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Acetobacter sacchari sp. nov., for a plant growth-promoting acetic acid bacterium isolated in Vietnam

Acetobacter sacchari sp. nov., for a plant growth-promoting acetic acid bacterium isolated in... Purpose Two bacterial strains, designated as isolates VTH-Ai14 and VTH-Ai15, that have plant growth-promoting ability were isolated during the study on acetic acid bacteria diversity in Vietnam. The phylogenetic analysis based on 16S rRNA gene sequences showed that the two isolates were located closely to Acetobacter nitrogenifigens RG1 but formed an independent cluster. Methods The phylogenetic analysis based on 16S rRNA gene and three housekeeping genes’ (dnaK, groEL, and rpoB) sequences T T were analyzed. The genomic DNA of the two isolates, VTH-Ai14 and VTH-Ai15, Acetobacter nitrogenifigens RG1 , the closest phylogenetic species, and Acetobacter aceti NBRC 14818 were hybridized and calculated the %similarity. Then, phenotypic and chemotaxonomic characteristics were determined for species’ description using the conventional method. Results The 16S rRNA gene and concatenated of the three housekeeping genes phylogenetic analysis suggests that the two isolates were constituted in a species separated from Acetobacter nitrogenifigens, Acetobacter aceti,and Acetobacter sicerae. The two isolates VTH-Ai14 and VTH-Ai15 showed 99.65% and 98.65% similarity of 16S rRNA gene when compared with Acetobacter nitrogenifigens and Acetobacter aceti and they were so different from Acetobacter nitrogenifigens RG1 with 56.99 ± 3.6 and 68.15 ± 1.8% in DNA-DNA hybridization, when isolates VTH-Ai14 and VTH-Ai15 were respectively labeled. Moreover, the two isolates were phenotypically distinguished from Acetobacter nitrogenifigens in growth in the presence of 0.35% acetic acid (v/v), on nitrogen-free LGI medium and D-mannitol, and in no ability to solubilize phosphate. T T T T T Conclusion Therefore, the two isolates, VTH-Ai14 (= VTCC 910031 = BCC 67843 = TBRC 11175 =NRIC0977 ) and VTH- Ai15 (= VTCC 910032 = BCC 67844 = TBRC 11176 = NRIC 0978), can be assigned to an independent species within the genus Acetobacter,and thenameof Acetobacter sacchari sp. nov. is proposed for the two isolates. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13213-019-01497-0) contains supplementary material, which is available to authorized users. * Pattaraporn Yukphan Yuki Muramatsu pattaraporn@biotec.or.th muramatsu-yuki@nite.go.jp Naoto Tanaka Huong Thi Lan Vu n3tanaka@nodai.ac.jp huong_vu14@yahoo.com.vn Somboon Tanasupawat Van Thi Thu Bui somboon.t@chula.ac.th buithuvan86@gmail.com Binh Thanh Le ltbinh24@gmail.com Piyanat Charoenyingcharoen piyanat.cha@biotec.or.th Yasuyoshi Nakagawa nakagawa-yasuyoshi@nite.go.jp Sukunphat Malimas malimas45@hotmail.com Yuzo Yamada ymdy333@kdt.biglobe.ne.jp Linh Khanh Nguyen mtn.nkl1209@gmail.com Extended author information available on the last page of the article 1163 1156 Ann Microbiol (2019) 69:1155–1163 . . . Keywords Acetobacter sacchari sp. nov. Acetic acid bacteria Plant growth-promoting bacteria Vietnam Introduction comprised of D-glucose, ethanol, peptone, yeast extract, and calcium carbonate to select acetic acid bacteria (Vu et al. The genus Acetobacter is the largest in the acetous group of the 2013; Yamada et al. 1999). Acetobacter nitrogenifigens T T family Acetobacteraceae from the viewpoint of generic circum- TBRC 15 (= RG1 ) (Dutta and Gachhui 2006)and scription and includes 28 validly published species at present Acetobacter aceti NBRC 14818 were used as reference strains. (Ferrer et al. 2016; Komagata et al. 2014; Li et al. 2014; The 16S rRNA gene sequences of the two isolates were se- Malimas et al. 2017; Pitiwittayakul et al. 2016, 2015; Spitaels quenced, as described previously (Vu et al. 2013). Sequenced et al. 2014; Yamada 2016). The genus was divided into two were 1,419–1,420 bases for the two isolates. Multiple sequence groups, i.e., the Acetobacter aceti group and the Acetobacter alignments were made with MUSCLE (Edgar 2004). Sequence pasteurianus group phylogenetically (Yamada and Yukphan gaps and ambiguous bases were excluded. A phylogenetic tree 2008). The species of the genus were characterized by the oxi- based on 16S rRNA gene sequences of 1,275 bases was con- dation of acetate and lactate, acetic acid production from ethanol, structed by the maximum likelihood method based on DNA no production of 2,5-diketo-D-gluconic acid from D-glucose, substitution model selected under the Bayesian Information and UQ-9 as major (Komagata et al. 2014; Malimas et al. 2017). Criterion (Kumar et al. 2016) using the program MEGA7 version Acetobacter diazotrophicus was first reported for acetic 5.05 (Kumar et al. 2016). In the phylogenetic tree constructed, acid bacteria capable of nitrogen fixation (Gillis et al. 1989). the type strains of Gluconobacter oxydans, Saccharibacter However, this species was later transferred to the genus floricola, Neokomagataea tanensis,and Swingsia samuiensis Gluconacetobacter as Gluconacetobacter diazotrophicus were used as outgroups. The confidence values of individual (Yamada et al. 1997). branches were calculated by the use of bootstrap analysis of The acetic acid bacteria with plant growth-promoting char- Felsenstein (Felsenstein 1985). acteristics were additionally reported in the genus Acetobacter. Calculation of sequence similarity levels was calculated Muthukumarasamy et al. (2005) isolated some nitrogen-fixing using the EzBioCloud server by pairwise sequence alignment, acetic acid bacteria from wetland rice cultivated in India and in which all gaps were not considered (Kim et al. 2014;Kim identified them as Acetobacter peroxydans. The type strain of et al. 2012; Tindall et al. 2010;Yoonetal. 2017). Acetobacter peroxydans (LMG 1635 ) was also proved to have Extraction and isolation of chromosomal DNAs were made the same characteristics as the isolates mentioned above by the use of the modified method of Marmur (Ezaki et al. (Muthukumarasamy et al. 2005; Pedraza 2008, 2016). 