Helianthous Verma

Reconstructing an ancestral genotype of two hexachlorocyclohexane-degrading Sphingobium species using metagenomic sequence data

Over the last 60 years, the use of hexachlorocyclohexane (HCH) as a pesticide has resulted in the production of >4 million tons of HCH waste, which has been dumped in open sinks across the globe. Here, the combination of the genomes of two genetic subspecies (Sphingobium japonicum UT26 and Sphingobium indicum B90A; isolated from two discrete geographical locations, Japan and India, respectively) capable of degrading HCH, with metagenomic data from an HCH dumpsite (∼450 mg HCH per g soil), enabled the reconstruction and validation of the last-common ancestor (LCA) genotype. Mapping the LCA genotype (3128 genes) to the subspecies genomes demonstrated that >20% of the genes in each subspecies were absent in the LCA. This includes two enzymes from the ‘upper’ HCH degradation pathway, suggesting that the ancestor was unable to degrade HCH isomers, but descendants acquired lin genes by transposon-mediated lateral gene transfer. In addition, anthranilate and homogentisate degradation traits were found to be strain (selectively retained only by UT26) and environment (absent in the LCA and subspecies, but prevalent in the metagenome) specific, respectively. One draft secondary chromosome, two near complete plasmids and eight complete lin transposons were assembled from the metagenomic DNA. Collectively, these results reinforce the elastic nature of the genus Sphingobium, and describe the evolutionary acquisition mechanism of a xenobiotic degradation phenotype in response to environmental pollution. This also demonstrates for the first time the use of metagenomic data in ancestral genotype reconstruction, highlighting its potential to provide significant insight into the development of such phenotypes.

Comparative genomic analysis of nine Sphingobium strains: insights into their evolution and hexachlorocyclohexane (HCH) degradation pathways

BackgroundSphingobium spp. are efficient degraders of a wide range of chlorinated and aromatic hydrocarbons. In particular, strains which harbour the lin pathway genes mediating the degradation of hexachlorocyclohexane (HCH) isomers are of interest due to the widespread persistence of this contaminant. Here, we examined the evolution and diversification of the lin pathway under the selective pressure of HCH, by comparing the draft genomes of six newly-sequenced Sphingobium spp. (strains LL03, DS20, IP26, HDIPO4, P25 and RL3) isolated from HCH dumpsites, with three existing genomes (S. indicum B90A, S. japonicum UT26S and Sphingobium sp. SYK6).ResultsEfficient HCH degraders phylogenetically clustered in a closely related group comprising of UT26S, B90A, HDIPO4 and IP26, where HDIPO4 and IP26 were classified as subspecies with ANI value >98%. Less than 10% of the total gene content was shared among all nine strains, but among the eight HCH-associated strains, that is all except SYK6, the shared gene content jumped to nearly 25%. Genes associated with nitrogen stress response and two-component systems were found to be enriched. The strains also housed many xenobiotic degradation pathways other than HCH, despite the absence of these xenobiotics from isolation sources. Additionally, these strains, although non-motile, but posses flagellar assembly genes. While strains HDIPO4 and IP26 contained the complete set of lin genes, DS20 was entirely devoid of lin genes (except linKLMN) whereas, LL03, P25 and RL3 were identified as lin deficient strains, as they housed incomplete lin pathways. Further, in HDIPO4, linA was found as a hybrid of two natural variants i.e., linA1 and linA2 known for their different enantioselectivity.ConclusionThe bacteria isolated from HCH dumpsites provide a natural testing ground to study variations in the lin system and their effects on degradation efficacy. Further, the diversity in the lin gene sequences and copy number, their arrangement with respect to IS6100 and evidence for potential plasmid content elucidate possible evolutionary acquisition mechanisms for this pathway. This study further opens the horizon for selection of bacterial strains for inclusion in an HCH bioremediation consortium and suggests that HDIPO4, IP26 and B90A would be appropriate candidates for inclusion.

Parapedobacter indicus sp. nov., isolated from hexachlorocyclohexane-contaminated soil

Strain RK1(T), a Gram-stain-negative, non-spore-forming, rod-shaped, non-motile bacterium was isolated from a hexachlorocyclohexane (HCH) dumpsite, Lucknow, India. 16S rRNA gene sequence analysis revealed that strain RK1(T) belongs to the family Sphingobacteriaceae and showed highest sequence similarity to Parapedobacter koreensis Jip14(T) (95.63%). The major cellular fatty acids of strain RK1(T) were iso-C15:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c), iso-C17:0 3-OH, summed feature 9 (10-methyl C16:0 and/or iso-C17:1ω9c), iso-C15:0 3-OH and C16 : 0. The major respiratory pigment and polyamine of RK1(T) were menaquinone (MK-7) and homospermidine, respectively. The main polar lipids were phosphatidylethanolamine and sphingolipid. The G+C content of the DNA was 44.5 mol%. The results of physiological and biochemical tests and 16S rRNA sequence analysis clearly demonstrated that strain RK1(T) represents a novel species of the genus Parapedobacter, for which the name Parapedobacter indicus sp. nov. is proposed. The type strain is RK1(T) ( = DSM 28470(T) =MCC 2546(T)).

