Amycolatopsis mediterranei: A Sixty-Year Journey from Strain Isolation to Unlocking Its Potential of Rifamycin Analogue Production by Combinatorial Biosynthesis.

Ever since the isolation of Amycolatopsis mediterranei in 1957, this strain has been the focus of research worldwide. In the last 60 years or more, our understanding of the taxonomy, development of cloning vectors and conjugation system, physiology, genetics, genomics, and biosynthetic pathway of rifamycin B production in A. mediterranei has substantially increased. In particular, the development of cloning vectors, transformation system, characterization of the rifamycin biosynthetic gene cluster, and the regulation of rifamycin B production by the pioneering work of Heinz Floss have made the rifamycin polyketide biosynthetic gene cluster (PKS) an attractive target for extensive genetic manipulations to produce rifamycin B analogues which could be effective against multi-drug-resistant tuberculosis. Additionally, a better understanding of the regulation of rifamycin B production and the application of newer genomics tools, including CRISPR-assisted genome editing systems, might prove useful to overcome the limitations associated with low production of rifamycin analogues.

Weaponising microbes for peace.

There is much human disadvantage and unmet need in the world, including deficits in basic resources and services considered to be human rights, such as drinking water, sanitation and hygiene, healthy nutrition, access to basic healthcare, and a clean environment. Furthermore, there are substantive asymmetries in the distribution of key resources among peoples. These deficits and asymmetries can lead to local and regional crises among peoples competing for limited resources, which, in turn, can become sources of discontent and conflict. Such conflicts have the potential to escalate into regional wars and even lead to global instability. Ergo: in addition to moral and ethical imperatives to level up, to ensure that all peoples have basic resources and services essential for healthy living and to reduce inequalities, all nations have a self-interest to pursue with determination all available avenues to promote peace through reducing sources of conflicts in the world. Microorganisms and pertinent microbial technologies have unique and exceptional abilities to provide, or contribute to the provision of, basic resources and services that are lacking in many parts of the world, and thereby address key deficits that might constitute sources of conflict. However, the deployment of such technologies to this end is seriously underexploited. Here, we highlight some of the key available and emerging technologies that demand greater consideration and exploitation in endeavours to eliminate unnecessary deprivations, enable healthy lives of all and remove preventable grounds for competition over limited resources that can escalate into conflicts in the world. We exhort central actors: microbiologists, funding agencies and philanthropic organisations, politicians worldwide and international governmental and non-governmental organisations, to engage - in full partnership - with all relevant stakeholders, to 'weaponise' microbes and microbial technologies to fight resource deficits and asymmetries, in particular among the most vulnerable populations, and thereby create humanitarian conditions more conducive to harmony and peace.

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.

Expanding Culturomics from Gut to Extreme Environmental Settings.

With the advent of metagenomics, a quest began to identify the dynamics of the microbial communities in different ecological niches. Altogether, this has resulted in identification of microorganisms but is limited to only a small number of phylogenetic groups that can be easily cultured. The majority of metagenomic sequencing data remains unassigned to any known microbial group and is regarded as the "microbial dark matter." Our group is now working on integrating culturomics (isolation of pure cultures) and metagenomics from extreme environments, particularly from hot water springs and chemically contaminated soils. Our target is to culture the rare extremophiles with biotechnological significance by designing culture media based on inputs from metagenomics. While culturomics integrated with metagenomics has been extensively employed for updating the microbial catalog from the human gut, there is a need to extend this approach to extreme environmental settings to explore the microbial dark matter.

