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Limibaculum Halophilum Gen. Nov., Sp. Nov., a New Member of the Family Rhodobacteraceae

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Limibaculum Halophilum Gen. Nov., Sp. Nov., a New Member of the Family Rhodobacteraceae TAXONOMIC DESCRIPTION Shin et al., Int J Syst Evol Microbiol 2017;67:3812–3818 DOI 10.1099/ijsem.0.002200 Limibaculum halophilum gen. nov., sp. nov., a new member of the family Rhodobacteraceae Yong Ho Shin,1 Jong-Hwa Kim,1 Ampaitip Suckhoom,2 Duangporn Kantachote2 and Wonyong Kim1,* Abstract A Gram-stain-negative, cream-pigmented, aerobic, non-motile, non-spore-forming and short-rod-shaped bacterial strain, designated CAU 1123T, was isolated from mud from reclaimed land. The strain’s taxonomic position was investigated by using a polyphasic approach. Strain CAU 1123T grew optimally at 37 C and at pH 7.5 in the presence of 2 % (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain CAU 1123T formed a monophyletic lineage within the family Rhodobacteraceae with 93.8 % or lower sequence similarity to representatives of the genera Rubrimonas, Oceanicella, Pleomorphobacterium, Rhodovulum and Albimonas. The major fatty acids were C18 : 1 !7c and 11-methyl C18 : 1 !7c and the predominant respiratory quinone was Q-10. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, two unidentified phospholipids, one unidentified aminolipid and one unidentified lipid. The DNA G+C content was 71.1 mol%. Based on the data from phenotypic, chemotaxonomic and phylogenetic studies, it is proposed that strain CAU 1123T represents a novel genus and novel species of the family Rhodobacteraceae, for which the name Limibaculumhalophilum gen. nov., sp. nov. The type strain is CAU 1123T (=KCTC 52187T, =NBRC 112522T). The family Rhodobacteraceae was first established by Garr- chemotaxonomic properties along with a detailed phyloge- ity et al. [1] as a member of the class Alphaproteobacteria netic analysis based on 16S rRNA gene sequences. (order Rhodobacterales, phylum Proteobacter). The names Selective isolation of strain CAU 1123T was performed were validated in Validation List 107 [2]. The class Alphap- according to Gordon and Mihm [17] by using the standard roteobacteria contains the most abundant bacteria from dilution plate technique. The appropriate dilutions were marine environments and the family Rhodobacteraceae is spread on marine agar 2216 (MA; Difco) plates and incu- also exclusively marine or hypersaline, with described iso- ˚ lates demonstrating salt tolerance [1, 3]. There are approxi- bated under aerobic conditions at 30 C for 10 days. A single colony of strain CAU 1123T was purified by streaking sev- mately 105 recognized genera in the family at the time of writing (www.bacterio.net/). Most members of the family eral times until pure culture was formed on MA at 30 C. À ˚ originate from marine environments, such as seawater, sedi- The strain was preserved at 80 C in marine broth 2216 (MB; Difco) supplemented with 25 % (v/v) glycerol. Strain ments, marine snails, marine hot springs, marine phyto- T plankton and marine sponges. Only 14 genera, including CAU 1123 has been deposited in the Korean Collection for Actibacterium [4], Hasllibacter [5], Huaishuia [6], Lenti- Type Cultures and the Biological Resource Centre, National T bacter [7], Oceanicella [8], Planktotalea [9], Poseidonocella Institute of Technology and Evaluation as KCTC 52187 T [10], Defluviimonas [11], Epibacterium [12], Litorisedimini- and NBRC 112522 , respectively. The type strains of type cola [13], Pararhodobacter [11], Pelagimonas [14], Pleomor- species of the most closely related genera, Rubrimonas clifto- T T phobacterium [15] and Profundibacterium [16], have been nensis OCh317 (=NBRC 100047 ), Oceanicella actignis T T described in the last 5 years. In the course of screening the PRQ-67 (=DSM 22673 ), Pleomorphobacterium xiame- bacteria off the west coast of the Korean peninsula, strain nense CLWT (=DSM 24423T), Rhodovulum sulfidophilum CAU 1123T was isolated from a mud sample from reclaimed Hansen W4T (=KCCM 41788T) and Albimonas donghaensis land in Modo (37 32¢12.28¢ N, 126 24¢51.47¢ E), Republic of DST (=KCTC 12586T), were obtained from the National Korea. The aim of the study was to establish the taxonomic Institute of Technology and Evaluation, the Deutsche position of strain CAU 1123T by using a polyphasic study Sammlung von Mikroorganismen und Zellkulturen, the that included the determination of phenotypic and Korean Culture Centre of Microorganisms and the Korean Author affiliations: 1Department of Microbiology, Chung-Ang University College of Medicine, Seoul 06974, Republic of Korea; 2Department of Microbiology, Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand. *Correspondence: Wonyong Kim, [email protected] Keywords: Limibaculum halophilum; Rhodobacteraceae; Alphaproteobacteria. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CAU 1123T is KX774334. Three figures are available with the online Supplementary Material. 002200 ã 2017 IUMS Downloaded from www.microbiologyresearch.org by IP: 165.194.103.153812 On: Thu, 25 Apr 2019 00:59:01 Shin et al., Int J Syst Evol Microbiol 2017;67:3812–3818 Collection for Type Cultures, and were used as reference The nearly complete 16S rRNA gene sequence (1403 bp) strains for fatty acid and biochemical analyses was obtained and compared with reference sequences avail- T able in the GenBank database. Phylogenetic analysis based The 16S rRNA gene of strain CAU 1123 was amplified T on the 16S rRNA gene showed that strain CAU 1123 using PCR with 27F and 1525R primers [18]. Multiple belonged to the family Rhodobacteraceae and formed a sepa- alignments with sequences of a broad selection of Rhodo- rate deep lineage with the recognized species of Rubrimonas, bacteraceae and calculation of sequence similarity levels Oceanicella, Pleomorphobacterium, Rhodovulum and Albi- were determined by using the CLUSTAL_X 2.1 program monas (Fig. 1). The trees reconstructed by using the least- [19] and the EzTaxon-e server (http://www.ezbiocloud. square, maximum-likelihood and maximum-parsimony net). Evolutionary distance matrices were created using algorithms presented a similar topology (data not shown). the neighbour-joining method [20]. Phylogenetic trees Strain CAU 1123T showed very low levels of 16S rRNA gene were generated by using three different methods: the sequence similarity to type strains of the most closely phylo- neighbour-joining [21], least-squares [22] and maximum- genetically related species in the family Rhodobacteraceae: likelihood [23] algorithms in the PHYLIP package [24]. R. cliftonensis OCh317T (similarity 93.8), O. actignis PRQ- The bootstrap resampling values, based on 1000 repli- 67T (similarity 93.0 %), P. xiamenense CLWT (similarity cates, were calculated using SEQBOOT and CONSENSE 93.0 %), Rhodovulum marinum JA128T (similarity 92.9 %) programs from the PHYLIP package [25]. The G+C con- and Albimonas pacifica P-50-3T (similarity 92.8 %). 16S tent of the genomic DNA was determined as described rRNA gene sequence similarities to all other species of the by Tamaoka and Komagata [26] using reversed-phase family Rhodobacteraceae were below 92.8 %. high-performance liquid chromatography (HPLC). The G T +C content was calculated by the ratio of deoxyguanosine Strain CAU 1123 and five reference strains, R. cliftonensis to thymidine using standard DNAs according to the OCh317T, O. actignis PRQ-67T, P. xiamenense CLWT, R. method of Mesbah et al. [27]. sulfidophilum Hansen W4T, and A. donghaensis DST were Rhodovulum steppense A-20sT (EU741680) 100 Rhodovulum strictum MB-G2T (D16419) 85 Rhodovulum viride JA756T (HE983843) 100 Rhodovulum sulfidophilum Hansen W4T (D16423) Rhodovulum mangrovi AK41T (HG529993) Rhodovulum iodosum N1 T (Y15011) 96 100 Confluentimicrobium lipolyticum SSK1-4T (KJ889015) 0.01 Rhodovulum marinum JA128T (AJ891122) Pseudoruegeria aestuarii MME-001T (KP410678) 100 T 99 Pseudoruegeria sabulilitoris GJMS-35 (KJ729032) 99 Pseudoruegeria aquimaris SW-255T (DQ675021) 95 Oceanicola litoreus M-M22T (JX291104) Rhodobacter veldkampii Hansen 51T (D16421) 91 Albidovulum xiamenense YBY-7T (HQ709061) Albimonas donghaensis DS2T (DQ280370) 79 Amaricoccus kaplicensis Ben101T (U88041) Oceanicella actignis PRQ-67T (JQ864435) 100 Pleomorphobacterium xiamenense CLW T (HQ709062) Limibaculum halophilum CAU 1123T (KX774334) Rubrimonas cliftonensis OCh317 T (D85834) 100 ‘Rubrimonas shengliensis’ SL014B-28A2 (GU125651) Escherichia coli DSM 30083T (X80725) Fig. 1. Phylogenetic tree derived from nearly complete 16S rRNA gene sequences showing the position of strain CAU 1123T among members of the family Rhodobacteraceae. The tree was reconstructed by using the neighbour-joining method and rooted Escherichia coli DSM 30083T (X80725) as the outgroup. Dots indicate that the corresponding nodes were also recovered in the trees generated with the maximum-likelihood and least-squares algorithms. Values of bootstrap (1000 resamplings) are indicated at the nodes and only values >70 % are shown. Bar, 0.01 substitutions per nucleotide position. Downloaded from www.microbiologyresearch.org by IP: 165.194.103.153813 On: Thu, 25 Apr 2019 00:59:01 Shin et al., Int J Syst Evol Microbiol 2017;67:3812–3818 cultivated on MA at their optimal growth temperatures to incubation. The pH value for growth was examined at 37 C determine their morphological, physiological and biochemi- in MB adjusted within a pH range of 4.5–11.5 (at intervals cal properties. Cell morphology was observed by light (DM of 0.5 pH unit) and values were confirmed again and 1000; Leica) and transmission electron (TEM; JEM 1010, adjusted using sodium acetate/acetic acid,
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