Puneet Kohli

Modification of Rifamycin Polyketide Backbone Leads to Improved Drug Activity against Rifampicin-resistant Mycobacterium tuberculosis

Background: The emergence of drug-resistant tuberculosis has called for the discovery of new antitubercular drugs. Results: We successfully generated 24-desmethylrifampicin by modifying the rifamycin polyketide backbone. Conclusion: 24-Desmethylrifamycin showed better antibacterial activity than rifampicin against multidrug-resistant strains of Mycobacterium tuberculosis. Significance: The combined genetic-synthetic strategy used in the study has opened up new avenues for generating more rifamycin analogs. Rifamycin B, a product of Amycolatopsis mediterranei S699, is the precursor of clinically used antibiotics that are effective against tuberculosis, leprosy, and AIDS-related mycobacterial infections. However, prolonged usage of these antibiotics has resulted in the emergence of rifamycin-resistant strains of Mycobacterium tuberculosis. As part of our effort to generate better analogs of rifamycin, we substituted the acyltransferase domain of module 6 of rifamycin polyketide synthase with that of module 2 of rapamycin polyketide synthase. The resulting mutants (rifAT6::rapAT2) of A. mediterranei S699 produced new rifamycin analogs, 24-desmethylrifamycin B and 24-desmethylrifamycin SV, which contained modification in the polyketide backbone. 24-Desmethylrifamycin B was then converted to 24-desmethylrifamycin S, whose structure was confirmed by MS, NMR, and X-ray crystallography. Subsequently, 24-desmethylrifamycin S was converted to 24-desmethylrifampicin, which showed excellent antibacterial activity against several rifampicin-resistant M. tuberculosis strains.

Draft Genome Sequence of Sphingobium ummariense Strain RL-3, a Hexachlorocyclohexane-Degrading Bacterium

ABSTRACT Here, we report the draft genome sequence of the hexachlorocyclohexane (HCH)-degrading bacterium Sphingobium ummariense strain RL-3, which was isolated from the HCH dumpsite located in Lucknow, India (27°00′N and 81°09′E). The annotated draft genome sequence (4.75 Mb) of strain RL-3 consisted of 139 contigs, 4,645 coding sequences, and 65% G+C content.

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

A halotolerant, Gram-stain-negative, rod-shaped and light-pink-pigmented bacterial strain, PB3T, was isolated from a pond sediment near a hexachlorocyclohexane-producing factory, located at Chinhat, Lucknow, India. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain PB3T formed a distinct phyletic clade along with the members of the genus Pontibacter. The 16S rRNA gene sequence similarity with other members of the genus Pontibacter ranged from 94.5 to 98.9 %. The cells were motile, aerobic, and catalase- and oxidase-positive. The major fatty acids were iso-C15:0, iso-C15:0 3-OH, iso-C17:0 3-OH, C16:1ω5c, summed feature 3 (C16:1ω6c/C16:1ω7c) and summed feature 4 (iso-C17:1I/ anteiso-C17:1 B). The polar lipid profile of strain PB3T showed the presence of phosphatidylethanolamine, an unidentified aminophospholipid, unknown aminolipids and other unknown polar lipids. DNA-DNA hybridization based homology of strain PB3T with respect to its most closely related species, Pontibacter chinhatensis LP51T, was 44.7 %. The DNA G+C content was 53.5 mol%. On the basis of these data, it is proposed that the isolate belongs to the genus Pontibacter and represents a novel species, for which the name Pontibacter mucosus is proposed. The type strain is PB3T (=DSM 100162T=KCTC 42942T).

Fictibacillus halophilus sp. nov., from a microbial mat of a hot spring atop the Himalayan Range.

