the fermentation, with a rapid drop after 12 h until D-glucose was completely depleted after 36 h (Fig 3). The biomass formation during fermentation was limited [from 4.5 to 5.9 log (CFU/mL)], which was probably due to the nutrient-limited composition of the medium used. The pH value of the medium decreased from 7.0 to 3.5 during fermentation, which was in accordance with the acid production from D-glucose .
Glucose was consumed rapidly after 12h and was completely used after 36h. Growth of cells stabilized after some 27h.
Bumble bees feed on nectar, which comprises sucrose, fructose, glucose, and in some plants also D-mannitol  as the main components; glucose and mannitol are also often used as main carbon source in AAB growth media [1, 14]. Therefore, these carbohydrates may serve as carbon source for B. intestini during its endosymbiotic lifestyle. As the digestive tract of bees is a micro-aerobic environment, growth on sucrose, D-glucose, D-fructose, and D-mannitol was checked under both aerobic and micro-aerobic conditions in 50-mL glass bottle experiments. Bombella intestini LMG 28161T was capable to utilize these four carbohydrates; under both aerobic and micro-aerobic conditions, D-glucose was consumed the most, followed by sucrose, however, sucrose and D-glucose were more consumed under aerobic than under micro-aerobic conditions, whereas D-fructose was utilized more under micro-aerobic conditions; for mannitol, no difference was seen (Table 3). In addition, 2-keto-D-gluconic acid was produced under both incubation conditions when sucrose and D-glucose were used as the sole carbon source. Gluconic acid and acetic acid were not found. The cultivation experiments therefore confirmed that B. intestini LMG 28161T was able to produce 2-keto-D-gluconic acid through oxidation of glucose (and sucrose) under both aerobic and micro-aerobic conditions.
Means are tested for difference by Least Significant Difference (LSD) test. Means indicated by the same letter in a column do not differ (P = 0.05) according to LSD test.
During the 10-L fermentation experiment (aerobic conditions), D-glucose was depleted completely after 36 h of incubation, whereas during the 50-mL glass bottle experiments (aerobic conditions) none of the four carbohydrates investigated was depleted. In the latter case, growth mainly occurred at the surface. The difference in aeration might explain the difference in carbohydrate depletion during the two test systems. The lack of effective aeration in small-scale carbohydrate consumption experiments may explain the lack of reproducibility of such results often reported in taxonomic studies of AAB [14, 30, 31].
B. intestini LMG 28161T, an endosymbiotic acetic acid bacterium occurring in bumble bees, carries a small genome of 2.02 Mb. The reconstructed metabolic pathways were congruent with the results of the fermentation experiments and with phenotypic features determined previously . Compared to four other AAB genomes analyzed, the genome of B. intestini lost 69 orthologs that were shared among the other 4 strains. Simultaneously it included 89 unique genes, many of which were hypothetical. The unique genes in B. intestini included genes encoding for several type IV secretion system proteins, amino acid transporter/permeases and membrane proteins, which might play a role in the interaction with the bumble bee host. B. intestini LMG 28161T was capable to oxidize sucrose, D-glucose, D-fructose, and D-mannitol, which are all present in nectar or honey, while it was incapable of oxidizing ethanol or glycerol, which are not available in the bumble bee gut. In addition, B. intestini showed a different preference in carbohydrates consumption under aerobic and micro-aerobic conditions, where sucrose and D-glucose were more preferred under aerobic condition, while D-fructose was utilized more under micro-aerobic conditions.
Materials and Methods
Strain cultivation, DNA extraction, genome sequencing, assembly, and annotation
Bombella intestini strain LMG 28161T was cultivated in LMG medium 404 [5%, w/v, D-glucose; 1%, w/v, yeast extract (Oxoid) and 1.5%, w/v, agar] for DNA extraction at large scale, using the method of Wilson as modified previously . The integrity of the DNA was evaluated on a 1.0% (w/v) agarose gel and the purity was checked by spectrophotometric measurements at 234, 260 and 280 nm. The DNA concentration was estimated with a Quantus™ fluorometer using a QuantiFluor®ONE ds DNA system kit (Promega Corporation, Madison, WI, USA). Library preparation and genome sequencing were performed by BaseClear BV (Leiden, The Netherlands). Paired-end sequence reads were generated using the Illumina HiSeq2500 system (Illumina Inc., San Diego, CA, USA). The initial de novo assembly of the raw reads into contigs was performed using the CLCgenomic workbench v6.5.1 (CLC Inc, Aarhus, Denmark).