Draft Genome Sequence of a Hexachlorocyclohexane-Degrading Bacterium, Sphingobium baderi Strain LL03T

ABSTRACT Sphingobium baderi strain LL03T was isolated from hexachlorocyclohexane (HCH)-contaminated soil from Spolana, Czech Republic. Strain LL03T is a mutant that is deficient in linB and linC (genes that encode hexachlorocyclohexane haloalkane dehalogenase and dehydrogenase, respectively). The draft genome sequence of LL03T (~4.85 Mb) consists of 92 contigs and 4,914 coding sequences, with a G+C content of 63.5%.

Comparative genomic analysis reveals habitat-specific genes and regulatory hubs within the genus Novosphingobium

This study highlights the significant role of the genetic repertoire of a microorganism in the similarity between Novosphingobium strains. The results suggest that the phylogenetic relationships were mostly influenced by metabolic trait enrichment, which is possibly governed by the microenvironment of each microbe’s respective niche. Using core genome analysis, the enrichment of a certain set of genes specific to a particular habitat was determined, which provided insights on the influence of habitat on the distribution of metabolic traits for Novosphingobium strains. We also identified habitat-specific protein hubs, which suggested delineation of Novosphingobium strains based on their habitat. Examining the available genomes of ecologically diverse bacterial species and analyzing the habitat-specific genes are useful for understanding the distribution and evolution of functional and phylogenetic diversity in the genus Novosphingobium. ABSTRACT Species belonging to the genus Novosphingobium are found in many different habitats and have been identified as metabolically versatile. Through comparative genomic analysis, we identified habitat-specific genes and regulatory hubs that could determine habitat selection for Novosphingobium spp. Genomes from 27 Novosphingobium strains isolated from diverse habitats such as rhizosphere soil, plant surfaces, heavily contaminated soils, and marine and freshwater environments were analyzed. Genome size and coding potential were widely variable, differing significantly between habitats. Phylogenetic relationships between strains were less likely to describe functional genotype similarity than the habitat from which they were isolated. In this study, strains (19 out of 27) with a recorded habitat of isolation, and at least 3 representative strains per habitat, comprised four ecological groups—rhizosphere, contaminated soil, marine, and freshwater. Sulfur acquisition and metabolism were the only core genomic traits to differ significantly in proportion between these ecological groups; for example, alkane sulfonate (ssuABCD) assimilation was found exclusively in all of the rhizospheric isolates. When we examined osmolytic regulation in Novosphingobium spp. through ectoine biosynthesis, which was assumed to be marine habitat specific, we found that it was also present in isolates from contaminated soil, suggesting its relevance beyond the marine system. Novosphingobium strains were also found to harbor a wide variety of mono- and dioxygenases, responsible for the metabolism of several aromatic compounds, suggesting their potential to act as degraders of a variety of xenobiotic compounds. Protein-protein interaction analysis revealed β-barrel outer membrane proteins as habitat-specific hubs in each of the four habitats—freshwater (Saro_1868), marine water (PP1Y_AT17644), rhizosphere (PMI02_00367), and soil (V474_17210). These outer membrane proteins could play a key role in habitat demarcation and extend our understanding of the metabolic versatility of the Novosphingobium species. IMPORTANCE This study highlights the significant role of a microorganism’s genetic repertoire in structuring the similarity between Novosphingobium strains. The results suggest that the phylogenetic relationships were mostly influenced by metabolic trait enrichment, which is possibly governed by the microenvironment of each microbe’s respective niche. Using core genome analysis, the enrichment of a certain set of genes specific to a particular habitat was determined, which provided insights on the influence of habitat on the distribution of metabolic traits in Novosphingobium strains. We also identified habitat-specific protein hubs, which suggested delineation of Novosphingobium strains based on their habitat. Examining the available genomes of ecologically diverse bacterial species and analyzing the habitat-specific genes are useful for understanding the distribution and evolution of functional and phylogenetic diversity in the genus Novosphingobium.

