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.

Modification of rifamycin polyketide backbone leads to improved drug activity against rifampicin-resistant Mycobacterium tuberculosis.

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.

Bacterial diversity and real-time PCR based assessment of linA and linB gene distribution at hexachlorocyclohexane contaminated sites.

The disposal of hexachlorocyclohexane (HCH) muck has created large number of HCH dumpsites all over the world from where the harmful HCH isomers are leaking into the environment. Bacteria have evolved at such contaminated sites that have the ability to degrade HCH. Degradation of various HCH isomers in bacterial strains is mediated primarily by two genes: linA and linB which encode dehydrochlorinase and haloalkane dehalogenase respectively. In this study we explored one such highly contaminated HCH dumpsite located in Lucknow, Uttar Pradesh, India. To assess the biostimulation potential of the contaminated site, microbial diversity study and real-time PCR based quantification of lin genes was carried out. The soil samples from dumpsite and surrounding areas were found to be highly contaminated with HCH residue levels as high as 1.8 × 10(5) mg kg(-1). The residues were detected in areas upto 13 km from the dumpsite. Sphingomonads, Chromohalobacter, and Marinobacter were the dominant genera present at the dump-site. Role of Sphingomonads in HCH degradation has been well documented. The highest copy numbers of linA and linB genes as determined using real-time PCR were 6.2 × 10(4) and 5.3 × 10(5), respectively, were found in sample from the dump site. The presence of Sphingomonads, linA, and linB genes from HCH contaminated soil indicates the presence of indigenous bacterial communities capable of HCH degradation.

Thermus parvatiensis RL(T) sp. nov., Isolated from a Hot Water Spring, Located Atop the Himalayan Ranges at Manikaran, India.

A Gram negative, yellow pigmented, rod shaped bacterium designated as RL(T) was isolated from a hot water spring (90-98 °C) located at Manikaran in Northern India. The isolate grows at 60-80 °C (optimum, 70 °C) and at pH 7.0-9.0 (optimum pH 7.2). Phylogenetic analysis of 16S rRNA gene sequences and levels of DNA-DNA relatedness together indicate that the new isolate represents a novel species of the genus Thermus with closest affinity to Thermus thermophilus HB8(T) (99.5 %) followed by Thermus arciformis (96.4 %). A comparative analysis of partial sequences of housekeeping genes (HKG) further revealed that strain RL(T) is a novel species belonging to the genus Thermus. The melting G+C content of strain RL(T) was calculated as 68.7 mol%. The DNA-DNA relatedness value of strain RL(T) with its nearest neighbours (>97 %) was found to be less than 70 % indicating that strain RL(T) represents a novel species of the genus Thermus. MK-8 was the predominant respiratory quinone. The presence of characteristic phospholipid and glycolipid further confirmed that strain RL(T) belongs to the genus Thermus. The predominant fatty acids of strain RL(T) were iso-C17:0 (23.67 %) and iso-C15:0 (24.50 %). The results obtained after DNA-DNA hybridization, biochemical and physiological tests clearly distinguished strain RL(T) from its closely related species. Thus, strain RL(T) represents a novel species of the genus Thermus for which the name Thermus parvatiensis is proposed (=DSM 21745(T)= MTCC 8932(T)).

Draft genome sequence of Thermus sp. strain RL, isolated from a hot water spring located atop the Himalayan ranges at Manikaran, India.

Thermus sp. strain RL was isolated from a hot water spring (90°C to 98°C) at Manikaran, Himachal Pradesh, India. Here we report the draft genome sequence (20,36,600 bp) of this strain. The draft genome sequence consists of 17 contigs and 1,986 protein-coding sequences and has an average G+C content of 68.77%.

Whole genome sequence of the rifamycin B-producing strain Amycolatopsis mediterranei S699.

Amycolatopsis mediterranei S699 is an actinomycete that produces an important antibiotic, rifamycin B. Semisynthetic derivatives of rifamycin B are used for the treatment of tuberculosis, leprosy, and AIDS-related mycobacterial infections. Here, we report the complete genome sequence (10.2 Mb) of A. mediterranei S699, with 9,575 predicted coding sequences.

Pseudomonas sp. to Sphingobium indicum: a journey of microbial degradation and bioremediation of Hexachlorocyclohexane.

