Systematic Study of Gluconobacter Asai 1935

Malimas, T., Yukphan, P., Muramatsu, Y., Potacharoen, W., Tanasupawat, S., Nakagawa, Y., Tanticharoen, M. and Yamada, Y.

BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand

Biological Resource Center (NBRC), National Institute of Technology and Evaluation (NITE), Japan

Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Thailand

Outline• History of classification of the genus

Gluconobacter Asai 1935• Sequence and restriction analyses of the 16S-23S rDNA ITS regions

• Several new species of Gluconobacter• An additional new species, Gluconobacter

kanchanaburiensis sp. nov., for strains isolated in Thailand

Classification of Gluconobacter Asai 1935 (I)

• Skerman et al. (1980) The only species, Gluconobacter oxydans (Henneberg 1897) De Ley 1961 was listed in the Approved Lists.

• Gosselé et al. (1983) A single species, G. oxydans was recognized in spite of being a wide range of 8.6 mol% G+C (54.2-62.8 mol% G+C).

• Yamada and Akita (1984) The Gluconobacter strains tested were divided into two groups or species: G. oxydans and the new species, Gluconobacter cerinus.

• Mason and Claus (1989) Three species were recognized: G. oxydans, and the two new species, Gluconobacter frateurii and Gluconobacter asaii. Gluconobacter cerinus was not accepted, because it was genetically different.

Classification of Gluconobacter Asai 1935 (II)• Swings (1992)

In The Prokaryotes, 2nd Ed., three species were recognized: G. oxydans, G. cerinus and G. asaii. Gluconobacter frateurii was not accepted.

• Holt et al. (1994) In Bergey’s Manual of Determinative Bacteriology, 9th Ed., three species were recognized: G. oxydans, G. frateurii and G. asaii. Gluconobacter cerinus was not accepted.

• Sievers et al. (1995) On the basis of the resulting 16S rDNA phylogenetic tree, all the four species were recognized: G. oxydans, G. cerinus, G. frateurii and G. asaii.

• Yamada et al. (1999) The type strain of G. asaii showed high DNA-DNA similarities (84-96%) to that of G. cerinus.

Classification of Gluconobacter Asai 1935 (III)

• Tanaka et al. (1999) The type strain of G. asaii showed high DNA-DNA similarities (88-95%) to that of G. cerinus.

• Katsura et al. (2002) Gluconobacter asaii is a junior subjective synonym of G. cerinus. The three species, G. oxydans, G. cerinus and G. frateurii were recognized.

• Tanasupawat et al. (2004) The new species, Gluconobacter thailandicus was proposed.

In total, four species were recognized in 2004.

Genetic and restriction analyses of the 16S-23S rDNA internal transcribed spacer

regions of the acetic acid bacteria(Trček and Teuber, 2002)

The 16S-23S rDNA ITS regions of 57 strains of acetic acid bacteria were sequenced and evaluated for molecular identification. The 16S-23S rDNA ITS region PCR products were digested with HaeIII and HpaII. In acetic acid bacteria, twelve distinct restriction groups were recognized, and three restriction groups were seen in the genus Gluconobacter.

Group and speciesIdentified by 16S rDNA seq

Digested with

HaeIII HpaII

Group G. oxydans G. oxydans G. oxydans G1 type

O1 type

Group G. asaii G. asaii G. asaii G2 type

T1 type

Group G. cerinus/ G. frateurii G. cerinus G. cerinus G2 type

U1 type

G. frateurii G. frateurii G2 typeU1

type

Identification of strains assigned to the genus Gluconobacter Asai 1935 based on the sequence and the restriction analyses of

the 16S-23S rDNA ITS regions (1) (Yukphan et al., 2004a)

tRNA

16S rDNA 23S rDNA

16S-23S rDNA ITS regions

tRNA

PCR amplification of 16S-23S rDNA ITS regions

Sequencing of 16S-23S rDNA ITS regionsRestriction analysis of 16S-23S rDNA ITS regionsPhylogenetic tree analysis- Multiple alignments: CLUSTAL X (version 1.81)- Distance matrices: Kimura’s two-parameter method- Phylogenetic tree: Neighbor-joining method

