Bombella intestini gen. nov., sp. nov., a novel acetic acid bacterium isolated from bumble bee crop

Leilei Li1, Jessy Praet1, Wim Borremans3, Olga C. Nunes4, Célia M. Manaia5, Ilse Cleenwerck2, Ivan Meeus6, Guy Smagghe6, Luc De Vuyst3, Peter Vandamme1

1Laboratory of Microbiology, Faculty of Sciences, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium

2BCCM/LMG Bacteria Collection, Faculty of Sciences, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium

3Research Group of Industrial Microbiology and Food Biotechnology, Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium

4LEPAE – Departamento de Engenharia Química, Faculdade de Engenharia, Universidade do Porto, 4200-465 Porto, Portugal

5CBQF – Escola Superior de Biotecnologia, Universidade Católica Portuguesa, 4200-072 Porto, Portugal

6Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Belgium

Corresponding author: Prof. Dr. Peter Vandamme

Peter.Vandamme@ugent.be

Tel: + 32 9 264 51 13

Fax: + 32 9 264 50 92

Keywords: Acetobacteraceae classification, MALDI-TOF MS

Running title: Bombella intestini gen. nov. sp. nov.

The Contents Category: New Taxa, subsection Proteobacteria

IJSEM Papers in Press. Published October 21, 2014 as doi:10.1099/ijs.0.068049-0

The NCBI accession numbers for the 16S rRNA, 16S-23S rRNA ITS, groEL gene sequences of Bombella intestini LMG 28161T, groEL gene sequences of Neokomagataea tanensis LMG 27020T and Neokomagataea thailandica LMG 27021T, are KJ584313, KM377627, KJ584315, KM504940 and KM504941, respectively.

Abstract 1

In the frame of a bumble bee gut microbiota study, acetic acid bacteria (AAB) were isolated 2

using a combination of direct isolation methods and enrichment procedures. MALDI-TOF MS 3

profiling of the isolates and a comparison of these profiles with profiles of established AAB 4

species identified most isolates as Asaia astilbes or as “Commensalibacter intestini", except for 5

two isolates (R-52486 and LMG 28161T) that showed an identical profile. A nearly complete 16S 6

rRNA gene sequence of LMG 28161T was determined and showed the highest pairwise similarity 7

to Saccharibacter floricola (96.5 %), which corresponded with genus level divergence in the 8

Acetobacteraceae family. Isolate LMG 28161T was subjected to whole-genome shotgun 9

sequencing; a 16S-23S rRNA internal transcribed spacer (ITS) sequence as well as partial 10

sequences of the housekeeping genes dnaK, groEL and rpoB were extracted for phylogenetic 11

analyses. The data obtained confirmed that this isolate is best classified into a novel genus in the 12

family Acetobacteraceae. Its DNA G+C content was 54.9 mol%. The fatty acid compositions of 13

R-52486 and LMG 28161T were similar to those of established AAB species [with C18:1ω7c (43.3 14

%) as major component], but the amounts of fatty acids such as C19:0 cyclo ω8c, C14:0 and C14:0 2-15

OH enabled to differentiate them. The major ubiquinone was Q-10. Both isolates could also be 16

differentiated from the known genera of AAB by means of biochemical characteristics, such as 17

their lack of ability to oxidize ethanol to acetic acid, negligible acid production from melibiose, 18

and notable acid production from D-fructose, sucrose and D-mannitol. In addition, they produced 19

2-keto-D-gluconic acid, but not 5-keto-D-gluconic acid from D-glucose. Therefore, the name 20

Bombella intestini is proposed for this new taxon, with LMG 28161T (= DSM 28636T = R-21

52487T) as the type strain. 22

Recently, acetic acid bacteria (AAB) have been isolated from various sources, mainly sugar-rich 23

or alcoholic niches, such as vinegar, wine and flowers (De Vuyst et al., 2008; Kersters et al., 24

2006; Muthukumarasamy et al., 2005). Some AAB species have symbiotic relationships with 25

insects (Crotti et al., 2010). In particular, Acetobacter, Gluconacetobacter, Gluconobacter, Asaia 26

and Saccharibacter species have frequently been associated with honey bees (Apis mellifera) 27

(Babendreier et al., 2007; Crotti et al., 2009; Crotti et al., 2010; Gilliam, 1997; Martinson et al., 28

2011; Mohr & Tebbe, 2006), but only rarely with bumble bees (Bombus spp.) (Koch & Schmid-29