Automated gene prediction and annotation of the assembled genome sequences were performed with GenDB v2.2 , the Rapid Annotations using Subsystems Technology (RAST) server , and the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP; http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html). The PGAAP gene predictions and annotations were used as basis for the final annotation. They were manually curated for the coding sequences (CDSs) of interest using BLASTp (http://blast.ncbi.nlm.nih.gov/blast) and UniProt (http://www.uniprot.org), taking also into account the information from RAST and GenDB. Metabolic pathways were manually reconstructed using the information from the final annotation. The KEGG database  aided in the reconstruction of the pathways. CRISPRs were searched for using CRISPR Finder  and considered if they were classified as ‘confirmed’.
Four genomes of AAB were selected for the ortholog analysis, including three insect associated AAB strains, Asaia platycodi SF2.1, Commensalibacter intestini A911, Saccharibacter sp. AM169 and the nearest phylogenetic neighbor of B. intestini, Saccharibacter floricola DSM 15669T (an organism isolated from the pollen of Japanese flowers) (Table 4). The analysis was carried out using OrthoMCL .
Carbohydrate consumption experiments
To determine the growth rate and glucose consumption rate of strain LMG 28161T, one 10-L monoculture fermentation experiment was carried out in basal medium [yeast extract 0.5%, w/v, ] supplemented with 1% (w/v) D-glucose. The fermentation was performed in a 15-L BiostatC fermentor (Sartorius AG, Melsungen, Germany) at 28°C, free pH, and 300 rpm for 36 h. Aerobic conditions during the fermentation were ensured by continuously sparging the medium with 5 liters min-1 of air. The inoculum for the fermentation experiment was prepared as follows. Strain LMG 28161T was cultivated in 100 mL of LMG medium 404 [5%, w/v, D-glucose and 1%, w/v, yeast extract (Oxoid)] and subsequently propagated twice in 400 mL of basal medium supplemented with 1% (w/v) D-glucose. During the inoculum buildup, the transferred volume was always 5% (v/v). Incubation was done at 28°C for 48 h on a rotary shaker. The inoculum was added to the fermentation vessel aseptically. During the fermentation experiment, the pH was monitored automatically. Samples were withdrawn at regular time intervals for offline analysis.
To verify metabolic pathways, the oxidation of the carbohydrates sucrose, D-glucose, D-fructose, and D-mannitol was verified under aerobic and micro-aerobic conditions (80% N2, 4% O2, 8% H2, and 8% CO2). The experiments were conducted in triplicate, using the same method as described previously for acid production from different carbon sources , in 50-mL glass bottles filled with 20 mL of basal medium supplemented with 1% (w/v) of the carbon source, but without bromocresol purple added to the medium. The bottles were incubated at 28°C for 7 days on a rotary shaker (aerobic conditions), and in a jar for the micro-aerobic conditions experiments. A medium sample was collected before inoculation and after 7 days of incubation with the culture. The samples were centrifuged and the supernatants were stored at -20C until further analyses were carried out.
Analysis of bacterial growth, carbohydrate consumption and metabolite production
Growth of LMG 28161T during fermentation [expressed in log (CFU/mL)] was quantified through plating of 10-fold serial dilutions of the samples in physiological solution [0.85% (w/v) NaCl] onto LMG medium M404. Determination of glucose consumption rate was calculated based on the time of glucose depletion, by measuring glucose concentration every three hours as described previously . Determination of carbohydrate, acetic acid, D-gluconic acid, and 2-keto-D-gluconic acid concentrations in the samples taken from the 50-mL bottles, was done using a Focus gas chromatograph (Interscience, Breda, The Netherlands) as described previously . Statistical analysis of carbohydrates consumption was carried out using SPSS version 15.0 (SPSS, Inc., Chicago, IL, USA).
S1 Table. Group of core orthologs only found among Asaia platycodi SF2.1, Commensalibacter intestini A911, Saccharibacter sp. AM169 and Saccharibacter floricola DSM 15669T but not in Bombella intestini LMG 28161T.
Abbrevation “com, sfl, sac” and “asp” before “|” represent Commensakibacter intestini A911, Saccharibacter floricola DSM 15669T, Saccharibacter sp. AM169 and Asaia platycodi SF2.1, respectively. The number after “|” refers to sequence locus in each of the genome.
- Conceptualization: LL LDV PV.
- Formal analysis: LL KI SVK WB.
- Funding acquisition: LL GS LDV PV.
- Investigation: LL KI SVK WB.
- Methodology: LL KI SVK WB.
- Project administration: LL PV.
- Resources: GS LDV PV.
- Software: LL KI.
- Supervision: IC PV.
- Visualization: LL KI.
- Writing – original draft: LL KI.