Selection of restriction endonucleases utilized for the species-level identificationNEBcutter V1.0

(http://tools.neb.com/NEBcutter2/index.php)

Digestion of PCR products with selected restriction

endonucleases2.5% Agarose gel electrophoresis at 100 V for 40 min in 1xTris-acetate running buffer

Identification of strains assigned to the genus Gluconobacter Asai 1935 based on the sequence and the restriction analyses of

the 16S-23S rDNA ITS regions (2) (Yukphan et al., 2004a)

5 = G. oxydans NBRC 32446 = G. oxydans NBRC 32937 = G. cerinus NBRC 32758 = G. cerinus IAM 18329 = G. frateurii NBRC 325110 = G. frateurii NBRC 325411 = G. frateurii NBRC 326512 = G. frateurii NBRC 326813 = G. frateurii NBRC 3270M = 50-bp DNA marker

Bsp1286I MboII

Bsp1286I

MboII

1 = G. oxydans NBRC 14819T

2 = G. cerinus NBRC 3267T

3 = G. asaii NBRC 3276T

4 = G. frateurii NBRC 3264T

Re-identification of Gluconobacter strains based on restriction analysis of 16S-23S rDNA internal transcribed spacer regions

(Yukphan et al., 2004b) G. cerinus IAM 1832 (AB111903) G. cerinus NBRC 3267T (AB111899)

G. cerinus NBRC 3274 (AB163849) G. cerinus NBRC 3276 (AB11190) G. frateurii NBRC 3271 (AB162709)

G. frateurii NBRC 3251 (AB111904) G. frateurii NBRC 3265 (AB111905)

G. frateurii NBRC 3264T (AB111898) G. oxydans NBRC 3990 (AB163865)

G. oxydans NBRC 14819T (AB111901) Gluconobacter sp. NBRC 3266 (AB162712)

Gluconobacter sp. NBRC 3273 (AB162711) Gluconobacter sp. NBRC 3250 (AB162710)

A. aceti NBRC 14818T (AB111902)

10059

73

99

9686

99

100

98

6572

0.05 Knuc

2 new species

Heterogeneity of strains assigned to Gluconobacter frateurii Mason and Claus 1989

based on restriction analysis of 16S-23S rDNA internal transcribed spacer

regions (Malimas et al., 2006)

Group and subgroup

Restriction pattern by digestion with

Bsp1286I MboII AvaII TaqI BsoBI BstNI

Group IV G. frateurii G. cerinus a c e h

Group III-1 Subgroup

III-1aG.

frateurii

G. frateurii b c e h

Subgroup III-1b

G. frateurii

G. frateurii b c f g

Group III-2G.

frateurii

G. frateurii a c e h

Group III-3G.

frateurii

G. frateurii b c f h

Group III-4 Subgroup

III-4aG.

frateurii

G. frateurii a d e i

Subgroup III-4b

G. frateurii

G. frateurii a d e h

Group III-5G.

frateurii

G. frateurii b d e i

A phylogenetic tree based on 16S-23S rDNA ITS sequences for strains assigned to Gluconobacter

frateurii (Malimas et al., 2006)

G. japonicus

III-3

III-1b

III-2

IV

III-1a

III-4

III-5

NBRC 3254 (AB206582) NBRC 3255 (AB206583)

G. thailandicus BCC 14116T (AB127941) NBRC 3289 (AB206588)

G. frateurii NBRC 3264T (AB111898) G. frateurii NBRC 3265 (AB111905)

G. frateurii NBRC 3251 (AB111904) NBRC 3268 (AB206586)

NBRC 3263 (AB206585) NBRC 3271 (AB162709)

G. oxydans NBRC 14819T (AB111901)

NBRC 3260 (AB206584) NBRC 3269 (AB206587)

597697

697695

9390

0.02Knuc

Intrageneric structure of the genus Gluconobacter analyzed by the 16S rRNA gene and 16S-23S rRNA gene internal transcribed

spacer sequences (Takahashi et al., 2006)Phylogenetic tree based on 16S-23S rDNA ITS sequences

