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.

Phylogenomic Framework for Taxonomic Delineation of Paracoccus spp. and Exploration of Core-Pan Genome.

The taxonomic classification of metabolically versatile Paracoccus spp. has been so far performed using polyphasic approach. The topology of single gene phylogenies, however, has highlighted ambiguous species assignments. In the present study, genome based multi-gene phylogenies and overall genome related index were used for species threshold assessment. Comprehensive phylogenomic analysis of Paracoccus genomes (n = 103) showed concordant clustering of strains across multi-gene marker set phylogenies (nMC = 0.08-0.14); as compared to 16S rDNA phylogeny (nMC = 0.37-0.42) suggesting robustness of multi gene phylogenies in drawing phylogenetic inferences. Functional gene content distribution across the genus showed that only 1.7% gene content constitutes the core genome highlighting the significance of extensive genomic variability in the evolution of Paracoccus spp. Further, genome metrics were used to validate characterized strains, identifying classification anomalies (n = 13), and based on this, genome derived taxonomic amendments were notified in present study. Conclusively, validated metric tools can be employed on whole genome sequences, including draft assemblies, for the assessment and assignment of uncharacterized strains and species level ascription of newly isolated Paracoccus strains in future.

Differential mass spectrometry-based proteome analyses unveil major regulatory hubs in rifamycin B production in Amycolatopsis mediterranei.

Rifamycin B is produced by Amycolatopsis mediterranei S699 as a secondary metabolite. Its semi-synthetic derivatives have been used for curing tuberculosis caused by Mycobacterium tuberculosis. But the emergence of rifampicin-resistant strains required analogs of rifamycin B to be developed by rifamycin biosynthetic gene cluster manipulation. In 2014 genetic engineering of the rifamycin polyketide synthase gene cluster in S699 led to a mutant, A. mediterranei DCO#34, that produced 24-desmethylrifamycin B. Unfortunately, the productivity was strongly reduced to 20 mgL-1 as compared to 50 mgL-1 of rifamycin B. To understand the mechanisms leading to reduced productivity and rifamycin biosynthesis by A. mediterranei S699 during the early and late growth phase we performed a proteome study for wild type strain S699, mutant DCO#34, and the non-producer strain SCO2-2. Proteins identification and relative label-free quantification were performed by nLC-MS/MS. Data are available via ProteomeXchange with identifier PXD016416. Also, in-silico protein-protein interaction approach was used to determine the relationship between different structural and regulatory proteins involved in rifamycin biosynthesis. Our studies revealed RifA, RifK, RifL, Rif-Orf19 as the major regulatory hubs. Relative abundance expression values revealed that genes encoding RifC-RifI and the transporter RifP, down-regulated in DCO#34 and genes encoding RifR, RifZ, other regulatory proteins up-regulated. SIGNIFICANCE: The study is designed mainly to understand the underlying mechanisms of rifamycin biosynthesis in Amycolatopsis mediterranei. This resulted in the identification of regulatory hubs which play a crucial role in regulating secondary metabolism. It elucidates the complex mechanism of secondary metabolite biosynthesis and their conversion and extracellular transportation in temporal correlation with the different growth phases. The study also elucidated the mechanisms leading to reduced production of analog, 24-desmethylrifamycin B by the genetically modified strain DCO#34, derivatives of which have been found effective against rifampicin-resistant strains of Mycobacterium tuberculosis. These results can be useful while carrying out genetic manipulations to improve the strains of Amycolatopsis to produce better analogs/drugs and promote the eradication of TB. Thus, this study is contributing significantly to the growing knowledge in the field of the crucial drug, rifamycin B biosynthesis by an economically important bacterium Amycolatopsis mediterranei.

Chicken Gut Microbiome and Human Health: Past Scenarios, Current Perspectives, and Futuristic Applications.

