International Journal of Systematic and Evolutionary Microbiology (2014), 64, 2009–2016
DOI 10.1099/ijs.0.055285-0
Rivicola pingtungensis gen. nov., sp. nov., a new member of the family Neisseriaceae isolated from a freshwater river Shih-Yi Sheu,1 Jhen-Ci Chen,1 Chiu-Chung Young2 and Wen-Ming Chen3 Correspondence
1
Wen-Ming Chen [email protected]
2
Department of Marine Biotechnology, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd. Nan-Tzu, Kaohsiung City 811, Taiwan, ROC College of Agriculture and Natural Resources, Department of Soil and Environmental Sciences, National Chung Hsing University, Taichung 402, Taiwan, ROC
3
Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd. Nan-Tzu, Kaohsiung City 811, Taiwan, ROC
A bacterial strain, designated Npb-03T, was isolated from a freshwater river in Taiwan and was characterized using a polyphasic taxonomic approach. The cells were Gram-reaction-negative, straight rod-shaped, non-motile, non-spore-forming and facultatively anaerobic. Growth occurred at 10–37 6C (optimum, 30–35 6C), at pH 6.0–8.0 (optimum, pH 6.0–7.0) and with 0–1.0 % NaCl (optimum, 0 %). The predominant fatty acids were summed feature 3 (comprising C16 : 1v7c and/or C16 : 1v6c) and C16 : 0. The major isoprenoid quinone was Q-8 and the DNA G+C content was 64.1 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an uncharacterized aminolipid and three uncharacterized phospholipids. The major polyamines were putrescine, 2-hydroxyputrescine, cadaverine and spermidine. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain Npb-03T forms a distinct lineage with respect to closely related genera within the family Neisseriaceae of the class Betaproteobacteria, most closely related to the genera Aquaspirillum, Laribacter, Leeia and Microvirgula, and the levels of 16S rRNA gene sequence similarity with respect to the type species of related genera are less than 93 %. On the basis of the genotypic and phenotypic data, strain Npb-03T represents a novel genus and species of the family Neisseriaceae, for which the name Rivicola pingtungensis gen. nov., sp. nov. is proposed. The type strain is Npb-03T (5BCRC 80376T5LMG 26668T5KCTC 23712T).
The family Neisseriaceae is a member of the order Neisseriales within the class Betaproteobacteria (Tønjum et al., 2005). This family has 32 identified genera at the time of writing (http://www.bacterio.net/classifgenerafamilies. html#Neisseriaceae), with representatives isolated from various habitats, such as a freshwater river, freshwater pond, rice field, forest soil, wetland and patient sample (Hylemon et al., 1973; Yuen et al., 2001; Chern et al., 2004; Yang et al., 2005; Chang et al., 2007; Lim et al., 2007; Stackebrandt et al., 2007; Weon et al., 2007; Chen et al., 2010; Yoon et al., 2010). In this study, a novel freshwater member of the family Neisseriaceae, strain Npb-03T, is described. Abbreviations: AL, uncharacterized aminolipid; DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PL uncharacterized phospholipid. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Rivicola pingtungensis Npb-03T is JN104394. Two supplementary figures are available with the online version of this paper.
055285 G 2014 IUMS
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During our investigations of the biodiversity of microorganisms associated with the Beishi River (GPS location: 22u 229 130 N 120u 359 310 E) located in Pingtung County, Taiwan, several cream-coloured bacterial colonies were isolated on R2A agar (BD Difco). Strains showing similar colony morphology were selected and a representative strain, Npb-03T, was selected for detailed analysis. Strain Npb-03T was maintained on R2A agar at 25 uC. Subcultivation was performed on R2A agar at 25 uC for 48–72 h. The isolate was preserved at 280 uC in R2A broth with 20 % (v/v) glycerol or by lyophilization. Genomic DNA was isolated by using a bacterial genomic kit (DP02-150; GeneMark Technology) and the 16S rRNA gene sequence was analysed as described by Chen et al. (2001). Primers FD1 (59-AGAGTTTGATCCTGGCTCAG39; nucleotide positions 8–27 of the Escherichia coli 16S rRNA gene) and RD1 (59-AAGGAGGTGATCCAGCC-39; nucleotide positions 1524–1540 of the E. coli 16S rRNA gene) were used for amplification of bacterial 16S rRNA 2009
S.-Y. Sheu and others
genes by PCR. The PCR product was purified, and direct sequencing was performed by using sequencing primers FD1, RD1, 520F and 800R (Weisburg et al., 1991; Anzai et al., 1997) with a DNA sequencer (ABI Prism 3730; Applied Biosystems). An almost-complete 16S rRNA gene sequence (1424 nt) of strain Npb-03T was compared against 16S rRNA gene sequences available from the EzTaxon-e (Kim et al., 2012), Ribosomal Database Project (Cole et al., 2009) and GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi) databases. Analysis of the sequence data were performed by using the software packages BioEdit (Hall, 1999) and MEGA, version 5 (Tamura et al., 2011), after multiple alignments of the data by CLUSTAL X (Thompson et al., 1997). Distances (corrected according to Kimura’s two-parameter model; Kimura, 1983) were calculated and clustering was performed with the neighbour-joining method (Saitou & Nei, 1987). The maximum-likelihood (Felsenstein, 1981) and maximumparsimony (Kluge & Farris, 1969) trees were generated by using the treeing algorithms contained in the PHYLIP software package (Felsenstein, 1993). In each case bootstrap values were calculated based on 1000 replications. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain Npb-03T formed a distinct phylogenetic lineage with respect to the closely related genera Aquaspirillum (Hylemon et al., 1973), Laribacter (Yuen et al., 2001), Leeia (Lim et al., 2007) and Microvirgula (Patureau et al., 1998) in the family Neisseriaceae of the order Neisseriales within the class Betaproteobacteria in the neighbour-joining tree (Fig. 1). The overall topologies of the phylogenetic trees obtained with the maximum-likelihood and maximumparsimony methods were similar. Sequence similarity calculations (over 1400 bp) indicated that strain Npb-03T was closely related to Aquaspirillum serpens ATCC 12638T (92.2 % 16S rRNA gene sequence similarity), Leeia oryzae HW7T (91.6 %), Aquitalea magnusonii TRO-001DR8T (91.4 %), Laribacter hongkongensis HKU1T (91.2 %), Vogesella indigofera ATCC 19706T (91.2 %) and Microvirgula aerodenitrificans SGLY2T (90.5 %). Aquaspirillum serpens ATCC 12638T and V. indigofera ATCC 19706T were obtained from the American Type Culture Collection (ATCC), Leeia oryzae DSM 17879T was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), and Aquitalea magnusonii LMG 23054T and M. aerodenitrificans LMG 18919T were obtained from the Belgian Coordinated Collections of Microorganisms (BCCM/LMG). All five type strains of type species were used as reference strains for phenotypic and genotypic tests. The morphology of bacterial cells was observed by phasecontrast microscopy (DM 2000; Leica) and by scanning electron microscopy (S-3500N; Hitachi) (Fig. S1 available in the online Supplementary Material) using cells grown in R2A agar at 30 uC for lag, exponential and stationary phases of growth. Cellular motility was tested by the hanging drop method (Beveridge et al., 2007). The Gram Stain Set S (BD Difco) kit and the Ryu non-staining KOH method (Powers, 2010
1995) were used for testing the Gram reaction. Colony morphology was observed on R2A agar using a stereoscopic microscope (SMZ 800; Nikon). The pH range for growth was determined by measuring the optical densities (wavelength 600 nm) of R2A broth cultures. The pH was adjusted prior to sterilization to pH 4–9 (at intervals of 1.0 pH unit) using appropriate biological buffers (Breznak & Costilow, 2007): citrate/ Na2HPO4 buffer, pH 4.0–5.0; phosphate buffer, pH 6.0– 7.0; Tris buffer, pH 8.0–9.0. Verification of the pH after autoclaving revealed only minor changes. The temperature range for growth was determined in R2A broth at 4–50 uC (4, 10, 15, 20, 25, 30, 35, 37, 40, 45 and 50 uC). To investigate the tolerance to NaCl, R2A broth was prepared according to the formula of the BD Difco medium with NaCl concentration adjusted to 0, 0.5 and 1.0–6.0 %, w/v (at intervals of 1.0 %). Growth under anaerobic conditions was determined after incubating strain Npb-03T on R2A agar in the Oxoid AnaeroGen system. Strain Npb-03T was examined for a broad range of phenotypic properties. Activities of catalase, oxidase, DNase, urease and lipase (corn oil), and hydrolysis of starch, casein, gelatin and Tweens 20, 40, 60 and 80 were determined using standard methods (Tindall et al., 2007). Chitin hydrolysis activity was determined by chitinase-detection agar (CDA) plates. Chitin hydrolysis was visualized by the formation of a clear zone around the colonies on CDA plates. CDA plates were prepared as described by Wen et al. (2002). Hydrolysis of CM-cellulose was tested using the method described by Bowman (2000) using R2A agar as the basal medium. Additional, biochemical tests were performed using API ZYM and API 20NE kits (bioMe´rieux) and carbon source utilization was evaluated using the GN2 microplate (Biolog). All commercial phenotypic tests were performed according to the manufacturers’ recommendations. Sensitivity of strain Npb-03T to antibiotics was tested by the disc diffusion method after spreading cell suspensions (0.5 McFarland) on R2A agar (BD Difco) plates. The discs (Oxoid) contained the following antibiotics: ampicillin (10 mg), chloramphenicol (30 mg), gentamicin (10 mg), kanamycin (30 mg), nalidixic acid (30 mg), novobiocin (30 mg), rifampicin (5 mg), penicillin G (10 U), streptomycin (10 mg), sulfamethoxazole (23.75 mg) plus trimethoprim (1.25 mg) and tetracycline (30 mg). The effect of antibiotics on cell growth was assessed after 2 days at 30 uC. The diameter of the antibiotic discs was 8 mm. The strain was considered susceptible when the diameter of the inhibition zone was .13 mm, intermediate at 10–12 mm and resistant at ,10 mm as described by Nokhal & Schlegel (1983). The fatty acid profiles of strain Npb-03T, Aquaspirillum serpens ATCC 12638T, Leeia oryzae DSM 17879T, M. aerodenitrificans LMG 18919T, V. indigofera ATCC 19706T and Aquitalea magnusonii LMG 23054T were analysed using cells grown on R2A agar at 30 uC for 3 days. Fatty acid methyl esters were prepared and separated according to the standard protocol described in the Microbial International Journal of Systematic and Evolutionary Microbiology 64
Rivicola pingtungensis gen. nov., sp. nov.
Amantichitinum ursilacus IGB-41T (FN994890) Silvimonas terrae KM-45T (AB194302) Chitiniphilus shinanonensis SAY3T (AB453176) Deefgea rivuli WB 3.4-79T (AM397080) 71 Andreprevotia chitinilytica JS11-7T (DQ836355) Jeongeupia naejangsanensis BIO-TAS4-2T (FJ669217) Chitinibacter tainanensis BCRC 17254T (AY264287) 74 Chitinilyticum aquatile c14T (DQ314581) Iodobacter fluviatilis NBRC 102505T (AB681839) Formivibrio citricus DSM 6150T (Y17602) Rivicola pingtungensis Npb-03T (JN104394) Aquaspirillum serpens IAM 13944T (AB074518) Laribacter hongkongensis HKU1T (AF389085) Leeia oryzae HW7T (DQ280369) Microvirgula aerodenitrificans SGLY2T (U89333) Vogesella indigofera ATCC 19706T (AB021385) Aquitalea magnusonii TRO-001DR8T (DQ018117) Chromobacterium violaceum ATCC 12472T (AE016825) Gulbenkiania mobilis E4FC31T (AM295491) Paludibacterium yongneupense 5YN8-15T (AM396358) Pseudogulbenkiania subflava BP-5T (EF626692) Vitreoscilla stercoraria ATCC 15218T (L06174) Uruburuella suis CCUG 47806T (AJ586614) Conchiformibius steedae IAM 14972T (AB087261) Kingella kingae ATCC 23330T (AY551999) Alysiella filiformis ATCC 15532T (AF487710) Stenoxybacter acetivorans TAM-DN1T (EF212897) Morococcus cerebrosus CIP 81.93T (JN175352) Neisseria gonorrhoeae NCTC 8375T (X07714) Eikenella corrodens JCM 12952T (AB525415) Bergeriella denitrificans IAM 14975T (AB087265) Simonsiella muelleri ATCC 29453T (AF328147) Proteus hauseri DSM 14437T (KC210868) 99 93
0.01
99 98
70 89 89
78
79
88 91 91
98 73
99 82
99
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the position of strain Npb-03T and type strains of genera in the family Neisseriaceae of the class Betaproteobacteria. Numbers at nodes are bootstrap percentages §70 % based on the neighbour-joining (above nodes) and maximum-parsimony (below nodes) tree-making algorithms. Filled circles indicate branches of the tree that were also recovered using the maximum-likelihood and maximum-parsimony treemaking algorithms. Open circles indicate that the corresponding nodes were also recovered in the tree generated with the maximum-parsimony algorithm. Proteus hauseri DSM 14437T was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.