1983;Marmur 1961;Saito andMiura 1963). DNA base com- Acetobacter nitrogenifigens was the second species position was determined by the method of Tamaoka and possessing a nitrogen-fixing ability and considered to be a Komagata (Tamaoka and Komagata 1984). DNA-DNA hy- plant growth-promoting bacterium as well (Dutta and bridization was done with five wells for each reciprocal reac- Gachhui 2006; Pedraza 2008, 2016). The two nitrogen- tions (e.g., A × B and B × A) by the photobiotin-labeling fixing species are quite distance phylogenetically; the former method using microplate wells, as described by Ezaki and was classified in the A. pasteurianus group, and the latter was coauthors (Ezaki et al. 1989). Percent similarities in DNA- in the A. aceti group. DNA hybridization were determined colorimetrically Previously, the two Acetobacter strains, namely isolates (Verlander 1992). The color density was measured at A VTH-Ai14 and VTH-Ai15, were isolated and phylogenetical- on a Synergy™ HTX Multi-Mode Microplate Reader ly located in the A. aceti group and closely related to (BioTek Instruments Inc., USA). Isolated, single-stranded, Acetobacter nitrogenifigens (Vu et al. 2016b). and labeled DNAwas hybridized with DNAs from test strains This paper describes Acetobacter sacchari sp. nov., as an in 2 × SSC containing 50% formamide at 48 °C. The highest additional plant growth-promoting species of the genus and the lowest values were excluded, and the mean of the Acetobacter, for the two isolates that were isolated in Binh remaining three values was taken as a similarity value and Phuoc Province, Vietnam, on January 28th, 2013. calculation of standard derivation. The housekeeping genes dnaK (encoding the heat shock 70 kDa protein), groEL (encoding a 60-kDa chaperonin), and Materials and methods rpoB (encoding the DNA-directed RNA polymerase subunit beta) of the two isolates, VTH-Ai14 and VTH-Ai15, were Two strains, designated as isolates VTH-Ai14 and VTH-Ai15, partially sequenced (Cleenwerck et al. 2010;Li et al. 2014; were isolated from the stems of sugar cane (Saccharum species) Pitiwittayakul et al. 2015). The phylogenetic position based by an enrichment culture approach at pH 3.5 (Vu et al. 2013; on the concatenated sequences of these housekeeping genes Yamada et al. 1999). When microbial growth was seen in the was compared with the type strains of the genus Acetobacter. medium, one loop of the culture was streaked onto an agar plate The respectively concatenated sequences of 528, 579, and 1157 Ann Microbiol (2019) 69:1155–1163 573 bp of partial dnaK, groEL, and rpoB were used for con- The DNA G+C contents of isolates VTH-Ai14 and VTH- structed the phylogenetic analysis. Accession numbers of Ai15 were 59.9 and 59.9 mol%, respectively (Table 1). The dnaK, groEL, and rpoB sequence of the type strains are ac- calculated values were so different from that of cording to the previous study (Cleenwerck et al. 2010;Lietal. A. nitrogenifigens RG1 (64.1 mol%). The DNA-DNA simi- 2014; Pitiwittayakul et al. 2015). larities when the isolate VTH-Ai14 was reciprocally hybrid- Whole-cell fatty acid methyl esters (FAME) of the two ized with VTH-Ai15 were high level at 100.00 ± 7.9% and isolates, VTH-Ai14 and VTH-Ai15, and type strains of 73.10 ± 9.3%. The DNA-DNA similarities of isolates VTH- T T Acetobacter nitrogenifigens TBRC 15 were extracted and Ai14 andVTH-Ai15withthe labeled Acetobacter analyzed as described by Vu et al. (2016a) after grown on nitrogenifigens TBRC 15 were 61.39 ± 8.0% and 44.21 ± GECA medium for 48 h at 28 °C under aerobic conditions. 6.9%. While the isolates VTH-Ai14 and VTH-Ai15 were Additionally, the two isolates, VTH-Ai14 and VTH-Ai15, labeled, the DNA-DNA similarities with Acetobacter T T and Acetobacter nitrogenifigens TBRC 15 were subjected to nitrogenifigens TBRC 15 were 56.99 ± 3.6% and 68.15 ± matrix-assisted laser desorption/ionization time-of-flight mass 1.8%, respectively. All the reciprocal DNA-DNA similarity spectrometry (MALDI-TOF MS) after grown in glucose-yeast between VTH-Ai14 and VTH-Ai15 with Acetobacter aceti extracted-peptone medium consisting of 2.0% glucose, 1.2% NBRC 14818 was between 4.16 ± 0.4% and 15.80 ± 2.1%. peptone, and 0.3% yeast extracted at 30 °C for 18 h with The concatenated sequences of the three housekeeping genes shaking at 200 rpm. The samples were prepared using the (1,676 bp) were constructed with MEGA7 using the maxi- standard extraction method as described by Matsuda et al. mum likelihood model. The DNA substitution GTR+G+I (2012). Then, applied to a MicroFlex LT mass spectrometer was selected under the Bayesian Information Criterion (Bruker Daltonik), and the results were analyzed by MALDI (Kumar et al. 2016). The isolates VTH-Ai14 and VTH- Biotyper 4.1 software (Bruker Daltonik). Escherichia coli Ai15 were grouped together and separated from Acetobacter DH5α was used as a quality control as recommended by the nitrogenifigens TBRC 15 as same as the topologies of the manufacturer on each experiment. phylogenetic tree based on the 16S rRNA gene (Fig. 2). Phenotypic characteristics were determined by the conven- The major cellular fatty acid of the isolates VTH-Ai14 , tional methods (Asai et al. 1964; Gosselé et al. 1980; Kersters VTH-Ai15, and Acetobacter nitrogenifigens TBRC 15 was et al. 2006;Lisdiyanti etal. 2000;Swings et al. 1992; Yamada C18:1ω7c at 55.94%, 57.04%, and 57.94%, respectively et al. 1999, 1976; Yukphan et al. 2011). A major isoprenoid (Table 2). quinone was extracted and quantitatively analyze by HPLC The MALDI-TOF MS profiles of the isolate VTH-Ai14 , (Komagata and Suzuki 1988; Tamaoka et al. 1983; Yamada VTH-Ai15, and Acetobacter nitrogenifigens TBRC 15 were et al. 1969). compared to each other and expressed by identification scores. The isolates VTH-Ai14 and VTH-Ai15 showed identifica- tion scores of 2.66 and 2.67, when comparing with VTH-Ai15 and VTH-Ai14 , respectively. While the identification score Results of the isolates VTH-Ai14 and VTH-Ai15 when comparing with Acetobacter nitrogenifigens TBRC 15 was only 1.88 In a phylogenetic tree deduced from the maximum likelihood and 1.92. The two isolates were discriminated from T T method, isolates VTH-Ai14 and VTH-Ai15 formed a cluster, Acetobacter nitrogenifigens TBRC 15 according to the man- which was connected to the cluster of Acetobacter ufacturer’s recommended log score identification criteria nitrogenifigens RG1 with the bootstrap value of 100% (Matsuda et al. 2012). All the results obtained suggested that (Fig. 1). The resulting cluster was then connected to a cluster the isolates are genetically separated from either Acetobacter containing Acetobacter aceti NBRC 14818 and Acetobacter nitrogenifigens or Acetobacter aceti. T T sicerae LMG 1531 with the bootstrap value of 67%. The The phenotypic characteristics of isolates VTH-Ai14 and pairwise sequence similarities of isolate VTH-Ai14 were VTH-Ai15 were given in the species description of 100, 99.65, 98.65, 98.09, 97.87, 97.87, 97.59, 96.59, and Acetobacter sacchari sp.nov.(Table 1). 