Paenirhodobacter Enshiensis Gen. Nov., Sp. Nov., a Non-Photosynthetic

%paper no. ije050351 charlesworth ref: ije050351& New Taxa - Proteobacteria

International Journal of Systematic and Evolutionary Microbiology (2014), 63, 000–000 DOI 10.1099/ijs.0.050351-0

Paenirhodobacter enshiensis gen. nov., sp. nov., a non-photosynthetic bacterium isolated from soil, and emended descriptions of the genera Rhodobacter and Haematobacter

Dan Wang, Hongliang Liu, Shixue Zheng and Gejiao Wang

Correspondence State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Gejiao Wang Huazhong Agricultural University, Wuhan, Hubei 430070, PR China [email protected]

A Gram-reaction-negative, facultatively anaerobic, non-motile, rod-shaped, non-photosynthetic bacterial strain, DW2-9T, was isolated from soil. The highest 16S rRNA gene sequence similarities were found to Rhodobacter capsulatus ATCC 11166T (97.1 %), Rhodobacter viridis JA737T (96.4 %), Rhodobacter maris JA276T (96.2 %), Rhodobacter veldkampii ATCC 35703T (96.0 %), Haematobacter massiliensis CCUG 47968T (96.0 %), Haematobacter missouriensis CCUG 52307T (95.9 %) and Rhodobacter aestuarii JA296T (95.7 %). The genomic DNA G+C content was 67.2 mol% and the major respiratory quinone was ubiquinone 10 (Q-10). The major

cellular fatty acids (.5 %) were C18 : 1v7c,C16 : 0,C19 : 0 cyclo v8c and summed feature 3 (one

or more of iso-C15 : 0 2-OH, C16 : 1v6c and C16 : 1v7c). However, unlike species of the genus Rhodobacter, strain DW2-9T neither formed internal photosynthetic membranes nor produced photosynthetic pigments. DNA–DNA hybridization between strain DW2-9T and R. capsulatus JCM 21090T showed a relatedness of 33 %. Strain DW2-9T contained phosphatidylethanolamine, phosphatidylglycerol and an unknown aminophospholipid as major polar lipids, which differed from those of species of the genera Rhodobacter and Haematobacter. In addition to the differences in phylogenetic position and polar lipid types, strain DW2-9T could be distinguished from species of the genus Haematobacter by the cultivation conditions. On the basis of our polyphasic taxonomic analysis, strain DW2-9T is considered to represent a novel genus and species, for which the name Paenirhodobacter enshiensis gen. nov., sp. nov. is proposed. The type strain of Paenirhodobacter enshiensis is DW2-9T (5CCTCC AB 2011145T5KCTC 15169T). Emended descriptions of the genera Rhodobacter and Haematobacter are also proposed.

At the time of writing, the family Rhodobacteraceae contains contain ubiquinone 10 (Q-10) as the major respiratory 105 genera (http://www.bacterio.net/classifgenerafamilies. quinone (Q-10 is common within the Rhodobacteraceae; html#Rhodobacteraceae). Members of the genus Rhodobacter Tanaka et al., 2004; Sorokin et al., 2005; Yu et al., 2011). The are photosynthetic; the genus was proposed by Imhoff et al. predominant cellular fatty acid is C18 : 1 (Imhoff, 2005). Polar (1984), and the genus was reorganized by Hiraishi & Ueda lipid profiles have been reported only for Rhodobacter (1994). At the time of writing, the genus Rhodobacter has 17 capsulatus ATCC 11166T and R. viridis JA737T,andthese ; species with validly published names, of which five have been reports showed phosphatidylglycerol, phosphatidylethanola- reclassified in other genera; the remaining species are all mine and phosphatidylcholine as the major polar lipids Gram-reaction-negative, rod-shaped, photosynthetic bacteria. (Shalem Raj et al., 2013). Haematobacter is another genus of Phototrophically grown cells contain vesicular or lamellar the family Rhodobacteraceae, and it contains only two species. internal photosynthetic membranes and bacteriochlorophyll a Haematobacter massiliensis was originally proposed as Rhodo- and carotenoids as photosynthetic pigments (Eckersley & bacter massiliensis by Greub & Raoult (2003); later, it was Dow, 1980; Hiraishi & Ueda, 1994). Members of the genus reclassified in Haematobacter,togetherwithHaematobacter missouriensis (Helsel et al.,2007).Distinctfromtheother The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene genera of the Rhodobacteraceae, the members of the genus sequence of strain DW2-9T is JN797511. Haematobacter were isolated from clinical samples and differ Six supplementary figures are available with the online version of this from members of the genus Rhodobacter in cultivation con- paper. ditions, the absence of internal photosynthetic membranes,