1-1

2

3

4-1

4-2

5

1-2

NBRC 3267T (G. cerinus)NBRC 3274

NBRC 3275NBRC 3276

NBRC 3260NBRC 3269NBRC 3272NBRC 3263NBRC 3271NBRC 3172NBRC 3289NBRC 3291F142-1 (AB127942)NBRC 3256

F149-1T (G. thailandicus, AB127941)NBRC 3257NBRC 3255NBRC 3258NBRC 3254

NBRC 16669NBRC 3264T (G. frateurii)NBRC 3265NBRC 3270

NBRC 3268NBRC 3251NBRC 3262NBRC 3253NBRC 3286NBRC 3171NBRC 3285NBRC 3290

699

737

989

978

997

0.01 Knuc

621

NBRC 3266NBRC 3273NBRC 3250T (G. albidus)1000 NBRC 3990NBRC 12467NBRC 3293NBRC 3292NBRC 3244NBRC 3294NBRC 3243

NBRC 3287NBRC 3464NBRC 3130NBRC 14819T (G. oxydans)NBRC 3189NBRC 3432NBRC 12118NBRC 3462NBRC 3431NBRC 12528

844

848

738

976

1000

973

1000

NBRC 3266

NBRC 3273

NBRC 3250T (G. albidus)1000NBRC 3990NBRC 12467NBRC 3293

NBRC 3292

NBRC 3244

NBRC 3294

NBRC 3243

NBRC 3287

NBRC 3464

NBRC 3130

NBRC 14819T (G. oxydans)

NBRC 3189

NBRC 3432

NBRC 12118

NBRC 3462

NBRC 3431

NBRC 12528

844

848

738

976

973

new species

A phylogenetic tree based on 16S-23S rDNA ITS sequences of the suggested

five new species G. oxydans NBRC 3244 (AB163827) G. oxydans NBRC 3292 (AB163858) G. oxydans NBRC 3294 (AB163860) G. oxydans NBRC 14819T (AB111901) G. sphaericus NBRC 12467T (AB163867) G. roseus NBRC 3990T (AB163865)

G. albidus NBRC 3250T (AB162710) G. kondonii NBRC 3266T (AB162712) G. cerinus NBRC 3267T (AB111899)

G. japonicus NBRC 3271T (AB162709) G. frateurii NBRC 3264T (AB111898) G. thailandicus strain F149-1T

(AB128050)A. aceti NBRC 14818T (AB111902)62

100100

92

100946679100

0.05 Knuc

Gluconobacter albidus (ex Kondo and Ameyama 1958) sp. nov., an acetic acid bacterium

in the Alphaproteobacteria

Labeled DNA from Level of DNA-DNA hybridization (%) of

G. oxydans NBRC 14819T 100 11 13 10 27 17G. cerinus NBRC 3267T 13 100 23 7 21 12G. frateurii NBRC 3264T 9 21 100 35 8 11G. thailandicus BCC 14116T 15 16 48 100 16 23G. albidus NBRC 3250T 42 30 9 12 100 89G. albidus NBRC 3273 37 29 21 16 93 100

DNA-DNA hybridization for G. albidus NBRC 3250T.[Yukphan et al., J. Gen. Appl. Microbiol., 50, 235-

242, 2004c]

G. o

xyda

ns

NBR

C 1481

9TG.

cer

inus

NBR

C 32

67T

G. a

lbid

us

NBR

C 3250

T

G. fr

ateu

rii

NBR

C 3264

T

G. th

aila

ndic

us

BCC

141

16T

G. a

lbid

us

NBRC

327

3

Gluconobacter kondonii sp. nov., an acetic acid bacterium in the Alphaproteobacteria

Labeled DNA from DNA-DNA similarity (%) of

G. kondonii NBRC 3266T 100 37 53 7 9 17 5

G. oxydans NBRC 14819T 41 100 37 8 11 11 6

G. albidus NBRC 3250T 51 37 100 7 10 11 3

G. frateurii NBRC 3264T 8 13 11 100 23 5 5

G. thailandicus BCC 14116T 7 18 11 21 100 10 9

DNA-DNA hybridization of G. kondonii NBRC 3266T.[Malimas et al., J. Gen. Appl. Microbiol., 53, 301-

307, 2007]

G. o

xyda

ns

N

BRC

1481

9TG.

