Microbial Journey: Mount Everest to Mars.

A rigorous exploration of microbial diversity has revealed its presence on Earth, deep oceans, and vast space. The presence of microbial life in diverse environmental conditions, ranging from moderate to extreme temperature, pH, salinity, oxygen, radiations, and altitudes, has provided the necessary impetus to search for them by extending the limits of their habitats. Microbiology started as a distinct science in the mid-nineteenth century and has provided inputs for the betterment of mankind during the last 150 years. As beneficial microbes are assets and pathogens are detrimental, studying both have its own merits. Scientists are nowadays working on illustrating the microbial dynamics in Earth's subsurface, deep sea, and polar regions. In addition to studying the role of microbes in the environment, the microbe-host interactions in humans, animals and plants are also unearthing newer insights that can help us to improve the health of the host by modulating the microbiota. Microbes have the potential to remediate persistent organic pollutants. Antimicrobial resistance which is a serious concern can also be tackled only after monitoring the spread of resistant microbes using disciplines of genomics and metagenomics The cognizance of microbiology has reached the top of the world. Space Missions are now looking for signs of life on the planets (specifically Mars), the Moon and beyond them. Among the most potent pieces of evidence to support the existence of life is to look for microbial, plant, and animal fossils. There is also an urgent need to deliberate and communicate these findings to layman and policymakers that would help them to take an adequate decision for better health and the environment around us. Here, we present a glimpse of recent advancements by scientists from around the world, exploring and exploiting microbial diversity.

Transformation of ε-HBCD with the Sphingobium Indicum enzymes LinA1, LinA2 and LinATM, a triple mutant of LinA2.

Hexabromocyclododecanes (HBCDs) were used as flame-retardants until their ban in 2013. Among the 16 stereoisomers known, ε-HBCD has the highest symmetry. This makes ε-HBCD an interesting substrate to study the selectivity of biotransformations. We expressed three LinA dehydrohalogenase enzymes in E. coli bacteria, two wild-type, originating from Sphingobium indicum B90A bacteria and LinATM, a triple mutant of LinA2, with mutations of L96C, F113Y and T133 M. These enzymes are involved in the hexachlorocyclohexane (HCH) metabolism, specifically of the insecticide γ-HCH (Lindane). We studied the reactivity of those eight HBCD stereoisomers found in technical HBCD. Furthermore, we compared kinetics and selectivity of these LinA variants with respect to ε-HBCD. LC-MS data indicate that all enzymes converted ε-HBCD to pentabromocyclododecenes (PBCDens). Transformations followed Michaelis-Menten kinetics. Rate constants kcat and enzyme specificities kcat/KM indicate that ε-HBCD conversion was fastest and most specific with LinA2. Only one PBCDen stereoisomer was formed by LinA2, while LinA1 and LinATM produced mixtures of two PBCDE enantiomers at three times lower rates than LinA2. In analogy to the biotransformation of (-)ß-HBCD, with selective conversion of dibromides in R-S-configuration, we assume that 1E,5S,6R,9S,10R-PBCDen is the ε-HBCD transformation product from LinA2. Implementing three amino acids of the LinA1 substrate-binding site into LinA2 resulted in a triple mutant with similar kinetics and product specificity like LinA1. Thus, point-directed mutagenesis is an interesting tool to modify the substrate- and product-specificity of LinA enzymes and enlarge their scope to metabolize other halogenated persistent organic pollutants regulated under the Stockholm Convention.

Comparative Genomic Analysis of Rapidly Evolving SARS-CoV-2 Reveals Mosaic Pattern of Phylogeographical Distribution.

The outbreak of coronavirus disease 2019 (COVID-19) that started in Wuhan, China, in December 2019 has spread worldwide, emerging as a global pandemic. The severe respiratory pneumonia caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has so far claimed more than 0.38 million lives and has impacted human lives worldwide. However, as the novel SARS-CoV-2 virus displays high transmission rates, the underlying genomic severity is required to be fully understood. We studied the complete genomes of 95 SARS-CoV-2 strains from different geographical regions worldwide to uncover the pattern of the spread of the virus. We show that there is no direct transmission pattern of the virus among neighboring countries, suggesting that its spread is a result of travel of infected humans to different countries. We revealed unique single nucleotide polymorphisms (SNPs) in nonstructural protein 13 (nsp13), nsp14, nsp15, and nsp16 (ORF1b polyproteins) and in the S-protein within 10 viral isolates from the United States. These viral proteins are involved in RNA replication and binding with the human receptors, indicating that the viral variants that are circulating in the population of the United States are different from those circulating in the populations of other countries. In addition, we found an amino acid addition in nsp16 (mRNA cap-1 methyltransferase) of a U.S. isolate (GenBank accession no. MT188341.1) leading to a shift in the amino acid frame from position 2540 onward. Through comparative structural analysis of the wild-type and mutant proteins, we showed that this addition of a phenylalanine residue renders the protein in the mutant less stable, which might affect mRNA cap-1 methyltransferase function. We further analyzed the SARS-CoV-2-human interactome, which revealed that the interferon signaling pathway is targeted by orf1ab during infection and that it also interacts with NF-κB-repressing factor (NKRF), which is a potential regulator of interleukin-8 (IL-8). We propose that targeting this interaction may subsequently improve the health condition of COVID-19 patients. Our analysis also emphasized that SARS-CoV-2 manipulates spliceosome machinery during infection; hence, targeting splicing might affect viral replication. In conclusion, the replicative machinery of SARS-CoV-2 is targeting interferon and the notch signaling pathway along with spliceosome machinery to evade host challenges.IMPORTANCE The COVID-19 pandemic continues to storm the world, with over 6.5 million cases worldwide. The severity of the disease varies with the territories and is mainly influenced by population density and age factor. In this study, we analyzed the transmission pattern of 95 SARS-CoV-2 genomes isolated from 11 different countries. Our study also revealed several nonsynonymous mutations in ORF1b and S-proteins and the impact on their structural stability. Our analysis showed the manipulation of host system by viral proteins through SARS-CoV-2-human protein interactome, which can be useful to understand the impact of virus on human health.