The unusual process of production of hexachlorocyclohexane (HCH) and extensive use of technical HCH and lindane has created a very serious problem of HCH contamination. While the use of technical HCH and lindane has been banned all over the world, India still continues producing lindane. Bacteria, especially Sphingomonads have been isolated that can degrade HCH isomers. Among all the bacterial strains isolated so far, Sphingobium indicum B90A that was isolated from HCH treated rhizosphere soil appears to have a better potential for HCH degradation. This conclusion is based on studies on the organization of lin genes and degradation ability of B90A. This strain perhaps can be used for HCH decontamination through bioaugmentation.

Comparative metagenomic analysis of soil microbial communities across three hexachlorocyclohexane contamination levels.

This paper presents the characterization of the microbial community responsible for the in-situ bioremediation of hexachlorocyclohexane (HCH). Microbial community structure and function was analyzed using 16S rRNA amplicon and shotgun metagenomic sequencing methods for three sets of soil samples. The three samples were collected from a HCH-dumpsite (450 mg HCH/g soil) and comprised of a HCH/soil ratio of 0.45, 0.0007, and 0.00003, respectively. Certain bacterial; (Chromohalobacter, Marinimicrobium, Idiomarina, Salinosphaera, Halomonas, Sphingopyxis, Novosphingobium, Sphingomonas and Pseudomonas), archaeal; (Halobacterium, Haloarcula and Halorhabdus) and fungal (Fusarium) genera were found to be more abundant in the soil sample from the HCH-dumpsite. Consistent with the phylogenetic shift, the dumpsite also exhibited a relatively higher abundance of genes coding for chemotaxis/motility, chloroaromatic and HCH degradation (lin genes). Reassembly of a draft pangenome of Chromohalobacter salaxigenes sp. (∼8X coverage) and 3 plasmids (pISP3, pISP4 and pLB1; 13X coverage) containing lin genes/clusters also provides an evidence for the horizontal transfer of HCH catabolism genes.

Sphingomonas histidinilytica sp. nov., isolated from a hexachlorocyclohexane dump site.

A Gram-negative, non-spore-forming, cream-coloured bacterial strain, UM2(T), was isolated from an open hexachlorocyclohexane (HCH) dump site at Ummari village in Lucknow, India. Data generated from a polyphasic approach including phenotypic, genotypic and chemotaxonomic analyses confirmed that strain UM2(T) belonged to the genus Sphingomonas. The highest similarity found to the 16S rRNA gene sequence of strain UM2(T) was 99.4 %, with Sphingomonas wittichii DSM 6014(T), whereas the DNA-DNA relatedness value between these strains was 31 %, indicating that they represent separate species. The DNA G+C content of UM2(T) was 66.9 mol%. The respiratory pigment ubiquinone Q-10 was present. The predominant fatty acids were summed feature 8 (C(18 : 1)omega6c and/or C(18 : 1)omega7c; 32.9 %), C(19 : 0) cyclo omega8c (15.5 %) and C(16 : 0) (12.1 %). The major polar lipids were phosphatidylcholine, phosphatidylglycerol and phosphatidyldimethylethanolamine. sym-Homospermidine was the major polyamine observed. On the basis of the data reported, it was concluded that UM2(T) represents a novel species of the genus Sphingomonas, for which the name Sphingomonas histidinilytica sp. nov. is proposed. The type strain is UM2(T) (=MTCC 9473(T) =CCM 7545(T)).

Sphingopyxis ummariensis sp. nov., isolated from a hexachlorocyclohexane dump site.

A Gram-negative, motile, rod-shaped, yellow-pigmented bacterium, strain UI2(T), was isolated from a hexachlorocyclohexane (HCH) dump site located in Ummari, in northern India. 16S rRNA gene sequence analysis revealed that strain UI2(T) belongs to the genus Sphingopyxis and showed highest 16S rRNA gene sequence similarity with Sphingopyxis terrae IFO 15098(T) (98.6 %). DNA-DNA relatedness values between strain UI2(T) and Sphingopyxis terrae IFO 15098(T) and other related strains were found to be less than 46 %. The major cellular fatty acids of strain UI2(T) were C(18 : 1)omega7c (28.3 %), C(16 : 0) (14.4 %), 11-methyl C(18 : 1)omega7c (10.9 %), C(17 : 1)omega6c (9.6 %) and summed feature 3 (consisting of C(16 : 1)omega7c and/or C(15 : 0) iso 2-OH; 14.7 %). The DNA G+C content of strain UI2(T) was 68 mol%. On the basis of DNA-DNA hybridization, phenotypic characteristics and phylogenetic analysis, strain UI2(T) is found to represent a novel species of the genus Sphingopyxis, for which the name Sphingopyxis ummariensis sp. nov. is proposed. The type strain is UI2(T) (=CCM 7428(T) =MTCC 8591(T)).