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D. Wang and others non-production of photosynthetic pigments, the presence of A phylogenetic tree reconstructed using the maximum- T C19 : 0 cyclo v8c and phylogenetic position (Greub & Raoult, likelihood algorithm revealed that strains DW2-9 and 2003; Helsel et al., 2007). The other genera belonging to the DW2-13 grouped together within the cluster containing R. family Rhodobacteraceae are phenotypically, metabolically and capsulatus ATCC 11166T, R. viridis JA737T, R. maris ecologically diverse (Imhoff, 2005). In this study, a soil JA276T, R. aestuarii JA296T and R. vinaykumarii JA123T, bacterial isolate, designated strain DW2-9T,wasfoundtobe but strains DW2-9T and DW2-13 formed a separate closely related to species of the genera Rhodobacter and branch. Strain DW2-9T was separated from H. massi- Haematobacter based on 16S rRNA gene sequence analysis; liensis CCUG 47968T, H. missouriensis CCUG 52307T, however, on the basis of phenotypic and phylogenetic analyses, Roseicitreum antarcticum ZS2-28T and T. pacifica TL2T it is proposed that strain DW2-9T represents a novel genus and (Fig. 1). The neighbour-joining and maximum-parsimony species. trees (Figs S1 and S2, available in IJSEM Online) both supported the phylogenetic position obtained with the The soil sample was collected from a sewage outlet of the maximum-likelihood tree. Bafeng pharmaceutical factory (29u 529 550 N 110u 039 210 E) located in Enshi, Hubei province, PR China. Soil For analyses of morphological, physiological and biochem- texture, organic matter, nitrogen, phosphorus, sulfur, iron ical characteristics, strain DW2-9T and the five most closely T and pH were analysed as described by Okkenhaug et al. related type strains, R. capsulatus JCM 21090 , R. maris T T (2011) and the nitrate concentration was analysed as JCM 14794 , R. aestuarii JCM 14887 (from the Japan described by Andrews et al. (2002). The soil texture was Collection of Microorganisms), H. missouriensis CCUG T T heavy loam type with a pH of 6.9. The organic matter, N, 52307 and H. massiliensis CCUG 47968 (from the P, S, Fe and NO concentrations were 59.7, 5.8, 1.8, 3.4, Culture Collection of the University of Go¨teborg), were 3 T 96.7 and 398.4 g?kg21, respectively. For bacterial isolation, analysed in parallel. Strain DW2-9 , R. capsulatus JCM T T T 1 g soil was added to 99 ml sterilized water and then 21090 , R. maris JCM 14794 , R. aestuarii JCM 14887 serially diluted and cultured on 1/5 TSBA plates (tryptic were cultured on modified Biebl & Pfennig’s medium 21 soy broth agar, pH 7.0; Difco BD). containing (l ) 0.5 g KH2PO4, 0.2 g MgSO4 .7H2O, 0.4 g NaCl, 0.6 g NH4Cl, 0.05 g CaCl2 .2H2O, 3.0 g sodium A nearly full-length 16S rRNA gene sequence was amplified pyruvate, 0.3 g yeast extract, 5 ml ferric citrate solution as described by Fan et al. (2008) and compared with (0.1 %, w/v) and 1 ml micronutrient solution [l21:1ml sequences available in the EzTaxon-e server (Kim et al., HCl (25 %, v/v), 0.07 g ZnCl2, 0.1 g MnCl2 .4H2O, 0.06 g 2012). Multiple alignments were performed using the H3BO3, 0.2 g CoCl2 .6H2O, 0.02 g CuCl2 .H2O, 0.02 g CLUSTAL_X program (Thompson et al., 1997). Phylogenetic ] NiCl2 .6H2O and 0.04 g NaMoO4 .2H2O (Biebl & analysis was carried out using MEGA 4.0 (Tamura et al., Pfennig, 1981), unless otherwise mentioned. H. mis- 2007) and the PHYML online web server (Guindon et al., souriensis CCUG 52307T and H. massiliensis CCUG 2010). Phylogenetic trees, viewed with MEGA 4.0, were 47968T were grown under clinical culture conditions using reconstructed using the neighbour-joining (Saitou & Nei, brain-heart infusion (BHI) (Helsel et al., 2007) or buffered 1987), maximum-parsimony (Kluge & Farris, 1969) and charcoal yeast extract (BCYE) agar (Greub & Raoult, 2003), maximum-likelihood (Felsenstein, 1981) methods with respectively. For all analyses, strain DW2-9T, R. capsulatus bootstrap analyses based on 1000 replications. Type strains JCM 21090T, R. maris JCM 14794T, R. aestuarii JCM of all species of the genera Rhodobacter and Haematobacter 14887T were incubated at 30 uC, while H. missouriensis and representative strains from 12 other genera of the CCUG 52307T and H. massiliensis CCUG 47968T were family Rhodobacteraceae were included in the phyloge- incubated at 35 uC, unless otherwise mentioned. netic trees. Strain DW2-13 (GenBank/EMBL/DDBJ no. KC820799), isolated from the same soil, was also included Cell morphology was observed using light microscopy in the phylogenetic analyses. (Olympus; 61000) and transmission electron microscopy (H-7650; Hitachi) (Grossart et al., 2000) with cells cultured The nearly full-length (1428 bp) 16S rRNA gene sequence of for 48 h. Motility tests were performed using the strain DW2-9T shared sequence identities in the range 97.1– appropriate medium supplemented with 0.3 % agar. 94.7 % with type strains of species of Rhodobacter and Gram staining was determined using the method described Haematobacter. The highest 16S rRNA gene sequence by Dussault (1955). Growth at 4, 10, 28, 30, 37, 42 and T similarities were found with R. capsulatus ATCC 11166 45 uC and 0–5 % (w/v) NaCl was assessed after 7 days of (97.1 %), R. viridis JA737T (96.4 %), R. maris JA276T incubation. Growth at pH 4–10 (at intervals of 1 pH unit) (96.2 %), R. veldkampii ATCC 35703T (96.0 %), H. massi- was determined using the following buffer systems: pH 4– T liensis CCUG 47968 (96.0 %), H. missouriensis CCUG 7, 0.1 M citric acid/0.2 M Na2HPO4; pH 8–9, 0.1 M Tris/ T T 52307 (95.9 %) and R. aestuarii JA296 (95.7 %). The only 0.2 M HCl; pH 10, 0.05 M NaHCO3/0.1 M NaOH. other 16S rRNA gene sequence identities higher than 95 % Anaerobic growth was determined in an anaerobic were observed with Roseicitreum antarcticum ZS2-28T chamber (Mitsubishi Gas Chemical Co.) after 7 days of (95.4 %) and Thioclava pacifica TL2T (95.3 %). Strain DW2- incubation at 30 uC on modified Biebl & Pfennig’s medium 13, isolated from the same soil, showed 100 % 16S rRNA gene plates. Carbon, sulfur and nitrogen source utilization tests sequence identity to strain DW2-9T. for strain DW2-9T, R. capsulatus JCM 21090T, R. maris