Identification System (Microbial ID), and identified by MIDI version 6.0 and the RTSBA6.00 database. The complete fatty acid compositions are shown in Table 1. The predominant cellular fatty acids (.10 %) of strain Npb-03T were summed feature 3 (comprising C16 : 1v7c and/or C16 : 1v6c; 52.0 %) and C16 : 0 (22.4 %). The major cellular hydroxy fatty acid was C12 : 0 3-OH (7.7 %). The dominant fatty acids were summed feature 3 and C16 : 0 for the novel species and all phylogenetically closely related species, which is consistent with those of previous publications (Tønjum et al., 2005; Lau et al., 2006; Lim et al., 2007; Subhash et al., 2013). However, the fatty acid profile of strain Npb-03T was distinct from those of all phylogenetically closely related species. In contrast to strain Npb-03T, Aquaspirillum serpens ATCC 12638T had a much higher proportion of fatty acid C18 : 0. The fatty acids C12 : 0 3-OH and C18 : 0 were detected in strain Npb-03T but not in Leeia oryzae DSM 17879T or V. http://ijs.sgmjournals.org
indigofera ATCC 19706T. Contrarily, the fatty acids C10 : 0 3-OH and C10 : 0 were detected in Leeia oryzae DSM 17879T and V. indigofera ATCC 19706T but not in strain Npb-03T. Furthermore, the fatty acid profile of strain Npb-03T differed markedly from that of M. aerodenitrificans LMG 18919T by higher amounts of fatty acid summed feature 3 and absence of fatty acid C17 : 0 cyclo, and from that of Aquitalea magnusonii LMG 23054T by higher amounts of fatty acid summed feature 3, lower amounts of fatty acid C18 : 0 and absence of fatty acid C10 : 0 3-OH. Strain Npb-03T was grown on R2A broth at 30 uC for 2 days and was examined for the determination of isoprenoid quinones, G+C content of genomic DNA, polar lipids and polyamines. Isoprenoid quinones were extracted and purified according to the method of Collins (1994) and were analysed by HPLC. Strain Npb-03T had Q-8 as the major respiratory quinone, which is the same as that of its closest phylogenetic relatives (Tønjum et al., 2005; Lim 2011
S.-Y. Sheu and others
Table 1. Cellular fatty acid compositions of strain Npb-03T and phylogenetically closely related species Strains: 1, Rivicola pingtungensis gen. nov. sp. nov. Npb-03T; 2, Aquaspirillum serpens ATCC 12638T; 3, Leeia oryzae DSM 17879T; 4, M. aerodenitrificans LMG 18919T; 5, V. indigofera ATCC 19706T; 6, Aquitalea magnusonii LMG 23054T. All data were obtained from this study. All strains were grown on R2A agar at 30 uC for 3 days. Values are percentages of the total fatty acids; fatty acids that make up ,1 % of the total are not shown. For unsaturated fatty acids, the position of the double bond is located by counting from the methyl (v) end of the carbon chain. Fatty acid
1
C8 : 0 3-OH – – C10 : 0 – C10 : 0 3-OH C12 : 0 6.3 7.7 C12 : 0 3-OH 1.5 C14 : 0 22.4 C16 : 0 – C17 : 0 cyclo C18 : 0 1.7 7.1 C18 : 1v7c Summed feature 3* 52.0
2
3
4
5
6
– – – 5.0 5.2 1.0 16.5 – 9.3 4.7 57.1
1.3 2.5 2.5 2.9 – 2.0 40.4 3.4 – 2.5 37.7
– – – 7.4 7.8 2.3 25.7 8.8 5.3 14.9 23.7
– 2.7 5.1 3.3 – 1.8 24.4 – – 4.5 52.9
– – 3.9 6.8 2.3 2.8 24.5 – 9.6 8.6 37.2
*Summed features are groups of two or three fatty acids that cannot be separated by GLC using the MIDI system. Summed feature 3 comprises C16 : 1v7c and/or C16 : 1v6c.
et al., 2007; Sheu et al., 2013). The DNA G+C content of strain Npb-03T, determined by HPLC according to Mesbah et al. (1989), was 64.1±1.0 mol%. Polar lipids of cells of strain Npb-03T, Aquaspirillum serpens ATCC 12638T, M. aerodenitrificans LMG 18919T and Aquitalea magnusonii LMG 23054T were extracted and analysed by two-dimensional TLC according to Embley & Wait (1994). Molybdophosphoric acid was used for the detection of the total polar lipids, ninhydrin for amino lipids, Zinzadze reagent for phospholipids, Dragendorff reagent for choline-containing lipids and a-naphthol reagent for glycolipids. Strain Npb-03T exhibited a complex polar lipid profile consisting of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), an uncharacterized aminolipid (AL) and three uncharacterized phospholipids (PL1–PL3) (Fig. S2).
uncharacterized glycolipid was detected in Leeia oryzae DSM 17879T (Chen et al., 2012) but not in the other species. These results suggested that there are some differences in the polar lipid profiles among strain Npb-03T and these phylogenetically closely related species, although they have very similar profiles. Polyamines were extracted from strain Npb-03T and identification was carried out as described by Busse & Auling (1988) and Busse et al. (1997). Cells were homogenized in 0.2 M perchloric acid (HClO4) and centrifuged. Polyamines in the resultant supernatant were treated with dansyl chloride solution (7.5 mg ml21 in acetone) and were then analysed by HPLC on a D-7000 high-speed liquid chromatograph (Hitachi) with a UV-VIS detector L-7420 (Hitachi). The polyamine pattern of strain Npb-03T contained putrescine (42.6 %), 2-hydroxyputrescine (32.8 %), cadaverine (24.2 %) and spermidine (0.4 %). However, the most closely related species, Aquaspirillum serpens ATCC 12638T had only putrescine and 2-hydroxyputrescine as the major components (Tønjum et al., 2005). These data indicated that polyamine patterns are suitable for the discrimination of strain Npb-03T from this close phylogenetic neighbour. Detailed results of physiological, biochemical and morphological characterization of strain Npb-03T are provided in the genus and species descriptions and Table 2. Several chemotaxonomic and physiological properties, such as colony pigmentation, straight rod shape, absence of cellular motility, facultatively anaerobic growth, inability to grow at more than 37 uC, ability to reduce nitrate to nitrite, and the DNA G+C content, distinguished strain Npb-03T from all members of the phylogenetically related genera in the family Neisseriaceae, such as Aquaspirillum, Laribacter, Leeia, Microvirgula, Vogesella and Aquitalea (Table 2).