- Writing – review & editing: IC GS LDV PV.
- 1. Cleenwerck I, González A, Camu N, Engelbeen K, De Vos P, De Vuyst L. Acetobacter fabarum sp. nov., an acetic acid bacterium from a Ghanaian cocoa bean heap fermentation. Int J Syst Evol Microbiol. 2008; 58:2180–2185. pmid:18768626
- 2. Illeghems K, De Vuyst L, Weckx S. Complete genome sequence and comparative analysis of Acetobacter pasteurianus 386B, a strain well-adapted to the cocoa bean fermentation ecosystem. BMC Genomics. 2013; 14:526. pmid:23902333
- 3. Gullo M, Giudici P. Acetic acid bacteria in traditional balsamic vinegar: phenotypic traits relevant for starter cultures selection. Int J Food Microbiol. 2008; 125:46–53. pmid:18177968
- 4. Raspor P, Goranovič D. Biotechnological applications of acetic acid bacteria. Crit Rev Biotechnol. 2008; 28:101–124. pmid:18568850
- 5. Fuentes-Ramirez LE, Bustillos-Cristales R, Tapia-Hernandez A, Jimenez-Salgado T, Wang ET, Martinez-Romero E, et al. Novel nitrogen-fixing acetic acid bacteria, Gluconacetobacter johannae sp. nov. and Gluconacetobacter azotocaptans sp. nov, associated with coffee plants. Int J Syst Evol Microbiol. 2001; 51:1305–1314. pmid:11491326
- 6. Crotti E, Rizzi A, Chouaia B, Ricci I, Favia G, Alma A, et al. Acetic acid bacteria, newly emerging symbionts of insects. Appl Environ Microbiol. 2010; 76:6963–6970. pmid:20851977
- 7. Wieme AD, Spitaels F, Aerts M, De Bruyne K, Van Landschoot A, Vandamme P. Identification of beer-spoilage bacteria using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Int J Food Microbiol. 2014; 185:41–50. pmid:24929682
- 8. Crotti E, Damiani C, Pajoro M, Gonella E, Rizzi A, Ricci I, et al. Asaia, a versatile acetic acid bacterial symbiont, capable of cross-colonizing insects of phylogenetically distant genera and orders. Environ Microbiol. 2009; 11:3252–3264. pmid:19735280
- 9. Cariveau DP, Powell JE, Koch H, Winfree R, Moran NA. Variation in gut microbial communities and its association with pathogen infection in wild bumble bees (Bombus). ISME J. 2014; 1–11.
- 10. Mohr KI, Tebbe CC. Diversity and phylotype consistency of bacteria in the guts of three bee species (Apoidea) at an oilseed rape field. Environ Microbiol. 2006; 8:258–272. pmid:16423014
- 11. Martinson VG, Danforth BN, Minckley RL, Rueppell O, Tingek S, Moran NA. A simple and distinctive microbiota associated with honey bees and bumble bees. Mol Ecol. 2011; 20:619–628. pmid:21175905
- 12. Cox-Foster DL, Conlan S, Holmes EC, Palacios G, Evans JD, Moran NA, et al. A metagenomic survey of microbes in honey bee colony collapse disorder. Science 2007; 318:283–287. pmid:17823314
- 13. Corby-Harris V, Snyder LA, Schwan MR, Maes P, McFrederick QS, Anderson KE. Origin and effect of Alpha 2.2 Acetobacteraceae in honey bee larvae and description of Parasaccharibacter apium gen. nov., sp. nov. Appl. Environ. Microbiol. 2014; 80:7460–7472. pmid:25239902
- 14. Li L, Praet J, Borremans W, Nunes OC, Manaia CM, Cleenwerck I, et al. Bombella intestini gen. nov., sp. nov., an acetic acid bacterium isolated from bumble bee crop. Int J Syst Evol Microbiol. 2015; 65:267–273. pmid:25336723
- 15. Stoop J, Williamson J, Masonpharr D. Mannitol metabolism in plants: a method for coping with stress. Trends Plant Sci. 1996; 1:139–144.
- 16. Mesbah M, Premachandran U, Whitman WB. Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Evol Microbiol. 1989; 39:159–167.
