J. Gen. Appl. Microbiol., 59, 153‒166 (2013)

Short Communication

Nguyenibacter vanlangensis gen. nov., sp. nov., an unusual acetic acid bacterium in the α-Proteobacteria

Huong Thi Lan Vu,1 Pattaraporn Yukphan,2 Winai Chaipitakchonlatarn,2 Taweesak Malimas,2 Yuki Muramatsu,3 Uyen Thi Tu Bui,1 Somboon Tanasupawat,4 Kien Cong Duong,1 Yasuyoshi Nakagawa,3 Ho Thanh Pham,1 and Yuzo Yamada2,*,**

1 Department of Microbiology, Faculty of Biology, University of Science, Vietnam National University-HCM City, 227 Nguyen Van Cu Street, Ward 4, District 5, Hochiminh City, Vietnam 2 BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phaholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand 3 NITE Biological Resource Center, National Institute of Technology and Evaluation, Kisarazu, Chiba 292‒0818, Japan 4 Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai Road, Wangmai, Pathumwan, Bangkok 10330, Thailand

(Received October 4, 2012; Accepted January 17, 2013)

Key Words—acetic acid bacteria; Acetobacteraceae; Acidomonas; Gluconacetobacter; Komaga- taeibacter; Nguyenibacter vanlangensis gen. nov., sp. nov.