Strain Npb-03T exhibited a very similar polar lipid profile to the three phylogenetically closely related species; they all contained PE, PG, DPG, AL and PL1. However, PL2 was only detected in strain Npb-03T and an uncharacterized phospholipid (PL4) was only present in M. aerodenitrificans LMG 18919T. In addition, the other two phylogenetically closely related species, Leeia oryzae DSM 17879T and V. indigofera ATCC 19706T, exhibited very similar polar lipid profiles, in which PE, PG and DPG were also predominant (Chen et al., 2012; Sheu et al., 2013). However, an
Some features of strain Npb-03T, such as straight rod shape, absence of cellular motility, facultatively anaerobic growth and inability to grow at more than 37 uC, may be helpful for separating the novel strain from the phylogenetically related genera Aquaspirillum and Leeia. Phenotypic properties such as colony pigmentation, straight rod shape, absence of cellular motility and inability to grow at more than 37 uC distinguished strain Npb-03T from all members of the phylogenetically related genera Microvirgula and Aquitalea. In addition, strain Npb-03T could also be differentiated from the phylogenetically related genus Laribacter on the basis of colony pigmentation, straight rod shape, inability to grow at more than 37 uC, inability to grow with more than 1 % NaCl and the DNA G+C content. Furthermore, all species of the phylogenetically related genus Vogesella except Vogesella alkaliphila JC141T are motile and exhibit aerobic growth. Thus, facultatively anaerobic growth and the absence of cellular motility in strain Npb-03T could distinguish this novel strain from these motile species. In addition, phenotypic properties such as colony pigmentation, inability to grow at pH greater than pH 8.0 and the ability of strain Npb-03T to
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Table 2. Characteristics that differentiate strain Npb-03T from other phylogenetically related genera in the family Neisseriaceae Taxa: 1, Rivicola pingtungensis gen. nov., sp. nov. (strain Npb-03); 2, Aquaspirillum (Hylemon et al., 1973; Tønjum et al., 2005); 3, Laribacter (Yuen et al., 2001); 4, Leeia (Lim et al., 2007); 5, Microvirgula (Patureau et al., 1998); 6, Vogesella (Grimes et al., 1997; Chou et al., 2008, 2009; Jørgensen et al., 2010; Sheu et al., 2013; Subhash et al., 2013); 7, Aquitalea (Lau et al., 2006; Lee et al., 2009). +, Positive reaction; V, variable; 2, negative reaction; ND, no data available. Characteristic Source Colony pigmentation Cell shape Motility Relation to O2
1
2
3
4
5
6
7
Freshwater river
Freshwater
Patient
Rice field
Activated sludge
Cream
Cream
Grey
Cream
Translucent
Humic lake, wetland Tan, yellow
Straight rods
Spiral rods
Seagull-shaped or spiral rods 2 Facultatively anaerobic 25–42
Short rods
Curved rods
Pond sediment, freshwater, soil Blue, colourless to white, slightly greyish Straight to slightly curved rods
+ Strictly aerobic
+ Facultatively anaerobic 15–45 (35) 6.0–7.0 (7.0) 0–1.0* + 65
2 Facultatively anaerobic Temperature range for growth (uC) (optimal) 10–37 (30–35) pH range for growth (optimal) 6.0–8.0 (6.0–7.0) NaCl range for growth (%) 0–1.0 Nitrate reduction + DNA G+C content (mol%) 64.1
8–44 (30–32) 5.5–9.0 (6.5–7.5) ,3.0 V
49–66
ND
0–2.0 + 68
10–40 (32-35) 5.0–8.5 (6.0) 0–0.5* + 56
V
Strictly aerobic 4–45 (20–37) 6.0–10.0 (6.0–8.5) 0–3.0 V
61.2–65.4
2013
Rivicola pingtungensis gen. nov., sp. nov.
*Results were obtained in this study.
+ Aerobic
Straight to slightly curved rods + Facultatively anaerobic 10–40 (30) 5.0–9.0 (6.0–9.0) 0–1.0 + 59.2–64.0
S.-Y. Sheu and others
reduce nitrate distinguish it from V. alkaliphila JC141T. Therefore, strain Npb-03T could be separated from all species of the genus Vogesella by phenotypic differences. It is now generally accepted that 16S rRNA gene sequence similarities of less than 95 % between two bacteria are an indication of affiliation to different genera (Ludwig et al., 1998). Strain Npb-03T most likely represents a species of a new genus, since the 16S rRNA gene sequence similarity to its closest relatives with validly published names, Aquaspirillum serpens ATCC 12638T, Leeia oryzae HW7T, Aquitalea magnusonii TRO-001DR8T, Laribacter hongkongensis HKU1T, V. indigofera ATCC 19706T and M. aerodenitrificans SGLY2T, is 90.5–92.2 %. Moreover, strain Npb-03T can be readily distinguished from these close phylogenetic neighbours by fatty acid composition, polar lipid profiles and physiological and biochemical characteristics. Therefore, based on phenotypic and phylogenetic criteria, we are of the opinion that strain Npb-03T should be assigned to a new genus and novel species, for which the name Rivicola pingtungensis gen. nov., sp. nov. is proposed. Description of Rivicola gen. nov. Rivicola [Ri.vi9co.la. L. masc. n. rivus creek or small river; L. suff. -cola (from L. masc. or fem. n. incola) inhabitant, dweller; N.L. masc. n. Rivicola river dweller]. Cells are Gram-reaction-negative, straight rod-shaped, non-motile, non-spore-forming and facultatively anaerobic. Oxidase and catalase are positive. The predominant quinone is Q-8. The predominant fatty acids are summed feature 3 (comprising C16 : 1v7c and/or C16 : 1v6c) and C16 : 0. PE, PG, DPG, an AL and three PLs are present in the polar lipid profile. The polyamine pattern contains putrescine, 2-hydroxyputrescine, cadaverine and spermidine. The DNA G+C content of the type strain of the type species is 64.1 mol%. The type species is Rivicola pingtungensis.
gluconate, caprate, adipate and malate, and negative reactions for indole production, glucose fermentation, aesculin and gelatin hydrolysis, arginine dihydrolase and bgalactosidase activities, and assimilation of citrate and phenylacetate. In the API ZYM kit, C4 esterase, C8 esterase lipase, acid phosphatase and naphthol-AS-BI-phosphohydrolase activities are present and alkaline phosphatase, C14 lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, bgalactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase activities are absent. The following compounds are utilized as sole carbon sources in the GN2 microplate: alactose, lactulose, D-mannitol, pyruvic acid methyl ester, acetic acid, cis-aconitic acid, citric acid, formic acid, phydroxyphenylacetic acid, itaconic acid, DL-lactic acid, propionic acid, succinic acid, bromosuccinic acid, succinamic acid, D-alanine, L-alanine, L-alanyl glycine, L-asparagine, L-glutamic acid, L-proline, L-serine and urocanic acid. All other substrates in the GN2 microplate are not utilized. Sensitive to penicillin G, ampicillin, chloramphenicol, gentamicin, rifampicin, kanamycin, tetracycline, novobiocin, streptomycin, sulfamethoxazole plus trimethoprim and nalidixic acid. The type strain is Npb-03T (5BCRC 80376T5LMG 26668T5 KCTC 23712T), isolated from a freshwater sample collected from the Jin-Shih Lake, Kaoshiung County, Taiwan.
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Chryseomonas, Flavimonas, and Pseudomonas supports synonymy of these three genera. Int J Syst Bacteriol 47, 249–251. Beveridge, T. J., Lawrence, J. R. & Murray, R. G. E. (2007). Sampling
and staining for light microscopy. In Methods for General and Molecular Bacteriology, 3rd edn, pp. 19–33. Edited by C. A. Reddy, T. J. Beveridge, J. A. Breznak, G. A. Marzluf, T. M. Schmidt & L. R. Snyder. Washington, DC: American Society for Microbiology.
Description of Rivicola pingtungensis sp. nov.
Bowman, J. P. (2000). Description of Cellulophaga algicola sp. nov.,
Rivicola pingtungensis (ping.tung.en9sis. N.L. masc. adj. pingtungensis pertaining to Pingtung, a town in Southern Taiwan).
isolated from the surfaces of Antarctic algae, and reclassification of Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Cellulophaga uliginosa comb. nov. Int J Syst Evol Microbiol 50, 1861– 1868.