Sphingopyxis flava sp. nov., isolated from a hexachlorocyclohexane (HCH)-contaminated soil

A Gram-negative-staining, aerobic, non-motile, non-spore-forming, rod-shaped and yellow-pigmented bacterium, designated R11HT, was isolated from a soil sample collected from a hexachlorocyclohexane dumpsite located at Ummari village, Lucknow, Uttar Pradesh, India. The 16S rRNA gene sequence similarity between strain R11HT and the type strains of species of genus Sphingopyxis with validly published names ranged from 93.75 to 97.85 %. Strain R11HT showed the highest 16S rRNA gene sequence similarity to Sphingopyxis indica DS15T (97.85 %), followed by Sphingopyxis soli JCM15910T (97.79 %), Sphingopyxis ginsengisoli KCTC 12582T (97.77 %) and Sphingopyxis panaciterrulae KCTC 22112T (97.34 %). The DNA G+C content of strain R11HT was 63.5 mol%. DNA-DNA relatedness between strain R11HT and its closest phylogenetic neighbours was well below the threshold value of 70 %, which suggested that strain R11HT represents a novel species of the genus Sphingopyxis. The major polar lipids of strain R11HT were sphingoglycolipid and other lipids commonly reported in this genus, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol and phosphatidylmonomethylethanolamine. Spermidine was detected as the major polyamine. The chemotaxonomic markers in strain R11HT confirmed its classification in the genus Sphingopyxis, i.e. Q-10 as the major ubiquinone and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and C14 : 0 2-OH as the predominant fatty acids. Results obtained from DNA-DNA hybridization and chemotaxonomic and phenotypic analyses clearly distinguished strain R11HT from its closest phylogenetic neighbours. Thus, strain R11HT represents a novel species of the genus Sphingopyxis, for which the name Sphingopyxis flava sp. nov. is proposed. The type strain is R11HT ( = DSM 28472T = MCC 2778T).

Draft Genome Sequence of Deinococcus sp. Strain RL Isolated from Sediments of a Hot Water Spring

ABSTRACT Deinococcus sp. strain RL, a moderately thermophilic bacterium, was isolated from sediments of a hot water spring in Manikaran, India. Here, we report the draft genome (2.79 Mbp) of this strain, which contains 62 contigs and 2,614 coding DNA sequences, with an average G+C content of 69.4%.

Luteimonas tolerans sp. nov., isolated from hexachlorocyclohexane-contaminated soil.

A Gram-stain-negative, aerobic, rod-shaped, non-spore-forming, yellow pigmented bacterial strain (UM1T) was isolated from the hexachlorocyclohexane (HCH)-contaminated dumpsite located at Ummari village in Lucknow, India. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain UM1T belongs to the genus Luteimonas with Luteimonas aestuarii B9T as the closest neighbour (97.2% 16S rRNA gene sequence similarity). The DNA G+C content of strain UM1T was 64.3 mol%. The major polar lipids were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). Main fatty acids were iso-C15:0, iso-C11:0, iso-C11:0 3-OH, iso-C17:0 and summed feature 9 (C16:0 10-methyl and/or iso-C17:1ω9c). Ubiquinone (Q-8) was the only respiratory quinone. Spermidine was detected as the major polyamine. The DNA-DNA relatedness value of strain UM1T with respect to its closest neighbour Luteimonas aestuarii B9T was well below 70 % (∼49%). Thus, data obtained from phylogenetic analysis, DNA-DNA hybridization, and chemotaxonomical and biochemical analyses supports classification of strain UM1T as representative of a novel species of the genus Luteimonas, for which the name Luteimonas tolerans sp. nov. is proposed. The type strain is UM1T (=DSM 28473T=MCC 2572T=KCTC 42936T).

Microbial taxonomy in the era of OMICS: application of DNA sequences, computational tools and techniques

The current prokaryotic taxonomy classifies phenotypically and genotypically diverse microorganisms using a polyphasic approach. With advances in the next-generation sequencing technologies and computational tools for analysis of genomes, the traditional polyphasic method is complemented with genomic data to delineate and classify bacterial genera and species as an alternative to cumbersome and error-prone laboratory tests. This review discusses the applications of sequence-based tools and techniques for bacterial classification and provides a scheme for more robust and reproducible bacterial classification based on genomic data. The present review highlights promising tools and techniques such as ortho-Average Nucleotide Identity, Genome to Genome Distance Calculator and Multi Locus Sequence Analysis, which can be validly employed for characterizing novel microorganisms and assessing phylogenetic relationships. In addition, the review discusses the possibility of employing metagenomic data to assess the phylogenetic associations of uncultured microorganisms. Through this article, we present a review of genomic approaches that can be included in the scheme of taxonomy of bacteria and archaea based on computational and in silico advances to boost the credibility of taxonomic classification in this genomic era.

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.