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Paenirhodobacter enshiensis gen. nov., sp. nov., from soil

Rhodospirillum rubrum ATCC 11170T (D30778) Rhodospirillum photometricum E-11T (D30777) Paracoccus homiensis DD-R11T (DQ342239) Thioclava pacifica TL 2T (AY656719) Ruegeria lacuscaerulensis ITI-1157T (ACNX01000031) 886 T 1000 Roseovarius pacificus 2-81 (DQ120726) Pararhodobacter aggregans D1-19T (AM403160) Roseicitreum antarcticum ZS2-28T (FJ196006) 526 Roseibaca ekhonensis EL-50T (AJ605746) 1000 Rhodobaca bogoriensis LBB1T (AF248638) 993 Rhodobaca barguzinensis VKM B-2406T (EF554833) Rhodobacter veldkampii ATCC 35703T (D16421) 988 Rhodobacter capsulatus ATCC 11166T (D16428) Rhodobacter viridis JA737T (HE572577) T Paenirhodobacter enshiensis DW2-9 (JN797511) 1000 Strain DW2-13 (KC820799) T Rhodobacter vinaykumarii JA123 (AM408117) Rhodobacter maris JA276T (AM745438) 781 Rhodobacter aestuarii JA296T (AM748926) JA297T (AM748927) 640 Rhodovulum kholense Rhodovulum euryhalinum DSM 4868T (D16426) 838 Rhodovulum strictum MB-G2T (D16419) T 1000 Haematobacter massiliensis CCUG 47968 (DQ342309) Haematobacter missouriensis CCUG 52307T (DQ342315) Falsirhodobacter halotolerans JA744T (HE662814) 797 T Rhodobacter sphaeroides ATH 2.4.1 (CP000143) T 816 Rhodobacter azotoformans KA25 (D70846) 798 T Rhodobacter ovatus JA234 (AM690348) 287 Rhodobacter johrii JA192T (AM398152)

Rhodobacter megalophilus JA194T (AM421024) T Rhodobacter blasticus ATCC 33485 (D16429) 0.05 521 T Catellibacterium caeni DCA-1 (FJ386516)

Fig. 1. Maximum-likelihood phylogenetic tree reconstructed on the basis of 16S rRNA gene sequences. Numbers at nodes indicate bootstrap values from 1000 replications; only values above 500 are shown. Bar, 5 substitutions per 100 nucleotide ? positions.