Displays the following properties in addition to those given in the genus description. Cells are approximately 0.3– 0.6 mm in diameter and 1.4–3.2 mm in length after 48 h of incubation on R2A agar at 30 uC. Colonies are cream, convex and circular with irregular, curled margins. Colony size is approximately 0.7–1.2 mm in diameter on R2A agar after 48 h of incubation at 30 uC. Growth occurs at 10– 37 uC (optimum, 30–35 uC), at pH 6.0–8.0 (optimum, pH 6.0–7.0) and with 0–1.0 % NaCl (optimum, 0 %). Positive for urease activity, and hydrolysis of Tweens 20 and 60. Negative for DNase activity, and hydrolysis of starch, chitin, casein, corn oil, CM-cellulose and Tweens 40 and 80. In API 20NE tests, positive reactions for nitrate reduction, urease activity, assimilation of glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose,
Breznak, J. A. & Costilow, R. N. (2007). Physicochemical factors in
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2016
International Journal of Systematic and Evolutionary Microbiology 64
DOI 10.1099/ijs.0.055285-0
Rivicola pingtungensis gen. nov., sp. nov., a new member of the family Neisseriaceae isolated from a freshwater river Shih-Yi Sheu,1 Jhen-Ci Chen,1 Chiu-Chung Young2 and Wen-Ming Chen3 Correspondence
1
Wen-Ming Chen [email protected]
2
Department of Marine Biotechnology, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd. Nan-Tzu, Kaohsiung City 811, Taiwan, ROC College of Agriculture and Natural Resources, Department of Soil and Environmental Sciences, National Chung Hsing University, Taichung 402, Taiwan, ROC
3
Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd. Nan-Tzu, Kaohsiung City 811, Taiwan, ROC
A bacterial strain, designated Npb-03T, was isolated from a freshwater river in Taiwan and was characterized using a polyphasic taxonomic approach. The cells were Gram-reaction-negative, straight rod-shaped, non-motile, non-spore-forming and facultatively anaerobic. Growth occurred at 10–37 6C (optimum, 30–35 6C), at pH 6.0–8.0 (optimum, pH 6.0–7.0) and with 0–1.0 % NaCl (optimum, 0 %). The predominant fatty acids were summed feature 3 (comprising C16 : 1v7c and/or C16 : 1v6c) and C16 : 0. The major isoprenoid quinone was Q-8 and the DNA G+C content was 64.1 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an uncharacterized aminolipid and three uncharacterized phospholipids. The major polyamines were putrescine, 2-hydroxyputrescine, cadaverine and spermidine. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain Npb-03T forms a distinct lineage with respect to closely related genera within the family Neisseriaceae of the class Betaproteobacteria, most closely related to the genera Aquaspirillum, Laribacter, Leeia and Microvirgula, and the levels of 16S rRNA gene sequence similarity with respect to the type species of related genera are less than 93 %. On the basis of the genotypic and phenotypic data, strain Npb-03T represents a novel genus and species of the family Neisseriaceae, for which the name Rivicola pingtungensis gen. nov., sp. nov. is proposed. The type strain is Npb-03T (5BCRC 80376T5LMG 26668T5KCTC 23712T).
The family Neisseriaceae is a member of the order Neisseriales within the class Betaproteobacteria (Tønjum et al., 2005). This family has 32 identified genera at the time of writing (http://www.bacterio.net/classifgenerafamilies. html#Neisseriaceae), with representatives isolated from various habitats, such as a freshwater river, freshwater pond, rice field, forest soil, wetland and patient sample (Hylemon et al., 1973; Yuen et al., 2001; Chern et al., 2004; Yang et al., 2005; Chang et al., 2007; Lim et al., 2007; Stackebrandt et al., 2007; Weon et al., 2007; Chen et al., 2010; Yoon et al., 2010). In this study, a novel freshwater member of the family Neisseriaceae, strain Npb-03T, is described. Abbreviations: AL, uncharacterized aminolipid; DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PL uncharacterized phospholipid. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Rivicola pingtungensis Npb-03T is JN104394. Two supplementary figures are available with the online version of this paper.
055285 G 2014 IUMS
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During our investigations of the biodiversity of microorganisms associated with the Beishi River (GPS location: 22u 229 130 N 120u 359 310 E) located in Pingtung County, Taiwan, several cream-coloured bacterial colonies were isolated on R2A agar (BD Difco). Strains showing similar colony morphology were selected and a representative strain, Npb-03T, was selected for detailed analysis. Strain Npb-03T was maintained on R2A agar at 25 uC. Subcultivation was performed on R2A agar at 25 uC for 48–72 h. The isolate was preserved at 280 uC in R2A broth with 20 % (v/v) glycerol or by lyophilization. Genomic DNA was isolated by using a bacterial genomic kit (DP02-150; GeneMark Technology) and the 16S rRNA gene sequence was analysed as described by Chen et al. (2001). Primers FD1 (59-AGAGTTTGATCCTGGCTCAG39; nucleotide positions 8–27 of the Escherichia coli 16S rRNA gene) and RD1 (59-AAGGAGGTGATCCAGCC-39; nucleotide positions 1524–1540 of the E. coli 16S rRNA gene) were used for amplification of bacterial 16S rRNA 2009
S.-Y. Sheu and others
genes by PCR. The PCR product was purified, and direct sequencing was performed by using sequencing primers FD1, RD1, 520F and 800R (Weisburg et al., 1991; Anzai et al., 1997) with a DNA sequencer (ABI Prism 3730; Applied Biosystems). An almost-complete 16S rRNA gene sequence (1424 nt) of strain Npb-03T was compared against 16S rRNA gene sequences available from the EzTaxon-e (Kim et al., 2012), Ribosomal Database Project (Cole et al., 2009) and GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi) databases. Analysis of the sequence data were performed by using the software packages BioEdit (Hall, 1999) and MEGA, version 5 (Tamura et al., 2011), after multiple alignments of the data by CLUSTAL X (Thompson et al., 1997). Distances (corrected according to Kimura’s two-parameter model; Kimura, 1983) were calculated and clustering was performed with the neighbour-joining method (Saitou & Nei, 1987). The maximum-likelihood (Felsenstein, 1981) and maximumparsimony (Kluge & Farris, 1969) trees were generated by using the treeing algorithms contained in the PHYLIP software package (Felsenstein, 1993). In each case bootstrap values were calculated based on 1000 replications. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain Npb-03T formed a distinct phylogenetic lineage with respect to the closely related genera Aquaspirillum (Hylemon et al., 1973), Laribacter (Yuen et al., 2001), Leeia (Lim et al., 2007) and Microvirgula (Patureau et al., 1998) in the family Neisseriaceae of the order Neisseriales within the class Betaproteobacteria in the neighbour-joining tree (Fig. 1). The overall topologies of the phylogenetic trees obtained with the maximum-likelihood and maximumparsimony methods were similar. Sequence similarity calculations (over 1400 bp) indicated that strain Npb-03T was closely related to Aquaspirillum serpens ATCC 12638T (92.2 % 16S rRNA gene sequence similarity), Leeia oryzae HW7T (91.6 %), Aquitalea magnusonii TRO-001DR8T (91.4 %), Laribacter hongkongensis HKU1T (91.2 %), Vogesella indigofera ATCC 19706T (91.2 %) and Microvirgula aerodenitrificans SGLY2T (90.5 %). Aquaspirillum serpens ATCC 12638T and V. indigofera ATCC 19706T were obtained from the American Type Culture Collection (ATCC), Leeia oryzae DSM 17879T was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), and Aquitalea magnusonii LMG 23054T and M. aerodenitrificans LMG 18919T were obtained from the Belgian Coordinated Collections of Microorganisms (BCCM/LMG). All five type strains of type species were used as reference strains for phenotypic and genotypic tests. The morphology of bacterial cells was observed by phasecontrast microscopy (DM 2000; Leica) and by scanning electron microscopy (S-3500N; Hitachi) (Fig. S1 available in the online Supplementary Material) using cells grown in R2A agar at 30 uC for lag, exponential and stationary phases of growth. Cellular motility was tested by the hanging drop method (Beveridge et al., 2007). The Gram Stain Set S (BD Difco) kit and the Ryu non-staining KOH method (Powers, 2010
1995) were used for testing the Gram reaction. Colony morphology was observed on R2A agar using a stereoscopic microscope (SMZ 800; Nikon). The pH range for growth was determined by measuring the optical densities (wavelength 600 nm) of R2A broth cultures. The pH was adjusted prior to sterilization to pH 4–9 (at intervals of 1.0 pH unit) using appropriate biological buffers (Breznak & Costilow, 2007): citrate/ Na2HPO4 buffer, pH 4.0–5.0; phosphate buffer, pH 6.0– 7.0; Tris buffer, pH 8.0–9.0. Verification of the pH after autoclaving revealed only minor changes. The temperature range for growth was determined in R2A broth at 4–50 uC (4, 10, 15, 20, 25, 30, 35, 37, 40, 45 and 50 uC). To investigate the tolerance to NaCl, R2A broth was prepared according to the formula of the BD Difco medium with NaCl concentration adjusted to 0, 0.5 and 1.0–6.0 %, w/v (at intervals of 1.0 %). Growth under anaerobic conditions was determined after incubating strain Npb-03T on R2A agar in the Oxoid AnaeroGen system. Strain Npb-03T was examined for a broad range of phenotypic properties. Activities of catalase, oxidase, DNase, urease and lipase (corn oil), and hydrolysis of starch, casein, gelatin and Tweens 20, 40, 60 and 80 were determined using standard methods (Tindall et al., 2007). Chitin hydrolysis activity was determined by chitinase-detection agar (CDA) plates. Chitin hydrolysis was visualized by the formation of a clear zone around the colonies on CDA plates. CDA plates were prepared as described by Wen et al. (2002). Hydrolysis of CM-cellulose was tested using the method described by Bowman (2000) using R2A agar as the basal medium. Additional, biochemical tests were performed using API ZYM and API 20NE kits (bioMe´rieux) and carbon source utilization was evaluated using the GN2 microplate (Biolog). All commercial phenotypic tests were performed according to the manufacturers’ recommendations. Sensitivity of strain Npb-03T to antibiotics was tested by the disc diffusion method after spreading cell suspensions (0.5 McFarland) on R2A agar (BD Difco) plates. The discs (Oxoid) contained the following antibiotics: ampicillin (10 mg), chloramphenicol (30 mg), gentamicin (10 mg), kanamycin (30 mg), nalidixic acid (30 mg), novobiocin (30 mg), rifampicin (5 mg), penicillin G (10 U), streptomycin (10 mg), sulfamethoxazole (23.75 mg) plus trimethoprim (1.25 mg) and tetracycline (30 mg). The effect of antibiotics on cell growth was assessed after 2 days at 30 uC. The diameter of the antibiotic discs was 8 mm. The strain was considered susceptible when the diameter of the inhibition zone was .13 mm, intermediate at 10–12 mm and resistant at ,10 mm as described by Nokhal & Schlegel (1983). The fatty acid profiles of strain Npb-03T, Aquaspirillum serpens ATCC 12638T, Leeia oryzae DSM 17879T, M. aerodenitrificans LMG 18919T, V. indigofera ATCC 19706T and Aquitalea magnusonii LMG 23054T were analysed using cells grown on R2A agar at 30 uC for 3 days. Fatty acid methyl esters were prepared and separated according to the standard protocol described in the Microbial International Journal of Systematic and Evolutionary Microbiology 64
Rivicola pingtungensis gen. nov., sp. nov.
Amantichitinum ursilacus IGB-41T (FN994890) Silvimonas terrae KM-45T (AB194302) Chitiniphilus shinanonensis SAY3T (AB453176) Deefgea rivuli WB 3.4-79T (AM397080) 71 Andreprevotia chitinilytica JS11-7T (DQ836355) Jeongeupia naejangsanensis BIO-TAS4-2T (FJ669217) Chitinibacter tainanensis BCRC 17254T (AY264287) 74 Chitinilyticum aquatile c14T (DQ314581) Iodobacter fluviatilis NBRC 102505T (AB681839) Formivibrio citricus DSM 6150T (Y17602) Rivicola pingtungensis Npb-03T (JN104394) Aquaspirillum serpens IAM 13944T (AB074518) Laribacter hongkongensis HKU1T (AF389085) Leeia oryzae HW7T (DQ280369) Microvirgula aerodenitrificans SGLY2T (U89333) Vogesella indigofera ATCC 19706T (AB021385) Aquitalea magnusonii TRO-001DR8T (DQ018117) Chromobacterium violaceum ATCC 12472T (AE016825) Gulbenkiania mobilis E4FC31T (AM295491) Paludibacterium yongneupense 5YN8-15T (AM396358) Pseudogulbenkiania subflava BP-5T (EF626692) Vitreoscilla stercoraria ATCC 15218T (L06174) Uruburuella suis CCUG 47806T (AJ586614) Conchiformibius steedae IAM 14972T (AB087261) Kingella kingae ATCC 23330T (AY551999) Alysiella filiformis ATCC 15532T (AF487710) Stenoxybacter acetivorans TAM-DN1T (EF212897) Morococcus cerebrosus CIP 81.93T (JN175352) Neisseria gonorrhoeae NCTC 8375T (X07714) Eikenella corrodens JCM 12952T (AB525415) Bergeriella denitrificans IAM 14975T (AB087265) Simonsiella muelleri ATCC 29453T (AF328147) Proteus hauseri DSM 14437T (KC210868) 99 93
0.01
99 98
70 89 89
78
79
88 91 91
98 73
99 82
99
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the position of strain Npb-03T and type strains of genera in the family Neisseriaceae of the class Betaproteobacteria. Numbers at nodes are bootstrap percentages §70 % based on the neighbour-joining (above nodes) and maximum-parsimony (below nodes) tree-making algorithms. Filled circles indicate branches of the tree that were also recovered using the maximum-likelihood and maximum-parsimony treemaking algorithms. Open circles indicate that the corresponding nodes were also recovered in the tree generated with the maximum-parsimony algorithm. Proteus hauseri DSM 14437T was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.
