Roshan Kumar

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.

Pontibacter ramchanderi sp. nov., isolated from hexachlorocyclohexane-contaminated pond sediment

A Gram-stain-negative, motile, red pigmented, rod-shaped bacterium, designated strain LP43(T), was isolated from hexachlorocyclohexane (HCH)-contaminated soil sediment (Lucknow, India). Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate formed a cluster with the genus Pontibacter in the phylum Bacteroidetes with sequence similarities ranging from 92.9 to 97.0 % with species of the genus Pontibacter. The DNA G+C content of strain LP43(T) was 59.1 mol%. The polar lipid profile of strain LP43(T) showed the presence of phosphatidylethanolamine, an unidentified aminophospholipid, unknown aminolipids and unknown polar lipids. Strain LP43(T) contained MK-7 as the predominant menaquinone and sym-homospermidine as the major polyamine. The major cellular fatty acids of strain LP43(T) were, iso-C15 : 0 (15.74 %), iso-C15 : 0 3-OH (7.57 %), iso-C17 : 0 3-OH (7.32 %), summed feature 4 (iso-C17 : 1 I/anteiso-C17 : 1 B) (31.22 %) and summed feature 8 (C18 : 1ω7c/ C18 : 1ω6c) (7.60 %). Based on the results of DNA-DNA hybridization and phenotypic and genotypic characteristics, strain LP43(T) represents a novel species of the genus Pontibacter, for which the name Pontibacter ramchanderi is proposed. The type strain is LP43(T) (= CCM 8406(T) = MCC 2019(T)).

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

An orange-pigmented bacterial strain, designated LP100(T), was isolated from hexachlorocyclohexane-contaminated soil (Lucknow, India). A neighbour-joining tree based on 16S rRNA gene sequences showed that strain LP100(T) occupied a distinct phylogenetic position in the Pontibacter species cluster, showing highest similarity with Pontibacter lucknowensis DM9(T) (97.4 %). Levels of similarity to strains of other Pontibacter species ranged between 94.0 and 96.8 %. Strain LP100(T) contained MK-7 as the predominant menaquinone and sym-homospermidine was the major polyamine in the cell. The major cellular fatty acids of strain LP100(T) were anteiso-C17 : 0 A, iso-C15 : 0 and iso-C18 : 1 H. The polar lipid profile of strain LP100(T) showed the presence of phosphatidylethanolamine, an unidentified aminophospholipid, three unknown aminolipids and two unknown polar lipids. The G+C content of strain LP100(T) was 58.2 mol%. The results of DNA-DNA hybridization, biochemical and physiological tests clearly distinguish the novel strain from closely related species of the genus Pontibacter. Therefore, strain LP100(T) represents a novel species of the genus Pontibacter for which the name Pontibacter indicus is proposed. The type strain is LP100(T) ( = CCM8435(T) = MCC2027(T)).

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.

Draft Genome Sequence of Sphingobium sp. Strain HDIPO4, an Avid Degrader of Hexachlorocyclohexane.

ABSTRACT Sphingobium sp. strain HDIPO4 was isolated from a hexachlorocyclohexane (HCH) dumpsite and degraded HCH isomers rapidly. The draft genome sequence of HDIPO4 (~4.7 Mbp) contains 143 contigs and 4,646 coding sequences with a G+C content of 65%.

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)).

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).

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.

Draft Genome Sequence of Sphingobium lactosutens Strain DS20T, Isolated from a Hexachlorocyclohexane Dumpsite.

ABSTRACT Sphingobium lactosutens DS20T has been isolated from the hexachlorocyclohexane (HCH) dumpsite in Lucknow, India, but does not degrade any of the HCH isomers. Here, we present the ~5.36-Mb draft genome sequence of strain DS20T, which consists of 110 contigs and 5,288 coding sequences, with a G+C content of 63.1%.