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Description of the Family Flammeo- for Strain Isolation, the Soil Sample Was Thoroughly Virgaceae Was Formally Established by Yoon Et Al

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Description of the Family Flammeo- for Strain Isolation, the Soil Sample Was Thoroughly Virgaceae Was Formally Established by Yoon Et Al International Journal of Systematic and Evolutionary Microbiology (2013), 63, 1639–1645 DOI 10.1099/ijs.0.044651-0 Nafulsella turpanensis gen. nov., sp. nov., a member of the phylum Bacteroidetes isolated from soil Lei Zhang,1,2 Xihui Shen,1 Yingbao Liu1 and Shiqing Li1,2,3 Correspondence 1State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau and College of Shiqing Li. Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, PR China [email protected] 2State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Institute of Soil and Water Conservation, Chinese Academy of Sciences and Northwest A&F University, Yangling, Shaanxi 712100, PR China 3 College of Resource and Environment, Northwest A&F University, Yangling, Shaanxi 712100, PR China A Gram-staining-negative, rod-shaped, gliding and pale-pink-pigmented bacterium, designated strain ZLM-10T, was isolated from a soil sample collected from an arid area in Xinjiang province, China, and characterized in a taxonomic study using a polyphasic approach. The novel strain grew optimally at 30–37 6C and in the presence of 2 % (w/v) sea salts. The only respiratory quinone detected was MK-7 and the major cellular fatty acids were summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1v7c), iso-C15 : 0 and iso-C17 : 0 3-OH. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, an unidentified aminolipid and two unidentified aminophospholipids. The DNA G+C content was 45.4 mol%. Flexirubin-type pigments were not produced. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZLM-10T was a member of the phylum Bacteroidetes and appeared most closely related to Cesiribacter roseus 311T (90.2 % sequence similarity), Marivirga sericea LMG 13021T (89.2 %), Cesiribacter andamanensis AMV16T (89.1 %) and Marivirga tractuosa DSM 4126T (89.1 %). On the basis of phenotypic and genotypic data and phylogenetic inference, strain ZLM-10T should be classified as a novel species of a new genus in the family Flammeovirgaceae, for which the name Nafulsella turpanensis gen. nov., sp. nov. is proposed. The type strain of the type species is ZLM-10T (5CCTCC AB 208222T5KCTC 23983T). The family Flammeovirgaceae belongs to the phylum Bac- phenotypic and genotypic data and phylogenetic analysis, it teroidetes and was first proposed by Garrity & Holt (2001) is proposed that the new isolate represents a novel species in the second edition of Bergey’s Manual of Systematic Bac- in a new genus of the family Flammeovirgaceae. teriology. Recently, thehttp://ir.iswc.ac.cn description of the family Flammeo- For strain isolation, the soil sample was thoroughly virgaceae was formally established by Yoon et al. (2011a). suspended in sterile water and serial dilutions were spread At the time of writing, the family Flammeovirgaceae incor- on marine agar 2216 (MA; Difco). Isolation was achieved porates the genera Aureibacter, Cesiribacter, Fabibacter, after incubation at 30 uC for 1 week. Strain ZLM-10T was Flammeovirga, Flexithrix, Fulvivirga, Limibacter, Marivirga, routinely cultivated on the same medium at 30 uC and Perexilibacter, Persicobacter,‘Porifericola’, Rapidithrix, Rei- preserved at 280 uC in marine broth 2216 (MB; Difco) chenbachiella, Roseivirga, Sediminitomix and ‘Tunicatimo- supplemented with 20 % (v/v) glycerol. nas’ (Ludwig et al., 2010; Nedashkovskaya et al., 2010; Srinivas et al., 2011; Yoon et al., 2011a, b, 2012). In the Genomic DNA of strain ZLM-10T was prepared according present study, we report on the taxonomic characterization to the procedure of Wilson (1987) with the modification of a pale-pink-pigmented bacterium, designated strain by Cleenwerck et al. (2002). Genomic DNA G+C content ZLM-10T, that was isolated from a soil sample collected was determined by HPLC (UltiMate 3000; Dionex) from an arid area in Xinjiang province, China. Based on according to the method of Mesbah et al. (1989). The 16S rRNA gene sequence of strain ZLM-10T was amplified The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene by PCR with the bacterial universal primers 27f and 1527r sequence of strain ZLM-10T is JN899241. (Lane, 1991), and the PCR products were sequenced A supplementary figure is available with the online version of this paper. commercially, by Invitrogen Biotechnology. Identification Downloaded from www.microbiologyresearch.org by 044651 G 2013 IUMS Printed in Great Britain 1639 IP: 221.11.67.252 On: Wed, 23 Dec 2015 02:05:16 L. Zhang and others of phylogenetic neighbours and calculation of pairwise 16S the maximum-parsimony tree was essentially the same as rRNA gene sequence similarities were achieved using the that of the neighbour-joining tree (data not shown). The EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim genomic DNA G+C content of strain ZLM-10T was et al., 2012). Phylogenetic analysis was performed using 45.4 mol%, which differs from the DNA