Shailly Anand

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.

Acinetobacter indicus sp. nov., isolated from a hexachlorocyclohexane dump site.

The taxonomic position of a Gram-negative, non-motile, oxidase negative and catalase positive strain, A648(T), isolated from a hexachlorocyclohexane (HCH) dump site located in Lucknow, India, was ascertained by using a polyphasic approach. A comparative analysis of a partial sequence of the rpoB gene and the 16S rRNA gene sequence revealed that strain A648(T) belonged to the genus Acinetobacter. DNA-DNA relatedness values between strain A648(T) and other closely related members (16S rRNA gene sequence similarity greater than 97%), namely Acinetobacter radioresistens DSM 6976(T), A. venetianus ATCC 31012(T), A. baumannii LMG 1041(T), A. parvus LMG 21765(T) A. junii LMG 998(T) and A. soli JCM 15062(T), were found to be less than 8%. The major cellular fatty acids of strain A648(T) were 18:1ω9c (19.6%), summed feature 3 (15.9%), 16:0 (10.6%) and 12:0 (6.4%). The DNA G+C content was 40.4 mol%. The polar lipid profile of strain A648(T) indicated the presence of diphosphatidylglycerol, phosphatidylethanolamine, followed by phosphatidylglycerol and phosphatidylcholine. The predominant polyamine of strain A648(T) was 1,3-diaminopropane and moderate amounts of putrescine, spermidine and spermine were also detected. The respiratory quinone consisted of ubiquinone with nine isoprene units (Q-9). On the basis of DNA-DNA hybridization, phenotypic characteristics and chemotaxonomic and phylogenetic comparisons with other members of the genus Acinetobacter, strain A648(T) is found to be a novel species of the genus Acinetobacter, for which the name Acinetobacter indicus sp. nov. is proposed. The type strain is A648(T) ( = DSM 25388(T) = CCM 7832(T)).

Genome Sequence of Sphingobium indicum B90A, a Hexachlorocyclohexane-Degrading Bacterium

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.

Whole Genome Sequence of the Rifamycin B-Producing Strain Amycolatopsis mediterranei S699

Amycolatopsis mediterranei S699 is an actinomycete that produces an important antibiotic, rifamycin B. Semisynthetic derivatives of rifamycin B are used for the treatment of tuberculosis, leprosy, and AIDS-related mycobacterial infections. Here, we report the complete genome sequence (10.2 Mb) of A. mediterranei S699, with 9,575 predicted coding sequences.

Novosphingobium lindaniclasticum sp. nov., a hexachlorocyclohexane (HCH)-degrading bacterium isolated from an HCH dumpsite

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)).

Phylogenetic analyses of phylum Actinobacteria based on whole genome sequences

Actinobacteria constitute one of the largest and ancient taxonomic phylum within the domain bacteria and are well known for their secondary metabolites. Considerable variation in the metabolic properties, genome size and GC content of the members of this phylum has been observed. Therefore, the placement of new or existing species based on 16S rRNA gene sometimes becomes problematic due to the low congruence level. In the present study, phylogeny of ninety actinobacterial genomes was reconstructed using single gene and whole genome based data. Where alignment-free phylogenetic method was found to be more robust, the concatenation of 94 proteins improved the resolution which all single gene based phylogenies failed to resolve. The comprehensive analysis of 94 conserved proteins resulted in a total of 42,447 informative sites, which is so far the largest meta-alignment obtained for this phylum. But the ultimate resolved phylogeny was obtained by generating a consensus tree by combining the information from single gene and genome based phylogenies. The present investigation clearly revealed that the consensus approach is a useful tool for phylogenetic inference and the taxonomic affiliations must be based on this approach. The consensus approach suggested that there is a need for taxonomic amendments of the orders Frankiales and Micrococcales.

Microbacterium amylolyticum sp. nov., isolated from soil from an industrial waste site.

A Gram-staining-positive, heterotrophic, aerobic, non-motile, non-endospore-forming, yellow-coloured rod, designated strain N5(T), was isolated from a soil sample collected at an industrial waste site in Noida, on the outskirts of Delhi, India. In phylogenetic analyses based on 16S rRNA gene sequences, strain N5(T) was most closely related to members of established species in the genus Microbacterium (with sequence similarities of approximately 94.0-97.6 %), particularly Microbacterium indicum LMG 23459(T) (97.59 %) and Microbacterium gubbeenense LMG 19263(T) (97.18 %). In DNA-DNA hybridization studies, however, none of the DNA-DNA relatedness values between strain N5(T) and members of the genus Microbacterium exceeded 11.3 %. The genomic DNA G+C content of the novel strain was 68 mol%. The chemotaxonomic characteristics of strain N5(T), which had MK-11 and MK-10 as its major menaquinones and anteiso-C(15 : 0) (45 %), anteiso-C(17 : 0) (37 %), iso-C(16 : 0) (8.5 %) and C(16 : 0) (4.5 %) as its predominant fatty acids, were consistent with classification in the genus Microbacterium. Peptidoglycan in the novel strain, which contained ornithine, alanine, glycine, homoserine, glutamic acid, 3-hydroxyglutamic acid, muramic acid and traces of N-glycolyl residues, was of type B2β. The polar lipid profile of strain N5(T) comprised diphosphatidylglycerol, phosphatidylglycerol and an unknown glycolipid. The novel strains major cell-wall sugars were glucose and galactose. Based on the phylogenetic, DNA-DNA hybridization, chemotaxonomic and phenotypic data, strain N5(T) represents a novel species within the genus Microbacterium for which the name Microbacterium amylolyticum sp. nov. is proposed; the type strain is N5(T) (= DSM 24221(T) = CCM 7881(T)).

