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.
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.
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 symhomospermidine as the major polyamine. The major cellular fatty acids of strain LP43 T were, iso-C 15 : 0 (15.74 %), iso-C 15 : 0 3-OH (7.57 %), iso-C 17 : 0 3-OH (7.32 %), summed feature 4 (iso-C 17 : 1 I/anteiso-C 17 : 1 B) (31.22 %) and summed feature 8 (C 18 : 1 v7c/ C 18 : 1 v6c) (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 (5CCM 8406 T 5MCC 2019 T
BackgroundPhylogenetic heterogeneity across Pseudomonas genus is complemented by its diverse genome architecture enriched by accessory genetic elements (plasmids, transposons, and integrons) conferring resistance across this genus. Here, we sequenced a stress tolerant genotype i.e. Pseudomonas sp. strain RL isolated from a hexachlorocyclohexane (HCH) contaminated pond (45 mg of total HCH g−1 sediment) and further compared its gene repertoire with 17 reference ecotypes belonging to P. stutzeri, P. mendocina, P. aeruginosa, P. psychrotolerans and P. denitrificans, representing metabolically diverse ecosystems (i.e. marine, clinical, and soil/sludge). Metagenomic data from HCH contaminated pond sediment and similar HCH contaminated sites were further used to analyze the pan-genome dynamics of Pseudomonas genotypes enriched across increasing HCH gradient.ResultsAlthough strain RL demonstrated clear species demarcation (ANI ≤ 80.03%) from the rest of its phylogenetic relatives, it was found to be closest to P. stutzeri clade which was further complemented functionally. Comparative functional analysis elucidated strain specific enrichment of metabolic pathways like α-linoleic acid degradation and carbazole degradation in Pseudomonas sp. strain RL and P. stutzeri XLDN-R, respectively. Composition based methods (%codon bias and %G + C difference) further highlighted the significance of horizontal gene transfer (HGT) in evolution of nitrogen metabolism, two-component system (TCS) and methionine metabolism across the Pseudomonas genomes used in this study. An intact mobile class-I integron (3,552 bp) with a captured gene cassette encoding for dihydrofolate reductase (dhfra1) was detected in strain RL, distinctly demarcated from other integron harboring species (i.e. P. aeruginosa, P. stutzeri, and P. putida). Mobility of this integron was confirmed by its association with Tnp21-like transposon (95% identity) suggesting stress specific mobilization across HCH contaminated sites. Metagenomics data from pond sediment and recently surveyed HCH adulterated soils revealed the in situ enrichment of integron associated transposase gene (TnpA6100) across increasing HCH contamination (0.7 to 450 mg HCH g−1 of soil).ConclusionsUnlocking the potential of comparative genomics supplemented with metagenomics, we have attempted to resolve the environment and strain specific demarcations across 18 Pseudomonas gene complements. Pan-genome analyses of these strains indicate at astoundingly diverse metabolic strategies and provide genetic basis for the cosmopolitan existence of this taxon.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1488-2) contains supplementary material, which is available to authorized users.
An orange-pigmented bacterial strain, designated LP100T, was isolated from hexachlorocyclohexane-contaminated soil (Lucknow, India). A neighbour-joining tree based on 16S rRNA gene sequences showed that strain LP100T occupied a distinct phylogenetic position in the Pontibacter species cluster, showing highest similarity with Pontibacter lucknowensis DM9T (97.4 %). Levels of similarity to strains of other Pontibacter species ranged between 94.0 and 96.8 %. Strain LP100T contained MK-7 as the predominant menaquinone and sym-homospermidine was the major polyamine in the cell. The major cellular fatty acids of strain LP100T were anteiso-C17 : 0 A, iso-C15 : 0 and iso-C18 : 1 H. The polar lipid profile of strain LP100T showed the presence of phosphatidylethanolamine, an unidentified aminophospholipid, three unknown aminolipids and two unknown polar lipids. The G+C content of strain LP100T 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 LP100T represents a novel species of the genus Pontibacter for which the name Pontibacter indicus is proposed. The type strain is LP100T ( = CCM8435T = MCC2027T).
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