Hempel, 2011; Martinson et al., 2011; Olofsson & Vasquez, 2009). Yet, a recent study has 30

revealed two Acetobacteraceae as relevant bacteria of the Bombus bimaculatus gut microbiota 31

(Cariveau et al., 2014). 32

In the frame of a bumble bee gut microbiota study, bumble bees were caught in the field, in 33

Ghent, Belgium, frozen at -20 °C for 10 min and surface-sterilized with 2.5 % Umonium38® 34

Master (Laboratoire Huckert’s International, Brussels, Belgium) before dissecting their honey 35

crop and whole gut. The crops and guts were homogenized in 500 µl saline solution (Olofsson & 36

Vasquez, 2008) with a sterile micro-pestle and stored at -20 °C until further use. Isolation of 37

AAB from these suspensions was carried out using a combination of direct isolation methods and 38

enrichment procedures. For the latter, 50 µl of suspension was added to 25 ml of both enrichment 39

medium I (EM I) and II (EM II), as described by (Lisdiyanti et al., 2003), except that the pH was 40

not adjusted; the final pH was therefore 6.8 ± 0.2. The inoculated media were shaken at 100 rpm 41

and incubated at 28 °C for 7 days. Enrichment media showing bacterial growth were 42

subsequently plated onto four solid agar media, i.e. LMG medium 13 [2.5 %, w/v, D-mannitol; 43

0.5 %, w/v, yeast extract (Oxoid, Basingstoke, UK); 0.3 %, w/v, bacteriological peptone (Oxoid) 44

and 1.5 %, w/v, agar], LMG medium 404 [5 %, w/v, D-glucose; 1 %, w/v, yeast extract (Oxoid) 45

and 1.5 %, w/v, agar], LMG medium 405 [5 %, w/v, D-glucose; 1 %, w/v, yeast extract (Oxoid); 46

2 %, v/v, ethanol; 1 %, v/v, acetic acid and 1.5 %, w/v, agar] and modified deoxycholate-47

mannitol-sorbitol (mDMS) medium [1 %, w/v, bacteriological peptone (Oxoid); 0.3 %, w/v, yeast 48

extract (Oxoid); 0.63 %, w/v, lactic acid; 0.5 %, w/v, ethanol; 0.1 %, w/v, D-glucose; 0.1 %, w/v, 49

sorbitol; 0.1 %, w/v, mannitol; 0.1 %, w/v, potassium hydrogen phosphate; 0.01 %, w/v, sodium 50

deoxycholate; 0.002 %, w/v, magnesium sulphate heptahydrate; 0.003 %, w/v, bromocresol 51

purple and 1.8 %, w/v, agar; pH 4.5 (Papalexandratou et al., 2013)], each containing 100 ppm 52

cycloheximide to inhibit yeast growth. For direct isolation, the cell suspensions were serially 53

diluted to 10-7 in physiological water (0.85 %, w/v, NaCl), and 50 µl of each dilution was plated 54

directly onto the same four solid agar media as mentioned above. All inoculated plates were 55

incubated aerobically at 28 °C for 7 days. The LMG media 13 and 404 were the sole media that 56

yielded growth. Colonies were picked up from agar media that were inoculated both directly as 57

well as after enrichment in EM II. Isolates (Supplementary Table 1) were dereplicated by matrix-58

assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS), as 59

described by Wieme et al. (2012). Cluster analysis of the MALDI-TOF MS profiles of these 60

isolates revealed eight groups, of which two could be identified as Asaia astilbes and 61

“Commensalibacter intestini” after comparing their profiles with profiles of established AAB 62

species (Supplementary Fig. 1). The other six groups could not be identified, and representative 63

isolates were selected for 16S rRNA gene sequence analysis using the method described 64

previously (Snauwaert et al., 2013). NCBI blast analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) 65

of the 16S rRNA gene sequences obtained (1353–1431 nt) revealed that all isolates, except for 66

one, namely LMG 28161T (=R-52487T), were lactic acid bacteria (data not shown). The 16S 67

rRNA gene sequence of isolate LMG 28161T (1450 nt) showed 99 % similarity to several 68

uncultured Acetobacteraceae from the honey bee gut, crop or larvae (Anderson et al., 2013; Koch 69

et al., 2013; Martinson et al., 2011; Mohr & Tebbe, 2007), which indicated that this strain was a 70

member of the family Acetobacteraceae. Isolate LMG 28161T formed a single MALDI-TOF MS 71

cluster with isolate R-52486, which showed an identical mass spectrum (Supplementary Fig. 2) 72

and which was isolated from the same bumble bee crop sample. The 16S rRNA gene sequence of 73