Comparative Genomics and Integrated Network Approach Unveiled Undirected Phylogeny Patterns, Co-mutational Hot Spots, Functional Cross Talk, and Regulatory Interactions in SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has resulted in 92 million cases in a span of 1 year. The study focuses on understanding population-specific variations attributing its high rate of infections in specific geographical regions particularly in the United States. Rigorous phylogenomic network analysis of complete SARS-CoV-2 genomes (245) inferred five central clades named a (ancestral), b, c, d, and e (subtypes e1 and e2). Clade d and subclade e2 were found exclusively comprised of U.S. strains. Clades were distinguished by 10 co-mutational combinations in Nsp3, ORF8, Nsp13, S, Nsp12, Nsp2, and Nsp6. Our analysis revealed that only 67.46% of single nucleotide polymorphism (SNP) mutations were at the amino acid level. T1103P mutation in Nsp3 was predicted to increase protein stability in 238 strains except for 6 strains which were marked as ancestral type, whereas co-mutation (P409L and Y446C) in Nsp13 were found in 64 genomes from the United States highlighting its 100% co-occurrence. Docking highlighted mutation (D614G) caused reduction in binding of spike proteins with angiotensin-converting enzyme 2 (ACE2), but it also showed better interaction with the TMPRSS2 receptor contributing to high transmissibility among U.S. strains. We also found host proteins, MYO5A, MYO5B, and MYO5C, that had maximum interaction with viral proteins (nucleocapsid [N], spike [S], and membrane [M] proteins). Thus, blocking the internalization pathway by inhibiting MYO5 proteins which could be an effective target for coronavirus disease 2019 (COVID-19) treatment. The functional annotations of the host-pathogen interaction (HPI) network were found to be closely associated with hypoxia and thrombotic conditions, confirming the vulnerability and severity of infection. We also screened CpG islands in Nsp1 and N conferring the ability of SARS-CoV-2 to enter and trigger zinc antiviral protein (ZAP) activity inside the host cell.IMPORTANCE In the current study, we presented a global view of mutational pattern observed in SARS-CoV-2 virus transmission. This provided a who-infect-whom geographical model since the early pandemic. This is hitherto the most comprehensive comparative genomics analysis of full-length genomes for co-mutations at different geographical regions especially in U.S. strains. Compositional structural biology results suggested that mutations have a balance of opposing forces affecting pathogenicity suggesting that only a few mutations are effective at the translation level. Novel HPI analysis and CpG predictions elucidate the proof of concept of hypoxia and thrombotic conditions in several patients. Thus, the current study focuses the understanding of population-specific variations attributing a high rate of SARS-CoV-2 infections in specific geographical regions which may eventually be vital for the most severely affected countries and regions for sharp development of custom-made vindication strategies.

Genome-based reclassification of Amycolatopsis eurytherma as a later heterotypic synonym of Amycolatopsis thermoflava.

The present study was carried out to clarify the taxonomic assignment of two closely related Amycolatopsis species. Genomic information for 48 type strains was available at the time of conducting this analysis. Our analysis showed that two species, viz. Amycolatopsis eurytherma Kim et al. 2002 and Amycolatopsis thermoflava Chun et al. 1999, are conspecific. The 16S rRNA gene sequences of the two species possess 98.85 % sequence similarity. Further, whole-genome comparisons showed that A. eurytherma DSM 44348T and A. thermoflava N1165T shared 98.75 % average nucleotide identity, 98.63 % average amino acid identity and 87.8 % digital DNA-DNA hybridization values. These values exceed the threshold values for bacterial species delineation, indicating that they belong to the same species. Further, the phylogenomic analysis based on the core genome of the strains under study confirmed that A. eurytherma DSM 44348T and A. thermoflava N1165T formed a monophyletic clade. Based on this evidence we propose the reclassification of Amycolatopsis eurytherma Kim et al. 2002 as a later heterotypic synonym of Amycolatopsis thermoflava Chun et al. 1999.

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.

Taxonomically Characterized and Validated Bacterial Species Based on 16S rRNA Gene Sequences from India During the Last Decade.

Microbial taxonomy dealing with identification and characterization of prokaryotes like bacteria and archaea has always been a major area of research all over the world. Exploring diversity of microbes and description of novel species with different genes and secondary compounds is of utmost importance for better future and sustenance of life. India having an enormous range of ecosystems and diverse species inhabiting these niches is considered to be one of the richest biodiversity regions of the world. During the last decade, with newer methodologies and better technology, the prokaryotic taxonomy from India has extended our inventory of microbial communities in specific niches. However, there still exist some limitations in classifying the microbes from India as compared to that is done world-over. This review enlists the taxonomic description of novel taxa of prokaryotes from India in the past decade. A total of 378 new bacterial species have been classified from different habitats in India in the last ten years and no descriptions of archaeal species is documented till date.

