Downloaded from www.microbiologyresearch.org by IP: 54.224.212.167 On: Fri, 05 Aug 2016 15:14:46 Flavobacterium lindanitolerans sp. nov., isolated from hexachlorocyclohexane-contaminated soil Simran Jit, Mandeep Dadhwal, Om Prakash and Rup Lal Correspondence Rup Lal duzdel@vsnl.com Molecular Biology Laboratory, Department of Zoology, University of Delhi, Delhi 110 007, India A Gram-negative, non-spore-forming, cream–yellow-pigmented bacterial strain, IP-10 T , was isolated from soil samples from a waste site highly contaminated with hexachlorocyclohexane in Ummari village, India. The organism showed the highest 16S rRNA gene sequence similarity of 92.7 % with Flavobacterium soli KCTC 12542 T and levels of 87–92 % with the type strains of other recognized species of the genus Flavobacterium. The DNA G+C content of strain IP-10 T was 31 mol%. The predominant fatty acids were iso-C 15 : 0 (22.1 %), iso-C 17 : 0 3-OH (18.5 %) and summed feature 3 (comprising C 16 : 1 v7c and/or iso-C 15 : 0 2-OH; 13.2 %). Strain IP-10 T could be differentiated from recognized species of the genus Flavobacterium based on a number of phenotypic features. Strain IP-10 T is therefore considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium lindanitolerans sp. nov. is proposed. The type strain is IP-10 T (5MTCC 8597 T 5CCM 7424 T ). The genus Flavobacterium, the type genus of the family Flavobacteriaceae, was proposed by Bergey et al. (1923) and the description was considerably emended by Bernardet et al. (1996). It belongs to the phylum Bacteroidetes (Bernardet & Bowman, 2006) (formerly the Flavo- bacterium–Flexibacter–Bacteroides group; Woese et al., 1990). Members of the genus are widely distributed in nature and are routinely isolated from soil (Yoon et al., 2006), sediments, freshwater and marine water ecosystems, microbial mats (Van Trappen et al., 2003, 2004), sea ice (Humphry et al., 2001) and infected fish. The genus Flavobacterium comprises Gram-negative, non-spore- forming, strictly aerobic, predominantly gliding, pigmen- ted bacteria with menaquinone-6 as the major respiratory quinone and a DNA G+C content of 30–41 mol% (Bernardet & Bowman. 2006; Park et al., 2006). Flavobacterium aquatile is the type species of the genus. The present study deals with the taxonomic characteriza- tion of a novel species of the genus Flavobacterium isolated from soil from a recently created waste site for a lindane manufacturing unit situated in Ummari village, Lucknow city, in the state of Uttar Pradesh, India. Lindane, c-hexachlorocyclohexane (HCH), is the only HCH isomer that has insecticidal properties. Strain IP-10 T was isolated after 7 days incubation at 28 u C by plating serially diluted soil samples on Luria–Bertani agar plates (10 g tryptone, 5 g yeast extract, 5 g NaCl, 1 g D-glucose, 15 g agar per litre) containing nystatin (30 mg ml 21 ) and streptomycin (200 mg ml 21 ), used to isolate HCH-degrading sphingo- monads (Vanbroekhoven et al., 2004). 16S rRNA gene sequence analysis, fatty acid profiling and other phenotypic characteristics revealed that the new isolate belonged to the genus Flavobacterium but exhibited sufficient significant differences from other closely related members of the genus to be considered as representing a novel species. 16S rRNA gene sequence analysis was carried out as described by Prakash et al. (2007). Briefly, a single colony of strain IP-10 T was picked with a sterile toothpick, suspended in 50 ml of sterile deionized water and heated at 95 uC for 10 min. Following centrifugation at 9000 g for 5 min and ten-fold dilution with sterile deionized water, the 16S rRNA gene sequence was amplified by using the universal primer pair 27F (59-AGAGTTTGATCCTGG- CTCAG-39) and 1492R (59-TACGGTTACCTTGTTA- CGACTT-39). The amplified product was purified and used for cycle sequencing with BigDye terminator chem- istry and run on a 3100 Avant Genetic Analyzer (Applied Biosystems). The stretch of the 16S rRNA gene sequence obtained (1359 bp) was checked manually for quality and gaps. Similarity searches were conducted by using the Sequence Match tool of the Ribosomal Database Project (http:// rdp.cme.msu.edu/seqmatch/seqmatch_intro.jsp) and the BLAST program of the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov). For construc- tion of phylogenetic trees, sequences of the 19 most closely Abbreviations: FAME, fatty acid methyl ester; HCH, hexachlorocyclo- hexane. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain IP-10 T is EF424395. A phylogenetic tree obtained by using the EzTaxon server is available with the online version of this paper. International Journal of Systematic and Evolutionary Microbiology (2008), 58, 1665–1669 DOI 10.1099/ijs.0.65578-0 65578 G 2008 IUMS Printed in Great Britain 1665