JCM 14794T and R. aestuarii JCM 14887T were performed determined using 1 % (w/v) tetramethyl b-phenylenedia- in the light (2000 lx, in 25 ml fully filled, screw-capped test mine (bioMe´rieux). Production of H2S was tested tubes) and in the dark (for strain DW2-9T only). according to the method of Dong & Cai (2001). For Physiological and biochemical properties, enzyme activities testing carbon source utilization, 0.1 % (v/v) each of and sole carbon substrate utilization tests were examined formate, propionate, butyrate, caproate, valerate, lactate, < using the API 20NE and API ZYM systems (bioMe´rieux) glycerol, methanol and ethanol was used; while the other according to the manufacturer’s instructions combined compounds were used at 0.3 % (w/v) (Dong & Cai, 2001). with traditional methods. Substrate utilization tests using For testing sulfur sources, MgSO4 .7H2O was replaced with traditional methods were not done for H. missouriensis MgCl2 .5H2O (0.2 %) and 0.5 mM each of sodium sulfate, CCUG 52307T and H. massiliensis CCUG 47968T, since cysteine, thiosulfate, sulfite, sulfide and methionine was = they did not grow on modified Biebl & Pfennig’s medium. used. Nitrogen source utilization was tested by replacing Catalase activity was determined by assessing bubble NH4Cl with 0.06 % (w/v) each of glutamate, peptone, production in 3 % (v/v) H2O2, and oxidase activity was nitrate, arginine, urea, aspartate and nitrite (Dong & Cai, > http://ijs.sgmjournals.org 3 %paper no. ije050351 charlesworth ref: ije050351&

D. Wang and others

2001). Growth factor requirements were tested by replacing it is not appropriate to classify strain DW2-9T within this yeast extract with biotin, vitamin B3, vitamin B12, niacin, p- genus. Cellular fatty acid and polar lipid analyses revealed aminobenzoic acid and thiamine, either alone or in further differences between strain DW2-9T and the type combination, as described by Srinivas et al. (2007). strains of species of the genus Rhodobacter. Growth was also tested on nutrient agar (NA), full-strength DNA–DNA hybridization was performed by the thermal trypticase soy agar (TSA), Luria–Bertani (LB) agar and denaturation and renaturation method (Huss et al., 1983). MacConkey agar (all from Difco). In order to test for the The DNA G+C content was determined by HPLC presence of photosynthetic pigments, bacterial cells were according to the method of Tamaoka & Komagata (1984). suspended in 60 % (w/v) sucrose solution and the absorp- Respiratory quinone analysis was performed by HPLC as tion spectrum was measured using a Beckman DU800 described by Minnikin et al. (1984). For cellular fatty acid spectrophotometer as described by Biebl & Drews (1969). analysis, fatty acids of strain DW2-9T, R. capsulatus JCM The presence of the pufL and pufM genes, respectively 21090T, R. maris JCM 14794T, R. aestuarii JCM 14887T, H. encoding subunits L and M of the photosynthetic reaction missouriensis CCUG 52307T and H. massiliensis CCUG centre proteins, was tested by PCR amplification as 47968T were analysed by GC (Hewlett Packard 6890) described by Uchino et al. (2002). Primers pufL1F (59- according to the instructions of the Sherlock Microbial TTCGACTTCTGGGT-39)/pufL3R (59-CCGATCGAATAG- Identification System (MIDI Sherlock version 4.5; MIDI CC-39) and pufM2F (59-CAGATCGGGCCGATCTA-39)/ database TSBA40 4.10). Polar lipids were analysed by two pufM4R (59-CCAGACGTACCAGTTGTC-39) were used to dimensional TLC as described by Tindall (1990). amplify pufL and pufM, respectively (Uchino et al., 2002). Antibiotic-susceptibility tests for strain DW2-9T were DNA–DNA relatedness between strain DW2-9T and R. performed as described by Luo et al. (2012); inhibition capsulatus JCM 21090T (81.2 uC hybridization temper- zone diameters above 10 mm were considered to indicate ature) was 33±1 %. The DNA G+C content of strain susceptibility. DW2-9T was 67.2 mol%. In accordance with members of the genera Rhodobacter and Haematobacter, Q-10 was the Cells of strain DW2-9T were ovoid or rod-shaped, 0.9– major respiratory quinone. The major cellular fatty acids 1.2 mm long and 0.3–0.6 mm in diameter, non-motile, (.5 %) were C v7c,C ,C cyclo v8c and divided by binary fission and formed chains (Fig. S3). 18 : 1 16 : 0 19 : 0 summed feature 3 (one or more of iso-C 2-OH, Gram staining showed a negative reaction. Growth 15 : 0 C v6c and C v7c). Of these fatty acids, C cyclo occurred at 4–42 uC and pH 5–8, with optimum growth 16 : 1 16 : 1 19 : 0 v8c was absent from members of the genus Rhodobacter, at 28 uC and pH 7. NaCl was not required for growth of but present in H. missouriensis CCUG 52307T and H. strain DW2-9T, but it was tolerated up to 3 % (w/v). massiliensis CCUG 47968T in addition to strain DW2-9T Multiple growth factors, including biotin, thiamine, niacin, (Tables 1 and 2). The major polar lipids found in strain pantothenate and p-aminobenzoic acid, were required for DW2-9T were phosphatidylglycerol, phosphatidylethanola- growth. Detailed results are given in the species descrip- mine and an unknown aminophospholipid; in R. capsulatus tion. Distinct from all the reference type strains, strain JCM 21090T, R. maris JCM 14794T and R. aestuarii JCM DW2-9T was positive for oxidase activity and negative for 14887T, the major polar lipids were phosphatidylglycerol, catalase activity; the reference strains were positive for both phosphatidylethanolamine and phosphatidylcholine, while, activities. H S was produced by all tested strains. Distinct 2 in H. missouriensis CCUG 52307T and H. massiliensis CCUG from the strains of Rhodobacter, colonies of strain DW2-9T 47968T, they were phosphatidylglycerol, phosphatidyletha- grown under photoheterotrophic conditions were white. In nolamine, an unknown aminolipid and two unknown addition, in vivo absorption spectra showed no absorption phospholipids (Table 1 and Fig. S6). by strain DW2-9T or the two strains of Haematobacter, while R. capsulatus JCM 21090T, R. maris JCM 14794T and The main phenotypic characteristics of strain DW2-9T and R. aestuarii JCM 14887T all exhibited absorption maxima the type strains of the genera Rhodobacter and Haema- at 377, 590, 803 and 860 nm. This result suggested that tobacter are shown in Table 1. In addition to its distinct strain DW2-9T does not contain any photosynthetic phylogenetic position and polar lipid composition, strain pigments. In addition, using transmission electron micro- DW2-9T could be distinguished from species of the genus scopy, internal photosynthetic membrane structures were Haematobacter by the cultivation conditions, since all observed in ultrathin sections of R. capsulatus JCM 21090T, strains of Haematobacter need the addition of serum or but not in strain DW2-9T (Fig. S4). Furthermore, the multiple cell factors to grow (Greub & Raoult, 2003; Helsel photosynthesis genes pufL and pufM were detected in R. et al., 2007). Furthermore, strain DW2-9T could be capsulatus JCM 21090T but not strain DW2-9T (Fig. S5). distinguished from members of the genus Thioclava by Based on the distinct phototrophism characteristics of its inability to oxidize sulfur and to grow chemoautotro- strain DW2-9T and the type strains of species of the genus phically (Sorokin et al., 2005) and from species of the Rhodobacter, we propose that strain DW2-9T is a non- genus Roseicitreum by the absence of internal photosyn- photosynthetic bacterium, although it shows a close thetic membranes and photosynthetic pigments and by its phylogenetic relationship with Rhodobacter. Since the non-halophilic character (Yu et al., 2011). Therefore, on genus Rhodobacter contains only photosynthetic bacteria, the basis of its distinctive phenotypic and phylogenetic