alb

idus

NBR

C 32

50T

G. k

ondo

nii

NBR

C 32

66T

G. f

rate

urii

NBRC

326

4T

G. th

aila

ndic

us

BCC

141

16T

G. c

erin

us

NBR

C 32

67T

A. a

ceti

NBR

C 14

818T

Gluconobacter roseus (ex Asai 1935) sp. nov., nom. rev., a pink-colored acetic acid bacterium in the Alphaproteobacteria

DNA-DNA hybridization of G. roseus NBRC 3990T.Labeled DNA from DNA-DNA similarity (%) of

G. roseus NBRC 3990T 100 49 47 37 8 9 10 3G. oxydans NBRC 14819T 34 100 37 41 8 8 8 3G. albidus NBRC 3250T 32 37 100 51 11 9 10 3G. kondonii NBRC 3266T 38 37 52 100 16 7 9 4

[Malimas et al., J. Gen. Appl. Microbiol., 54, 119-125, 2008]

G. o

xyda

ns

NBR

C 14

819T

G. a

lbid

us

NBRC

3250

T

G. k

ondo

nii

NBR

C 32

66T

G. ro

seus

NBR

C 39

90T

G. fr

ateu

rii

NBR

C 32

64T

G. th

aila

ndic

us

BCC

141

16T

G. c

erin

us

NBR

C 32

67T

A. a

ceti

NBR

C 14

818T

Gluconobacter sphaericus (Ameyama 1975) comb. nov., a brown pigment-producing

acetic acid bacterium in the Alphaproteobacteria

DNA-DNA hybridization of G. sphaericus NBRC 12467T.Labeled DNA from Level of DNA-DNA hybridization (%) of

G. sphaericus NBRC 12467T 100 49 31 33 38 10 9 15 17 2G. oxydans NBRC 14819T 56 100 31 41 55 13 12 15 19 4G. albidus NBRC 3250T 40 37 100 42 27 8 6 8 10 3G. kondonii NBRC 3266T 40 41 50 100 39 18 11 15 24 2G. roseus NBRC 3990T 44 39 33 31 100 11 13 11 15 2

[Malimas et al., J. Gen. Appl. Microbiol., 54, 211-220, 2008]

G. s

phae

ricus

NBRC

124

67T

G. o

xyda

ns

NBR

C 14

819T

G. a

lbid

us

NBR

C 32

50T

G. k

ondo

nii

NBR

C 32

66T

G. ro

seus

NBR

C 39

90T

G. fr

ateu

rii

NBR

C 32

64T

G. th

aila

ndic

us

BCC

141

16T

G. j

apon

icus

NB

RC 3

271T

G. c

erin

us

NBR

C 32

67T

A. a

ceti

NB

RC 148

18T

Gluconobacter japonicus sp. nov., an acetic acid bacterium in the Alphaproteobacteria

Labeled DNA fromLevel of DNA-DNA hybridization (%) of

G. japonicus NBRC 3271T

100

100 100 100 77 48 49 29 23 17 16 6

G. japonicus NBRC 3272 100

100 94 91 80 41 42 25 22 18 19 5

G. japonicus NBRC 3263 97 92 100 90 70 34 33 19 21 12 14 5

G. japonicus NBRC 3260 76 84 76 100 76 39 43 24 19 19 13 4

G. japonicus NBRC 3269 96 100 93 100 100 48 51 28 19 17 15 4

G. frateurii NBRC 3264T 53 51 50 57 49 100 54 27 15 15 11 4

G. thailandicus BCC 14116T

49 43 39 44 40 48 100 24 16 15 12 5

DNA-DNA hybridization of Gluconobacter japonicus NBRC 3271T.[Malimas et al., Int J Syst Evol Microbiol., 2008, in

press]

G. o

xyda

ns

NB

RC

1481

9TG.

alb

idus

NBR

C 32

50T

G. k

ondo

nii

NBR

C 32

66T

G. fr

ateu

rii

NBR

C 32

64T

G. th

aila

ndic

us

BCC 1

4116

T

G. ja

poni

cus

NBR

C 32

71T

G. c

erin

us

NBR

C 32

67T

A. a

ceti

NBR

C 14

818T

G. ja

poni

cus

NBR

C 32

72G.