Sustainable poultry practices are needed to maintain an adequate supply of poultry products to the increasing human population without compromising human wellbeing. In order to achieve the understanding of the core microbiome that assumes an imperative role in digestion, absorption, and assimilation of feed as well as restrict the growth of pathogenic strains, a proper meta-data survey is required. The dysbiosis of the core microbiome or any external infection in chickens leads to huge losses in the poultry production worldwide. Along with this, the consumption of infected meat also impacts on human health as chicken meat is a regular staple in many diets as a vital source of protein. To tackle these losses, sub-therapeutic doses of antibiotics are being used as a feed additive along with other conventional approaches including selective breeding and modulation in feed composition. Altogether, these conventional approaches have improved the yield and quality of poultry products, however, the use of antibiotics encompasses the risk of developing multi-drug resistant pathogenic strains that can be harmful to human beings. Thus, there is an urgent need to understand the chicken microbiome in order to modulate chicken gut microbiome and provide alternatives to the conventional methods. Although there is now emerging literature available on some of these important microbiome aspects, in this article, we have analysed the relevant recent developments in understanding the chicken gut microbiome including the establishment of integrated gene catalogue for chicken microbiome. We have also focussed on novel strategies for the development of a chicken microbial library that can be used to develop novel microbial consortia as novel probiotics to improve the poultry meat production without compromising human health. Thus, it can be an alternative and advanced step compared to other conventional approaches to improve the gut milieu and pathogen-mediated loss in the poultry industry.

Enhanced biodegradation of hexachlorocyclohexane (HCH) in contaminated soils via inoculation with Sphingobium indicum B90A.

Soil pollution with hexachlorocyclohexane (HCH) has caused serious environmental problems. Here we describe the targeted degradation of all HCH isomers by applying the aerobic bacterium Sphingobium indicum B90A. In particular, we examined possibilities for large-scale cultivation of strain B90A, tested immobilization, storage and inoculation procedures, and determined the survival and HCH-degradation activity of inoculated cells in soil. Optimal growth of strain B90A was achieved in glucose-containing mineral medium and up to 65% culturability could be maintained after 60 days storage at 30 degrees C by mixing cells with sterile dry corncob powder. B90A biomass produced in water supplemented with sugarcane molasses and immobilized on corncob powder retained 15-20% culturability after 30 days storage at 30 degrees C, whereas full culturability was maintained when cells were stored frozen at -20 degrees C. On the contrary, cells stored on corncob degraded gamma-HCH faster than those that had been stored frozen, with between 15 and 85% of gamma-HCH disappearance in microcosms within 20 h at 30 degrees C. Soil microcosm tests at 25 degrees C confirmed complete mineralization of [(14)C]-gamma-HCH by corncob-immobilized strain B90A. Experiments conducted in small pits and at an HCH-contaminated agricultural site resulted in between 85 and 95% HCH degradation by strain B90A applied via corncob, depending on the type of HCH isomer and even at residual HCH concentrations. Up to 20% of the inoculated B90A cells survived under field conditions after 8 days and could be traced among other soil microorganisms by a combination of natural antibiotic resistance properties, unique pigmentation and PCR amplification of the linA genes. Neither the addition of corncob nor of corncob immobilized B90A did measurably change the microbial community structure as determined by T-RFLP analysis. Overall, these results indicate that on-site aerobic bioremediation of HCH exploiting the biodegradation activity of S. indicum B90A cells stored on corncob powder is a promising technology.

Organization of lin genes and IS6100 among different strains of hexachlorocyclohexane-degrading Sphingomonas paucimobilis: evidence for horizontal gene transfer.