The genus Gluconacetobacter corrig. Yamada et al. In the systematic and ecological studies of acetic 1998 was recently divided into two genera (Yamada acid bacteria, the present authors found that a certain and Yukphan, 2008; Yamada et al., 2012a, b, 2013). strain isolated from the natural environment of Vietnam One is the genus Gluconacetobacter, in which the was located in a unique phylogenetic position, name- type species is retained as Gluconacetobacter liquefa- ly, outside of the Gluconacetobacter cluster (Yamada ciens (Asai 1935) Yamada et al. 1998, and the other is et al., 2012b). the genus Komagataeibacter Yamada et al. 2013, in This paper describes Nguyenibacter vanlangensis which the type species is newly designated as Komaga- gen. nov., sp. nov., for isolate TN01LGIT as well as iso- taeibacter xylinus (Brown 1898) Yamada et al. 2013. In late VTH-AC01. The former was isolated from the rhi- the genus Gluconacetobacter, seven species are now- zosphere of Asian rice collected at Long Thanh Trung adays accommodated (Franke et al., 1999; Fuentes- Commune, Hoa Thanh District, Tay Ninh Province, Ramírez et al., 2001; Gillis et al., 1989; Tazato et al., Vietnam on March 6, 2011, and the latter was isolated 2012; Yamada and Yukphan, 2008). from the root of Asian rice collected at Hoc Mon Dis- trict, Ho Chi Minh City, Vietnam on November 12, 2011. * Corresponding author: Dr. Yuzo Yamada, 2‒3‒21 Seinan-cho, The two isolates were obtained by an enrichment Fujieda, Shizuoka 426‒0063, Japan. culture approach using nitrogen-free LGI medium that Tel/Fax: +81‒54‒635‒2316 contained 10.0% sucrose w/v, 0.06% KH PO w/v, E-mail: [email protected] 2 4 ** JICA Senior Overseas Volunteer, Japan International Co- 0.02% K2HPO4 w/v, 0.02% MgSO4 w/v, 0.002% CaCl2 operation Agency, Shibuya-ku, Tokyo 151‒8558, Japan; Profes- w/v. 0.001% FeCl3 w/v, and 0.0002% Na2MoO4 w/v, sor Emeritus, Shizuoka University, Suruga-ku, Shizuoka, Shi- pH 6.0 (Cavalcante and Döbereiner, 1988). When mi- zuoka 422‒8529, Japan. crobial growth was seen in the LGI medium, the cul- 154 VU et al. Vol. 59 ture was transferred to pH-3.5 medium that contained were calculated by use of the bootstrap analysis of 1.0% D-glucose w/v, 1.0% ethanol v/v, 0.3% peptone Felsenstein (1985) based on 1,000 replications. The w/v, and 0.2% yeast extract w/v. One loop of the pH- pair-wise 16S rRNA gene sequence similarities were 3.5 medium showing microbial growth was streaked calculated for 1,403 bases. onto an agar plate comprised of 2.0% D-glucose w/v, In a 16S rRNA gene sequence phylogenetic tree 0.5% ethanol v/v, 0.3% peptone w/v, 0.3% yeast extract constructed by the neighbor-joining method, the two w/v, 0.7% calcium carbonate w/v, and 1.5% agar w/v, isolates that formed a small cluster with a bootstrap and the resulting colonies that dissolved calcium car- value of 100% were located on the outside but not on bonate on the agar plate were picked up and exam- the inside of the Gluconacetobacter cluster, and the ined again for growth at pH 3.5 (Yamada and Yukphan, resulting cluster formed a large cluster along with the 2008). type strains of Komagataeibacter species (Fig. 1). The Gluconacetobacter liquefaciens NBRC 12388T, Glu- large cluster was then connected to the type strain of conacetobacter diazotrophicus LMG 7603T, Komaga- Acidomonas methanolica. The calculated bootstrap taeibacter xylinus NBRC 15237T, Komagataeibacter han- value at the branching point of the two clusters was senii NBRC 14820T, Acidomonas methanolica NRIC 65%. The data obtained indicated that the two isolates 0498T, and Acetobacter aceti NBRC 14818T were used were not very tightly coupled to the type strains of Glu- as reference strains. conacetobacter species phylogenetically. The 16S rRNA gene sequences of the two isolates In a 16S rRNA gene sequence phylogenetic tree were determined, as described previously (Murama- constructed by the maximum parsimony method, the tsu et al., 2009; Yamada et al., 2000; Yukphan et al., two isolates constituted a cluster first along with the 2004, 2011). Gene fragments for 16S rRNA gene- type strain of Acidomonas methanolica, and then the encoding regions were amplified by PCR with the follow- resulting cluster was connected to the Gluconaceto- ing two primers; 20F (5′-GAGTTTGATCCTGGCTCAG-3′, bacter cluster (Fig. 2). The calculated bootstrap values positions 9‒27) and 1500R (5′-GTTACCTTGTTACGACT- at the respective branching points were 41 and 16%. T- 3 ′, positions 1509‒1492) (the numbering of positions The data obtained indicated that the two isolates were was based on the Escherichia coli numbering system, related phylogenetically to the Acidomonas cluster rather Brosius et al., 1978, 1981; accession number V00348). than the Gluconacetobacter cluster. Amplified 16S rRNA genes were sequenced with the four In a 16S rRNA gene sequence phylogenetic tree primers, 27F (5′-AGAGTTTGATCMTGGCTCAG-3′, posi- constructed by the maximum likelihood method, the two tions 8‒27), 1492R (5′- TACGGYTACCTTGTTACGACT- isolates were located on the outside but not on the inside T- 3 ′, positions 1513-1492), 518F (5′-CCAGCAGCCGC- of the Gluconacetobacter cluster, as found in the phylo- GGTAATACG-3′, positions 518‒537), and 800R (5′-TAC- genetic tree constructed by the neighbor-joining method CAGGGTATCTAATCC-3′, positions 802‒785). Other 16S (Fig. 3). The calculated bootstrap value was 27%. rRNA gene sequences were cited from the GenBank/ The calculated pair-wise 16S rRNA gene sequence EMBL/DDBJ databases. similarities of isolate TN01LGIT were 97.9, 97.7, 96.5, Multiple sequence alignments were made with the 96.9, 95.5, 94.1, 96.2, 96.0, 95.6, 92.0, 96.3, 95.1, 96.2, program Clustal X (version 1.81) (Thompson et al., 97.1, and 94.0% respectively to the type strains of Glu- 1997). Sequence gaps and ambiguous bases were conacetobacter liquefaciens, Gluconacetobacter diaz- excluded. Distance matrices were calculated by the otrophicus, Acidomonas methanolica, Komagataeibacter two-parameter method of Kimura (1980). Phylogenetic xylinus, Acetobacter aceti, Gluconobacter oxydans, Asaia trees based on 16S rRNA gene sequences of 1,216 bogorensis, Kozakia baliensis, Swaminathania salitoler- bases were constructed by the neighbor-joining meth- ans, Saccharibacter floricola, Neoasaia chiangmaiensis, od (Saitou and Nei, 1987), the maximum parsimony Granulibacter bethesdensis, Tanticharoenia sakaeraten- method (Felsenstein, 1983), and the maximum likeli- sis, Ameyamaea chiangmaiensis, and Neokomagataea hood method (Felsenstein, 1981) using the program thailandica. Between the two isolates, the 16S rRNA gene MEGA 5 (version 5.05, Tamura et al., 2011). In the phylo- sequence similarity was 99.9%. On the other hand, the genetic trees constructed, the type strain of Acidocella 16S rRNA gene sequence similarity was 98.5% between facilis was designated as an outgroup. The confidence the type strains of Gluconacetobacter liquefaciens and values of individual branches in the phylogenetic trees Gluconacetobacter diazotrophicus. 2013 Nguyenibacter vanlangensis gen. nov., sp. nov. 155