japo

nicu

s

N

BRC

3263

G. ja

poni

cus

NBR

C 32

60G.

japo

nicu

s

NB

RC 3

269

Phenotypic characteristics differentiating the nine species (I)

Characteristics

Water soluble brown pigment - - - - + - - - -

Ketogluconic acid 2-keto-D-gluconate + + + + + + + + + 5-keto-D-gluconate + + + + + + + + + 2,5-diketo-D-

gluconate - - - - + - - - -

Dihydroxyacetone from glycerol + + + - + + + + w

Acid production from meso-Erythritol + w + vw vw + w + + Maltose + - - vw - - w - - Raffinose w + + + + w + w +Growth on D-arabitol - - w w w + + + + L-arabitol - - - - vw - + w w meso-ribitol - - - - - w + w +Growth without nicotinic

acid - - - - - + + + +

G. o

xyda

ns

NBRC

14819T

G. a

lbid

usNB

RC 325

0T

G. k

ondo

nii

NBRC

3266T

G. fr

ateu

rii

NBRC

326

4T

G. ja

poni

cus

NBRC

327

1T

G. c

erin

us

NBRC

3267T

G. sp

haer

icus

NBRC

124

67T

G. ro

seus

NBRC

3990T

G. th

aila

ndic

usBCC

14116T

Restriction pattern of 16S-23S rDNA ITS regions

differentiating the nine species

Bsp1286I Go Go Go Gr Gk Gc Gf Gf Gf

MboII Go Ga Go Go Gk Gc Gf Gf Gf

BstNI Go Ga Ga Ga Gk nd h h i

BsoBI Go Ga Ga Ga Gk nd e f e

BsaJI Go Ga Gs Gr Gk nd nd nd nd

Restriction endonucleases

G. o

xyda

ns

NBRC

14819

T

G. a

lbid

us

N

BRC 32

50T

G. k

ondo

nii

NBRC

3266

T

G. fr

ateu

rii

N

BRC

3264

T

G. ja

poni

cus

N

BRC

3271

T

G. c

erin

us

NBR

C 32

67T

G. sp

haer

icus

NB

RC

12467T

G. ro

seus

NBRC

3990

T

G. th

aila

ndic

us

BCC

14116

T

nd = No determined

Gluconobacter kanchanaburiensis sp. nov., a brown pigment-producing acetic acid bacterium for Thai isolates in the

Alphaproteobacteria [Malimas et al., J. Gen. Appl. Microbiol., 2008, submitted]

G. oxydans NBRC14819T (AB111901) G. sphaericus NBRC 12467T (AB163867) G. roseus NBRC 3990T (AB163865)

G. albidus NBRC 3250T (AB162710) G. kondonii NBRC 3266T (AB162712)G. kanchanaburiensis BCC 15889T (AB459532)G. kanchanaburiensis BCC 15890 (AB459533) G. cerinus NBRC 3267T (AB111899)

G. japonicus NBRC 3271T (AB162709) G. thailandicus F149-1T (AB127941)

G. frateurii NBRC 3264T (AB111898) A. aceti NBRC 14818T (AB111902)

53100

100

100

96

99

79

5091

0.05Knuc

Phylogenetic tree based on 16S-23S rDNA ITS sequences

Recently, 14 Gluconobacter species

recognized • Roh et al. 2008 “Gluconobacter morbifer” was proposed, but not validation approved list

Thai isolated• Malimas et al. 2009 Gluconobacter kanchanaburiensis was proposed• Yukphan et al., 2010 Gluconobacter wancherniae was proposed • Kommanee et al., 2011 Gluconobacter nephelii was proposed• Tanasupawat et al., 1012 Gluconobacter uchimurae was proposed

Conclusion- Restriction and phylogenetic analyses of 16S-23S rDNA ITS regions were useful for dividing Gluconobacter strains into the species groups.

- Twenty-three strains assigned to Gluconobacter frateurii Mason and Claus 1989 had a heterogeneous nature taxonomically and can be referred to the so-called G. frateurii complex.

- Among thirty NBRC strains of Gluconobacter, several new species and a new combination were proposed: G. albidus, G. kondonii, G. roseus, G. sphaericus and G. japonicus.

Thank you for your kind attention.