Genome-Wide Transcriptome Profiling Provides Insight on Cholesterol and Lithocholate Degradation Mechanisms in Nocardioides simplex VKM Ac-2033D.

Steroid microbial degradation plays a significant ecological role for biomass decomposition and removal/detoxification of steroid pollutants. In this study, the initial steps of cholesterol degradation and lithocholate bioconversion by a strain with enhanced 3-ketosteroid dehydrogenase (3-KSD) activity, Nocardioides simplex VKM Ac-2033D, were studied. Biochemical, transcriptomic, and bioinformatic approaches were used. Among the intermediates of sterol sidechain oxidation cholest-5-en-26-oic acid and 3-oxo-cholesta-1,4-dien-26-oic acid were identified as those that have not been earlier reported for N. simplex and related species. The transcriptomic approach revealed candidate genes of cholesterol and lithocholic acid (LCA) catabolism by the strain. A separate set of genes combined in cluster and additional 3-ketosteroid Δ1-dehydrogenase and 3-ketosteroid 9α-hydroxylases that might be involved in LCA catabolism were predicted. Bioinformatic calculations based on transcriptomic data showed the existence of a previously unknown transcription factor, which regulates cholate catabolism gene orthologs. The results contribute to the knowledge on diversity of steroid catabolism regulation in actinobacteria and might be used at the engineering of microbial catalysts for ecological and industrial biotechnology.

Genome-wide response on phytosterol in 9-hydroxyandrostenedione-producing strain of Mycobacterium sp. VKM Ac-1817D.

BACKGROUND: Aerobic side chain degradation of phytosterols by actinobacteria is the basis for the industrial production of androstane steroids which are the starting materials for the synthesis of steroid hormones. A native strain of Mycobacterium sp. VKM Ac-1817D effectively produces 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) from phytosterol, but also is capable of slow steroid core degradation. However, the set of the genes with products that are involved in phytosterol oxidation, their organisation and regulation remain poorly understood. RESULTS: High-throughput sequencing of the global transcriptomes of the Mycobacterium sp. VKM Ac-1817D cultures grown with or without phytosterol was carried out. In the presence of phytosterol, the expression of 260 genes including those related to steroid catabolism pathways significantly increased. Two of the five genes encoding the oxygenase unit of 3-ketosteroid-9α-hydroxylase (kshA) were highly up-regulated in response to phytosterol (55- and 25-fold, respectively) as well as one of the two genes encoding its reductase subunit (kshB) (40-fold). Only one of the five putative genes encoding 3-ketosteroid-∆1-dehydrogenase (KstD_1) was up-regulated in the presence of phytosterol (61-fold), but several substitutions in the conservative positions of its product were revealed. Among the genes over-expressed in the presence of phytosterol, several dozen genes did not possess binding sites for the known regulatory factors of steroid catabolism. In the promoter regions of these genes, a regularly occurring palindromic motif was revealed. The orthologue of TetR-family transcription regulator gene Rv0767c of M. tuberculosis was identified in Mycobacterium sp. VKM Ac-1817D as G155_05115. CONCLUSIONS: High expression levels of the genes related to the sterol side chain degradation and steroid 9α-hydroxylation in combination with possible defects in KstD_1 may contribute to effective 9α-hydroxyandrost-4-ene-3,17-dione accumulation from phytosterol provided by this biotechnologically relevant strain. The TetR-family transcription regulator gene G155_05115 presumably associated with the regulation of steroid catabolism. The results are of significance for the improvement of biocatalytic features of the microbial strains for the steroid industry.

Genome Sequencing of Steroid-Producing Bacteria with Illumina Technology.

Illumina technology is widely used for bacterial whole-genome sequencing due to its simplicity, cheapness, reliability, and abundant software for manipulation with raw data. Illumina technology belongs to a second generation of whole genome sequencing that yields great amount of short reads for genome regions. Genomic DNA is fragmented to short pieces. DNA fragments are amplified for signal increasing, and are read using sequencing-by-synthesis. Millions of short reads up to 100-300 bp in length are assembled in continuous sequences. Mate-pair technology allows resolving a long repeat.Here, we describe the principles of standard and mate-pair library preparation from DNA samples, library quality control, sequencing with MiSeq instrument and following data bioinformatics treatment. Software for genome assembly and completion are listed that assemble, map, annotate, visualize, edit and allow doing other manipulations with genomic sequences. The whole genomes sequencing of the steroid-producing Actinobacteria using these protocols is exemplified.

Effect of methyl-ß-cyclodextrin on gene expression in microbial conversion of phytosterol.