LMG 28161T was then compared with 16S rRNA gene sequences of type strains of all AAB 74

species retrieved from the EMBL and NCBI databases, using the BioNumerics version 5.1 75

software (Applied Maths, Sint-Martens-Latem, Belgium). Isolate LMG 28161T showed the 76

highest pairwise 16S rRNA gene sequence similarity to Saccharibacter floricola (96.5 %; 77

accession number, AB110421). This rather low nearest neighbor similarity value suggested that 78

this isolate should be classified into a new genus of the Acetobacteraceae family. The 16S rRNA 79

gene sequence of LMG 28161T and of type strains of species of this family, representing all 80

known genera, were aligned against the SILVA bacteria database using the Mothur pipeline 81

(Quast et al., 2013; Schloss et al., 2009). Subsequently, phylogenetic trees were constructed with 82

MEGA6, using the neighbor-joining (NJ) and maximum-likelihood (ML) methods (Felsenstein, 83

1981; Saitou & Nei, 1987). The DNA substitution TN93+G+I was selected under the Bayesian 84

Information Criterion (Nei & Kumar, 2000; Tamura et al., 2013). The robustness of the topology 85

of both trees was estimated by bootstrap analysis with 1000 replicates (Felsenstein, 1985). Both 86

trees showed highly similar topologies, and therefore only the maximum likelihood tree is shown 87

(Fig. 1). 88

Genomic DNA of isolate LMG 28161T was obtained, as described previously (Cleenwerck et al., 89

2002), and subjected to whole-genome shotgun sequencing (Li et al., unpublished results). A 90

16S-23S rRNA internal transcribed spacer (ITS) sequence and sequences of the housekeeping 91

gene groEL was extracted for phylogenetic analyses, as these sequences were proven useful to 92

refine the phylogeny of members of the family Acetobacteraceae (Cleenwerck et al., 2010; Li et 93

al., 2014; Trcek & Teuber, 2002). A groEL sequence was also extracted from the draft genome of 94

S. floricola DSM 15669T, available online (accession number, NZ_ARJS00000000.1). A 95

phylogenetic analysis based on 16S-23S rRNA ITS sequences was performed using MEGA 6 96

software package; a phylogenetic tree was constructed using the neighbor-joining method. The 97

robustness of the tree topology was estimated by bootstrap analysis with 1000 replicates. The 98

16S-23S rRNA ITS analysis confirmed that isolate LMG 28161T could clearly be differentiated 99

from its close neighbors (Supplementary Fig. 3). The phylogenetic position of isolate LMG 100

28161T, based on partial sequences of the housekeeping gene groEL (720 nt, Supplementary Fig. 101

4), and based on the corresponding amino acid sequences (240 aa, Supplementary Fig. 5), was 102

also determined as described before (Li et al., 2014) using the MEGA 6 software. Again, isolate 103

LMG 28161T formed a very distinct branch and the branch lengths obtained in the various trees 104

further confirmed that isolate LMG 28161T should be best classified into a new genus of this 105

family. 106

Random amplified polymorphic DNA (RAPD) analysis was performed on LMG 28161T and R-107

52486, as described previously (Williams et al., 1990). Two primers (RAPD-270 and RAPD-108

272) were used, which both yielded indistinguishable profiles, suggesting that LMG 28161T and 109

R-52486 are re-isolates of the same strain (Supplementary Fig. S6). 110

The DNA G+C content of isolate LMG 28161T was determined as described previously 111

(Cleenwerck et al., 2008) and was 54.9 mol%. This value falls within the range reported for the 112

family Acetobacteraceae (Greenberg et al., 2006; Sievers & Swings, 2005), and is also consistent 113

with the whole-genome shotgun sequencing data, which revealed a value of 55.0 %. 114

The whole-cell fatty acid methyl esters (FAME) composition was determined for isolate LMG 115

28161T and for the type strains of the type species of nearly all genera of the family 116

Acetobacteraceae, using an Agilent Technologies 6890N gas chromatograph (Santa Clara, CA, 117

USA). Cultivation of the strains and extraction of the FAME were performed according to the 118

recommendations of the Microbial Identification System, Sherlock version 3.10 (MIDI, Hewlett 119