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Paenirhodobacter enshiensis gen. nov., sp. nov., from soil

Table 1. Differential phenotypic characteristics of strain DW2-9T and type strains of the genera Rhodobacter and Haematobacter

Strains: 1, DW2-9T;2,R. capsulatus JCM 21090T (type species); 3, R. maris JCM 14794T;4,R. aestuarii JCM 14887T;5,H. massiliensis CCUG 47968T;6,H. missouriensis CCUG 52307T (type species). Data are from this study unless otherwise indicated. Cells of all strains are ovoid to rod- T shaped. +, Positive; 2, negative; W, weak growth; ND, not done (substrate utilization tests for H. massiliensis CCUG 47968 and H. missouriensis CCUG 52307T were only performed using the API 20NE system, since the strains did not grow on modified Biebl & Pfennig’s medium).

Characteristic 1 2 3 4 5 6

Isolation source Soil Stagnant water Seawater Water Clinical sample Clinical sample Formation of chains of cells ++++22 Motility 2 +++ 22 Colour of colonies White Yellow brown Yellow brown Yellow brown Pale white Yellow Photosynthetic pigmentsD 2 +++ 22 Internal membrane systemd 2 +++ 22

Vitamin(s) required§ b, n, t, p-ABA, B3 t, b, n t t NA NA @ pH range 5.0–8.0 6.0–8.0 5.0–8.0 6.0–8.0 6.0–8.0 6.0–8.0 pH optimum 7.0 6.0–7.0 6.0–7.0 7.0 7.0 7.0 Temperature optimum (uC) 28 30 30 30 35 35 DNA G+C content (mol%) 67.2 68.8a|| 62.9a 65.5a 65.0–65.5b 65b Major polar lipids PG, PE, APL PG, PE, PC PG, PE, PC PG, PE, PC PG, PE, AL, PL1, PG, PE, AL, PL2 PL1, PL2