Modified ß-cyclodextrins are widely used for the enhancement of microbial conversions of lipophilic compounds such as steroids. Multiple mechanisms of cyclodextrin-mediated enhancement of phytosterol bioconversion by mycobacteria had previously been shown to include steroid solubilization, alterations in the cell wall permeability for both steroids and nutrients, facilitation of protein leaking, and activity suppression of some steroid-transforming enzymes.In this work, we studied whether cyclodextrins might affect expression of the genes involved in the steroid catabolic pathway. Phytosterol bioconversion with 9α-hydroxy-androst-4-ene-3,17-dione accumulation by Mycobacterium sp. VKM Ac-1817D in the presence of methylated ß-cyclodextrin (MCD) was investigated. RNA sequencing of the whole transcriptomes in different combinations of phytosterol and MCD showed a similar expression level of the steroid catabolism genes related to the KstR-regulon and was responsible for side chain and initial steps of steroid core oxidation; whereas, induction levels of the genes related to the KstR2-regulon were attenuated in the presence of MCD in this strain. The data were attenuated with quantitative real-time PCR.The results contribute to the understanding of cyclodextrin effects on microbial steroid conversion and provide a basis for the use of cyclodextrins as expression enhancers for studies of sterol catabolism in actinobacteria.

Comparative analysis of plastid genomes of non-photosynthetic Ericaceae and their photosynthetic relatives.

Although plastid genomes of flowering plants are typically highly conserved regarding their size, gene content and order, there are some exceptions. Ericaceae, a large and diverse family of flowering plants, warrants special attention within the context of plastid genome evolution because it includes both non-photosynthetic and photosynthetic species with rearranged plastomes and putative losses of "essential" genes. We characterized plastid genomes of three species of Ericaceae, non-photosynthetic Monotropa uniflora and Hypopitys monotropa and photosynthetic Pyrola rotundifolia, using high-throughput sequencing. As expected for non-photosynthetic plants, M. uniflora and H. monotropa have small plastid genomes (46 kb and 35 kb, respectively) lacking genes related to photosynthesis, whereas P. rotundifolia has a larger genome (169 kb) with a gene set similar to other photosynthetic plants. The examined genomes contain an unusually high number of repeats and translocations. Comparative analysis of the expanded set of Ericaceae plastomes suggests that the genes clpP and accD that are present in the plastid genomes of almost all plants have not been lost in this family (as was previously thought) but rather persist in these genomes in unusual forms. Also we found a new gene in P. rotundifolia that emerged as a result of duplication of rps4 gene.

Genome-wide bioinformatics analysis of steroid metabolism-associated genes in Nocardioides simplex VKM Ac-2033D.

Actinobacteria comprise diverse groups of bacteria capable of full degradation, or modification of different steroid compounds. Steroid catabolism has been characterized best for the representatives of suborder Corynebacterineae, such as Mycobacteria, Rhodococcus and Gordonia, with high content of mycolic acids in the cell envelope, while it is poorly understood for other steroid-transforming actinobacteria, such as representatives of Nocardioides genus belonging to suborder Propionibacterineae. Nocardioides simplex VKM Ac-2033D is an important biotechnological strain which is known for its ability to introduce ∆(1)-double bond in various 1(2)-saturated 3-ketosteroids, and perform convertion of 3ß-hydroxy-5-ene steroids to 3-oxo-4-ene steroids, hydrolysis of acetylated steroids, reduction of carbonyl groups at C-17 and C-20 of androstanes and pregnanes, respectively. The strain is also capable of utilizing cholesterol and phytosterol as carbon and energy sources. In this study, a comprehensive bioinformatics genome-wide screening was carried out to predict genes related to steroid metabolism in this organism, their clustering and possible regulation. The predicted operon structure and number of candidate gene copies paralogs have been estimated. Binding sites of steroid catabolism regulators KstR and KstR2 specified for N. simplex VKM Ac-2033D have been calculated de novo. Most of the candidate genes grouped within three main clusters, one of the predicted clusters having no analogs in other actinobacteria studied so far. The results offer a base for further functional studies, expand the understanding of steroid catabolism by actinobacteria, and will contribute to modifying of metabolic pathways in order to generate effective biocatalysts capable of producing valuable bioactive steroids.

Comparative analysis reveals similarities between cultured submandibular salivary gland cells and liver progenitor cells.

Mouse submandibular salivary gland cells and liver progenitor cells from long-term in vitro cultures with a high proliferation potential were side-by-side compared by methods of immunocytochemistry, quantitative real-time PCR, flow cytometry, and transcriptome analysis. The two cell types were found to be similar in expressing cell markers such as EpCAM, CD29, c-Kit, Sca-1, and c-Met. In addition, both cell types expressed cytokeratins 8, 18, and 19, alpha-fetoprotein, and (weakly) albumin. Unlike the liver cells, however, the salivary gland cells in culture showed high-level expression of cytokeratin 14 and CD49f, which was indicative of their origin from salivary gland ducts. Quantitative real-time PCR and deep-sequencing transcriptome analysis revealed similarities in the expression pattern of transcription factors between the two cell types. In this respect, however, the cultured salivary gland cells proved to be closer to exocrine cells of the pancreas than to the liver progenitor cells. Thus, ductal cells of postnatal submandibular salivary glands in culture show phenotypic convergence with progenitor cells of endodermal origin, suggesting that these glands may serve as a potential cell source for cellular therapy of hepatic and pancreatic disorders. The results of this study provide a deeper insight into the molecular features of salivary gland cells and may help optimize procedures for stimulating their differentiation in a specified direction.