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

Among sphingomonads, Sphingobium indicum B90A is widely investigated for its ability to degrade a manmade pesticide, γ-hexachlorocyclohexane (γ-HCH) and its isomers (α-, ß-, 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.

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 aurantiacus sp. nov. isolated from hexachlorocyclohexane (HCH) contaminated soil.

Strain NP1T, a Gram-stain-negative, orange, rod-shaped bacterium was isolated from hexacholorocyclohexane (HCH)-contaminated soil sediment samples collected from Ummari village, Lucknow, Uttar Pradesh, India. The results of 16S rRNA gene sequence analysis indicated that NP1T clustered with members of the genus Pontibacter of the order Cytophagales, family Cytophagaceae and phylum Bacteriodetes. The 16S rRNA gene sequence similarity with type strains of members of the genus Pontibacter ranged from 98.57 to 93.95 % with Pontibacter odishensis JC-130T (98.57 %), Pontibacter korlensis X14-1T (97.82 %), Pontibacter litorisediminisYKTF-7T (97.42 %) and Pontibacter virosus W-14T (97.01 %) as the closest neighbours. Cells of NP1T were aerobic, motile and oxidase- and catalase-positive. NP1T was capable of hydrolysis of gelatin, aesculin and starch and reduced nitrates to nitrogen. The major fatty acids of NP1T were summed feature 4 and iso-C15 : 0. The polar lipid profile of NP1T showed the presence of phosphatidylethanolamine (PE), unknown glycolipids and unknown aminolipids. Menaquinone-7 (MK-7) was the predominant respiratory quinone and sym-homospermidine was found to be the predominant polyamine in NP1T. The DNA G+C content of NP1T was 52.1±0.7 mol%. The levels of DNA-DNA relatedness of NP1T to P. odishensis JC-130T, P. korlensis X14-1T, P. litorisediminis YKTF-7T and P. virosus W14T were 44.9±0.6 %, 40.5±0.4 %, 34.4±0.7 % and 33.4±0.5 % respectively. Based on the phenotypic, chemotaxonomic, physiological and biochemical evidence and DNA-DNA hybridization results, it is proposed that NP1T represents a novel species of the genus Pontibacter, for which the name Pontibacter aurantiacus sp. nov. is proposed. The type strain is NP1T (KCTC 42943T=CCM 8697T=MCC 2931T).

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).

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).

Laboratory and field scale bioremediation of hexachlorocyclohexane (HCH) contaminated soils by means of bioaugmentation and biostimulation.

Hexachlorocyclohexane (HCH) contaminated soils were treated for a period of up to 64 days in situ (HCH dumpsite, Lucknow) and ex situ (University of Delhi) in line with three bioremediation approaches. The first approach, biostimulation, involved addition of ammonium phosphate and molasses, while the second approach, bioaugmentation, involved addition of a microbial consortium consisting of a group of HCH-degrading sphingomonads that were isolated from HCH contaminated sites. The third approach involved a combination of biostimulation and bioaugmentation. The efficiency of the consortium was investigated in laboratory scale experiments, in a pot scale study, and in a full-scale field trial. It turned out that the approach of combining biostimulation and bioaugmentation was most effective in achieving reduction in the levels of α- and ß-HCH and that the application of a bacterial consortium as compared to the action of a single HCH-degrading bacterial strain was more successful. Although further degradation of ß- and δ-tetrachlorocyclohexane-1,4-diol, the terminal metabolites of ß- and δ-HCH, respectively, did not occur by the strains comprising the consortium, these metabolites turned out to be less toxic than the parental HCH isomers.

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).

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 Pseudomonas sp. Strain JMM, a Sediment-Hosted Environmental Isolate.

Pseudomonas sp. strain JMM was isolated from the sediments of a natural water reservoir (pH, 6 to 7) located at Chambyal village in Samba district of Jammu and Kashmir, India. Here we report the annotated draft genome sequence of strain JMM having 52 contigs with 5,884 genes and an average G+C content of 66.5%.

Hexachlorocyclohexane: persistence, toxicity and decontamination.

Hexachlorocyclohexane (HCH), a persistent organochlorine insecticide, has been extensively used in the past for control of agricultural pests and vector borne diseases. The use of HCH has indeed accrued benefits, however the unusual production of the insecticidal isomer; γ-HCH (lindane) and unregulated disposal of HCH muck has created various dumpsites all over the world, leading to serious environmental concerns. HCH isomers have been ranked as possible human carcinogens and endocrine disruptors with proven teratogenic, mutagenic and genotoxic effects, hence making its decontamination mandatory. Efforts in this direction have led to the isolation of various HCH degrading bacteria from the dumpsites, reflecting their role in HCH bioremediation. This review summarizes the problem of environmental persistence of HCH isomers along with their toxicity and possible solutions for their decontamination.

Genome Sequence of Novosphingobium lindaniclasticum LE124T, Isolated from a Hexachlorocyclohexane Dumpsite.

Novosphingobium lindaniclasticum LE124(T) is a hexachlorocyclohexane (HCH)-degrading bacterium isolated from a high-dosage-point HCH dumpsite (450 mg HCH/g soil) located in Lucknow, India (27°00'N and 81°09'E). Here, we present the annotated draft genome sequence of strain LE124(T), which has an estimated size of 4.86 Mb and is comprised of 4,566 coding sequences.