Packard, Newark, DE, USA), except that fatty acids were extracted from cultures grown on LMG 120

medium 404 at 28°C under aerobic conditions for 48 to 72 hours (depending on the strain). The 121

peaks of the profiles were identified using the TSBA50 identification library version 5.0 (MIDI). 122

The predominant fatty acid of strain LMG 28161T was C18:1ω7c (43.1%), while the following 123

fatty acids were present in lower percentages (above 1 %): C19:0 cyclo ω8c (16.7 %), C16:0 (12.7 124

%), C14:0 2-OH (8.5 %), C14:0 (9.3 %), C16:0 2-OH (1.1 %) and C16:0 3-OH (2.8 %) (Table 1). The 125

fatty acid composition of LMG 28161T was similar to that of known AAB species (with C18:1ω7c 126

as major component), but the amounts of fatty acids, such as C19:0 cyclo ω8c, C14:0 and C14:0 2-127

OH, enabled to differentiate it (Table 1, Supplementary Table S2). The analysis of respiratory 128

quinones of LMG 28161T was performed, as described previously (Vaz-Moreira et al., 2007) 129

using the method of Tindall (Tindall, 1989). The major ubiquinone present was Q-10, which 130

agreed with the major ubiquinone of most other genera in the family Acetobacteraceae, except 131

for Acetobacter species that have Q-9 as major ubiquinone; no other quinones were found. 132

Both isolates LMG 28161T and R-52486 were subjected to phenotypic tests, using methods 133

described previously (Cleenwerck et al., 2007; Cleenwerck et al., 2002). For microscopy and 134

colony morphology, isolates were grown aerobically at 28 °C on LMG medium 404 for 48 hours. 135

On this medium, both isolates also grew when micro-aerobic conditions (80 % N2, 4 % O2, 8 % 136

H2 and 8% CO2) were used. As acetic acid bacteria are well known as obligate aerobic organisms, 137

two close relatives (S. floricola LMG 23170T and Gluconobacter oxydans LMG 1408T) were also 138

tested for growth on LMG medium 404 after incubation at 28 °C under micro-aerobic conditions. 139

Both strains showed good growth, which seems logical as species from these genera have been 140

detected in the bee gut, which is micro-aerobic environment. The biochemical characteristics 141

examined for LMG 28161T and R-52486 included analysis of oxidase and catalase activity, 142

oxidation of acetate and lactate, production of acetic acid from ethanol, growth in the presence of 143

30 % (w/v) D-glucose, growth on methanol as carbon source, growth at 37 °C on LMG medium 144

404, assimilation of ammonium nitrogen on Frateur-Hoyer and Frateur-modified Hoyer medium 145

with D-glucose, D-mannitol and ethanol. The production of 2-keto-D-gluconate and 5-keto-D-146

gluconate from D-glucose was determined as reported previously (Spitaels et al., 2014). In 147

addition, acid production from various carbon sources was tested in triplicate, using the methods 148

described previously (Asai et al., 1964). S. floricola LMG 23170T was investigated concurrently 149

for all the tests conducted. Acetobacter aceti LMG 1504T and Gluconobacter roseus LMG 1418T 150

were included as controls for part of the acid production tests. Isolates R-52486 and LMG 28161T 151

could be differentiated from the genera of the family Acetobacteraceae by means of multiple 152

biochemical characteristics (Table 2, Supplementary Table S3), such as their lack of ability to 153

produce acetic acid and 5-keto-D-gluconate from ethanol and D-glucose, respectively; their 154

ability to grow at 37 °C on LMG medium 404; their negligible acid production from melibiose; 155

and notable acid production from D-fructose, sucrose and D-mannitol. For S. floricola LMG 156

23170T, the phenotypic test results were not always congruent with published data, while for A. 157

aceti LMG 1504T and Gl. roseus LMG 1418T the published data were confirmed (Table 2). 158

Therefore, a nearly complete 16S rRNA gene sequence was determined for strain LMG 23170T 159

(1436 nt, accession number, KJ850435) and compared to publicly available 16S rRNA gene 160

sequences of the type strain of S. floricola, including a sequence extracted from the draft genome. 161