Understanding alternative fluxes/effluxes through comparative metabolic pathway analysis of phylum actinobacteria using a simplified approach

Actinobacteria are known for their diverse metabolism and physiology. Some are dreadful human pathogens whereas some constitute the natural flora for human gut. Therefore, the understanding of metabolic pathways is a key feature for targeting the pathogenic bacteria without disturbing the symbiotic ones. A big challenge faced today is multiple drug resistance by Mycobacterium and other pathogens that utilize alternative fluxes/effluxes. With the availability of genome sequence, it is now feasible to conduct the comparative in silico analysis. Here we present a simplified approach to compare metabolic pathways so that the species specific enzyme may be traced and engineered for future therapeutics. The analyses of four key carbohydrate metabolic pathways, i.e., glycolysis, pyruvate metabolism, tri carboxylic acid cycle and pentose phosphate pathway suggest the presence of alternative fluxes. It was found that the upper pathway of glycolysis was highly variable in the actinobacterial genomes whereas lower glycolytic pathway was highly conserved. Likewise, pentose phosphate pathway was well conserved in contradiction to TCA cycle, which was found to be incomplete in majority of actinobacteria. The clustering based on presence and absence of genes of these metabolic pathways clearly revealed that members of different genera shared identical pathways and, therefore, provided an easy method to identify the metabolic similarities/differences between pathogenic and symbiotic organisms. The analyses could identify isoenzymes and some key enzymes that were found to be missing in some pathogenic actinobacteria. The present work defines a simple approach to explore the effluxes in four metabolic pathways within the phylum actinobacteria. The analysis clearly reflects that actinobacteria exhibit diverse routes for metabolizing substrates. The pathway comparison can help in finding the enzymes that can be used as drug targets for pathogens without effecting symbiotic organisms within the same host. This may help to prevail over the multiple drug resistance, for designing broad spectrum drugs, in food industries and other clinical research areas.

Genome Organization of Sphingobium indicum B90A: An Archetypal Hexachlorocyclohexane (HCH) Degrading Genotype

Abstract Among sphingomonads, Sphingobium indicum B90A is widely investigated for its ability to degrade a manmade pesticide, γ-hexachlorocyclohexane (γ-HCH) and its isomers (α-, β-, δ-, and ε-HCH). In this study, complete genome of strain B90A was constructed using Single Molecule Real Time Sequencing (SMRT) and Illumina platform. The complete genome revealed that strain B90A harbors four replicons: one chromosome (3,654,322 bp) and three plasmids designated as pSRL1 (139,218 bp), pSRL2 (108,430 bp) and pSRL3 (43,761 bp). The study determined the precise location of lin genes (genes associated with the degradation of HCH isomers), for example, linA2, linB, linDER, linF, linGHIJ, and linKLMN on the chromosome; linA1, linC, and linF on pSRL1 and linDEbR on pSRL3. Strain B90A contained 26 copies of IS6100 element and most of them (15 copies) was found to be associated with lin genes. Duplication of several lin genes including linA, linDER, linGHIJ, and linF along with two variants of linE, that is, linEa (hydroquinone 1,2-dioxygenase) and linEb (chlorohydroquinone/hydroquinone 1,2-dioxygenase) were identified. This suggests that strain B90A not only possess efficient machinery for upper and lower HCH degradation pathways but it can also act on both hydroquinone and chlorohydroquinone metabolites produced during γ-HCH degradation. Synteny analysis revealed the duplication and transposition of linA gene (HCH dehydrochlorinase) between the chromosome and pSRL1, possibly through homologous recombination between adjacent IS6100 elements. Further, in silico analysis and laboratory experiments revealed that incomplete tyrosine metabolism was responsible for the production of extracellular brown pigment which distinguished strain B90A from other HCH degrading sphingomonads. The precise localization of lin genes, and transposable elements (IS6100) on different replicons now opens up several experimental avenues to elucidate the functions and regulatory mechanism of lin genes acquisition and transfer that were not completely known among the bacterial population inhabiting the HCH contaminated environment.

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.