96.31% respectively to isolate VTH-Ai15, Acetobacter T T nitrogenifigens RG1 , Acetobacter aceti NBRC 14818 , Acetobacter musti Bo7 , Acetobacter estunensis NBRC Discussion T T 13751 , Acetobacter oeni B13 , Acetobacter sicerae LMG T T 1531 , Acetobacter peroxydans NBRC 13755 ,and Acetobacter nitrogenifigens was first reported as a nitrogen- Acetobacter pasteurianus LMD 22.1 . The phylogenetic data fixing bacterium equipped with polar flagellation and with a obtained suggested that the two isolates constitute a species high G+C content of DNA (64.1 mol% G + C). In general, the separate from either Acetobacter nitrogenifigens or G+C contents of DNA are ranged from 53.5 to 60.7 mol% in Acetobacter aceti. the genus Acetobacter (Komagata et al. 2014; Malimas et al. 1158 Ann Microbiol (2019) 69:1155–1163 Fig. 1 A phylogenetic Acetobacter orleanensis LMG 1583 (AJ419845) relationship of isolates VTH- 50 Acetobacter farinalis G360-1 (AB602333) Ai14 and VTH-Ai15. The Acetobacter malorum LMG 1746 (AJ419844) phylogenetic tree based on 16S Acetobacter cerevisiae LMG 1625 (AJ419843) rRNA gene sequences was constructed by the maximum Acetobacter persici T-120 (AB665070) likelihood method. The type Acetobacter indonesiens NRIC 0313 (AB032356) strains of Gluconobacter Acetobacter thailandicus AD25 (AB937775) oxydans, Saccharibacter Acetobacter cibinongensis 4H-1 (AB052710) floricola, Neokomagataea 81 Acetobacter orientalis 21F-2 (AB052706) tanensis,and Swingsia samuiensis were used as Acetobacter senegalensis CWBI-B418 (AY883036) outgroups. The numerals at the 88 Acetobacter tropicalis NRIC 0312 (AB032354) nodes of the respective branches Acetobacter estunensis LMG 1626 (AJ419838) indicate bootstrap values (%) Acetobacter oeni B13 (AY829472) derived from 1,000 replications. 97 Bar, 0.02% sequence divergence 65 Acetobacter musti Bo7 (HM162854) Acetobacter aceti NBRC 14818 (X74066) 75 T Acetobacter sicerae LMG 1531 (AJ419840) Acetobacter nitrogenifigens RG1 (AY669513) Acetobacter sacchari VTH-Ai15 (LC103251) 86 Acetobacter sacchari VTH-Ai14 (LC103252) Acetobacter pasteurianus LMD 22.1 (X71863) Acetobacter pomorum LMG 18848 (AJ419835) Acetobacter lambici LMG 27439T (HF969863) Acetobacter ghanensis 430A (EF030713) Acetobacter fabarum R-36330 (AM905849) Acetobacter lovaniensis LMG 1617 (AJ419837) Acetobacter syzygii 9H-2 (AB052712) Acetobacter okinawensis 1-35 (AB665068) Acetobacter suratthaniensis BCC 26087 (AB937774) Acetobacter peroxydans NBRC 13755T (AB032352) 77 Acetobacter papayae 1-25 (AB665066) Gluconobacter oxydans NBRC 14819 (X73820) Neokomagataea tanensis BCC 25711 (AB513364) 66 Swingsia samuiensis AH83 (AB786666) Saccharibacter floricola S-877 (AB110421) 0.02 2017). In addition, the peritrichous flagellation appears to be The two isolates were phenotypically distinguished widely distributed in the genus (Komagata et al. 2014; from Acetobacter nitrogenifigens in growth in the pres- Malimas et al. 2017). The present authors’ estimation of ence of 0.35% acetic acid (v/v), nitrogen-free LGI medi- 59.7 mol% G+C and observation of peritrichous flagellation um, and D-mannitol and in no ability to solubilize phos- in Acetobacter nitrogenifigens TBRC 15 may be reasonable phate and from Acetobacter aceti in growth in the pres- (Table 1). ence of 0.35% (v/v) acetic acid, nitrogen-free LGI medi- The result of fatty acid composition showed that C18:1ω7c um, and D-mannitol and in the absence of phosphate sol- is a major fatty acid and the data were consistent with those ubility and production of 2-keto-D-gluconic acid reported for the validly published species of the genus (Komagata et al. 2014; Malimas et al. 2017). In addition, Acetobacter reported by Ferrer et al. (2016). Although, the they were genetically and physiologically discriminated composition of minor fatty acid was slightly different and by DNA-DNA similarities obtained from the reciprocal cannot discriminate the two isolates from the closest known DNA-DNA hybridization, MALDI-TOF MS profiles of species (Li et al. 2014;Spitaels etal. 2014), but the result of the isolate, and the concatenated sequences of the three the two isolates is different from the other genera of acetic acid housekeeping genes from the type strain of Acetobacter bacteria (Li et al. 2015;Vuetal. 2013; Yukphan et al. 2011). nitrogenifigens (Ferrer et al. 2016;Lietal. 2014). 1159 Ann Microbiol (2019) 69:1155–1163 Table 1 Differential a b c b d Characteristic 1 2 3 4 5 6 7 characteristics of Acetobacter sacchari sp. nov., for isolates f,g Flagellation per per per nd per nd per VTH-Ai14 and VTH-Ai15, from the phylogenetically closest Ketogenesis from glycerol + + + + nd – nd species of the genus Acetobacter. Production of ketogluconate from D-glucose (1) A. sacchari isolate VTH- 5-Keto-D-gluconate + + + ++ – + Ai14 ;(2) A. sacchari isolate VTH-Ai15; (3) A. nitrogenifigens 2-Keto-D-gluconate –– – +++ – T T RG1 ;(4) A. aceti NBRC 14818 ; Production of levan-like polysaccharide from (5) A. sicerae LMG 1531 ;(6) f f D-Glucose + + + – nd nd nd A. estunensis NBRC 13751 ;(7) f f D-Fructose + + + – nd nd nd A. oeni B13 ;+,positive; −, f f negative; w, weakly positive; vw, D-Mannitol + + + – nd nd nd f f very weakly positive; per, Glycerol + + + – nd nd nd peritrichous; nd, not determined c c Growth on ammoniac nitrogen with ethanol + + + + ++ – c c Growth in presence of 10% ethanol (v/v)w w + – –– + c c Growth in presence of 30% D-glucose (w/v) –– + – + – – f f Growth in presence of 0.35% acetic (w/v)+ + w w nd nd nd Growth on f f D-Glucose + + w vw nd nd nd c c c D-Fructose w w + – ++ + c c c c D-Sorbitol –– – – ++ – f f D-Mannitol + + – – nd nd nd f f Ethanol + + w + nd nd – f f 2-Propanol w + vw vw nd nd nd Acid production from c c D-Arabinose –– + ww – + h h h h Ethanol + + ++ nd + nd Glycerol vw vw – – nd – nd h f h 1-Propanol ++ + + nd nd nd Solubilization of f f Ca (PO4 ) on Pikovskaya agar medium –– vw vw nd nd nd 3 4 2 ZnOonLGIagarmedium + + + nd ndndnd Production phytohormon IAA with the presence of + + + nd ndndnd L-tryptophan in LGI medium f a a a Growth on nitrogen-free LGI medium + + w – nd – – γ-Pyrone production from D-Fructose –– – – nd – nd D-Glucose vw vw + – nd – nd Ubiquinone system Q-9 Q-9 Q-9 Q-9 Q-9 Q-9 nd f,g DNA G + C content (mol%) 59.9 59.9 59.7 57.2 58.3 59.3 58.1 Dutta and Gachhui (2006) Lisdiyanti et al. (2000) Li et al. (2014) Silva et al. (2006) Spitaels et al. (2014) The present work According to the original authors of the species, flagellation is polar and DNA G+C content is 64.1 mol% (Dutta and Gachhui 2006) Ethanol (1%, v/v) was completely oxidized to carbon dioxide and water for 9, 9, and 5 days respectively in T T A. sacchari isolate VTH-Ai14 , A. sacchari isolate VTH-Ai15, and A. aceti NBRC 14818 , and 1-propanol (1%, v/v) was done for 10 days in A. aceti NBRC 14818 1160 Ann Microbiol (2019) 69:1155–1163 Acetobacter cerevisiae LMG 1625 (KF537424, KF537477, KF537492) Fig. 2 Maximum likelihood tree based on the concatenated T Acetobacter malorum LMG 1746 (KF537431, KF537476, KF537504) sequence (1,676 bp) of dnaK Acetobacter orleanensis LMG 1583 (KF537421, KF537473, KF537507) (528 bp), groEL (579 bp), and Acetobacter persici LMG 26458 (KF537423, KF537471, KF537531) rpoB (573 bp) showing the 63 phylogenetic position of isolates T Acetobacter indonesiensis LMG 19824 (KF537420, KF537463, KF537503) VTH-Ai14 and VTH-Ai15 Acetobacter tropicalis LMG 19825 (KF537411, KF537469, KF537527) within the genus Acetobacter.The Acetobacter pasteurianus LMG 1262 (KF537405, KF537450, KF537510) type strains of Gluconacetobacter liquefaciens and Granulibacter T Acetobacter ghanensis LMG 23848 (HG329535, HG329547, HG329559) bethesdensis were used as Acetobacter okinawensis LMG 26457 (HG329537, HG329549, HG329561) outgroup. Numbers at branching Acetobacter fabarum LMG 24244 (HG329536, HG329548, HG329560) points are percentage bootstrap values based on 1,000 T 100 Acetobacter lovaniensis LMG 1617 (HG329533, HG329545, HG329557) replications. Bar, 0.05% sequence Acetobacter aceti LMG 1504 (FN421342, FN421343, FN421344) divergence Acetobacter sicerae LMG 1531 (KF537395, KF537438, KF537524) Acetobacter estunensis LMG 1626 (KF537394, KF537437, KF537498) Acetobacter oeni LMG 21952 (KF537391, KF537434, KF537506) Acetobacter nitrogenifigens LMG 23498 (KF537390, KF537433, KF537505) VTH-Ai14 (MK751131, MK751133, MK751135) 100 VTH-Ai15 (MK751132, MK751134, MK751136) Gluconacetobacter liquefaciens LMG 1381 (FN391626, FN391699, FN391772) Granulibacter bethesdensis CGDNIH1 (CP000394: 25545-23647, 2432985-2431351, 628088-632263) 0.05 As described above, the two isolates VTH-Ai14 and can therefore be assigned to an independent species within the VTH-Ai15 were separated phenotypically, genetically, and genus Acetobacter, and the name of Acetobacter sacchari sp. physiologically from either Acetobacter nitrogenifigens or nov. is introduced for the two isolates (Table 1). Acetobacter aceti, the phylogenetically closest species (Komagata et al. 2014; Malimas et al. 2017). The two isolates Description of Acetobacter sacchari sp. nov. Table 2 Cellular fatty acid contents (%) of Acetobacter sacchari sp. Acetobacter sacchari (sac’cha,ri. L. gen. sacchari;L.neut. n. nov., for isolates VTH-Ai14 and VTH-Ai15, and the phylogenetically Saccharum sugar cane, from which the two isolates were closest species of the genus Acetobacter.(1) A. sacchari isolate VTH- T T isolated). Ai14 ;(2) A. sacchari isolate VTH-Ai15; (3) A. nitrogenifigens RG1 ; T T (4) A. aceti NBRC 14818 ;(5) A. sicerae LMG 1531 Gram-negative short rods and motile with peritrichous fla- gella, measuring 0.4–0.5 × 0.6–1.5 μm. Colonies are entire, a a Fatty acid 1 2 3 4 5 smooth, transparent, glistening, and creamy to slightly light pink. Catalase is positive, and oxidase is negative. Grows on C : 0.1 0.1 0.1 0.1 – 12 0 LGI medium. Oxidize acetate and lactate. Produces acetic acid Summed feature 2* 1.88 1.78 2.06 3.5 3.2 from ethanol. Does not grow in the presence of 30% D- Summed feature 3 0.44 0.39 0.42 0.3 – glucose (w/v) or 1% potassium nitrate (w/v) but in the presence C : 0.1 0.1 –– – 17 0 of 0.35% acetic acid (v/v). Does not hydrolyze starch and C : 2-OH 2.34 2.31 2.18 0.3 – 18 1 casein. Produces 5-keto-D-gluconate but not 2-keto-D- C : 1.77 1.64 1.5 0.5 1.2 14 0 gluconate and 2,5-diketo-D-gluconate from D-glucose. C : 2-OH 5.66 5.39 5.72 – 8.8 14 0 Produces levan-like polysaccharide in the presence of 3.5% C : 16.63 16.86 14.62 12.0 9.2 16 0 and 5% D-glucose (w/v), D-fructose, and glycerol. Produces C : 2-OH 10.37 9.89 10.73 21.0 5.8 16 0 dihydroxyacetone from glycerol. Very weak production of γ- C : 3-OH 1.67 1.65 1.47 2.9 3.7 16 0 pyrone compounds from D-glucose is shown. C : 1.03 0.91 0.76 0.6 1.2 18 0 Acid is produced from L-arabinose, D-xylose, D-galactose, C : ω7c 55.94 57.04 57.94 50.9 59.6* 18 1 D-glucose, D-mannose, glycerol very weakly, 1-propanol, C : 3-OH 0.68 0.69 0.61 2.6 1.9 18 0 and ethanol, but not from D-arabinose, D-fructose, L-sorbose, C : cyclo ω8c 0.68 0.48 0.94 1.7 3.6 19 0 L-rhamnose, D-mannitol, D-sorbitol, dulcitol, myo-inositol, Ferrer et al. (2016) maltose, lactose, melibiose, sucrose, raffinose, 2-propanol, 1161 Ann Microbiol (2019) 69:1155–1163 Felsenstein J (1985) Confidence limits on phylogenies: an approach using and methanol. Grows on L-arabinose weakly, D-galactose the bootstrap. Evolution 39:783–791. https://doi.org/10.2307/ very weakly, D-glucose, D-mannose very weakly, D- fructose weakly, D-mannitol, glycerol, 1-propanol weakly Ferrer S, Manes-Lazaro R, Benavent-Gil Y, Yepez A, Pardo I (2016) (VTH-Ai15 grows), 2-propanol weakly, but not on D-arabi- Acetobacter musti sp. nov., isolated from Bobal grape must. Int J Syst Evol Microbiol 66:957–961. https://doi.org/10.1099/ijsem.0. nose, L-arabinose, D-xylose, L-rhamnose, L-sorbose, D-sor- bitol, dulcitol, myo-inositol, maltose, sucrose, raffinose, and Gillis M et al (1989) Acetobacter diazotrophicus sp. nov., a nitrogen- methanol. fixing acetic acid bacterium associated with sugarcane. Int J Syst Growth occurs at 20–37 °C, and no growth is found at Evol Microbiol 39:361–364. https://doi.org/10.1099/00207713-39- 3-361 40 °C. Optimal growth temperature is at 25–33 °C. Optimal Gosselé F, Swings J, De Ley J (1980) A rapid, simple and simultaneous growth pH is from 3.0 to 8.0, and growth occurs at pH 2.5– detection of 2-keto-, 5-keto-and 2,5-diketogluconic acids by thin- 8.0. Optimum growth is from 0 to 0.5% NaCl, and no growth layer chromatography in culture media of acetic acid bacteria. at 2.0% NaCl. Zentralbl Bakteriol 1:178–181. https://doi.org/10.1016/S0172- 5564(80)80039-X A major isoprenoid quinone is Q-9. DNA G+C contents is Kersters K, Lisdiyanti P, Komagata K, Swings J (2006) The family 59.9 mol%. Major fatty acid composition is C18:1ω7c. The Acetobacteraceae:the genera Acetobacter, Acidomonas, Asaia, T T T type strain is VTH-Ai14 (= VTCC 910031 = BCC 67843 Gluconacetobacter, Gluconobacter, and Kozakia. In: Dworkin M, T T = TBRC 11175 =NRIC0977 ), which was isolated from the Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E (eds) The Prokaryotes. Springer, New York, pp 163–200. https://doi.org/10. stem of sugar cane (Saccharum species) collected in Thuận 1007/0-387-30745-1_9 Phú, Đồng Phú, Bình Phước (GPS location is 11.59, 106.85), Kim OS et al (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA Vietnam, and whose DNA G+C content is 59.9 mol%. gene sequence database with phylotypes that represent uncultured species. 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Academic Publisher’snote Springer Nature remains neutral with regard to Press, Boston, pp 185–201. https://doi.org/10.1016/B978-0-12- jurisdictional claims in published maps and institutional affiliations. 426296-6.50012-5 1163 Ann Microbiol (2019) 69:1155–1163 Affiliations 1,2 3 1 3 Huong Thi Lan Vu & Pattaraporn Yukphan & Van Thi Thu Bui & Piyanat Charoenyingcharoen & 4 1 5 6 7 Sukunphat Malimas & Linh Khanh Nguyen & Yuki Muramatsu & Naoto Tanaka & Somboon Tanasupawat & 2 5 3,8,9 Binh Thanh Le & Yasuyoshi Nakagawa & Yuzo Yamada 1 5 Department of Microbiology, Faculty of Biology and Biotechnology, NITE Biological Resource Center, National Institute of Technology University of Science, Vietnam National University-HCM City, 227 and Evaluation, 2-5-8 Kazusa-Kamatari, Kisarazu 292-0818, Japan Nguyen Van Cu Street, Ward 4, District 5, Ho Chi Minh Department of Molecular Microbiology, NODAI Culture Collection City, Vietnam Center, Faculty of Life Sciences, Tokyo University of Agriculture, 1- Graduate University of Science and Technology, Vietnam Academy 1-1 Sakuragaoka, Setagaya, Tokyo 156-8502, Japan of Science and Technology, 18 Hoang Quoc Viet Street, Cau Giay Department of Biochemistry and Microbiology, Faculty of District, Hanoi, Vietnam Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai National Center for Genetic Engineering and Biotechnology Road, Wangmai, Pathumwan, Bangkok 10330, Thailand (BIOTEC), National Science and Technology Development Agency Japan International Cooperation Agency (JICA Senior Overseas (NSTDA), 113 Thailand Science Park, Phahonyothin Road, Khlong Volunteer), Shibuya-ku, Tokyo 151-8558, Japan Nueng, Khlong Luang, Pathum Thani 12120, Thailand Laboratory of Applied Microbiology (Professor Emeritus), Rungrueng-Fertilizer Co., Ltd, 207/5 Moo1, Nong Ya, Muang Department of Applied Biological Chemistry, Faculty of Kanchanaburi, Kanchanaburi 71000, Thailand Agriculture, Shizuoka University, Suruga-ku, Shizuoka 422-8529, Japan http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Acetobacter sacchari sp. nov., for a plant growth-promoting acetic acid bacterium isolated in Vietnam