A Gram-stain-positive staining, motile, endospore forming and moderately halophilic bacterium, designated as strain AS8T, was isolated from a microbial mat deposited at thermal discharges of Manikaran hot spring (with surface water temperature ~95 °C) located in Himachal Pradesh, India. 16S rRNA gene sequence based phylogenetic analysis revealed that strain AS8T belonged to the genus Fictibacillus with the highest sequence similarity to Fictibacillus nanhaiensis DSM 23009T (99.9 %) and Fictibacillus phosphorivorans Ca7T (99.9 %), followed by Fictibacillus barbaricus V2-BIII-A2T (99.1 %) and Fictibacillus arsenicus Con a/3T (97.4 %). The polar lipids fraction consisted of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The cell-wall peptidoglycan was of the type A1γ based on directly cross-linked meso-diaminopimelic acid. The DNA G+C content of strain AS8T was found to be 46.9 mol%. The quinone system of strain AS8T consisted of MK-7 predominantly, and the polyamine pattern primarily contained spermidine and spermine. The major cellular fatty acids in strain AS8T were iso-C15:0, anteiso-C15:0 and iso-C16:0. The strain showed DNA-DNA relatedness of 52.7 % with F. nanhaiensis DSM 23009T, 50.7 % with F. phosphorivorans Ca7T, 34.8 % with F. barbaricus V2-BIII-A2T and 38.0 % with F. arsenicus Con a/3T. In spite of the high 16S rRNA gene sequence similarities, the DNA-DNA hybridization and gyr B gene sequencing results (≤87 %) supported by physiological and biochemical tests demonstrated that strain AS8T is a representative of a novel species, for which the name Fictibacillus halophilus sp. nov. is proposed. The type strain is AS8T (=MCC 2765T=DSM 100124T=KCTC 33758T).

Draft Genome Sequence of Agrobacterium sp. Strain UHFBA-218, Isolated from Rhizosphere Soil of Crown Gall-Infected Cherry Rootstock Colt

ABSTRACT We report here the draft genome sequence of the alphaproteobacterium Agrobacterium sp. strain UHFBA-218, which was isolated from rhizosphere soil of crown gall-infected cherry rootstock Colt. The draft genome of strain UHFBA-218 consists of 112 contigs (5,425,303 bp) and 5,063 coding sequences with a G+C content of 59.8%.

Algoriphagus roseus sp. nov., isolated from a hexachlorocyclohexane contaminated dumpsite.

A Gram-staining negative, reddish-pink, non-motile, rod-shaped bacterial strain designated W29T, was isolated from a hexachlorocyclohexane-contaminated dumpsite located in the northern part of India at Ummari Village, Lucknow. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain W29T formed a lineage within the genus Algoriphagusand exhibited highest sequence similarity to Algoriphagus trabzonensis MS7T (98.8 %), followed by Algoriphagusalkaliphilus AC-74T (97.1 %). The 16S rRNA gene sequence similarity between strain W29T and other species of the genus Algoriphagusranged from 93.3-98.8 %. The DNA-DNA relatedness between strain W29T and A. trabzonensisMS7T was 47 % and with other related strains was found to be less than 45 %, confirming strain W29T represents a novel species. The DNA G+C content of strain W29T was 46.2 mol%. Strain W29T was oxidase- and catalase-positive. The major fatty acids (>10 %) of strain W29T were iso-C15 : 0, summed feature 9 (comprising 10-methyl C16 : 0 and/or iso-C17 : 1ω9c) and summed feature 3 (comprising C16 : 1ω6c and/or C16 : 1ω7c). The respiratory quinone was MK-7. The polar lipid profile consisted of phosphatidylethanolamine, diphosphatidylglycerol, two unidentified aminolipids, an unidentified aminophospholipid, an unidentified phospholipid and unidentified lipids. On the basis of the results obtained from DNA-DNA hybridization, and biochemical and physiological tests in this study, strain W29T represents a novel species of the genus Algoriphagus for which the name Algoriphagus roseus sp. nov. is proposed. The type strain is W29T (=KCTC 42940T=MCC 2876T=DSM 100160T).

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.

Pontibacter virosus sp. nov., isolated from a hexachlorocyclohexane-contaminated dumpsite.