Fig. 1. Phylogenetic relationships of Nguyenibacter vanlangensis gen. nov., sp. nov. (1). The phylogenetic tree based on 16S rRNA gene sequences was construct- ed by the neighbor-joining method. The type strain of Acidocella facilis was used as an outgroup. The numer- als at the nodes of the respective branches indicate bootstrap values (%) derived from 1,000 replications. 156 VU et al. Vol. 59

Fig. 2. Phylogenetic relation- ships of Nguyenibacter vanlan- gensis gen. nov., sp. nov. (2). The phylogenetic tree based on 16S rRNA gene sequences was constructed by the maxi- mum parsimony method. The type strain of Acidocella facilis was used as an outgroup. The phylogenetic relationships were represented by tree #1 out of 28 most parsimonious trees. The fi- nal data set had a total of 1,216 positions (bases), of which 660 were parsimony informative. Consistency index=0.328, re- tention index=0.804, composite index=0.264, homoplasy index =0.672. The numerals at the nodes of the respective branches indicate bootstrap values (%) de- rived from 1,000 replications. 2013 Nguyenibacter vanlangensis gen. nov., sp. nov. 157

Fig. 3. Phylogenetic relationships of Nguy- enibacter vanlangensis gen. nov., sp. nov. (3). The phylogenetic tree based on 16S rRNA gene sequences was constructed by the maxi- mum likelihood method. The type strain of Ac- idocella facilis was used as an outgroup. The numerals at the nodes of the respective branch- es indicate bootstrap values (%) derived from 1,000 replications. 158 VU et al. Vol. 59

A phylogenetic tree based on the so-called partial methanolica, and Komagataeibacter xylinus. Between 16S rRNA gene 800R-region sequences of 562 bases isolates TN01LGIT and VTH-AC01, between the type (positions 139‒739) was constructed by the neighbor- strains of Gluconacetobacter liquefaciens and Gluco- joining method (Saitou and Nei, 1987). The confidence nacetobacter diazotrophicus, between the type strains values of individual branches in the phylogenetic tree of Gluconacetobacter liquefaciens and Komagataei- were calculated by use of the bootstrap analysis of bacter xylinus, and between the type strains of Gluco- Felsenstein (1985), as described above. The type strain nacetobacter liquefaciens and Acidomonas methanol- of Acetobacter aceti was used as an outgroup. Se- ica, the calculated pair-wise sequence similarities quence gaps and ambiguous bases were excluded as were respectively 100, 98.6, 96.3, and 95.2%. The data well. The pair-wise partial 16S rRNA gene 800R-region obtained indicated that the two isolates were phyloge- sequence similarities were calculated for 659 bases netically not very similar and not very related to the (positions 26‒739). type strains of Gluconacetobacter species in the so- In a phylogenetic tree based on the so-called partial called partial 16S rRNA gene 800R-region sequences. 16S rRNA gene 800R-region sequences constructed Extraction and isolation of chromosomal DNA were by the neighbor-joining method, the two isolates formed made by use of the modified method of Marmur (1961) a cluster with the type strain of Acidomonas methanoli- (Ezaki et al., 1983; Saito and Miura, 1963). DNA base ca, but not with the type strains of Gluconacetobacter composition was determined by the method of Tama- species (Fig. 4). The calculated bootstrap value was oka and Komagata (1984). 65%. The cluster including the two isolates and the The DNA G+C contents of isolates TN01LGIT and type strain of Acidomonas methanolica was connected VTH-AC01 were respectively 69.4 and 68.1 mol%, with to the cluster of Gluconacetobacter species with a a range of 1.3 mol%. The data obtained indicated that bootstrap value of 36%, and then to the Komagataei- the DNA base compositions of the two isolates were bacter cluster. The calculated pair-wise partial 16S much higher than those of the type strains of Glucona- rRNA gene 800R-region sequence similarities of iso- cetobacter species (58.0‒65 mol% with a range of late TN01LGIT were 96.9, 97.8, 96.0, and 96.6% re- 7 mol%; Yamada et al., 2012b) as well as the type spectively to the type strains of Gluconacetobacter lique- strain of Acidomonas methanolica (62 mol%; Yamashi- faciens, Gluconacetobacter diazotrophicus, Acidomonas ta et al., 2004). The calculated differences in the DNA