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Springer Journals
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Copyright © 2019 by Università degli studi di Milano
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/s13213-019-01497-0
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Abstract

Purpose Two bacterial strains, designated as isolates VTH-Ai14 and VTH-Ai15, that have plant growth-promoting ability were isolated during the study on acetic acid bacteria diversity in Vietnam. The phylogenetic analysis based on 16S rRNA gene sequences showed that the two isolates were located closely to Acetobacter nitrogenifigens RG1 but formed an independent cluster. Methods The phylogenetic analysis based on 16S rRNA gene and three housekeeping genes’ (dnaK, groEL, and rpoB) sequences T T were analyzed. The genomic DNA of the two isolates, VTH-Ai14 and VTH-Ai15, Acetobacter nitrogenifigens RG1 , the closest phylogenetic species, and Acetobacter aceti NBRC 14818 were hybridized and calculated the %similarity. Then, phenotypic and chemotaxonomic characteristics were determined for species’ description using the conventional method. Results The 16S rRNA gene and concatenated of the three housekeeping genes phylogenetic analysis suggests that the two isolates were constituted in a species separated from Acetobacter nitrogenifigens, Acetobacter aceti,and Acetobacter sicerae. The two isolates VTH-Ai14 and VTH-Ai15 showed 99.65% and 98.65% similarity of 16S rRNA gene when compared with Acetobacter nitrogenifigens and Acetobacter aceti and they were so different from Acetobacter nitrogenifigens RG1 with 56.99 ± 3.6 and 68.15 ± 1.8% in DNA-DNA hybridization, when isolates VTH-Ai14 and VTH-Ai15 were respectively labeled. Moreover, the two isolates were phenotypically distinguished from Acetobacter nitrogenifigens in growth in the presence of 0.35% acetic acid (v/v), on nitrogen-free LGI medium and D-mannitol, and in no ability to solubilize phosphate. T T T T T Conclusion Therefore, the two isolates, VTH-Ai14 (= VTCC 910031 = BCC 67843 = TBRC 11175 =NRIC0977 ) and VTH- Ai15 (= VTCC 910032 = BCC 67844 = TBRC 11176 = NRIC 0978), can be assigned to an independent species within the genus Acetobacter,and thenameof Acetobacter sacchari sp. nov. is proposed for the two isolates. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13213-019-01497-0) contains supplementary material, which is available to authorized users. * Pattaraporn Yukphan Yuki Muramatsu pattaraporn@biotec.or.th muramatsu-yuki@nite.go.jp Naoto Tanaka Huong Thi Lan Vu n3tanaka@nodai.ac.jp huong_vu14@yahoo.com.vn Somboon Tanasupawat Van Thi Thu Bui somboon.t@chula.ac.th buithuvan86@gmail.com Binh Thanh Le ltbinh24@gmail.com Piyanat Charoenyingcharoen piyanat.cha@biotec.or.th Yasuyoshi Nakagawa nakagawa-yasuyoshi@nite.go.jp Sukunphat Malimas malimas45@hotmail.com Yuzo Yamada ymdy333@kdt.biglobe.ne.jp Linh Khanh Nguyen mtn.nkl1209@gmail.com Extended author information available on the last page of the article 1163 1156 Ann Microbiol (2019) 69:1155–1163 . . . Keywords Acetobacter sacchari sp. nov. Acetic acid bacteria Plant growth-promoting bacteria Vietnam Introduction comprised of D-glucose, ethanol, peptone, yeast extract, and calcium carbonate to select acetic acid bacteria (Vu et al. The genus Acetobacter is the largest in the acetous group of the 2013; Yamada et al. 1999). Acetobacter nitrogenifigens T T family Acetobacteraceae from the viewpoint of generic circum- TBRC 15 (= RG1 ) (Dutta and Gachhui 2006)and scription and includes 28 validly published species at present Acetobacter aceti NBRC 14818 were used as reference strains. (Ferrer et al. 2016; Komagata et al. 2014; Li et al. 2014; The 16S rRNA gene sequences of the two isolates were se- Malimas et al. 2017; Pitiwittayakul et al. 2016, 2015; Spitaels quenced, as described previously (Vu et al. 2013). Sequenced et al. 2014; Yamada 2016). The genus was divided into two were 1,419–1,420 bases for the two isolates. Multiple sequence groups, i.e., the Acetobacter aceti group and the Acetobacter alignments were made with MUSCLE (Edgar 2004). Sequence pasteurianus group phylogenetically (Yamada and Yukphan gaps and ambiguous bases were excluded. A phylogenetic tree 2008). The species of the genus were characterized by the oxi- based on 16S rRNA gene sequences of 1,275 bases was con- dation of acetate and lactate, acetic acid production from ethanol, structed by the maximum likelihood method based on DNA no production of 2,5-diketo-D-gluconic acid from D-glucose, substitution model selected under the Bayesian Information and UQ-9 as major (Komagata et al. 2014; Malimas et al. 2017). Criterion (Kumar et al. 2016) using the program MEGA7 version Acetobacter diazotrophicus was first reported for acetic 5.05 (Kumar et al. 2016). In the phylogenetic tree constructed, acid bacteria capable of nitrogen fixation (Gillis et al. 1989). the type strains of Gluconobacter oxydans, Saccharibacter However, this species was later transferred to the genus floricola, Neokomagataea tanensis,and Swingsia samuiensis Gluconacetobacter as Gluconacetobacter diazotrophicus were used as outgroups. The confidence values of individual (Yamada et al. 1997). branches were calculated by the use of bootstrap analysis of The acetic acid bacteria with plant growth-promoting char- Felsenstein (Felsenstein 1985). acteristics were additionally reported in the genus Acetobacter. Calculation of sequence similarity levels was calculated Muthukumarasamy et al. (2005) isolated some nitrogen-fixing using the EzBioCloud server by pairwise sequence alignment, acetic acid bacteria from wetland rice cultivated in India and in which all gaps were not considered (Kim et al. 2014;Kim identified them as Acetobacter peroxydans. The type strain of et al. 2012; Tindall et al. 2010;Yoonetal. 2017). Acetobacter peroxydans (LMG 1635 ) was also proved to have Extraction and isolation of chromosomal DNAs were made the same characteristics as the isolates mentioned above by the use of the modified method of Marmur (Ezaki et al. (Muthukumarasamy et al. 2005; Pedraza 2008, 2016). 