Identification System (Microbial ID), and identified by MIDI version 6.0 and the RTSBA6.00 database. The complete fatty acid compositions are shown in Table 1. The predominant cellular fatty acids (.10 %) of strain Npb-03T were summed feature 3 (comprising C16 : 1v7c and/or C16 : 1v6c; 52.0 %) and C16 : 0 (22.4 %). The major cellular hydroxy fatty acid was C12 : 0 3-OH (7.7 %). The dominant fatty acids were summed feature 3 and C16 : 0 for the novel species and all phylogenetically closely related species, which is consistent with those of previous publications (Tønjum et al., 2005; Lau et al., 2006; Lim et al., 2007; Subhash et al., 2013). However, the fatty acid profile of strain Npb-03T was distinct from those of all phylogenetically closely related species. In contrast to strain Npb-03T, Aquaspirillum serpens ATCC 12638T had a much higher proportion of fatty acid C18 : 0. The fatty acids C12 : 0 3-OH and C18 : 0 were detected in strain Npb-03T but not in Leeia oryzae DSM 17879T or V. http://ijs.sgmjournals.org
indigofera ATCC 19706T. Contrarily, the fatty acids C10 : 0 3-OH and C10 : 0 were detected in Leeia oryzae DSM 17879T and V. indigofera ATCC 19706T but not in strain Npb-03T. Furthermore, the fatty acid profile of strain Npb-03T differed markedly from that of M. aerodenitrificans LMG 18919T by higher amounts of fatty acid summed feature 3 and absence of fatty acid C17 : 0 cyclo, and from that of Aquitalea magnusonii LMG 23054T by higher amounts of fatty acid summed feature 3, lower amounts of fatty acid C18 : 0 and absence of fatty acid C10 : 0 3-OH. Strain Npb-03T was grown on R2A broth at 30 uC for 2 days and was examined for the determination of isoprenoid quinones, G+C content of genomic DNA, polar lipids and polyamines. Isoprenoid quinones were extracted and purified according to the method of Collins (1994) and were analysed by HPLC. Strain Npb-03T had Q-8 as the major respiratory quinone, which is the same as that of its closest phylogenetic relatives (Tønjum et al., 2005; Lim 2011
S.-Y. Sheu and others
Table 1. Cellular fatty acid compositions of strain Npb-03T and phylogenetically closely related species Strains: 1, Rivicola pingtungensis gen. nov. sp. nov. Npb-03T; 2, Aquaspirillum serpens ATCC 12638T; 3, Leeia oryzae DSM 17879T; 4, M. aerodenitrificans LMG 18919T; 5, V. indigofera ATCC 19706T; 6, Aquitalea magnusonii LMG 23054T. All data were obtained from this study. All strains were grown on R2A agar at 30 uC for 3 days. Values are percentages of the total fatty acids; fatty acids that make up ,1 % of the total are not shown. For unsaturated fatty acids, the position of the double bond is located by counting from the methyl (v) end of the carbon chain. Fatty acid
1
C8 : 0 3-OH – – C10 : 0 – C10 : 0 3-OH C12 : 0 6.3 7.7 C12 : 0 3-OH 1.5 C14 : 0 22.4 C16 : 0 – C17 : 0 cyclo C18 : 0 1.7 7.1 C18 : 1v7c Summed feature 3* 52.0
2
3
4
5
6
– – – 5.0 5.2 1.0 16.5 – 9.3 4.7 57.1
1.3 2.5 2.5 2.9 – 2.0 40.4 3.4 – 2.5 37.7
– – – 7.4 7.8 2.3 25.7 8.8 5.3 14.9 23.7
– 2.7 5.1 3.3 – 1.8 24.4 – – 4.5 52.9
– – 3.9 6.8 2.3 2.8 24.5 – 9.6 8.6 37.2
*Summed features are groups of two or three fatty acids that cannot be separated by GLC using the MIDI system. Summed feature 3 comprises C16 : 1v7c and/or C16 : 1v6c.
et al., 2007; Sheu et al., 2013). The DNA G+C content of strain Npb-03T, determined by HPLC according to Mesbah et al. (1989), was 64.1±1.0 mol%. Polar lipids of cells of strain Npb-03T, Aquaspirillum serpens ATCC 12638T, M. aerodenitrificans LMG 18919T and Aquitalea magnusonii LMG 23054T were extracted and analysed by two-dimensional TLC according to Embley & Wait (1994). Molybdophosphoric acid was used for the detection of the total polar lipids, ninhydrin for amino lipids, Zinzadze reagent for phospholipids, Dragendorff reagent for choline-containing lipids and a-naphthol reagent for glycolipids. Strain Npb-03T exhibited a complex polar lipid profile consisting of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), an uncharacterized aminolipid (AL) and three uncharacterized phospholipids (PL1–PL3) (Fig. S2).
uncharacterized glycolipid was detected in Leeia oryzae DSM 17879T (Chen et al., 2012) but not in the other species. These results suggested that there are some differences in the polar lipid profiles among strain Npb-03T and these phylogenetically closely related species, although they have very similar profiles. Polyamines were extracted from strain Npb-03T and identification was carried out as described by Busse & Auling (1988) and Busse et al. (1997). Cells were homogenized in 0.2 M perchloric acid (HClO4) and centrifuged. Polyamines in the resultant supernatant were treated with dansyl chloride solution (7.5 mg ml21 in acetone) and were then analysed by HPLC on a D-7000 high-speed liquid chromatograph (Hitachi) with a UV-VIS detector L-7420 (Hitachi). The polyamine pattern of strain Npb-03T contained putrescine (42.6 %), 2-hydroxyputrescine (32.8 %), cadaverine (24.2 %) and spermidine (0.4 %). However, the most closely related species, Aquaspirillum serpens ATCC 12638T had only putrescine and 2-hydroxyputrescine as the major components (Tønjum et al., 2005). These data indicated that polyamine patterns are suitable for the discrimination of strain Npb-03T from this close phylogenetic neighbour. Detailed results of physiological, biochemical and morphological characterization of strain Npb-03T are provided in the genus and species descriptions and Table 2. Several chemotaxonomic and physiological properties, such as colony pigmentation, straight rod shape, absence of cellular motility, facultatively anaerobic growth, inability to grow at more than 37 uC, ability to reduce nitrate to nitrite, and the DNA G+C content, distinguished strain Npb-03T from all members of the phylogenetically related genera in the family Neisseriaceae, such as Aquaspirillum, Laribacter, Leeia, Microvirgula, Vogesella and Aquitalea (Table 2).