Fig. 4. Phylogenetic relationships of Nguyenibacter vanlangensis gen. nov., sp. nov. (4). The phylogenetic tree based on the so-called partial 16S rRNA gene 800R-region sequenc- es of 562 bases (positions 139‒739) was constructed by the neighbor-joining method. The type strain of Acetobacter aceti was used as an outgroup. The numerals at the nodes of the respective branches indicate bootstrap values (%) derived from 1,000 replications. 2013 Nguyenibacter vanlangensis gen. nov., sp. nov. 159

G+C contents were almost 4‒10 mol%. (2000, 2002, 2006), Yukphan et al. (2004, 2011), and DNA-DNA hybridization was carried out by the pho- Kommanee et al. (2011). Electron microscopy was done, tobiotin-labeling method using microplate wells, as as described previously (Kommanee et al., 2011). Major described by Ezaki et al. (1989). Percent similarities in ubiquinone homologue determination was made by DNA-DNA hybridization were determined colorimetri- the method of Yamada et al. (1969). cally (Verlander, 1992). The color intensity was mea- The phenotypic and chemotaxonomic features of sured at A450 on a model VersaMax microplate reader the two isolates are given in the genus and the species (Molecular Devices, Sunnyvale, California, USA). Iso- descriptions of Nguyenibacter vanlangensis gen. nov., lated, single-stranded, and labeled DNAs were hybrid- sp. nov. ized with DNAs from test strains in 2×SSC containing Phylogenetically, the two isolates occupied unique 50% formamide at 48.0°C for 15 h. The highest and positions. When a phylogenetic tree was constructed lowest values obtained in each sample were excluded, by the neighbor-joining method, the two isolates con- and the mean of the remaining three values was taken stituted a cluster along with the type strains of Gluco- as a similarity value. nacetobacter species. However, the position of the two A single-stranded and labeled DNA from isolate TN- isolates was not on the inside, but on the outside of the 01LGIT represented 100, 86, 26, 31, 3, 11, 10, and 4% cluster. The calculated bootstrap value showing 65% similarity respectively to isolates TN01LGIT and VTH- indicated that the two isolates were not very tightly AC01 and the type strains of Gluconacetobacter lique- coupled phylogenetically to the type strains of Gluco- faciens, Gluconacetobacter diazotrophicus, Komagataei- nacetobacter species. On the other hand, the two iso- bacter xylinus, Komagataeibacter hansenii, Acidomonas lates constituted a cluster firstly with the type strain of methanolica, and Acetobacter aceti, which was used as Acidomonas methanolica, when a phylogenetic tree a negative control. On the other hand, isolate VTH- was constructed by the maximum parsimony method. AC01 represented 94, 100, 23, 24, 3, 9, 9, and 2%, and The phylogenetic data obtained above suggested the the type strain of Gluconacetobacter liquefaciens rep- possibility that the two isolates are necessarily accom- resented 26, 15, 100, 22, 2, 7, 12, and 5% as well. The modated to neither the genus Gluconacetobacter nor data obtained indicated that the two isolates constitute the genus Acidomonas. a single species and are at least differentiated at the In the so-called partial 16S rRNA gene 800R-region species level from the above-mentioned species of the sequence phylogenetic tree constructed by the neigh- genera Gluconacetobacter, Acidomonas, and Kom- bor-joining method, the two isolates formed a cluster agataeibacter. with the type strain of Acidomonas methanolica, but The cellular fatty acid composition of the two iso- not with the type strains of Gluconacetobacter spe- lates was determined, as described previously (Yuk- cies. The phylogenetic data obtained above support- phan et al., 2011). Isolates TN01LGIT and VTH-AC01 ed that the two isolates are not very closely related to contained 63.6 and 62.3% monounsaturated fatty acid and not simply accommodated in the genus Glucona- of C18:1ω7c, respectively, as the major fatty acid (Table cetobacter. 1). Others were minor components of cellular fatty ac- In the yeast systematics, Yamada and Kawasaki ids; for example, straight chain fatty acids of C14:0, C16:0, (1989) once examined basidiomycetous yeasts for and C19:0cycloω8c, and hydroxy fatty acids of C14:02OH, three sets of partial 18S and 26S rRNA sequence de- C16:02OH, C16:03OH, C18:03OH, and C18:12OH were terminations. In the experiment, the three dendro- found. Differing from the type strain of Gluconaceto- grams obtained showed similar patterns in the three bacter liquefaciens, the two isolates contained a con- regions. Among the yeasts examined, similar patterns siderable amount of hydroxy fatty acid of C18:12OH. were not necessarily seen in the three dendrograms The two isolates were different from the type strain of (Jindamorakot et al., 2012; Yamada and Nagahama, Acidomonas methanolica in no finding of hydroxy fatty 1991; Yamada and Nogawa, 1995). Recently, the so- acid of C10:03OH. called D1/D2 region sequences of the large subunit of The phenotypic characteristics were determined by rRNA or 26S rRNA genes have been widely used in the methods of Asai et al. (1964), Yamada et al. (1976, yeast systematics (Fell, 1993; Kurtzman and Robnett, 1999, 2000), Swings et al. (1992), Navarro and Koma- 1998; Kurtzman et al., 2011). In future, the so-called gata (1999), Katsura et al. (2001, 2002), Lisdiyanti et al. partial 16S rRNA gene 800R-region sequences men-

