Draft genome sequence of soil isolate Mycolicibacterium fortuitum DVD-1301.

Some strains of Mycolicibacterium possess high sterol-oxidizing activity and are used in the pharmaceutical industry for the production of steroid precursors. Herein, we report a draft genome sequence of the soil-dwelling Mycolicibacterium fortuitum DVD-1301 isolated in the floodplain of the river Oka. The genome contains a full set of steroid catabolic genes.

Reconstruction of the Steroid 1(2)-Dehydrogenation System from Nocardioides simplex VKM Ac-2033D in Mycolicibacterium Hosts.

Microbial 1(2)-dehydrogenation of 3-ketosteroids is an important basis for the production of many steroid pharmaceuticals and synthons. When using the wild-type strains for whole cell catalysis, the undesirable reduction of the 20-carbonyl group, or 1(2)-hydrogenation, was observed. In this work, the recombinant strains of Mycolicibacterium neoaurum and Mycolicibacterium smegmatis were constructed with blocked endogenous activity of 3-ketosteroid-9α-hydroxylase, 3-ketosteroid-1(2)-dehydrogenase (3-KSD), and expressing 3-KSD encoded by the gene KR76_27125 (kstD2NS) from Nocardioides simplex VKM Ac-2033D. The in vivo activity of the obtained recombinant strains against phytosterol, 6α-methyl-hydrocortisone, and hydrocortisone was studied. When using M. smegmatis as the host strain, the 1(2)-dehydrogenation activity of the constructed recombinant cells towards hydrocortisone was noticeably higher compared to those on the platform of M. neoaurum. A comparison of the strengths of inducible acetamidase and constitutive hsp60 promoters in M. smegmatis provided comparable results. Hydrocortisone biotransformation by M. smegmatis BD/pMhsp_k expressing kstD2NS resulted in 95.4% prednisolone yield, and the selectivity preferred that for N. simplex. Mycolicibacteria showed increased hydrocortisone degradation at 35 °C compared to 30 °C. The presence of endogenous steroid catabolism in Mycolicibacterium hosts does not seem to confer an advantage for the functioning of KstD2NS. The results allow for the evaluation of the prospects for the development of simple technological methods for the selective 1(2)-dehydrogenation of 3-ketosteroids by growing bacterial cells.

Obtaining of 24-Norchol-4-ene-3,22-dione from Phytosterol with Mutants of Mycolicibacterium neoaurum.

Engineered mutants of Mycolicibacterium spp. are known producers of valuable steroid synthons with C19 or C22 skeleton. Here we describe a method for site-directed mutagenesis of Mycolicibacterium neoaurum strains, bioconversion from phytosterol, and selective purification of C23 steroid 24-norchol-4-ene-3,22-dione (24-NCED) and C22 steroid 20-hydroxymethylpregn-4-ene-3-one (20-HMP). The yields of crystalline products with 95% purity by the method here described are 2.74 ± 0.085 g for 24-NCED and 1.42 ± 0.085 g for 20-HMP from 10 g/L phytosterol. 20-HMP is recognized as the key precursor in chemical syntheses of pharmaceutical corticosteroids and 24-NCED is a promising synthon for the synthesis of valuable steroids and own potent biological activity.

In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties.

The incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein Aequorea victoria enhanced green fluorescent protein (EGFP) or Gaussia princeps luciferase (GLuc) into the tetradecameric quaternary structure of GroEL chaperonin and describe some physicochemical properties of the labeled chaperonin. Using size-exclusion and affinity chromatography, electrophoresis, fluorescent and electron transmission microscopy (ETM), small-angle X-ray scattering (SAXS), and bioluminescence resonance energy transfer (BRET), we show the following: (i) The GroEL14-EGFP is evenly distributed within normally divided E. coli cells, while gigantic undivided cells are characterized by the uneven distribution of the labeled GroEL14 which is mainly localized close to the cellular periplasm; (ii) EGFP and likely GLuc are located within the inner cavity of one of the two GroEL chaperonin rings and do not essentially influence the protein oligomeric structure; (iii) GroEL14 containing either EGFP or GLuc is capable of interacting with non-native proteins and the cochaperonin GroES.

Draft Genome Sequence of Mycolicibacterium smegmatis VKM Ac-1171 Contains Full Set of Sterol Catabolic Genes.

Mycolicibacterium smegmatis VKM Ac-1171 is a saprotrophic bacterium that was isolated several decades ago and is deposited in microbial collections around the world. We report here a draft genome sequence of the strain. Annotation of the genome revealed the presence of a complete set of genes related to the sterol catabolic pathway.