- 17. Chouaia B, Gaiarsa S, Crotti E, Comandatore F, Degli Esposti M, Ricci I, et al. Acetic acid bacteria genomes reveal functional traits for adaptation to life in insect guts. Genome Biol Evol. 2014; 6:912–920. pmid:24682158
- 18. Azuma Y, Hosoyama A, Matsutani M, Furuya N, Horikawa H, Harada T, et al. Whole-genome analyses reveal genetic instability of Acetobacter pasteurianus. Nucleic Acids Res. 2009; 37:5768–5783. pmid:19638423
- 19. Prust C, Hoffmeister M, Liesegang H, Wiezer A, Fricke WF, Ehrenreich A, et al. Complete genome sequence of the acetic acid bacterium Gluconobacter oxydans. Nat Biotechnol. 2005; 23:195–200. pmid:15665824
- 20. Bertalan M, Albano R, de Pádua V, Rouws L, Rojas C, Hemerly A, et al. Complete genome sequence of the sugarcane nitrogen-fixing endophyte Gluconacetobacter diazotrophicus Pal5. BMC Genomics. 2009; 10:450. pmid:19775431
- 21. Kwong WK, Engel P, Koch H, Moran NA. Genomics and host specialization of honey bee and bumble bee gut symbionts. Proc Natl Acad Sci USA. 2014; 111:11509–11514. pmid:25053814
- 22. Nilsson AI, Koskiniemi S, Eriksson S, Kugelberg E, Hinton J, Andersson DI. Bacterial genome size reduction by experimental evolution. Proc Natl Acad Sci USA. 2005; 102:12112–12116. pmid:16099836
- 23. Bossi RT, Negri A, Tedeschi G, Mattevi A. Structure of FAD-bound L-aspartate oxidase: insight into substrate specificity and catalysis. Biochemistry-US. 2002; 41:3018–3024.
- 24. Gosselé F, Swings J, Kersters K, Ley JD. Numerical analysis of phenotypic features and protein gel electropherograms of Gluconobacter Asai 1935 emend. mut. char. Asai, Iizuka, and Komagata 1964. Int J Syst Bacteriol. 1983; 33:65–81.
- 25. Hölscher T, Görisch H. Knockout and overexpression of pyrroloquinoline quinone biosynthetic genes in Gluconobacter oxydans 621H. J Bacteriol. 2006; 188:7668–7676. pmid:16936032
- 26. Matsutani M, Fukushima K, Kayama C, Arimitsu M, Hirakawa H, Toyama H, et al. Replacement of a terminal cytochrome c oxidase by ubiquinol oxidase during the evolution of acetic acid bacteria. Biochim Biophys Acta. 2014; 1837:1810–1820. pmid:24862920
- 27. Low HH, Gubellini F, Rivera-Calzada A, Braun N, Connery S, Dujeancourt A, et al. Structure of a type IV secretion system. Nature. 2014; 508:550–553. pmid:24670658
- 28. Backert S, Tegtmeyer N, Fischer W. Composition, structure and function of the Helicobacter pylori cag pathogenicity island encoded type IV secretion system. Future Microbiol. 2015; 10:955–965. pmid:26059619
- 29. Lohaus G, Schwerdtfeger M. Comparison of sugars, iridoid glycosides and amino acids in nectar and phloem sap of Maurandya barclayana, Lophospermum erubescens, and Brassica napus. PLoS ONE. 2014; 9:e87689. pmid:24489951
- 30. Cleenwerck I, De Vos P. Polyphasic taxonomy of acetic acid bacteria: an overview of the currently applied methodology. Int J Food Microbiol. 2008; 125:2–14. pmid:18237808
- 31. Spitaels F, Wieme A, Balzarini T, Cleenwerck I, Van Landschoot A, De Vuyst L, et al. Gluconobacter cerevisiae sp. nov., isolated from the brewery environment. Int J Syst Evol Microbiol. 2014; 64:1134–1141. pmid:24368694
- 32. Cleenwerck I, Vandemeulebroecke K, Janssens D, Swings J. Re-examination of the genus Acetobacter, with descriptions of Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov. Int J Syst Evol Microbiol. 2002; 52:1551–1558. pmid:12361257
- 33. Meyer F. GenDB—an open source genome annotation system for prokaryote genomes. Nucleic Acids Res. 2003; 31:2187–2195. pmid:12682369
- 34. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, et al. The RAST Server: rapid annotations using subsystems technology. BMC Genomics. 2008; 9:75. pmid:18261238
- 35. Kanehisa M, Goto S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000; 28:27–30. pmid:10592173
- 36. Grissa I, Vergnaud G, Pourcel C. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 2007; 35(Web Server): W52–W57. pmid:17537822
- 37. Li L, Stoeckert CJ Jr, Roos DS. OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res. 2003, 13:2178–2189. pmid:12952885
- 38. Moens F, Lefeber T, De Vuyst L. Oxidation of metabolites highlights the microbial interactions and role of Acetobacter pasteurianus during cocoa bean fermentation. Appl Environ Microbiol. 2014; 80:1848–1857. pmid:24413595