Rapid and solitary production of mono-rhamnolipid biosurfactant and biofilm inhibiting pyocyanin by a taxonomic outlier Pseudomonas aeruginosa strain CR1.

Biosurfactant - Rhamnolipids (RLs) and antibacterial toxin - pyocyanin (PYO) produced by Pseudomonas aeruginosa strains have great potential for biotechnological applications. Generally, RLs are produced as a mixture of di-rhamnolipids (di-RLs) and mono-rhamnolipids (mono-RLs). Mono-RLs possess superior emulsification and antimicrobial properties and are costlier than di-RLs. In this study, a taxonomic outlier P. aeruginosa strain CR1 isolated from rhizosphere soil was explored for mono-RLs and PYO production. Phylogenetically strain CR1 resembles avirulent outlier P. aeruginosa strain ATCC9027, lacks archetypical virulence genes and harbors unique pathways for the synthesis of solely mono-RLs and PYO. Strain CR1 produced RL biosurfactant which efficiently emulsified hydrocarbons, showed hemolysis and inhibited Bacillus subtilis. At 37 °C, strain CR1 exclusively produced 21.77 g L-1 and 19.22 g L-1 rhamnolipid in glycerol amended Luria Bertani (LB) medium and basal medium amended with rice bran oil, respectively after 54 h growth. Besides RL production was unaffected under varying nitrogen sources. Structural characterization using FTIR, TLC, and LC-MS confirmed that strain CR1 exclusively produced mono-RLs, majorly dominated by Rha-C10-C10, Rha-C10-C8, and CH3-Rha-C12:2-C10:1. The compound was stable over a wide pH range (4-12), salinity (25%) and 100 °C indicating its applicability under harsh environmental conditions. In addition, strain CR1 produced 4.5 µg mL-1 PYO, which could efficiently inhibit biofilm formation by Bacillus species. The environmental outlier strain CR1 can be used for the industrial production of biotechnologically important mono-RLs and PYO.

Comparative Genomic Analyses Reveal Core-Genome-Wide Genes Under Positive Selection and Major Regulatory Hubs in Outlier Strains of Pseudomonas aeruginosa.

Genomic information for outlier strains of Pseudomonas aeruginosa is exiguous when compared with classical strains. We sequenced and constructed the complete genome of an environmental strain CR1 of P. aeruginosa and performed the comparative genomic analysis. It clustered with the outlier group, hence we scaled up the analyses to understand the differences in environmental and clinical outlier strains. We identified eight new regions of genomic plasticity and a plasmid pCR1 with a VirB/D4 complex followed by trimeric auto-transporter that can induce virulence phenotype in the genome of strain CR1. Virulence genotype analysis revealed that strain CR1 lacked hemolytic phospholipase C and D, three genes for LPS biosynthesis and had reduced antibiotic resistance genes when compared with clinical strains. Genes belonging to proteases, bacterial exporters and DNA stabilization were found to be under strong positive selection, thus facilitating pathogenicity and survival of the outliers. The outliers had the complete operon for the production of vibrioferrin, a siderophore present in plant growth promoting bacteria. The competence to acquire multidrug resistance and new virulence factors makes these strains a potential threat. However, we identified major regulatory hubs that can be used as drug targets against both the classical and outlier groups.

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 Cellulosimicrobium sp. Strain MM, Isolated from Arsenic-Rich Microbial Mats of a Himalayan Hot Spring.

Microbial mats situated at the Manikaran hot springs (>95°C) are characterized by their high arsenic content (140 ppb), qualifying as a stressed niche. Here, we report the annotated draft genome (3.85 Mb) of Cellulosimicrobium sp. strain MM, isolated from these microbial mats, consisting of 3,718 coding sequences, with an average % G+C of 74.4%.