The organization of lin genes and IS6100 was studied in three strains of Sphingomonas paucimobilis (B90A, Sp+, and UT26) which degraded hexachlorocyclohexane (HCH) isomers but which had been isolated at different geographical locations. DNA-DNA hybridization data revealed that most of the lin genes in these strains were associated with IS6100, an insertion sequence classified in the IS6 family and initially found in Mycobacterium fortuitum. Eleven, six, and five copies of IS6100 were detected in B90A, Sp+, and UT26, respectively. IS6100 elements in B90A were sequenced from five, one, and one regions of the genomes of B90A, Sp+, and UT26, respectively, and were found to be identical. DNA-DNA hybridization and DNA sequencing of cosmid clones also revealed that S. paucimobilis B90A contains three and two copies of linX and linA, respectively, compared to only one copy of these genes in strains Sp+ and UT26. Although the copy number and the sequence of the remaining genes of the HCH degradative pathway (linB, linC, linD, and linE) were nearly the same in all strains, there were striking differences in the organization of the linA genes as a result of replacement of portions of DNA sequences by IS6100, which gave them a strange mosaic configuration. Spontaneous deletion of linD and linE from B90A and of linA from Sp+ occurred and was associated either with deletion of a copy of IS6100 or changes in IS6100 profiles. The evidence gathered in this study, coupled with the observation that the G+C contents of the linA genes are lower than that of the remaining DNA sequence of S. paucimobilis, strongly suggests that all these strains acquired the linA gene through horizontal gene transfer mediated by IS6100. The association of IS6100 with the rest of the lin genes further suggests that IS6100 played a role in shaping the current lin gene organization.

Development of cloning vectors and transformation methods for Amycolatopsis.

The genus Amycolatopsis is of industrial importance, as its species are known to produce commercial antibiotics. It belongs to the family Pseudonocardiaceae and has an eventful taxonomic history. Initially strains were identified as Streptomyces, then later as Nocardia. However, based on biochemical, morphological and molecular features, the genus Amycolatopsis, containing seventeen species, was created. The development of molecular genetic techniques for this group has been slow. The scarcity of molecular genetic tools including stable plasmids, antibiotic resistance markers, transposons, reporter genes, cloning vectors, and high efficiency transformation protocols has made progress slow, but efforts in the past decade have led to the development of cloning vectors and transformation methods for these organisms. Some of the cloning vectors have broad host range (pRL series) whereas others have limited host range (pMEA300 and pMEA100). The cloning vector pMEA300 has been completely sequenced, while only the minimal replicon (pA- rep) has been sequenced from pRL plasmids. Direct transformation of mycelia and electroporation are the most widely applicable methods for transforming species of Amycolatopsis. Conjugational transfer from Escherichia coli has been reported only in the species A. japonicum, and gene disruption and replacements using homologous recombination are now possible in some strains.

Cloning and characterization of lin genes responsible for the degradation of Hexachlorocyclohexane isomers by Sphingomonas paucimobilis strain B90.

Hexachlorocyclohexane (HCH) has been used extensively against agricultural pests and in public health programs for the control of mosquitoes. Commercial formulations of HCH consist of a mixture of four isomers, alpha, beta, gamma, and delta. While all these isomers pose serious environmental problems, beta-HCH is more problematic due to its longer persistence in the environment. We have studied the degradation of HCH isomers by Sphingomonas paucimobilis strain B90 and characterized the lin genes encoding enzymes from strain B90 responsible for the degradation of HCH isomers. Two nonidentical copies of the linA gene encoding HCH dehydrochlorinase, which were designated linA1 and linA2, were found in S. paucimobilis B90. The linA1 and linA2 genes could be expressed in Escherichia coli, leading to dehydrochlorination of alpha-, gamma-, and delta-HCH but not of beta-HCH, suggesting that S. paucimobilis B90 contains another pathway for the initial steps of beta-HCH degradation. The cloning and characterization of the halidohydrolase (linB), dehydrogenase (linC and linX), and reductive dechlorinase (linD) genes from S. paucimobilis B90 revealed that they share approximately 96 to 99% identical nucleotides with the corresponding genes of S. paucimobilis UT26. No evidence was found for the presence of a linE-like gene, coding for a ring cleavage dioxygenase, in strain B90. The gene structures around the linA1 and linA2 genes of strain B90, compared to those in strain UT26, are suggestive of a recombination between linA1 and linA2, which formed linA of strain UT26.