1983;Marmur 1961;Saito andMiura 1963). DNA base com- Acetobacter nitrogenifigens was the second species position was determined by the method of Tamaoka and possessing a nitrogen-fixing ability and considered to be a Komagata (Tamaoka and Komagata 1984). DNA-DNA hy- plant growth-promoting bacterium as well (Dutta and bridization was done with five wells for each reciprocal reac- Gachhui 2006; Pedraza 2008, 2016). The two nitrogen- tions (e.g., A × B and B × A) by the photobiotin-labeling fixing species are quite distance phylogenetically; the former method using microplate wells, as described by Ezaki and was classified in the A. pasteurianus group, and the latter was coauthors (Ezaki et al. 1989). Percent similarities in DNA- in the A. aceti group. DNA hybridization were determined colorimetrically Previously, the two Acetobacter strains, namely isolates (Verlander 1992). The color density was measured at A VTH-Ai14 and VTH-Ai15, were isolated and phylogenetical- on a Synergy™ HTX Multi-Mode Microplate Reader ly located in the A. aceti group and closely related to (BioTek Instruments Inc., USA). Isolated, single-stranded, Acetobacter nitrogenifigens (Vu et al. 2016b). and labeled DNAwas hybridized with DNAs from test strains This paper describes Acetobacter sacchari sp. nov., as an in 2 × SSC containing 50% formamide at 48 °C. The highest additional plant growth-promoting species of the genus and the lowest values were excluded, and the mean of the Acetobacter, for the two isolates that were isolated in Binh remaining three values was taken as a similarity value and Phuoc Province, Vietnam, on January 28th, 2013. calculation of standard derivation. The housekeeping genes dnaK (encoding the heat shock 70 kDa protein), groEL (encoding a 60-kDa chaperonin), and Materials and methods rpoB (encoding the DNA-directed RNA polymerase subunit beta) of the two isolates, VTH-Ai14 and VTH-Ai15, were Two strains, designated as isolates VTH-Ai14 and VTH-Ai15, partially sequenced (Cleenwerck et al. 2010;Li et al. 2014; were isolated from the stems of sugar cane (Saccharum species) Pitiwittayakul et al. 2015). The phylogenetic position based by an enrichment culture approach at pH 3.5 (Vu et al. 2013; on the concatenated sequences of these housekeeping genes Yamada et al. 1999). When microbial growth was seen in the was compared with the type strains of the genus Acetobacter. medium, one loop of the culture was streaked onto an agar plate The respectively concatenated sequences of 528, 579, and 1157 Ann Microbiol (2019) 69:1155–1163 573 bp of partial dnaK, groEL, and rpoB were used for con- The DNA G+C contents of isolates VTH-Ai14 and VTH- structed the phylogenetic analysis. Accession numbers of Ai15 were 59.9 and 59.9 mol%, respectively (Table 1). The dnaK, groEL, and rpoB sequence of the type strains are ac- calculated values were so different from that of cording to the previous study (Cleenwerck et al. 2010;Lietal. A. nitrogenifigens RG1 (64.1 mol%). The DNA-DNA simi- 2014; Pitiwittayakul et al. 2015). larities when the isolate VTH-Ai14 was reciprocally hybrid- Whole-cell fatty acid methyl esters (FAME) of the two ized with VTH-Ai15 were high level at 100.00 ± 7.9% and isolates, VTH-Ai14 and VTH-Ai15, and type strains of 73.10 ± 9.3%. The DNA-DNA similarities of isolates VTH- T T Acetobacter nitrogenifigens TBRC 15 were extracted and Ai14 andVTH-Ai15withthe labeled Acetobacter analyzed as described by Vu et al. (2016a) after grown on nitrogenifigens TBRC 15 were 61.39 ± 8.0% and 44.21 ± GECA medium for 48 h at 28 °C under aerobic conditions. 6.9%. While the isolates VTH-Ai14 and VTH-Ai15 were Additionally, the two isolates, VTH-Ai14 and VTH-Ai15, labeled, the DNA-DNA similarities with Acetobacter T T and Acetobacter nitrogenifigens TBRC 15 were subjected to nitrogenifigens TBRC 15 were 56.99 ± 3.6% and 68.15 ± matrix-assisted laser desorption/ionization time-of-flight mass 1.8%, respectively. All the reciprocal DNA-DNA similarity spectrometry (MALDI-TOF MS) after grown in glucose-yeast between VTH-Ai14 and VTH-Ai15 with Acetobacter aceti extracted-peptone medium consisting of 2.0% glucose, 1.2% NBRC 14818 was between 4.16 ± 0.4% and 15.80 ± 2.1%. peptone, and 0.3% yeast extracted at 30 °C for 18 h with The concatenated sequences of the three housekeeping genes shaking at 200 rpm. The samples were prepared using the (1,676 bp) were constructed with MEGA7 using the maxi- standard extraction method as described by Matsuda et al. mum likelihood model. The DNA substitution GTR+G+I (2012). Then, applied to a MicroFlex LT mass spectrometer was selected under the Bayesian Information Criterion (Bruker Daltonik), and the results were analyzed by MALDI (Kumar et al. 2016). The isolates VTH-Ai14 and VTH- Biotyper 4.1 software (Bruker Daltonik). Escherichia coli Ai15 were grouped together and separated from Acetobacter DH5α was used as a quality control as recommended by the nitrogenifigens TBRC 15 as same as the topologies of the manufacturer on each experiment. phylogenetic tree based on the 16S rRNA gene (Fig. 2). Phenotypic characteristics were determined by the conven- The major cellular fatty acid of the isolates VTH-Ai14 , tional methods (Asai et al. 1964; Gosselé et al. 1980; Kersters VTH-Ai15, and Acetobacter nitrogenifigens TBRC 15 was et al. 2006;Lisdiyanti etal. 2000;Swings et al. 1992; Yamada C18:1ω7c at 55.94%, 57.04%, and 57.94%, respectively et al. 1999, 1976; Yukphan et al. 2011). A major isoprenoid (Table 2). quinone was extracted and quantitatively analyze by HPLC The MALDI-TOF MS profiles of the isolate VTH-Ai14 , (Komagata and Suzuki 1988; Tamaoka et al. 1983; Yamada VTH-Ai15, and Acetobacter nitrogenifigens TBRC 15 were et al. 1969). compared to each other and expressed by identification scores. The isolates VTH-Ai14 and VTH-Ai15 showed identifica- tion scores of 2.66 and 2.67, when comparing with VTH-Ai15 and VTH-Ai14 , respectively. While the identification score Results of the isolates VTH-Ai14 and VTH-Ai15 when comparing with Acetobacter nitrogenifigens TBRC 15 was only 1.88 In a phylogenetic tree deduced from the maximum likelihood and 1.92. The two isolates were discriminated from T T method, isolates VTH-Ai14 and VTH-Ai15 formed a cluster, Acetobacter nitrogenifigens TBRC 15 according to the man- which was connected to the cluster of Acetobacter ufacturer’s recommended log score identification criteria nitrogenifigens RG1 with the bootstrap value of 100% (Matsuda et al. 2012). All the results obtained suggested that (Fig. 1). The resulting cluster was then connected to a cluster the isolates are genetically separated from either Acetobacter containing Acetobacter aceti NBRC 14818 and Acetobacter nitrogenifigens or Acetobacter aceti. T T sicerae LMG 1531 with the bootstrap value of 67%. The The phenotypic characteristics of isolates VTH-Ai14 and pairwise sequence similarities of isolate VTH-Ai14 were VTH-Ai15 were given in the species description of 100, 99.65, 98.65, 98.09, 97.87, 97.87, 97.59, 96.59, and Acetobacter sacchari sp.nov.