Neha Niharika

Comparative Metagenomic Analysis of Soil Microbial Communities across Three Hexachlorocyclohexane Contamination Levels

This paper presents the characterization of the microbial community responsible for the in-situ bioremediation of hexachlorocyclohexane (HCH). Microbial community structure and function was analyzed using 16S rRNA amplicon and shotgun metagenomic sequencing methods for three sets of soil samples. The three samples were collected from a HCH-dumpsite (450 mg HCH/g soil) and comprised of a HCH/soil ratio of 0.45, 0.0007, and 0.00003, respectively. Certain bacterial; (Chromohalobacter, Marinimicrobium, Idiomarina, Salinosphaera, Halomonas, Sphingopyxis, Novosphingobium, Sphingomonas and Pseudomonas), archaeal; (Halobacterium, Haloarcula and Halorhabdus) and fungal (Fusarium) genera were found to be more abundant in the soil sample from the HCH-dumpsite. Consistent with the phylogenetic shift, the dumpsite also exhibited a relatively higher abundance of genes coding for chemotaxis/motility, chloroaromatic and HCH degradation (lin genes). Reassembly of a draft pangenome of Chromohalobacter salaxigenes sp. (∼8X coverage) and 3 plasmids (pISP3, pISP4 and pLB1; 13X coverage) containing lin genes/clusters also provides an evidence for the horizontal transfer of HCH catabolism genes.

Pontibacter lucknowensis sp. nov., isolated from a hexachlorocyclohexane dump site

A Gram-negative, orange-pigmented, rod-shaped, motile and aerobic bacterial strain designated DM9(T) 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-C(17 : 0) 3-OH (6.00 %), iso-C(15 : 0) (21.54 %) and summed feature 4 (comprising C(17 : 1) iso I/anteiso B; 32.3 %). The polar lipid profile of strain DM9(T) showed the presence of phosphatidylethanolamine, an unidentified aminophospholipid, two unknown aminolipids and four unknown polar lipids. Strain DM9(T) 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 DM9(T) from closely related members of the genus Pontibacter. It is proposed that DM9(T) represents a novel species, Pontibacter lucknowensis sp. nov.; the type strain is DM9(T) (= CCM 7955(T) = MTCC 11079(T)).

Novosphingobium barchaimii sp. nov., isolated from hexachlorocyclohexane-contaminated soil.

A yellow-pigmented bacterial strain, designated LL02(T), was isolated from hexachlorocyclohexane-contaminated soil from Spolana Neratovice, a former Czech producer of lindane. A neighbour-joining tree based on 16S rRNA gene sequences showed that strain LL02(T) occupied a distinct phylogenetic position in the genus Novosphingobium and showed the highest sequence similarity with Novosphingobium resinovorum NCIMB 8767(T) (98.59 %). DNA-DNA relatedness between strain LL02(T) and its closest phylogenetic neighbours was <70 %, which indicated that strain LL02(T) represented a novel species of the genus Novosphingobium. The DNA G+C content of strain LL02(T) was 67.72±0 mol%. The major respiratory quinone was ubiquinone Q-10. The polar lipid profile of the isolate corresponded to those reported for other members of the genus Novosphingobium (phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylmonomethylethanolamine and sphingoglycolipids), thus supporting its classification in the genus. Spermidine was the major polyamine. The major fatty acids were summed feature 3 (consisting of C(16 : 1)ω7c and/or C(16 : 1)ω6c; 40.13 %), summed feature 8 (consisting of C(18 : 1)ω7c and/or C(18 : 1)ω6c; 31.09 %) and C(14 : 0) 2-OH (23.16 %). The results obtained from DNA-DNA hybridization and biochemical and physiological tests clearly distinguished the isolate from its closest phylogenetic neighbours. Thus, strain LL02(T) represents a novel species of the genus Novosphingobium, for which the name Novosphingobium barchaimii sp. nov. is proposed. The type strain is LL02(T) ( = CCM 7980(T)  = DSM 25411(T)).

Draft Genome Sequence of Thermus sp. Strain RL, Isolated from a Hot Water Spring Located atop the Himalayan Ranges at Manikaran, India

Thermus sp. strain RL was isolated from a hot water spring (90°C to 98°C) at Manikaran, Himachal Pradesh, India. Here we report the draft genome sequence (20,36,600 bp) of this strain. The draft genome sequence consists of 17 contigs and 1,986 protein-coding sequences and has an average G+C content of 68.77%.

Sphingomonas laterariae sp. nov., isolated from a hexachlorocyclohexane-contaminated dump site.

A Gram-staining-negative, non-motile, cream-coloured and rod-shaped bacterium, designated strain LNB2(T), was isolated from a hexachlorocyclohexane-contaminated dump site in the village of Ummari, in northern India. The taxonomic position of the novel strain was investigated by using a polyphasic approach. In a phylogenetic analysis based on 16S rRNA gene sequences, strain LNB2(T) appeared to be most closely related to Sphingomonas haloaromaticamans A175(T) (98.0% sequence similarity) and Sphingomonas histidinilytica UM2(T) (97.3%). In DNA-DNA hybridizations, the levels of DNA-DNA relatedness between the novel strain and S. haloaromaticamans A175(T) and S. histidinilytica UM2(T) were found to be low (8.6% and 5.6%, respectively). The genomic DNA G+C content of strain LNB2(T) was 61.0 mol%. The novel strains predominant fatty acids were summed feature 8 (C(18:1)ω7c and/or C(18:1)ω6c), C(16:0), summed feature 3 (C(16:1)ω7c and/or C(16:1)ω6c), C(14:0) 2-OH, C(17:1)ω6c and 11-methyl C(18:1)ω7c. The major ubiquinone was Q-10, the predominant polyamine was homospermidine, and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, sphingoglycolipid, phosphatidylethanolamine and phosphatidyldimethylethanolamine. Based on the phylogenetic, biochemical and chemotaxonomic evidence and the results of the DNA-DNA hybridizations, strain LNB2(T) represents a novel species of the genus Sphingomonas, for which the name Sphingomonas laterariae sp. nov. is proposed. The type strain is LNB2(T) ( = MTCC 10873(T) = CCM 7880(T) = DSM 25432(T)).