Presence of C19 : 0 cyclo v8c + 222 ++ Enzyme activities Catalase 2 +++ ++ Urease + 222 ++ Alkaline phosphatase 2 +++ ++ a-Glucosidase ++++22 Growth on MacConkey agar 2222+ 2 Utilization of: D-Glucose +++222 L-Arabinose + 222 22 Mannose 2222++ Adipate 2222++ Malate +++2 ++ Citrate + 222 22 Maltose + 2 + 222 b b Acetate ++W 2 + + b b Sucrose 2 + W 222 b b D-Xylose ++22 22 Formate 2 + 22 ND ND Propionate W + W 2 ND ND Butyrate 2 + W 2 ND ND Valerate 2 + W 2 ND ND Caproate 2 + W 2 ND ND Caprylate 2 + 22 ND ND Lactate 2 + W W ND ND Fumarate ++W 2 ND ND Succinate + 222 ND ND Glutamate 2 + 22 ND ND Fructose +++2 ND ND Glycerol 22W 2 ND ND Electron donors Hydrogen 2 + 22 ND ND Cysteine + 22+ ND ND Methionine 2 + 22 ND ND Sulfide 2 ++2 ND ND Thiosulfate 2 +++ ND ND Sulfite 2 ++2 ND ND

*C, Chains; O, ovoid; R, rod-shaped. http://ijs.sgmjournals.org 5 %paper no. ije050351 charlesworth ref: ije050351&

D. Wang and others

Table 1. cont.

DWhere present, photosynthetic pigments are bacteriochlorophyll a and carotenoids. dWhere present, a vesicular internal membrane system is observed.

§b, Biotin; B3, vitamin B3,B12, vitamin B12; n, niacin; p-ABA, p-aminobenzoic acid; t, thiamine. ||Data taken from: a, Shalem Raj et al. (2013); b, Helsel et al. (2007). PC, Phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; AL, unknown aminolipid; APL, unknown aminophospholipid; PL, unknown phospholipid.