(Table 1). 96.31% respectively to isolate VTH-Ai15, Acetobacter T T nitrogenifigens RG1 , Acetobacter aceti NBRC 14818 , Acetobacter musti Bo7 , Acetobacter estunensis NBRC Discussion T T 13751 , Acetobacter oeni B13 , Acetobacter sicerae LMG T T 1531 , Acetobacter peroxydans NBRC 13755 ,and Acetobacter nitrogenifigens was first reported as a nitrogen- Acetobacter pasteurianus LMD 22.1 . The phylogenetic data fixing bacterium equipped with polar flagellation and with a obtained suggested that the two isolates constitute a species high G+C content of DNA (64.1 mol% G + C). In general, the separate from either Acetobacter nitrogenifigens or G+C contents of DNA are ranged from 53.5 to 60.7 mol% in Acetobacter aceti. the genus Acetobacter (Komagata et al. 2014; Malimas et al. 1158 Ann Microbiol (2019) 69:1155–1163 Fig. 1 A phylogenetic Acetobacter orleanensis LMG 1583 (AJ419845) relationship of isolates VTH- 50 Acetobacter farinalis G360-1 (AB602333) Ai14 and VTH-Ai15. The Acetobacter malorum LMG 1746 (AJ419844) phylogenetic tree based on 16S Acetobacter cerevisiae LMG 1625 (AJ419843) rRNA gene sequences was constructed by the maximum Acetobacter persici T-120 (AB665070) likelihood method. The type Acetobacter indonesiens NRIC 0313 (AB032356) strains of Gluconobacter Acetobacter thailandicus AD25 (AB937775) oxydans, Saccharibacter Acetobacter cibinongensis 4H-1 (AB052710) floricola, Neokomagataea 81 Acetobacter orientalis 21F-2 (AB052706) tanensis,and Swingsia samuiensis were used as Acetobacter senegalensis CWBI-B418 (AY883036) outgroups. The numerals at the 88 Acetobacter tropicalis NRIC 0312 (AB032354) nodes of the respective branches Acetobacter estunensis LMG 1626 (AJ419838) indicate bootstrap values (%) Acetobacter oeni B13 (AY829472) derived from 1,000 replications. 97 Bar, 0.02% sequence divergence 65 Acetobacter musti Bo7 (HM162854) Acetobacter aceti NBRC 14818 (X74066) 75 T Acetobacter sicerae LMG 1531 (AJ419840) Acetobacter nitrogenifigens RG1 (AY669513) Acetobacter sacchari VTH-Ai15 (LC103251) 86 Acetobacter sacchari VTH-Ai14 (LC103252) Acetobacter pasteurianus LMD 22.1 (X71863) Acetobacter pomorum LMG 18848 (AJ419835) Acetobacter lambici LMG 27439T (HF969863) Acetobacter ghanensis 430A (EF030713) Acetobacter fabarum R-36330 (AM905849) Acetobacter lovaniensis LMG 1617 (AJ419837) Acetobacter syzygii 9H-2 (AB052712) Acetobacter okinawensis 1-35 (AB665068) Acetobacter suratthaniensis BCC 26087 (AB937774) Acetobacter peroxydans NBRC 13755T (AB032352) 77 Acetobacter papayae 1-25 (AB665066) Gluconobacter oxydans NBRC 14819 (X73820) Neokomagataea tanensis BCC 25711 (AB513364) 66 Swingsia samuiensis AH83 (AB786666) Saccharibacter floricola S-877 (AB110421) 0.02 2017). In addition, the peritrichous flagellation appears to be The two isolates were phenotypically distinguished widely distributed in the genus (Komagata et al. 2014; from Acetobacter nitrogenifigens in growth in the pres- Malimas et al. 2017). The present authors’ estimation of ence of 0.35% acetic acid (v/v), nitrogen-free LGI medi- 59.7 mol% G+C and observation of peritrichous flagellation um, and D-mannitol and in no ability to solubilize phos- in Acetobacter nitrogenifigens TBRC 15 may be reasonable phate and from Acetobacter aceti in growth in the pres- (Table 1). ence of 0.35% (v/v) acetic acid, nitrogen-free LGI medi- The result of fatty acid composition showed that C18:1ω7c um, and D-mannitol and in the absence of phosphate sol- is a major fatty acid and the data were consistent with those ubility and production of 2-keto-D-gluconic acid reported for the validly published species of the genus (Komagata et al. 2014; Malimas et al. 2017). In addition, Acetobacter reported by Ferrer et al. (2016). Although, the they were genetically and physiologically discriminated composition of minor fatty acid was slightly different and by DNA-DNA similarities obtained from the reciprocal cannot discriminate the two isolates from the closest known DNA-DNA hybridization, MALDI-TOF MS profiles of species (Li et al. 2014;Spitaels etal. 2014), but the result of the isolate, and the concatenated sequences of the three the two isolates is different from the other genera of acetic acid housekeeping genes from the type strain of Acetobacter bacteria (Li et al. 2015;Vuetal. 2013; Yukphan et al. 2011). nitrogenifigens (Ferrer et al. 2016;Lietal. 2014). 1159 Ann Microbiol (2019) 69:1155–1163 Table 1 Differential a b c b d Characteristic 1 2 3 4 5 6 7 characteristics of Acetobacter sacchari sp. nov., for isolates f,g Flagellation per per per nd per nd per VTH-Ai14 and VTH-Ai15, from the phylogenetically closest Ketogenesis from glycerol + + + + nd – nd species of the genus Acetobacter. Production of ketogluconate from D-glucose (1) A. sacchari isolate VTH- 5-Keto-D-gluconate + + + ++ – + Ai14 ;(2) A. sacchari isolate VTH-Ai15; (3) A. nitrogenifigens 2-Keto-D-gluconate –– – +++ – T T RG1 ;(4) A. aceti NBRC 14818 ; Production of levan-like polysaccharide from (5) A. sicerae LMG 1531 ;(6) f f D-Glucose + + + – nd nd nd A. estunensis NBRC 13751 ;(7) f f D-Fructose + + + – nd nd nd A. oeni B13 ;+,positive; −, f f negative; w, weakly positive; vw, D-Mannitol + + + – nd nd nd f f very weakly positive; per, Glycerol + + + – nd nd nd peritrichous; nd, not determined c c Growth on ammoniac nitrogen with ethanol + + + + ++ – c c Growth in presence of 10% ethanol (v/v)w w + – –– + c c Growth in presence of 30% D-glucose (w/v) –– + – + – – f f Growth in presence of 0.35% acetic (w/v)+ + w w nd nd nd Growth on f f D-Glucose + + w vw nd nd nd c c c D-Fructose w w + – ++ + c c c c D-Sorbitol –– – – ++ – f f D-Mannitol + + – – nd nd nd f f Ethanol + + w + nd nd – f f 2-Propanol w + vw vw nd nd nd Acid production from c c D-Arabinose –– + ww – + h h h h Ethanol + + ++ nd + nd Glycerol vw vw – – nd – nd h f h 1-Propanol ++ + + nd nd nd Solubilization of f f Ca (PO4 ) on Pikovskaya agar medium –– vw vw nd nd nd 3 4 2 ZnOonLGIagarmedium + + + nd ndndnd Production phytohormon IAA with the presence of + + + nd ndndnd L-tryptophan in LGI medium f a a a Growth on nitrogen-free LGI medium + + w – nd – – γ-Pyrone production from D-Fructose –– – – nd – nd D-Glucose vw vw + – nd – nd Ubiquinone system Q-9 Q-9 Q-9 Q-9 Q-9 Q-9 nd f,g DNA G + C content (mol%) 59.9 59.9 59.7 57.2 58.3 59.3 58.1 Dutta and Gachhui (2006) Lisdiyanti et al. (2000) Li et al. (2014) Silva et al. (2006) Spitaels et al. (2014) The present work According to the original authors of the species, flagellation is polar and DNA G+C content is 64.1 mol% (Dutta and Gachhui 2006) Ethanol (1%, v/v) was completely oxidized to carbon dioxide and water for 9, 9, and 5 days respectively in T T A. sacchari isolate VTH-Ai14 , A. sacchari isolate VTH-Ai15, and A. aceti NBRC 14818 , and 1-propanol (1%, v/v) was done for 10 days in A. aceti NBRC 14818 1160 Ann Microbiol (2019) 69:1155–1163 Acetobacter