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.
Sphingobium indicum B90A, an efficient degrader of hexachlorocyclohexane (HCH) isomers, was isolated in 1990 from sugarcane rhizosphere soil in Cuttack, India. Here we report the draft genome sequence of this bacterium, which has now become a model system for understanding the genetics, biochemistry, and physiology of HCH degradation.
A yellow-pigmented, Gram-negative, aerobic, non-motile, non-spore-forming, rod-shaped-bacterium, LE124(T), was isolated from a hexachlorocyclohexane (HCH) dumpsite located in Lucknow, India. The type strain LE124(T) grew well with hexachlorocyclohexane as a sole carbon source, degrading it within 24 h of incubation. Phylogenetic analysis of strain LE124(T) showed highest 16S rRNA gene sequence similarity to Novosphingobium barchaimii LL02(T) (98.5%), Novosphingobium panipatense SM16(T) (98.1%), Novosphingobium soli CC-TPE-1(T) (97.9%), Novosphingobium naphthalenivorans TUT562(T) (97.6%), Novosphingobium mathurense SM117(T) (97.5%) and Novosphingobium resinovorum NCIMB 8767(T) (97.5%) and lower sequence similarity (<97%) to all other members of the genus Novosphingobium. The DNA-DNA relatedness between strain LE124(T) and N. barchaimii LL02(T) and other related type strains was found to vary from 15% to 45% confirming that it represents a novel species. The genomic DNA G+C content of strain LE124(T) was 60.7 mol%. The predominant fatty acids were summed feature 8 (C18:1ω7c, 49.1%), summed feature 3 (C16:1ω7c/C16:1ω6c, 19.9%), C16:0 (6.7%), C17:1ω6c (4.9%) and a few hydroxyl fatty acids, C14:0 2-OH (9.4%) and C16:0 2-OH (2.1%). Polar lipids consisted mainly of phosphatidyldimethylethanolamine, phosphatidylcholine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, sphingoglycolipid and some unidentified lipids. The major respiratory quinone was ubiquinone Q-10. Spermidine was the major polyamine observed. Phylogenetic analysis, DNA-DNA hybridization, chemotaxonomic and phenotypic analysis support the conclusion that strain LE124(T) represents a novel species within the genus Novosphingobium for which we propose the name Novosphingbium lindaniclasticum sp. nov. The type strain is LE124(T) (=CCM 7976(T)=DSM 25409(T)).
bIn this study, Acinetobacter sp. strain HA was isolated from the midgut of a fifth-instar larva of Helicoverpa armigera. Here, we report the draft genome sequence (3,125,085 bp) of this strain that consists of 102 contigs, 2,911 predicted coding sequences, and a G؉C content of 41%.A cinetobacter sp. strain HA was isolated from the midgut of a fifth-instar larva of Helicoverpa armigera collected from an agricultural field in Maharashtra, India (7). The strain exhibited enhanced esterase activity and facilitated the metabolism of the insecticide cypermethrin, which can thus contribute to insect resistance (1). Here, we report the draft genome sequence of strain HA using the Roche 454 GS (FLX Titanium) system (3-kb pairedend library; 511,555 reads) that led to ϳ45-fold coverage of the entire genome. The reads generated were assembled into 102 contigs by using Ray de novo assembler version 0.0.3 set at a k-mer length of 25 (3). The final assembly was validated using paired-end constraints (N 50 contigs, 68.4 kb). The draft genome was annotated using RAST version 4.0 (2), NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP; http://www.ncbi.nlm.nih .gov/genomes/static/Pipeline.html), and the KEGG database (4).The draft genome of Acinetobacter sp. HA represents a genome size of 3,125,085 bp with an average GϩC content of 41%. A total of 2,911 protein-coding sequences and 932 hypothetical proteins were predicted on annotation. The genome contains 66 tandem repeats, and the coding density was calculated to be 84.83%. Sixtyfour predicted tRNA genes, representing all amino acids, were identified by using tRNAscan-SE (6). Four copies of 5S rRNA, 2 copies of 23S rRNA, and 2 copies of 16S rRNA were found in the sequence by using RNAmmer (5). In addition, 123 predicted transposase genes but no gene coding for pyruvate kinase, a key component in carbohydrate metabolism, were found. However, 3 polyglutamic acid (PGA) synthesis-related protein-coding genes responsible for biofilm formation were present. Also, the presence of genes coding for esterase enzyme have been observed. Annotations by RAST revealed 383 subsystems and 45 genes relevant to resistance to antibiotics, and toxic compounds were found. Among these, 9 genes coding for cobalt-zinc-cadmium resistance and 14 coding for multidrug resistance efflux pumps were found. RAST results also indicated Acinetobacter lwoffii SH145 (score, 520), Acinetobacter baumannii AB0057 (score, 518), and Acinetobacter baumannii ACICU (score, 518) as the closest neighbors of strain HA. Detailed analysis of the genome sequence of Acinetobacter sp. HA will further provide a comprehensive understanding toward predicting its role in metabolizing insecticides and hence conferring resistance to Helicoverpa armigera.Nucleotide sequence accession number. The genome sequence of Acinetobacter sp. strain HA is available in GenBank under accession number AJXD00000000. ACKNOWLEDGMENTS
Hexachlorocyclohexane (HCH) contaminated soils were treated for a period of up to 64 days in situ (HCH dumpsite, Lucknow) and ex situ (University of Delhi) in line with three bioremediation approaches. The first approach, biostimulation, involved addition of ammonium phosphate and molasses, while the second approach, bioaugmentation, involved addition of a microbial consortium consisting of a group of HCH-degrading sphingomonads that were isolated from HCH contaminated sites. The third approach involved a combination of biostimulation and bioaugmentation. The efficiency of the consortium was investigated in laboratory scale experiments, in a pot scale study, and in a full-scale field trial. It turned out that the approach of combining biostimulation and bioaugmentation was most effective in achieving reduction in the levels of α- and β-HCH and that the application of a bacterial consortium as compared to the action of a single HCH-degrading bacterial strain was more successful. Although further degradation of β- and δ-tetrachlorocyclohexane-1,4-diol, the terminal metabolites of β- and δ-HCH, respectively, did not occur by the strains comprising the consortium, these metabolites turned out to be less toxic than the parental HCH isomers.
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