A pairwise similarity of 100 % with accession number JF794031(1349 nt), 99.9% with a 162

sequence extracted from the draft genome (NZ_KB899360, 1485 nt) and 99.8% with accession 163

number AB110421 (1436 nt) was found, suggesting that LMG 23170T is a true subculture of the 164

type strain of S. floricola. 165

In conclusion, the present study demonstrated that the taxon represented by the isolates R-52486 166

and LMG 28161T can be differentiated genotypically and phenotypically from established species 167

and genera of the family Acetobacteraceae. The phylogenetic relationship between this taxon and 168

its nearest neighbor species, S. floricola, is considerably divergent (Fig. 1, Suppl. Fig. S3-S5). Its 169

fatty acid profile is unique (Table 1, Suppl. Table S2). Phenotypic characteristics that allow to 170

distinguish this taxon from S. floricola are its ability to grow on LMG medium 404 at 37 °C; its 171

inability to oxidize lactate and produce 5-keto-D-gluconate from D-glucose; and its ability to 172

produce acid from D-galactose, D-fructose, D-mannitol, L-arabinose and D-mannose (the latter 173

two weakly) (Table 2, Suppl. Table S3). Therefore, the classification of this taxon into a novel 174

species of a novel genus is warranted. We propose the name Bombella intestini gen. nov., sp. 175

nov., with strain LMG 28161T (= DSM 28636T) as the type strain. 176

177

Description of Bombella gen. nov. 178

Bombella (Bom.bel’la N.L. fem. dim. n. Bombella, named after the bumble bee genus, Bombus, 179

from which the first isolate of this genus was obtained). 180

Cells are Gram-negative, straight rods. Catalase activity is present, but no oxidase activity. The 181

predominant fatty acid is C18:1ω7c; other fatty acids in significant amounts are C19:0 cyclo ω8c, 182

C16:0, C14:0 2-OH, C14:0, C16:0 2-OH and C16:0 3-OH. The major quinone type is Q-10. The type 183

species is Bombella intestini. 184

The presence of 16S rRNA gene fragments in the gut metagenomes of honey bees and bumble 185

bees, which share 99% and 97% sequence similarity (query coverage of 34% and 48%, 186

respectively) (Cariveau et al., 2014; Martinson et al., 2011) with the nearly complete strain LMG 187

28161T 16S rRNA gene sequence suggests that the occurrence of members of this taxon is not 188

limited to bumble bees. 189

190

Description of Bombella intestini sp. nov. 191

Bombella intestini (in.tes.ti’ni. L. gen. n. intestini, of the gut) 192

Characteristics are the same as those described for the genus with the following additional 193

properties. Cells are non-motile, approximately 1 µm wide and 2~3 µm long. Cells occur singly 194

or in pairs. On LMG medium 404 agar [5 %, w/v, D-glucose; 1 %, w/v, yeast extract (Oxoid)], 195

colonies are round, smooth, brownish and slightly raised, with a diameter of approximately 1-2 196

mm after 3 days of incubation. Ethanol is not oxidized to acetic acid. Production of 2-keto-D-197

gluconate from D-glucose, but no 5-keto-D-gluconate. Production of acid from sucrose, D-198

fructose, D-glucose, D-mannitol and L-arabinose (weakly). Growth in the presence of 30 % (w/v) 199

D-glucose. Growth at 37 °C on LMG medium 404. No growth with ammonium as the sole 200

nitrogen source. The DNA G+C content of the type strain is 54.9 mol%. 201

The type strain LMG 28161T (= DSM 28636T = R-52487T) was isolated from the crop of a 202

bumble bee (Bombus lapidarius) in 2013 in Ghent, Belgium. 203

204

Acknowledgements 205

Leilei Li has a PhD grant from the Chinese Scholarship Council and Ghent University Co-206

Funding. The BCCM/LMG collection is supported by the Federal Public Planning Service - 207

Science Policy, Belgium. P. V. and L. D. V acknowledge their financial support of the University 208

Research Council, the Research Foundation-Flanders (FWO-Vlaanderen), and the Hercules 209

Foundation. 210

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Fig. 1. Maximum-likelihood tree based on nearly complete 16S rRNA gene sequences (1353-369

1431 nt), showing the phylogenetic position of Bombella intestini gen. nov., sp. nov. within the 370

family Acetobacteraceae. The robustness of the branching is indicated by bootstrap values 371

calculated for 1000 subsets. Bar, 2 % sequence divergence. 372

Table. 1. Cellular fatty acid contents (%) of Bombella intestini gen. nov., sp. nov. (data in bold) 373

and of the type strains of Saccharibacter floricola and Gluconobacter oxydans. 374