160 VU et al. Vol. 59 Neokomagataea 16 3.6 3.5 0.8 2.9 6.1 1.8

14.4 15.6 48.9 ; 4, Ac -

T Ameyamaea 15 4.8 1.5 1.1 8.6 5.7 1.6 2.2 0.5 0.7 1.9 1.0 1.4 68.0

; 8, Asaia bogorensis

chiangmaiensis strain T Tanticharoenia 14 1.2 8.1 1.0 3.6 1.0 1.0 2.5 5.0 1.5

11.1 64.1 ; 16, Neokomagataea thai -

T Granulibacter 13 1.1 2.2 2.1 1.1 1.3 2.1 1.8 2.2

10.2 17.6 57.4 ; 12, Neoasaia

T Neoasaia

12

2.7 1.7 5.5 1.6 2.0 3.1 1.3 2.4 14.4 10.7 53.2 Saccharibacter

11 0.4 0.4 5.9 2.1 2.1 1.5 0.2

20.0 34.3 30.7 Swaminathania ; 7, Gluconobacter oxydans NBRC 14819 T

10

0.7 9.8 9.1 1.3 3.4 3.1 1.1 1.2 2.8 13.0 52.6 Kozakia 9 ; 15, Ameyamaea chiangmaiensis strain AC04 4.2 5.5 1.2 1.8 1.7 0.7 3.2 1.6 16.1 61.8 T

; 11, Saccharibacter floricola strain S-877

T Asaia 8

1.9 0.6 0.4 9.5 4.6 7.5 0.6 0.7 3.0 1.1 1.0 2.3 66.0 Gluconobacter 7 8.8 2.6 1.0 1.5 1.4 2.2 2.1 10.1 69.6 acid and/or unknown acids. ; 6, Acetobacter aceti NBRC 14818