A Gram-staining-negative, red-pigmented, motile, rod-shaped bacterial strain, designated as W14T, was isolated from a hexachlorocyclohexane-contaminated dumpsite located in the northern part of India at Ummari Village, Lucknow. Phylogenetic analysis based on 16S rRNA gene sequence showed that the strain belongs to the genus Pontibacter with highest sequence similarity to Pontibacter lucknowensis DM9T (98.1 %). The 16S rRNA gene sequence similarity between strain W14T and members of other species of the genus Pontibacter ranged from 98.1 to 94.2 %. The DNA-DNA relatedness between strain W14T and P. lucknowensis DM9T was 33.7 % and with other closely related strains was found to be less than 20 %, confirming it to represent a novel species. The DNA G+C content of strain W14T was 51.3 mol%. Strain W14T was oxidase- and catalase-positive. The predominant cellular fatty acids were summed feature 4 (C17 : 1 iso I/anteiso B and C17 : 1 anteiso B/iso I), iso-C15 : 0 and iso-C17 : 0 3-OH. The polar lipid profile of strain W14T consisted of phosphatidylethanolamine, diphosphatidylglycerol, aminolipid and glycolipid. On the basis of the results obtained from DNA-DNA hybridization, biochemical and physiological tests in this study, strain W14T represents a novel species of the genus Pontibacter, for which the name Pontibacter virosus sp. nov. is proposed. The type strain is W14T (=MCC 2932T=DSM 100231T=KCTC 42941T).

Paracoccus sordidisoli sp. nov., isolated from an agricultural field contaminated with hexachlorocyclohexane isomers

A novel bacterial strain, designated LP91T, was isolated from an agricultural field contaminated with hexachlorocyclohexane (HCH) isomers at Ummari Village, Lucknow, Uttar Pradesh, India. Cells of the strain were aerobic, short rod or coccoid, Gram-stain-negative and non-motile. Colonies of the strain were initially transparent but with time changed to a creamy white colour. Phylogenetic analysis based on the 16S rRNA marker gene showed that it was closely associated with Paracoccus aestuariivivens GHD-30T (99.1 %) and Paracoccus limosus NB88T (98.0 %), followed by Paracoccus laeviglucosivorans 43PT (97.9 %) and Paracoccus marinus KKL-A5T (97.0 %). The DNA-DNA hybridization values of strain LP91T with the closely related type strains mentioned above were below 51.2±0.64 %, confirming it as a distinct species from other known species of the genus Paracoccus. The major cellular fatty acids of strain LP91T were C18 : 0 ω7c/C18 : 0 ω6c and C16 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and aminophospholipid, along with other lipids including glycolipids, aminolipids and other unknown phosphoglycolipids. Spermine was the major polyamine, along with putrescine in a minor amount. Ubiquinone (Q-10) was the sole isoprenoid quinone. Based on the results of phylogenetic, phenotypic and chemotaxonomic analysis, it is proposed that the isolate represents a new species of the genus Paracoccus, for which the name Paracoccus sordidisoli sp. nov. is proposed. The type strain is LP91T (=KCTC 42938T=CCM 8696T=MCC 3128T).

Compound-Specific Stable Isotope Analysis: Implications in Hexachlorocyclohexane in-vitro and Field Assessment

Assessment of biotic and abiotic degradation reactions by studying the variation in stable isotopic compositions of organic contaminants in contaminated soil and aquifers is being increasingly considered during the last two decades with development of Compound specific stable isotope analysis (CSIA) technique. CSIA has been recognized as a potential tool for evaluating both qualitative and quantitative degradation with measurement of shifts in isotope ratios of contaminants and their degradation products as its basis. Amongst a wide variety of environmental pollutants including monoaromatics, chlorinated ethenes and benzenes etc., it is only recently that its efficacy is being tested for assessing biodegradation of a noxious pollutant namely hexachlorocyclohexane (HCH), by pure microbial cultures as well as directly at the field site. Anticipating the increase in demand of this technique for monitoring the microbial degradation along with natural attenuation, this review highlights the basic problems associated with HCH contamination emphasizing the applicability of emerging CSIA technique to absolve the major bottlenecks in assessment of HCH. To this end, the review also provides a brief overview of this technique with summarizing the recent revelations put forward by both in vitro and in situ studies by CSIA in monitoring HCH biodegradation.