T traits, strain DW2-9 represents a novel genus and species, C16 : 0,C18 : 1v7c,C19 : 0 cyclo v8c and summed feature 3 for which the name Paenirhodobacter enshiensis gen. nov., (one or more of iso-C15 : 0 2-OH, C16 : 1v6c and C16 : 1v7c). sp. nov. is proposed. On the basis of the new data obtained Major polar lipids are phosphatidylglycerol, phosphatidy- in this study, emended descriptions of the genera lethanolamine and an unknown aminophospholipid. The Rhodobacter and Haematobacter are also proposed. genomic DNA G+C content of the type strain of the type species is 67.2 mol%. The type species is Paenirhodobacter Description of Paenirhodobacter gen. nov. enshiensis. Paenirhodobacter (Pa.e9ni.rho.do.bac9ter. L. adv. paene almost; -i- connecting vowel; N.L. masc. n. Rhodobacter a Description of Paenirhodobacter enshiensis bacterial genus name; N.L. masc. n. Paenirhodobacter sp. nov. almost a Rhodobacter). Paenirhodobacter enshiensis (en.shi.en9sis. N.L. masc. adj. Cells are ovoid or rod-shaped, divide by binary fission and enshiensis of or belonging to Enshi, where the type strain may form chains. Gram-reaction negative, non-motile, was isolated). without either internal photosynthetic membrane struc- Displays the following properties in addition to those tures or photosynthetic pigments. Positive for oxidase described for the genus. Cells are 0.3–0.660.9–1.2 mm. activity and negative for catalase activity. Facultatively Colonies are convex, circular, smooth and white after anaerobic and grow chemoheterotrophically. Growth 2 days of incubation on modified Biebl & Pfennig’s agar at factors are required. NaCl is not required for growth. Q- 30 uC. Also grows well on complex media such as LB agar, 10 is the major quinone. The major cellular fatty acids are TSBA and NA, but not on MacConkey agar. Temperature range for growth is 4–42 uC (optimum at 28 uC). Tolerates up to 3 % (w/v) NaCl. Biotin, thiamine, niacin, pantothe- Table 2. Fatty acid compositions of strain DW2-9T and its nate and p-aminobenzoic acid are needed as growth closest genetic relatives factors. The sole carbon sources utilized include acetate, Strains: 1, DW2-9T;2,R. capsulatus JCM 21090T;3,R. maris JCM propionate, pyruvate, fumarate, malate, citrate, succinate, 14794T;4,R. aestuarii JCM 14887T;5,H. massiliensis CCUG 47968T; D-glucose, D-fructose, D-xylose and maltose. Formate, 6, H. missouriensis CCUG 52307T. Values are percentages of total fatty valerate, caprylate, caproate, caprate, gluconate, benzoate, acids; 2, ,1 % or not detected. All data are from this study. Data for glutamate, propanol, sucrose, Casamino acids, mannose, strain DW2-9T are means of two tests. mannitol, methanol, ethanol, sorbitol, tartrate, adipate, phenylacetate and lactate cannot be utilized as sole carbon Fatty acid 123456 sources. Ammonium chloride, glutamate, peptone, nitrate, arginine and urea are utilized as nitrogen sources, but C10 : 0 3-OH 4.0 3.4 3.6 5.3 2.4 2.1 aspartate and nitrite are not. Sodium sulfate and cysteine C 20.5 5.7 8.0 6.2 3.1 4.8 16 : 0 are utilized as sulfur sources, but thiosulfate, sulfite, sulfide C17 : 0 22221.4 1.2 and methionine are not. Can produce H2S in complex C17 : 1v6c 22221.4 2 C 1.2 1.4 1.7 3.8 22 media containing cysteine. In API 20NE tests, shows 18 : 0 positive reactions for oxidase, glucose fermentation, C18 : 0 3-OH 2 1.5 1.4 1.7 22 arginine dihydrolase, urease and assimilation of L-arabi- C18 : 1v7c 55.2 70.7 70.3 75.0 69.5 82.7 nose, malic acid and trisodium citrate, but negative C19 : 0 cyclo v8c 6.6 22215.0 3.1 b 10-Methyl C19 : 0 2 1.2 2222 reactions for nitrate reduction, indole production, - Summed features* galactosidase and hydrolysis of aesculin and gelatin. In 2 4.2 2222.9 2.9 API ZYM tests, positive for esterase (C4), esterase lipase 3 5.5 11.7 9.8 6.3 1.3 1.0 (C8), a-glucosidase, leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but negative for *Summed features are groups of two or three fatty acids that cannot alkaline phosphatase, valine arylamidase, cystine arylami- be separated by GLC with the MIDI System. Summed feature 2 dase, trypsinase, a-chymotrypsin, a-galactosidase, b-galac- represented C14 : 0 3-OH and/or iso-C16 : 1 I; summed feature 3 tosidase, b-glucuronidase, b-glucosidase, N-acetyl-b-gluco- represented one or more of iso-C15 : 0 2-OH, C16 : 1v6c and C16 : 1v7c. saminidase, a-mannosidase and b-fucosidase. Resistant to