T

12:0 Acetobacter 6 4.9 0.3 1.6 5.5 1.6 2.1 1.7 4.8 1.5 16.8 58.7

; 2, Nguyenibacter vanlangensis isolate VTH-AC01; 3, Gluconacetobacter liquefaciens NBRC 12388

T Komagataeibacter a 5 1.8 6.7 1.6 0.2 2.6 1.2 0.4 12.2 10.4 60.6 comprised alde-C

b Table 1. Cellular fatty acid composition of the genus Nguyenibacter . Table Acidomonas Tanticharoenia sakaeratensis strain AC37 ; 14, Tanticharoenia 4 T 1.2 8.3 8.3 1.2 1.5 5.2 2.1 0.9 0.4 1.4 2.1

; 10, Swaminathania salitolerans strain PA51 65.8

T Gluconacetobacter 3 9.4 4.3 4.6 6.8 2.3 0.9 0.6 4.9 0.8 1.6 1.8 61.1 ; 5, Komagataeibacter xylinus JCM 7644 T a 2 0.7 1.0 6.2 1.9 1.0 7.9 0.2 0.8 0.4 4.4 1.4 10.6 62.3 a 1 1.1 0.9 5.5 1.9 1.0 7.5 0.2 0.6 0.2 3.2 1.4 10.1 63.6 Nguyenibacter b . ; 9, Kozakia baliensis NBRC 16664 T T cyclo ω 8c 3OH 2OH 2OH 3OH 3OH 2OH ω 6c ω 7c ; 13, Granulibacter bethesdensis CGDNIH1 Fatty acid Fatty T 14:0 16:0 17:0 18:0 19:0 10:0 14:0 16:0 16:0 18:0 18:1 17:1 18:1 Examined newly for cellular fatty acid composition; a C Monounsarurated Monounsarurated fatty acid C C C C C Hydroxy fatty acid C C Saturated fatty acid C C C C Summed feature 2 C landica AH11 idomonas methanolica NRIC 0498 NBRC 16594 Unknown fatty acids below 0.5% were not listed. AC28

The table was cited from Yukphan et al. (2011) with slight modifications. The table was cited from Yukphan Abbreviations: 1, Nguyenibacter vanlangensis isolate TN01LGI 2013 Nguyenibacter vanlangensis gen. nov., sp. nov. 161 tioned above may be utilized for bacterial systematics ferentiated them from the strains of the two genera. The as well. calculated G+C contents of the two isolates also sup- As described above, the two isolates were indepen- ported such a differentiation genetically. dent phylogenetically from the type strains of species It is quite unique that the production of acetic acid of the related genera, Gluconacetobacter, Acidomo- from ethanol was not found in the two isolates, just as nas, and Komagataeibacter. in most strains of Asaia species. It differentiated the Morphologically, the two isolates had peritrichous two isolates from strains of any other genera except for flagellation (Fig. 5), which was identical especially with the genus Asaia, which is discriminated by no produc- that of strains assigned to the genus Gluconaceto- tion of a water-soluble brown pigment as well as bacter, but different from polar flagellation of strains 2,5-diketo-D-gluconate (Table 2). In the acetate/lactate assigned to the genus Acidomonas (Table 2). Physio- oxidation pattern mentioned above, the two isolates logically/biochemically, the two isolates oxidized ace- were somewhat similar to strains of the genus Ameya- tate but not lactate. Such an oxidation pattern of ace- maea. However, the two isolates were also discrimi- tate/lactate was, on the other hand, identical especially nated by forming peritrichous but not polar flagellation with that of the genus Acidomonas, but different from and by production of a water-soluble brown pigment that of the genus Gluconacetobacter, in which both and 2,5-diketo-D-gluconate as well. acetate and lactate were oxidized to carbon dioxide The two isolates, TN01LGIT and VTH-AC01, are thus and water. The two isolates differed from strains of the distinguished at the generic level from strains of 14 genus Acidomonas in production of a water-soluble genera of acetic acid bacteria phylogenetically, genet- brown pigment and 2,5-diketo-D-gluconate and in no ically, chemotaxonomically, morphologically, physio- growth on methanol as a sole source of carbon. Che- logically, and biochemically, and are appropriately clas- motaxonomically, the two isolates differed from the type sified under a separate new genus (Table 2). The name strain of Gluconacetobacter liquefaciens by showing a of Nguyenibacter vanlangensis gen. nov., sp. nov. is considerable amount of hydroxy fatty acid of C18:12OH therefore introduced for the two isolates. and from the type strain of Acidomonas methanolica by showing no hydroxy fatty acid of C10:03OH. The Description of Nguyenibacter gen. nov. phenotypic data of the two isolates were therefore as- Nguyenibacter [Ngu.ye.ni.bac’ter. N. L. masc. n. Nguy- sumed to reflect a phylogenetic intermediary position enius Nguyen (the name of a famous Vietnamese micro- between the genera Gluconacetobacter and Acidomo- biologist); N. L. masc. n. bacter, a rod; N. L. masc. n. nas. In addition, no production of acetic acid from Nguyenibacter a rod, which is named in honor of Dr. ethanol being found in the two isolates decisively dif- Dung Lan Nguyen, Professor, Institute of Microbiology and Biotechnology, Vietnam National University-Ha- noi, Hanoi, Vietnam, who contributed to the study of microorganisms, especially of strains isolated in Viet- nam]. Gram-negative rods and motile with peritrichous fla- gella, measuring 0.6‒0.8×1.0‒1.6 µm. Colonies are entire, smooth, transparent, creamy to brownish. Cat- alase is positive, and oxidase is negative. Grows on nitrogen-free LGI medium. Oxidizes acetate to carbon dioxide and water but not lactate. Does not produce acetic acid from ethanol. Growth is weakly positive ei- ther on 30% D-glucose w/v or in the presence of 0.35% acetic acid v/v. Grows on glutamate agar and mannitol