1. Bombella intestini LMG 28161T; 2. Saccharibacter floricola LMG 23170T; 3. Gluconobacter 375

oxydans LMG 1408T; -, not detectable or trace amount (< 1 %). All data were generated in the 376

frame of this study. Cultivation conditions prior to fatty acid extraction were identical for all 377

strains. 378

1 2 3

C18:1ω7c 43.1 29.8 51

C19:0 cyclo ω8c 16.7 28.4 -

C16:0 12.7 24.4 9.6

C14:0 9.3 2.8 -

C14:0 2-OH 8.5 - -

C16:0 3-OH 2.8 2.9 6.7

C16:0 2-OH 1.1 6.4 20.8

C18:0 - 1.1 2.1

C18:0 3-OH - - 4.3

379

380

Table 2. Characteristics that differentiate Bombella gen. nov. from its phylogenetic close 381

relatives of the family Acetobacteraceae. 382

Genera: 1. Bombella gen. nov.; 2. Saccharibacter; 3. Gluconobacter; 4. Swingsia; 5. 383

Neokomagataea. +: positive, -: negative, w: weak positive, v: variable, ND: not determined. 384

Unless indicated otherwise, data for taxon 1 were obtained in this study; data for taxon 2 were 385

taken from Jojima et al. (2004); data for taxa 3 were taken from Lisdiyanti et al. (2002); data for 386

taxon 4 were taken from Malimas et al. (2013); data for taxon 5 were taken from Yukphan et al. 387

(2011). °: data obtained in the present study; * data obtained in the present study not 388

corresponding to published data. 389

Characteristic 1 2 3 4 5

motility and flagellation non-motile non-motile

polar or

non-motile non-motile non-motile

Production of water soluble brown pigment(s) - - v + -

Growth on LMG medium 404 at 37 + - - ND ND

Oxidation of

acetate - -° - - -

lactate - w° - - -

Production of acetic acid from ethanol - -* + w/+ w

Growth in the presence of 30% (w/v) D-glucose + +° - + +

Assimilation of ammonium nitrogen on Frateur-

modified Hoyer medium with

D-Glucose - -* + - ND

D-Mannitol - -* + + ND

Ethanol - -* - - ND

Production of keto-D-gluconates from D-glucose

2-keto-D-gluconate + +° + + +

5-keto-D-gluconate - +° + + +

Acid production from

L-Arabinose w -* +° w v(w/+)

D-Arabinose - -° + w/- -

D-Xylose - w* +° w +

D-Glucose + w* +° + +

D-Galactose + -* +° +/- v(w/+)

D-Mannose w -* +° +/w v(w/-)

D-Fructose + -* + w +

L-Sorbose - -° + - -

Melibiose - -* +° + v

Sucrose + + + +/w +

Raffinose - -° - w v

D-Mannitol + - +(w°) + -

D-Sorbitol - -° + -/w -

Glycerol - -° + -/w -

Ethanol - -° + - v(w/-)

Major ubiquinone Q-10 Q-10 Q-10 Q-10 Q-10

DNA G+C content (mol %) 54.8 52-53 52-64 46.9-47.3 51.2-56.8

390

Neokomagataea thailandica BCC 25710T (AB513363)

Neokomagataea tanensis BCC 25711T (AB513364)

Swingsia samuiensis AH83T (AB786666)

Saccharibacter floricola S-877T (AB110421)

Bombella intestini LMG 28161T (KJ584313)

Gluconobacter cerinus LMG 1368T (X80775)

Gluconobacter oxydans DSM 3503T (X73820)

Gluconobacter albidus NBRC 3250T (AB178392)

Acetobacter aceti NCIB 8621T (X74066)

Acetobacter estunensis IFO 13751T (AB032349)

Commensalibacter intestini A911T (EU409601)

Asaia bogorensis 71T (AB025928)

Asaia spathodeae GB23-2T (AB511277)

Kozakia baliensis Yo-3T (AB056321)

Neoasaia chiangmaiensis BCC15763T (AB208549)

Tanticharoenia sakaeratensis NBRC 103193T (AB304087)

Ameyamaea chiangmaiensis BCC 15744T (AB303366)

Acidomonas methanolica LMG 1668T (X77468)

Komagataeibacter xylinus NCIB 11664T (X75619)

Gluconacetobacter liquefaciens IFO 12388T (X75617)

Nguyenibacter vanlangensis TN01LGIT (AB739062)

Endobacter medicaginis M1MS02T (JQ436923)

Granulibacter bethesdensis CGDNIH1T (AY788950)

100

100

99

92

100

99

98

92

97

85

74

97

0.02