International Journal of Systematic and Evolutionary Microbiology (2013), 63, 309–313 DOI 10.1099/ijs.0.040147-0 Pontibacter lucknowensis sp. nov., isolated from a hexachlorocyclohexane dump site Vatsala Dwivedi, Neha Niharika and Rup Lal Correspondence Molecular Biology Laboratory, Department of Zoology, University of Delhi, Delhi 110007, India Rup Lal [email protected] A Gram-negative, orange-pigmented, rod-shaped, motile and aerobic bacterial strain designated DM9T was isolated from hexachlorocyclohexane (HCH)-contaminated soil (Lucknow, India) and its taxonomic position was determined using a polyphasic approach. 16S rRNA gene sequence analysis showed that the isolate belonged to the phylum Bacteroidetes and confirmed its placement in the genus Pontibacter, with sequence similarity ranging from 93.92 to 96.21 % with other members of the genus Pontibacter. The major cellular fatty acids of the novel strain were iso- C17 : 0 3-OH (6.00 %), iso-C15 : 0 (21.54 %) and summed feature 4 (comprising C17 : 1 iso I/ anteiso B; 32.3 %). The polar lipid profile of strain DM9T showed the presence of phosphatidylethanolamine, an unidentified aminophospholipid, two unknown aminolipids and four unknown polar lipids. Strain DM9T contained MK-7 as the predominant menaquinone and its DNA G+C content was 49.2 mol%. sym-Homospermidine was the major polyamine observed in the cell. The results obtained on the basis of phenotypic characteristics, phylogenetic analysis, biochemical and physiological tests clearly distinguished DM9T from closely related members of the genus Pontibacter. It is proposed that DM9T represents a novel species, Pontibacter lucknowensis sp. nov.; the type strain is DM9T (5CCM 7955T5MTCC 11079T). The genus Pontibacter belongs to the phylum Bacteroidetes agar plates. An orange-coloured colony, designated DM9T, and was first described by Nedashkovskaya et al. (2005). At appeared within 36 h of incubation at 28 uC which was the time of writing, there were seven species with validly picked and purified by streaking on LB agar. A polyphasic published names belonging to this genus, Pontibacter approach (Prakash et al., 2007) was adopted for taxonomic actiniarum (Nedashkovskaya et al., 2005), P. akesuensis classification of strain DM9T. (Zhou et al., 2007), P. korlensis (Zhang et al., 2008), P. 16S rRNA gene sequence determines the phylogenetic niistensis (Dastager et al., 2010), P. roseus (Suresh et al., relationship among bacteria based on its universal 2006), P. xinjiangensis (Wang et al., 2010) and P. populi distribution, highly conserved nature, the fundamental (Xu et al., 2012). In addition, ‘Pontibacter salisaro’ has also role of the ribosome in protein synthesis, no horizontal been described (Joung et al., 2011), but the name is not yet transfer and its rate of evolution, which represents an validly published. The type strains of the recognized species appropriate level of variation between organisms. The 16S of the genus are Gram-negative, rod-shaped, aerobic, rRNA gene sequence analysis of strain DM9T was carried heterotrophic and pigmented. Various species of the genus out as described by Prakash et al. (2007) using a 3100- Pontibacter have been isolated from marine actinians, Avant Genetic Analyzer sequencer at the Department of desert soil, muddy water and forest soil (Nedashkovskaya Zoology, University of Delhi, India. The sequence thus et al., 2005; Zhou et al., 2007; Suresh et al., 2006; Dastager obtained was assembled manually using Sequencing et al., 2010). In our ongoing effort (Kaur et al., 2011) to Analysis version 5.1.1 and Clone Manager software version identify the culturable and unculturable diversity at a 5. A continuous stretch of 1400 bp of the 16S rRNA gene of hexachlorocyclohexane (HCH) dump site, Lucknow, Uttar the strain DM9T was obtained and this sequence was Pradesh in North India (27u009 N and 81u099 E), we subjected to similarity search using the Seqmatch tool describe here a further species of the genus Pontibacter. For of the Ribosomal Database Project (http://rdp.cme.msu. this purpose, soil samples from the HCH dump site were edu/) and BLAST program of the National Centre for collected, serially diluted and plated on Luria–Bertani (LB) Biotechnology Information (http://www.ncbi.nlm.nih.gov/). A non-redundant BLASTN search of full sequences through Abbreviation: HCH, hexachlorocyclohexane. GenBank (Altschul et al., 1990), RDP II (Maidak et al., The GenBank accession number for 16S rRNA gene sequence of strain 2001) and EzTaxon Server version 2.1 (Chun et al., 2007) DM9T (5CCM 7955T5MTCC 11079T) is JN561788 identified its closest relatives. 