agar. Does not grow in the presence of 1.0% KNO3 w/v Fig. 5. A transmission electron micrograph of Nguyeni- or 3.0% NaCl w/v. Produces a water-soluble brown bacter vanlangensis isolate TN01LGIT. Bacterial cells were cultivated on a glucose/ethanol/peptone/ pigment on glucose/yeast extract/calcium carbonate yeast extract agar plate at 30°C for 24 h. Bar, 1 µm. Isolate VTH- medium. Production of dihydroxyacetone from glyc- AC01 had peritrichous flagellation (not shown). erol is negative. Levan-like polysaccharides are pro-

162 VU et al. Vol. 59 Neokomagataea g

n ------w + + + + + + +

vw vw 16 Ameyamaea f

------w w w + + + + + + +

vw vw vw 15 pol Tanticharoenia e n ------

w + + + + + + + + +

nd 14 Granulibacter d n - -

w w + + +

nd nd nd nd nd nd nd nd nd nd nd nd vw 13 Neoasaia c n ------

w w w + + + + + + + +

nd 12 Saccharibacter b l g n ------w

- + + + + + - nd nd

11 w/ - Swaminathania a

- w w + + + + + + + +

nd nd nd nd nd nd nd nd nd per 10 Kozakia

9 n ------w w w w w + + + + + + + Asaia 8

------w w w + + + + + + + + + +

per Gluconobacter m n n 7 ------w + + + + + + + + + + - -

pol Acetobacter m 6 ------w

+ + + + + + + + vw

per Komagataeibacter

5 n - - - - + + + + + + + + + + nd nd nd nd nd nd Acidomonas i i a

4 ------w w w w w + + + +

+ nd nd nd + pol Gluconacetobacter 3 ------+ + + + + + + + + + + + + + per 2 ------w w w + + + + + + + + + per Table 2. Characteristics differentiating the genus Nguyenibacter . Table 1 ------w w w + + + + + + + + + per Nguyenibacter -gluconate D (w/v) 3 -gluconate -gluconate D D -Glucose (w/v) D -Glucose (w/v) D -Mannitol -Glucose -Mannitol D D D Production of acetic acid from ethanol Acid production from Ethanol Production of 2-Keto- 2,5-Diketo- 1% Flagellation Raffinose Mannitol agar Production of levan-like polysaccharide Production of dihydroxyacetone from glycerol Water-soluble brown pigment production Water-soluble