Strain Npb-03T exhibited a very similar polar lipid profile to the three phylogenetically closely related species; they all contained PE, PG, DPG, AL and PL1. However, PL2 was only detected in strain Npb-03T and an uncharacterized phospholipid (PL4) was only present in M. aerodenitrificans LMG 18919T. In addition, the other two phylogenetically closely related species, Leeia oryzae DSM 17879T and V. indigofera ATCC 19706T, exhibited very similar polar lipid profiles, in which PE, PG and DPG were also predominant (Chen et al., 2012; Sheu et al., 2013). However, an
Some features of strain Npb-03T, such as straight rod shape, absence of cellular motility, facultatively anaerobic growth and inability to grow at more than 37 uC, may be helpful for separating the novel strain from the phylogenetically related genera Aquaspirillum and Leeia. Phenotypic properties such as colony pigmentation, straight rod shape, absence of cellular motility and inability to grow at more than 37 uC distinguished strain Npb-03T from all members of the phylogenetically related genera Microvirgula and Aquitalea. In addition, strain Npb-03T could also be differentiated from the phylogenetically related genus Laribacter on the basis of colony pigmentation, straight rod shape, inability to grow at more than 37 uC, inability to grow with more than 1 % NaCl and the DNA G+C content. Furthermore, all species of the phylogenetically related genus Vogesella except Vogesella alkaliphila JC141T are motile and exhibit aerobic growth. Thus, facultatively anaerobic growth and the absence of cellular motility in strain Npb-03T could distinguish this novel strain from these motile species. In addition, phenotypic properties such as colony pigmentation, inability to grow at pH greater than pH 8.0 and the ability of strain Npb-03T to
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Table 2. Characteristics that differentiate strain Npb-03T from other phylogenetically related genera in the family Neisseriaceae Taxa: 1, Rivicola pingtungensis gen. nov., sp. nov. (strain Npb-03); 2, Aquaspirillum (Hylemon et al., 1973; Tønjum et al., 2005); 3, Laribacter (Yuen et al., 2001); 4, Leeia (Lim et al., 2007); 5, Microvirgula (Patureau et al., 1998); 6, Vogesella (Grimes et al., 1997; Chou et al., 2008, 2009; Jørgensen et al., 2010; Sheu et al., 2013; Subhash et al., 2013); 7, Aquitalea (Lau et al., 2006; Lee et al., 2009). +, Positive reaction; V, variable; 2, negative reaction; ND, no data available. Characteristic Source Colony pigmentation Cell shape Motility Relation to O2
1
2
3
4
5
6
7
Freshwater river
Freshwater
Patient
Rice field
Activated sludge
Cream
Cream
Grey
Cream
Translucent
Humic lake, wetland Tan, yellow
Straight rods
Spiral rods
Seagull-shaped or spiral rods 2 Facultatively anaerobic 25–42
Short rods
Curved rods
Pond sediment, freshwater, soil Blue, colourless to white, slightly greyish Straight to slightly curved rods
+ Strictly aerobic
+ Facultatively anaerobic 15–45 (35) 6.0–7.0 (7.0) 0–1.0* + 65
2 Facultatively anaerobic Temperature range for growth (uC) (optimal) 10–37 (30–35) pH range for growth (optimal) 6.0–8.0 (6.0–7.0) NaCl range for growth (%) 0–1.0 Nitrate reduction + DNA G+C content (mol%) 64.1
8–44 (30–32) 5.5–9.0 (6.5–7.5) ,3.0 V
49–66
ND
0–2.0 + 68
10–40 (32-35) 5.0–8.5 (6.0) 0–0.5* + 56
V
Strictly aerobic 4–45 (20–37) 6.0–10.0 (6.0–8.5) 0–3.0 V
61.2–65.4
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Rivicola pingtungensis gen. nov., sp. nov.
*Results were obtained in this study.
+ Aerobic
Straight to slightly curved rods + Facultatively anaerobic 10–40 (30) 5.0–9.0 (6.0–9.0) 0–1.0 + 59.2–64.0
S.-Y. Sheu and others
reduce nitrate distinguish it from V. alkaliphila JC141T. Therefore, strain Npb-03T could be separated from all species of the genus Vogesella by phenotypic differences. It is now generally accepted that 16S rRNA gene sequence similarities of less than 95 % between two bacteria are an indication of affiliation to different genera (Ludwig et al., 1998). Strain Npb-03T most likely represents a species of a new genus, since the 16S rRNA gene sequence similarity to its closest relatives with validly published names, Aquaspirillum serpens ATCC 12638T, Leeia oryzae HW7T, Aquitalea magnusonii TRO-001DR8T, Laribacter hongkongensis HKU1T, V. indigofera ATCC 19706T and M. aerodenitrificans SGLY2T, is 90.5–92.2 %. Moreover, strain Npb-03T can be readily distinguished from these close phylogenetic neighbours by fatty acid composition, polar lipid profiles and physiological and biochemical characteristics. Therefore, based on phenotypic and phylogenetic criteria, we are of the opinion that strain Npb-03T should be assigned to a new genus and novel species, for which the name Rivicola pingtungensis gen. nov., sp. nov. is proposed. Description of Rivicola gen. nov. Rivicola [Ri.vi9co.la. L. masc. n. rivus creek or small river; L. suff. -cola (from L. masc. or fem. n. incola) inhabitant, dweller; N.L. masc. n. Rivicola river dweller]. Cells are Gram-reaction-negative, straight rod-shaped, non-motile, non-spore-forming and facultatively anaerobic. Oxidase and catalase are positive. The predominant quinone is Q-8. The predominant fatty acids are summed feature 3 (comprising C16 : 1v7c and/or C16 : 1v6c) and C16 : 0. PE, PG, DPG, an AL and three PLs are present in the polar lipid profile. The polyamine pattern contains putrescine, 2-hydroxyputrescine, cadaverine and spermidine. The DNA G+C content of the type strain of the type species is 64.1 mol%. The type species is Rivicola pingtungensis.
gluconate, caprate, adipate and malate, and negative reactions for indole production, glucose fermentation, aesculin and gelatin hydrolysis, arginine dihydrolase and bgalactosidase activities, and assimilation of citrate and phenylacetate. In the API ZYM kit, C4 esterase, C8 esterase lipase, acid phosphatase and naphthol-AS-BI-phosphohydrolase activities are present and alkaline phosphatase, C14 lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, bgalactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase activities are absent. The following compounds are utilized as sole carbon sources in the GN2 microplate: alactose, lactulose, D-mannitol, pyruvic acid methyl ester, acetic acid, cis-aconitic acid, citric acid, formic acid, phydroxyphenylacetic acid, itaconic acid, DL-lactic acid, propionic acid, succinic acid, bromosuccinic acid, succinamic acid, D-alanine, L-alanine, L-alanyl glycine, L-asparagine, L-glutamic acid, L-proline, L-serine and urocanic acid. All other substrates in the GN2 microplate are not utilized. Sensitive to penicillin G, ampicillin, chloramphenicol, gentamicin, rifampicin, kanamycin, tetracycline, novobiocin, streptomycin, sulfamethoxazole plus trimethoprim and nalidixic acid. The type strain is Npb-03T (5BCRC 80376T5LMG 26668T5 KCTC 23712T), isolated from a freshwater sample collected from the Jin-Shih Lake, Kaoshiung County, Taiwan.
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