cerevisiae LMG 1625 (KF537424, KF537477, KF537492) Fig. 2 Maximum likelihood tree based on the concatenated T Acetobacter malorum LMG 1746 (KF537431, KF537476, KF537504) sequence (1,676 bp) of dnaK Acetobacter orleanensis LMG 1583 (KF537421, KF537473, KF537507) (528 bp), groEL (579 bp), and Acetobacter persici LMG 26458 (KF537423, KF537471, KF537531) rpoB (573 bp) showing the 63 phylogenetic position of isolates T Acetobacter indonesiensis LMG 19824 (KF537420, KF537463, KF537503) VTH-Ai14 and VTH-Ai15 Acetobacter tropicalis LMG 19825 (KF537411, KF537469, KF537527) within the genus Acetobacter.The Acetobacter pasteurianus LMG 1262 (KF537405, KF537450, KF537510) type strains of Gluconacetobacter liquefaciens and Granulibacter T Acetobacter ghanensis LMG 23848 (HG329535, HG329547, HG329559) bethesdensis were used as Acetobacter okinawensis LMG 26457 (HG329537, HG329549, HG329561) outgroup. Numbers at branching Acetobacter fabarum LMG 24244 (HG329536, HG329548, HG329560) points are percentage bootstrap values based on 1,000 T 100 Acetobacter lovaniensis LMG 1617 (HG329533, HG329545, HG329557) replications. Bar, 0.05% sequence Acetobacter aceti LMG 1504 (FN421342, FN421343, FN421344) divergence Acetobacter sicerae LMG 1531 (KF537395, KF537438, KF537524) Acetobacter estunensis LMG 1626 (KF537394, KF537437, KF537498) Acetobacter oeni LMG 21952 (KF537391, KF537434, KF537506) Acetobacter nitrogenifigens LMG 23498 (KF537390, KF537433, KF537505) VTH-Ai14 (MK751131, MK751133, MK751135) 100 VTH-Ai15 (MK751132, MK751134, MK751136) Gluconacetobacter liquefaciens LMG 1381 (FN391626, FN391699, FN391772) Granulibacter bethesdensis CGDNIH1 (CP000394: 25545-23647, 2432985-2431351, 628088-632263) 0.05 As described above, the two isolates VTH-Ai14 and can therefore be assigned to an independent species within the VTH-Ai15 were separated phenotypically, genetically, and genus Acetobacter, and the name of Acetobacter sacchari sp. physiologically from either Acetobacter nitrogenifigens or nov. is introduced for the two isolates (Table 1). Acetobacter aceti, the phylogenetically closest species (Komagata et al. 2014; Malimas et al. 2017). The two isolates Description of Acetobacter sacchari sp. nov. Table 2 Cellular fatty acid contents (%) of Acetobacter sacchari sp. Acetobacter sacchari (sac’cha,ri. L. gen. sacchari;L.neut. n. nov., for isolates VTH-Ai14 and VTH-Ai15, and the phylogenetically Saccharum sugar cane, from which the two isolates were closest species of the genus Acetobacter.(1) A. sacchari isolate VTH- T T isolated). Ai14 ;(2) A. sacchari isolate VTH-Ai15; (3) A. nitrogenifigens RG1 ; T T (4) A. aceti NBRC 14818 ;(5) A. sicerae LMG 1531 Gram-negative short rods and motile with peritrichous fla- gella, measuring 0.4–0.5 × 0.6–1.5 μm. Colonies are entire, a a Fatty acid 1 2 3 4 5 smooth, transparent, glistening, and creamy to slightly light pink. Catalase is positive, and oxidase is negative. Grows on C : 0.1 0.1 0.1 0.1 – 12 0 LGI medium. Oxidize acetate and lactate. Produces acetic acid Summed feature 2* 1.88 1.78 2.06 3.5 3.2 from ethanol. Does not grow in the presence of 30% D- Summed feature 3 0.44 0.39 0.42 0.3 – glucose (w/v) or 1% potassium nitrate (w/v) but in the presence C : 0.1 0.1 –– – 17 0 of 0.35% acetic acid (v/v). Does not hydrolyze starch and C : 2-OH 2.34 2.31 2.18 0.3 – 18 1 casein. Produces 5-keto-D-gluconate but not 2-keto-D- C : 1.77 1.64 1.5 0.5 1.2 14 0 gluconate and 2,5-diketo-D-gluconate from D-glucose. C : 2-OH 5.66 5.39 5.72 – 8.8 14 0 Produces levan-like polysaccharide in the presence of 3.5% C : 16.63 16.86 14.62 12.0 9.2 16 0 and 5% D-glucose (w/v), D-fructose, and glycerol. Produces C : 2-OH 10.37 9.89 10.73 21.0 5.8 16 0 dihydroxyacetone from glycerol. Very weak production of γ- C : 3-OH 1.67 1.65 1.47 2.9 3.7 16 0 pyrone compounds from D-glucose is shown. C : 1.03 0.91 0.76 0.6 1.2 18 0 Acid is produced from L-arabinose, D-xylose, D-galactose, C : ω7c 55.94 57.04 57.94 50.9 59.6* 18 1 D-glucose, D-mannose, glycerol very weakly, 1-propanol, C : 3-OH 0.68 0.69 0.61 2.6 1.9 18 0 and ethanol, but not from D-arabinose, D-fructose, L-sorbose, C : cyclo ω8c 0.68 0.48 0.94 1.7 3.6 19 0 L-rhamnose, D-mannitol, D-sorbitol, dulcitol, myo-inositol, Ferrer et al. (2016) maltose, lactose, melibiose, sucrose, raffinose, 2-propanol, 1161 Ann Microbiol (2019) 69:1155–1163 Felsenstein J (1985) Confidence limits on phylogenies: an approach using and methanol. Grows on L-arabinose weakly, D-galactose the bootstrap. Evolution 39:783–791. https://doi.org/10.2307/ very weakly, D-glucose, D-mannose very weakly, D- fructose weakly, D-mannitol, glycerol, 1-propanol weakly Ferrer S, Manes-Lazaro R, Benavent-Gil Y, Yepez A, Pardo I (2016) (VTH-Ai15 grows), 2-propanol weakly, but not on D-arabi- Acetobacter musti sp. nov., isolated from Bobal grape must. 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Academic Publisher’snote Springer Nature remains neutral with regard to Press, Boston, pp 185–201. https://doi.org/10.1016/B978-0-12- jurisdictional claims in published maps and institutional affiliations. 426296-6.50012-5 1163 Ann Microbiol (2019) 69:1155–1163 Affiliations 1,2 3 1 3 Huong Thi Lan Vu & Pattaraporn Yukphan & Van Thi Thu Bui & Piyanat Charoenyingcharoen & 4 1 5 6 7 Sukunphat Malimas & Linh Khanh Nguyen & Yuki Muramatsu & Naoto Tanaka & Somboon Tanasupawat & 2 5 3,8,9 Binh Thanh Le & Yasuyoshi Nakagawa & Yuzo Yamada 1 5 Department of Microbiology, Faculty of Biology and Biotechnology, NITE Biological Resource Center, National Institute of Technology University of Science, Vietnam National University-HCM City, 227 and Evaluation, 2-5-8 Kazusa-Kamatari, Kisarazu 292-0818, Japan Nguyen Van Cu Street, Ward 4, District 5, Ho Chi Minh Department of Molecular Microbiology, NODAI Culture Collection City, Vietnam Center, Faculty of Life Sciences, Tokyo University of Agriculture, 1- Graduate University of Science and Technology, Vietnam Academy 1-1 Sakuragaoka, Setagaya, Tokyo 156-8502, Japan of Science and Technology, 18 Hoang Quoc Viet Street, Cau Giay Department of Biochemistry and Microbiology, Faculty of District, Hanoi, Vietnam Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai National Center for Genetic Engineering and Biotechnology Road, Wangmai, Pathumwan, Bangkok 10330, Thailand (BIOTEC), National Science and Technology Development Agency Japan International Cooperation Agency (JICA Senior Overseas (NSTDA), 113 Thailand Science Park, Phahonyothin Road, Khlong Volunteer), Shibuya-ku, Tokyo 151-8558, Japan Nueng, Khlong Luang, Pathum Thani 12120, Thailand Laboratory of Applied Microbiology (Professor Emeritus), Rungrueng-Fertilizer Co., Ltd, 207/5 Moo1, Nong Ya, Muang Department of Applied Biological Chemistry, Faculty of Kanchanaburi, Kanchanaburi 71000, Thailand Agriculture, Shizuoka University, Suruga-ku, Shizuoka 422-8529, Japan

Journal

Annals of Microbiology – Springer Journals

Published: Jul 18, 2019

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