6 International Journal of Systematic and Evolutionary Microbiology 63 %paper no. ije050351 charlesworth ref: ije050351&

Paenirhodobacter enshiensis gen. nov., sp. nov., from soil polymyxin B (300 U), novobiocin (5 mg), vancomycin Dong, X.-Z. & Cai, M.-Y. (2001). Determinative Manual for Routine (30 mg), rifampicin (5 mg), nalidixic acid (30 mg), cepha- Bacteriology. Beijing: Scientific Press. losporin IV (30 mg), oxazocillin (1 mg), lincomycin (2 mg), Dussault, H. P. (1955). An improved technique for staining red teicoplanin (30 mg) and trimethoprim (5 mg), but sensitive halophilic bacteria. J Bacteriol 70, 484–485. to chloramphenicol (30 mg), tetracycline (30 mg), kanamy- Eckersley, K. & Dow, C. S. (1980). Rhodopseudomonas blastica sp. nov.: cin (30 mg), neomycin (30 mg), nitrofurantoin (300 mg), a member of the Rhodospirillaceae. J Gen Microbiol 119, 465–473. cefoxitin (30 mg), ofloxacin (5 mg), erythromycin (15 mg), Fan, H., Su, C., Wang, Y., Yao, J., Zhao, K., Wang, Y. & Wang, G. cefotaxime (30 mg), streptomycin (10 mg), amoxicillin (2008). Sedimentary arsenite-oxidizing and arsenate-reducing bac- (10 mg), cephalosporin V (30 mg), penicillin (10 IU), teria associated with high arsenic groundwater from Shanyin, Northwestern China. J Appl Microbiol 105, 529–539. cephalothin (20 mg), ampicillin (10 mg) and carbenicillin (100 mg). Contains two unknown phospholipids as minor Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376. components. Small amounts of C10 : 0 3-OH and summed feature 2 (C 3-OH and/or iso-C I) are found. Greub, G. & Raoult, D. (2003). Rhodobacter massiliensis sp. nov., a 14 : 0 16 : 1 new amoebae-resistant species isolated from the nose of a patient. Res The type strain is DW2-9T (5CCTCC AB 2011145T5 Microbiol 154, 631–635. T KCTC 15169 ), isolated from soil near a sewage outlet of Grossart, H. P., Steward, G. F., Martinez, J. & Azam, F. (2000). A the Bafeng pharmaceutical factory, Enshi city, Hubei simple, rapid method for demonstrating bacterial flagella. Appl province, PR China. Environ Microbiol 66, 3632–3636. Guindon, S., Dufayard, J. F., Lefort, V., Anisimova, M., Hordijk, W. & Gascuel, O. (2010). New algorithms and methods to estimate Emended description of the genus Rhodobacter maximum-likelihood phylogenies: assessing the performance of Imhoff et al. 1984 PhyML 3.0. Syst Biol 59, 307–321. The description is as given previously (Imhoff et al., 1984; Helsel, L. O., Hollis, D., Steigerwalt, A. G., Morey, R. E., Jordan, J., Aye, T., Radosevic, J., Jannat-Khah, D., Thiry, D. & other authors Imhoff, 2005) with the following addition. The major polar (2007). Identification of ‘‘Haematobacter,’’ a new genus of aerobic lipids are phosphatidylglycerol, phosphatidylethanolamine Gram-negative rods isolated from clinical specimens, and reclassifica- and phosphatidylcholine. tion of Rhodobacter massiliensis as ‘‘Haematobacter massiliensis comb. nov.’’ J Clin Microbiol 45, 1238–1243. Emended description of the genus Hiraishi, A. & Ueda, Y. (1994). Intrageneric structure of the genus Haematobacter Helsel et al. 2007 Rhodobacter: transfer of Rhodobacter sulfidophilus and related marine species to the genus Rhodovulum gen. nov. Int J Syst Bacteriol 44, 15–23. The description is as given previously (Helsel et al., 2007) with Huss, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the the following addition. Major polar lipids are phosphatidyl- spectrophotometric determination of DNA hybridization from glycerol, phosphatidylethanolamine, an unidentified amino- renaturation rates. Syst Appl Microbiol 4, 184–192. lipid and two unidentified phospholipids. Imhoff, J. F. (2005). Genus Rhodobacter.InBergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2C, pp. 161–167. Edited by D. J. Brenner, N. R. Krieg, J. T. Staley & G. M. Garrity. New York: Springer. Acknowledgements Imhoff, J. F., Tru¨ per, H. G. & Pfennig, N. (1984). Rearrangement of the species and genera of the phototrophic ‘‘purple nonsulfur bacteria’’. We are grateful to Mr Liang Guo for sampling and Dr Christopher Int J Syst Bacteriol 34, 340–343. Rensing for editing of the manuscript. We also thank Professor Jean Euze´by (E´ cole National Ve´te´rinaire) for providing the etymology of Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., the new name and Dr Lingling Yang (Yunan University) for Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e: respiratory quinone and polar lipid analyses. This work was a prokaryotic 16S rRNA gene sequence database with phylotypes that supported by a Chinese 863 project (no. 2012AA101402-3) and by represent uncultured species. Int J Syst Evol Microbiol 62, 716–721. a fund of the Hubei Tobacco Industrial Corporation Ltd, Enshi Kluge, A. G. & Farris, J. S. (1969). Quantitative phyletics and the Branch, PR China. evolution of anurans. Syst Zool 18, 1–32. Luo, G., Shi, Z. & Wang, G. (2012). Lysobacter arseniciresistens sp. nov., an arsenite-resistant bacterium isolated from iron-mined soil. Int J References Syst Evol Microbiol 62, 1659–1665. Andrews, S. S., Mitchell, J. P., Mancinelli, R. D., Karlen, D. L., Hartz, Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., T. K., Horwath, W. R., Pettygrove, G. S., Scow, K. M. & Munk, D. S. Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated (2002). 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1 The genus Rhodobacter has 17 species with validly published names. Although proposals have been published to reclassify five of them in other genera, their original names in Rhodobacter remain validly published, as is the case for any new combination. Please confirm the edit from 12 to 17 is correct.

2 The ions formate, propionate, butyrate, caproate, valerate and lactate are not liquids, so it seems unlikely that they were used at 0.1% (v/v). Please clarify whether some salt of these various ions (e.g. sodium formate) was used, presumably at 0.1% (w/v), or whether the respective acids (e.g. formic acid) were used [Note that both lactic acid and lactate salts are solids at room temperature].

3 Please clarify which salts of thiosulfate, sulfite and sulfide were used.

4 Please clarify which salts of nitrate and nitrite were used.

5 Note that I have removed any bootstrap values under 50 %, as this low level of support is not statistically significant.

6 Please clarify the meaning of ‘‘NA’’ in the columns for vitamin requirement of the two Haematobacter strains. Is it the same explanation as for the carbon source utilization tests? Ordering reprints for SGM journals

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