Sphingobium baderi sp. nov., isolated from a hexachlorocyclohexane dump site

A Gram-stain-negative, rod-shaped and white-coloured bacterial strain, designated LL03(T), was isolated from hexachlorocyclohexane-contaminated soil at Spolana Neratovice, Czech Republic, where lindane was formerly produced. Strain LL03(T) was found to be a degrader of α-, γ- and δ-isomers of hexachlorocyclohexane, although no significant degradation activity was observed for the β-isomer. A neighbour-joining tree based on 16S rRNA gene sequences showed that strain LL03(T) occupied a distinct phylogenetic position in the Sphingobium cluster, showing the highest similarity with Sphingobium wenxiniae JZ-1(T) (99.2 %). The DNA G+C content of strain LL03(T) was 67.0 mol%. DNA-DNA relatedness values of strain LL03(T) with its close phylogenetic neighbours were below the threshold level of 70 %, supporting its identification as a representative of a novel species of the genus Sphingobium. The predominant respiratory quinone was ubiquinone Q-10. The polar lipid profile of strain LL03(T) also corresponded to those reported for other Sphingobium species (phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylmonomethylethanolamine and sphingoglycolipid), supporting its identification as a member of the genus Sphingobium. Spermidine was identified as the major polyamine. The predominant fatty acids were 16 : 0, summed feature 3 (16 : 1ω7c and/or 16 : 1ω6c), summed feature 8 (18 : 1ω7c and/or 18 : 1ω6c) and 14 : 0 2-OH. The polar lipid pattern, the presence of spermidine and ubiquinone Q-10, the predominance of the cellular fatty acids C(18 : 1)ω7c, C(16 : 0) and C(14 : 0) 2-OH and the G+C content of the genomic DNA supported the affiliation of the strain to the genus Sphingobium. The results obtained after DNA-DNA hybridization, biochemical and physiological tests clearly distinguished it from closely related species of the genus Sphingobium. Therefore, strain LL03(T) represents a novel species of the genus Sphingobium for which the name Sphingobium baderi LL03(T) sp. nov. is proposed; the type strain is LL03(T) ( = CCM 7981(T) = DSM 25433(T)).

Draft Genome Sequence of Sphingobium chinhatense Strain IP26T, Isolated from a Hexachlorocyclohexane Dumpsite

ABSTRACT Sphingobium chinhatense strain IP26T is a conducive hexachlorocyclohexane (HCH) degrader isolated from a heavily contaminated (450 mg HCH/g soil) HCH dumpsite. IP26T degrades α-, β-, γ-, and δ-HCH, which are highly persistent in the environment. Here we report the draft genome sequence (~5.8 Mbp) of this strain.

Sphingobium czechense sp nov., isolated from a hexachlorocyclohexane dump site

A yellow-pigmented bacterial strain, designated LL01(T), was isolated from hexachlorocyclohexane (HCH)-contaminated soil at Spolana Neratovice, a former Czech producer of lindane. A neighbour-joining tree based on 16S rRNA gene sequences showed that strain LL01(T) occupied a distinct phylogenetic position in the Sphingobium cluster, showing highest similarity to Sphingobium rhizovicinum CC-FH12-1(T) (98.5 %). The DNA G+C content of strain LL01(T) was 66.1 mol%. The predominant respiratory pigment was ubiquinone Q-10. The polar lipid profile of strain LL01(T) also corresponded to those reported for other Sphingobium species (phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, sphingoglycolipids), supporting its identification as a member of the genus Sphingobium. Spermidine was the major polyamine observed. The results obtained from DNA-DNA hybridization and biochemical and physiological tests clearly distinguished strain LL01(T) from closely related species of the genus Sphingobium. Therefore, strain LL01(T) represents a novel species of the genus Sphingobium, for which the name Sphingobium czechense sp. nov. is proposed (type strain LL01(T) = CCM 7979(T) = DSM 25410(T)).

Functional screening of enzymes and bacteria for the dechlorination of hexachlorocyclohexane by a high-throughput colorimetric assay

Two distinct microbial dehalogenases are involved in the first steps of degradation of hexachlorocyclohexane (HCH) isomers. The enzymes, LinA and LinB, catalyze dehydrochlorination and dechlorination reactions of HCH respectively, each with distinct isomer specificities. The two enzymes hold great promise for use in the bioremediation of HCH residues in contaminated soils, although their kinetics and isomer specificities are currently limiting. Here we report the functional screening of a library of 700 LinA and LinB clones generated from soil DNA for improved dechlorination activity by means of a high throughput colorimetric assay. The assay relies upon visual colour change of phenol red in an aqueous medium, due to the pH drop associated with the dechlorination reactions. The assay is performed in a microplate format using intact cells, making it quick and simple to perform and it has high sensitivity, dynamic range and reproducibility. The method has been validated with quantitative gas chromatographic analysis of promising clones, revealing some novel variants of both enzymes with superior HCH degrading activities. Some sphingomonad isolates with potentially superior activities were also identified.

Bioremediation of Hexachlorocyclohexane (HCH) Pollution at HCH Dump Sites

Globally, the period from early the 1950s to late 1980s has shown an increased use of primarily three pesticides namely DDT.