Oxidation of Acetate Growth on: 30% Growth in the presence of 0.35% acetic acid (w/v) Assimilation of ammoniac nitrogen on 5-Keto- Glutamate agar Lactate 1% KNO Characteristic 2013 Nguyenibacter vanlangensis gen. nov., sp. nov. 163

- Neokomagataea g - - - - - ; 12,

16 T

56.8 Q-10 Ameyamaea f Yukphan et Yukphan - - - w + g 15

66.0 Q-10 Tanticharoenia e - - w + + 14 65.6 Q-10

f Granulibacter d - - + nd

13 w/ - 59.1

Q-10 Neoasaia c ; 7, Gluconobacter oxydans NBRC w + + + Yukphan et al. (2009), Yukphan

f T 12

63.1 Q-10 + (d) Saccharibacter ; 15, Ameyamaea chiangmaiensis strain b T - - - - - 11 52.3 Q-10 ; 11, Saccharibacter floricola strain S-877

T Swaminathania a v + + + nd 10 Q-10 57.6 ‒ 59.9 Yukphan et al. (2008), Yukphan

e

k Kozakia 9 - - + + + Q-10 57.2 According to Jojima et al. (2004), growth was shown at 7% gluta - l

j Asaia 8 - + + ; 6, Acetobacter aceti NBRC 14818 T 60.2 Q-10 + (d) + (d)

h Gluconobacter 7 - w + + + Q-10 60.6

Greenberg et al. (2006), h Acetobacter d 6 - - - + + Q-9 58.6 Lisdiyanti et al. (2002). Tanticharoenia sakaeratensis strain AC37 ; 14, Tanticharoenia k

Some strains in the genus are positive. T

n Komagataeibacter ; 10, Swaminathania salitolerans strain PA51 5 - + T nd nd nd 62.5 Q-10

Table 2. Continued Table Acidomonas i 4 - - - + + 62 Q-10

h Gluconacetobacter et al. (2005), Yukphan c 3 - - - + + Q-10 64.5 ; 5, Komagataeibacter xylinus JCM 7644 T Yamada et al. (2000), and Yamada j 2 - - - - w 68.1 Q-10 1 - - - - w 69.4 Q-10 Nguyenibacter . T Jojima et al. (2004), b ; 9, Kozakia baliensis NBRC 16664 T ; 2, Nguyenibacter vanlangensis isolate VTH-AC01 (= VTCC-B-1190 = BCC 54775 = NBRC 109337); 3, Gluconacetobacter liquefa - Some strains in the genus are non-motile. T ; 13, Granulibacter bethesdensis CGDNIH1 m T Yamashita et al. (2004), Yamashita i ; 4, Acidomonas methanolica NRIC 0498 T Loganathan and Nair (2004), a

Yamada et al. (1981), Yamada h chiangmaiensis strain AC28 ; 8, Asaia bogorensis NBRC 16594 ; 16, Neokomagataea thailandica AH11 T T -Sorbitol D

DNA G+C (mol%) Dulcitol Raffinose Glycerol Ethanol Major isoprenoid quinone 14819 AC04 al. (2011), Cited from ciens NBRC 12388 mate w/v but not at 1% glutamate w/v. mate w/v but not at 1% glutamate w/v. Characteristic Neoasaia The table was cited from Yukphan et al. (2011) and Yamada et al. (2012b) with slight modifications. et al. (2011) and Yamada The table was cited from Yukphan variable; nd, not determined; 1, Nguy very weakly positive; d, delayed; v, weakly positive; vw, peritrichous; n, none; +, positive; - , negative; w, Abbreviations: pol, polar; per, enibacter vanlangensis isolate TN01LGI 164 VU et al. Vol. 59 duced, when grown on sucrose. Ammoniac nitrogen is Ezaki, T., Yamamoto, N., Ninomiya, K., Suzuki, S., and Yabuu- weakly assimilated on D-mannitol, but not on D-glucose chi, E. (1983) Transfer of Peptococcus indolicus, Pepto- coccus asaccharolyticus, Peptococcus prevotii, and Pep- or ethanol. 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