16S rRNA gene sequence T Two supplementary figures and a supplementary table are available with analysis (1400 bp) revealed that strain DM9 showed T the online version of this paper. highest similarity to P. korlensis X14-1 (96.21 %). The 040147 G 2013 IUMS Printed in Great Britain 309 V. Dwivedi, N. Niharika and R. Lal nearly full-length 16S rRNA gene sequences of all members Cantor (1969) and an evolutionary tree was constructed of the genus Pontibacter were retrieved for the construction using the neighbour-joining method of Saitou & Nei (1987). of a phylogenetic tree. The 16S rRNA gene sequence Statistical evaluation of the tree topology based on 1000 similarity between strain DM9T and member of the genus resamplings was done using the bootstrap option in the T Pontibacter ranged from 93.92 to 96.21 %. Strain DM9T TREECON software (Fig. 1). Strain DM9 , falls in the clade showed highest similarity to P. korlensis X14-1T (96.21 %) containing members exclusively belonging to the genus followed by ‘P. salisaro’ HMC5104 (95.89 %), P. actiniarum Pontibacter. T T KMM 6156 (95.35 %), P. niistensis NII-0905 (95.06 %), P. Cell morphology was examined using light (Eclipse E 600; roseus SRC-1T (94.43 %), P. populi HYL7-15T (94.06 %), P. T T Nikon) and electron microscopes (transmission electron akesuensis AKS 1 (94.01 %) and P. xinjiangensis 311-10 microscope, TEM 269D; Morgagni, FEI) (see Fig. S1 (93.92 %). A phylogenetic tree was constructed using available in IJSEM Online). Microscopic analysis revealed sequences of closely related species selected from GenBank that strain DM9T is non-flagellated. Gliding motility of the and EzTaxon Server version 2.1 (Chun et al., 2007). The 16S organism was tested on a fresh LB broth culture using the rRNA gene sequence of the Reichenbachiella agariperforans hanging-drop method (Bowman et al., 2003) and was T KMM 3525 was used as an outgroup and the selected found to be positive. Colonies of strain DM9T showed sequences were aligned using the program CLUSTAL_X optimum growth on LB agar and marine agar at 28 uC version 1.81b (Thompson et al., 1997). Trimming of within 36 h of incubation. DM9T colonies were orange- terminal nucleotides that were not common to all sequences coloured, circular and smooth. The Gram staining test was was carried out manually. Phylogenetic analysis was carried performed using a Gram staining kit (HiMedia); the strain out using the TREECONW software package version 1.3b (Van was found to be Gram-negative. Antibiotic sensitivity tests de Peer & De Wachter, 1994). The evolutionary distance were performed on Muller-Hinton II medium using matrix was calculated using the distance model of Jukes & Readymade Sensi-discs (HiMedia) with varying amounts T 0.02 757 Hymenobacter xinjiangensis X2-1g (DQ888329) 531 Hymenobacter rigui WPCB131T (DQ089669) T 571 Hymenobacter gelipurpurascens Txg1 (Y18836) Hymenobacter yonginensis HMD1010T (GU808562) Hymenobacter psychrotolerans Tibet-IIU11T (DQ177475) T 999 Hymenobacter aerophilus DSM13606 (EU155008) 552 Hymenobacter actinosclerus CCUG 39621T (Y17356) 756 Hymenobacter psychrophilus BZ33rT (GQ131579) 998 Hymenobacter tibetensis XTM003T(EU382214) 591 Hymenobacter norwichensis NS/50T (AJ549285) Hymenobacter roseosalivarius AA-718T (Y18833) 945 1000 Hymenobacter chitinivorans Txc1T (Y18837) Hymenobacter elongatus VUG-A112T (GQ454797) 981 Hymenobacter daecheongensis Dae14T (EU370958) 1000 1000 Hymenobacter soli PB17T (AB251884) Hymenobacter glaciei VUG-A130T (GQ454806) 1000 Hymenobacter ocellatus Myx 2105T (Y18835) Hymenobacter deserti ZLB-3T (EU325941) 1000 997 Adhaeribacter aerophilus 6424S-25T ( GQ421850) 965 Adhaeribacter aerolatus 6515J-31T (GQ421846) 895 Adhaeribacter aquaticus MBRG1.5T (AJ626894) T 834 Adhaeribacter terreus DNG6 (EU682684) Pontibacter populi HLY7-15T (HQ223078) 1000 Pontibacter xinjiangensis 311-10T (FJ004994) 743 ‘Pontibacter salisaro’ HMC5104 (FJ903180) 513 Pontibacter lucknowensis DM9T (JN561788) 711 T 497 Pontibacter korlensis X14-1 (DQ888330) Pontibacter actiniarum KMM 6156T (AY989908) 611 Pontibacter roseus SRC-1T (AM049256) Pontibacter akesuensis AKS 1T (DQ672723) Pontibacter niistensis NII-0905T (FJ897494) Reichenbachiella agariperforans KMM 3525T (AB058919) Fig. 1. Phylogenetic tree based on nearly complete 16S rRNA gene sequence data showing the evolutionary relationship of strain DM9T and representative members of the genera Pontibacter, Adhaeribacter and Hymenobacter. The tree was constructed by using the neighbour-joining (Jukes & Cantor, 1969) method of TREECONW software and the rooting was done by using Reichenbachiella agariperforans KMM 3525T as the outgroup. Bar, 0.02 nt substitutions per 1000 nt positions. The GenBank accession numbers for the 16S rRNA gene sequences of each strain are shown in parentheses. 310 International Journal of Systematic and Evolutionary Microbiology 63 Pontibacter lucknowensis sp. nov. of antibiotics.