Deletion of alpB Gene Influences Outer Membrane Vesicles Biogenesis of Lysobacter sp. XL1.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria constitute important factors in defining interactions with the extracellular milieu. Lysobacter sp. XL1 produces OMVs capable of lysing microbial cells due to the presence in their cargo of bacteriolytic protease L5 (AlpB). Although protein L5 has been functionally and biochemically characterized (including aspects of its packing into OMVs), its role in vesicle biogenesis through genetic deletion of alpB had not been studied previously. Here, we have successfully deleted alpB by allelic replacement and show that the alpB deletion mutant produces a significantly lower amount of OMVs that lack bacteriolytic activity and display altered ultrastructural characteristics in relation to the OMVs produced by the wild-type strain. These results confirm that, as previously proposed, protein L5 participates in OMV production through a mechanism that is not yet fully understood.

Back to GroEL-Assisted Protein Folding: GroES Binding-Induced Displacement of Denatured Proteins from GroEL to Bulk Solution.

The main events in chaperone-assisted protein folding are the binding and ligand-induced release of substrate proteins. Here, we studied the location of denatured proteins previously bound to the GroEL chaperonin resulting from the action of the GroES co-chaperonin in the presence of Mg-ATP. Fluorescein-labeled denatured proteins (α-lactalbumin, lysozyme, serum albumin, and pepsin in the presence of thiol reagents at neutral pH, as well as an early refolding intermediate of malate dehydrogenase) were used to reveal the effect of GroES on their interaction with GroEL. Native electrophoresis has demonstrated that these proteins tend to be released from the GroEL-GroES complex. With the use of biotin- and fluorescein-labeled denatured proteins and streptavidin fused with luciferase aequorin (the so-called streptavidin trap), the presence of denatured proteins in bulk solution after GroES and Mg-ATP addition has been confirmed. The time of GroES-induced dissociation of a denatured protein from the GroEL surface was estimated using the stopped-flow technique and found to be much shorter than the proposed time of the GroEL ATPase cycle.

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.

Comparison of the substrate specificity of two ring-hydroxylating dioxygenases from Sphingomonas sp. VKM B-2434 to polycyclic aromatic hydrocarbons.

The genes of two ring-hydroxylating dioxygenases (RHDs) of Sphingomonas sp. VKM B-2434 were cloned and expressed in Escherichia coli. The relative values of the RHD specificity constants were estimated for six polycyclic aromatic hydrocarbons (PAHs) based on the kinetics of PAH mixture conversion by the recombinant strains. The substrate specificity profiles of the enzymes were found to be very different. Dioxygenase ArhA was the most specific to acenaphthylene and showed a low specificity to fluoranthene. Dioxygenase PhnA was the most specific to anthracene and phenanthrene and showed a considerable specificity to fluoranthene. Knockout derivatives of Sphingomonas sp. VKM B-2434 lacking ArhA, PhnA, and both dioxygenases were constructed. PAH degradation by the single-knockout mutants was in agreement with the substrate specificity of the RHD remaining intact. Double-knockout mutant lacking both enzymes was unable to oxidize PAHs. A mutant form of dioxygenase ArhA with altered substrate specificity was described.

Complete Genome Sequence of Sterol-Transforming Mycobacterium neoaurum Strain VKM Ac-1815D.

Mycobacterium neoaurum strain VKM Ac-1815D produces 4-androstene-3,17-dione as a major compound from phytosterols. Here, we report the complete genome sequence of the strain. The genome consists of a single circular 5,438,190-bp chromosome, with a G+C content of 66.88%, containing 5,318 putative open reading frames (ORFs), 46 tRNAs, and 6 rRNAs. Arrays of cholesterol metabolism genes are randomly clustered throughout the chromosome.

Fusion of Aequorea victoria GFP and aequorin provides their Ca(2+)-induced interaction that results in red shift of GFP absorption and efficient bioluminescence energy transfer.

The bioluminescence emitted by Aequorea victoria jellyfish is greenish while its single bioluminescent photoprotein aequorin emits blue light. This phenomenon may be explained by a bioluminescence resonance energy transfer (BRET) from aequorin chromophore to green fluorescent protein (GFP) co-localized with it. However, a slight overlapping of the aequorin bioluminescence spectrum with the GFP absorption spectrum and the absence of marked interaction between these proteins in vitro pose a question on the mechanism providing the efficient BRET in A. victoria. Here we report the in vitro study of BRET between homologous Ca(2+)-activated photoproteins, aequorin or obelin (Obelia longissima), as bioluminescence energy donors, and GFP, as an acceptor. The fusions containing donor and acceptor proteins linked by a 19 aa peptide were purified after expressing their genes in Escherichia coli cells. It was shown that the GFP-aequorin fusion has a significantly greater BRET efficiency, compared to the GFP-obelin fusion. Two main factors responsible for the difference in BRET efficiency of these fusions were revealed. First, it is the presence of Ca(2+)-induced interaction between the donor and acceptor in the aequorin-containing fusion and the absence of the interaction in the obelin-containing fusion. Second, it is a red shift of GFP absorption toward better overlapping with aequorin bioluminescence induced by the interaction of aequorin with GFP. Since the connection of the two proteins in vitro mimics their proximity in vivo, Ca(2+)-induced interaction between aequorin and GFP may occur in A. victoria jellyfish providing efficient BRET in this organism.