Post on 28-Mar-2023
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.