G+C contents of version 4.1 of the MEGA package (Tamura et al., 2007), after its phylogenetically closest relatives (Table 1). These results T multiple alignment of the data within CLUSTAL_X (Thompson suggest that strain ZLM-10 represents a novel genus within et al., 1997). Phylogenetic distances were calculated by the the family Flammeovirgaceae. neighbour-joining method (Saitou & Nei, 1987), using Growth was evaluated at 30 uC on several standard bac- the Kimura two-parameter model (Kimura, 1980), and by teriological media: marine agar 2216 (MA; Difco), R2A the maximum-parsimony method (Fitch, 1971), using the agar (Difco), trypticase soy agar (TSA; Difco), nutrient agar close-neighbour-interchange algorithm (search level52, (NA; Difco) and MacConkey agar (Difco). Cell morpho- random additions5100). Each resultant tree was evaluated logy was examined by transmission electron microscopy by bootstrap analysis on the basis of 1000 replications (H-8100; Hitachi) using cells from the exponential growth (Felsenstein, 1985). phase. The cells were negatively stained with 1 % (w/v) The almost-complete 16S rRNA gene sequence (1441 nt) phosphotungstic acid and the grids were examined after of strain ZLM-10T was determined. Comparative 16S being air-dried. Gram staining of cells was carried out rRNA gene sequence analysis revealed that strain ZLM-10T according to the classical Gram procedure described by was affiliated with the family Flammeovirgaceae within the Doetsch (1981). Gliding motility was determined as phylum Bacteroidetes, and exhibited the highest sequence described by Bowman (2000). The presence of flexirubin- similarities to Cesiribacter roseus 311T (90.2 %), Marivirga type pigments was investigated using the bathochromatic sericea LMG 13021T (89.2 %), Cesiribacter andamanensis shift test, with 20 % (w/v) KOH (Bernardet et al., 2002; AMV16T (89.1 %) and Marivirga tractuosa DSM 4126T McCammon & Bowman, 2000). The temperature range (89.1 %). All other species of the family Flammeovirgaceae and optimum for growth were assessed at 4, 7, 10, 15, 20, with validly published names were more distantly related, 25, 30, 37, 40, 42, 45 and 50 uC on MA for up to 1 week. showing 16S rRNA gene sequence similarities of ,88.9 %. The pH range and optimum for growth were tested in MB In the phylogenetic tree based on the neighbour-joining medium at pH 4.0–11.0, by using 0.1 M citric acid/0.1 M T algorithm, strain ZLM-10 was clustered with members of sodium citrate (for pH 4.0–5.0), 0.1 M KH2PO4/0.1 M the genera Cesiribacter and Marivirga but was clearly sepa- NaOH (for pH 6.0–8.0), 0.1 M NaHCO3/0.1 M Na2CO3 rated from these two genera (Fig. 1). The overall topology of (for pH 9.0–10.0) or 0.05 M Na2HPO4/0.1 M NaOH (for 100 Roseivirga ehrenbergii KMM 6017 T (AY608410) 0.02 100 Roseivirga echinicomitans KMM 6058 T (AY753206) Roseivirga spongicola UST030701-084 T (DQ080996) 55 72 Fabibacter halotolerans UST030701-097 T (DQ080995) MRN461T (DQ660388) 61 Marinoscillum pacificum 100 Marinoscillum furvescens NBRC 15994 T (AB078079) Fulvivirga kasyanovii KMM 6220T (DQ836305) 99 Reichenbachiella agariperforans KMM 3525 T (AB058919) 100 Reichenbachiella faecimaris PCP11 T (GU143096) 99 ‘Porifericola rhodea’ N5EA6-3A2B (AB602436) ‘Tunicatimonas pelagia’ N5DB8-4 (AB602437) 100 http://ir.iswc.ac.cnMarivirga sericea LMG 13021 T (AB078081) T 56 Marivirga tractuosa NBRC 15989 (AB078072) 84 Nafulsella turpanensis ZLM-10 T (JN899241) T 79 Cesiribacter roseus 311 (HM775387) 100 Cesiribacter andamanensis AMV16 T (FN396961) T 56 95 Adhaeribacter aerophilus 6424S-25 (GQ421850) Adhaeribacter aerolatus 6515J-31 T (GQ421846) 100 Adhaeribacter terreus DNG6 T (EU682684) 59 Adhaeribacter aquaticus MBRG1.5 T (AJ626894) Aureibacter tunicatorum A5Q-118 T (AB572584) T 99 Persicobacter diffluens NBRC15940 (AB260929) 100 Persicobacter psychrovividus Asr22-19 T (AB260932) Fig. 1. Neighbour-joining phylogenetic tree 100 Flexithrix dorotheae NBRC 15987 T (AB078077) based on 16S rRNA gene sequences, show- T Rapidithrix thailandica TISTR 1750 (AB265192) ing the relationships between strain ZLM-10T 95 Sediminitomix flava NBRC 101625 T (AB255370) T and related taxa. Bootstrap values .50% 97 Limibacter armeniacum YM11-185 (AB359907) MBIC06993 T (AB276355) (based on 1000 replications) are shown at 51 Perexilibacter aurantiacus 73 Flammeovirga aprica NBRC 15941 T (AB247553) branching points. Thermonema lapsum ATCC T 100 Flammeovirga arenaria NBRC 15982 T (AB078078) 43542 was used as the outgroup. Bar, 0.02 Thermonema lapsum ATCC 43542 T (L11703) substitutions per nucleotide position. Downloaded from www.microbiologyresearch.org by 1640 International Journal of Systematic and Evolutionary Microbiology 63 IP: 221.11.67.252 On: Wed, 23 Dec 2015 02:05:16 Nafulsella turpanensis gen. nov., sp. nov. Table 1. Features that distinguish strain ZLM-10T from the pH 11.0) as buffer. Growth in the absence of NaCl and in closely related Cesiribacter roseus 311T and Marivirga the presence of 0.5–10.0 % (w/v) NaCl (at 0.5 % intervals) tractuosa DSM 4126T was investigated in peptone–yeast extract (PY) medium that contained (l21) 2.5 g Bacto peptone and 0.5 g yeast Strains: 1, ZLM-10T;2,C.
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