Expression of Synthetic cyp102A1-LG23 Gene and Functional Analysis of Recombinant Cytochrome P450 BM3-LG23 in the Actinobacterium Mycolicibacterium smegmatis.

Cytochrome CYP102A1 (P450 BM3) of Priestia megaterium (bas. Bacillus megaterium) has several unique functional features and thus provides an ideal object for directed evolution and other synthetic applications. Previously, the CYP102A1-LG23 mutant with 14 mutations in the heme part was obtained that hydroxylates several androstanes at C7ß with the formation of products with the anti-inflammatory and neuroprotective activities. In this study, synthetic cyp102A1-LG23 gene encoding the P450 BM3 mutant was expressed as a component of either monocistronic operon or bicistronic operon containing the gdh (glucose dehydrogenase, GDH) or zwf2 (glucose 6-phosphate dehydrogenase, G6PD) gene in Mycolicibacterium smegmatis BD cells. The recombinant bacteria were able hydroxylate androst-4-ene-3,17-dione (AD) into 7ß-OH-AD. Their biocatalytic activity was increased twice by increasing the solubility of CYP102A1-LG23 protein in the cells and supplementing the cells with the additional cofactor regeneration system by introducing GDH and G6PD. The maximum 7ß-OH-AD yield (37.68 mol%) was achieved by co-expression of cyp102A1-LG23 and gdh genes in M. smegmatis. These results demonstrate the possibility of using synthetic genes to obtain recombinant enzymes and expand our understanding of the processes involved in steroid hydroxylation by bacterial cytochromes. The data obtained can be used to develop new approaches for microbiological production of 7ß-hydroxylated steroids in genetically modified Mycolicibacterium species.

Playgrounds in City of Pushchino with Different Types of Coating as Reservoir of Antibiotic-Resistant Strains of Pseudomonas spp.

In this study, we investigated antibiotic-resistant microorganisms isolated by the direct plating method from 6 playgrounds in the city of Pushchino, Moscow Region, with different types of coating: sand, soil with sand, grass and a modern playground coating made of pressed rubber crumb. According to the results of the study, sand is the cleanest type of coating, both in terms of the total count of cultivated microorganisms (8 × 105/g of substrate) and in terms of the content of resistant strains. The most contaminated both in terms of the total count of cultivated microorganisms (1.2-1.9 × 109/g of substrate) and in terms of the content of antibiotic-resistant strains was the coating of pressed rubber crumb. We isolated 65 antibiotic-resistant strains of fluorescent pseudomonads. Nine Pseudomonas strains were found to contain antibiotic resistance plasmids (one belongs to P-1 incompatibility group, seven to IncP-7 and one to unidentified incompatibility group). For the first time, we discovered a conjugative plasmid pD4A-46 conferring tetracycline resistance and belonging to the P-7 incompatibility group. Taking into account the results obtained under this study, it can be recommended to periodically treat the crumb rubber coating with non-toxic antiseptics, i.e. hydrogen peroxide or chlorhexidine.

Removal of Phenol by Rhodococcus opacus 1CP after Dormancy: Insight into Enzymes' Induction, Specificity, and Cells Viability.

Biodegradation of phenol is an effective method for removing this toxicant from contaminated sites. Phenol is a toxic compound for living cells, so many bacteria degrade phenol in relatively low concentrations, up to 0.75 g L-1. The Rhodococcus opacus strain 1CP is an effective destructor of a wide range of pollutants. In the absence of a carbon source in the medium, cells of the R. opacus 1CP strain easily form cyst-like resting cells (CLC). The purpose of this work was to evaluate the viability of cells during long-term storage and the efficiency of the process of phenol destruction by R. opacus 1CP cells germinating after dormancy. Resting cells were obtained by simple cultivation in a rich medium followed by storage under static conditions. This is a simple approach to obtain a large amount of biomass. Decomposition of phenol proceeded via catechol followed by ortho-cleavage of aromatic ring. The induction of three phenol hydroxylases was detected by RT-PCR in cells germinated in a mineral medium with phenol as the carbon source. The stability of the genome of cells germinating after dormancy is shown by box-PCR. Dormant R. opacus 1CP cells, both suspended and immobilized, can be directly used for the decomposition of phenol after 4-12 months storage. In addition to phenol, after 9 months of storage, immobilized germinating cells easily metabolized 4-chlorophenol and 2,4,6-trichlorophenol. The results demonstrate a potential and simple approach toward achieving long-term storage of cells for further use in bioremediation.

Genome Analysis and Physiology of Pseudomonas sp. Strain OVF7 Degrading Naphthalene and n-Dodecane.

The complete genome of the naphthalene- and n-alkane-degrading strain Pseudomonas sp. strain OVF7 was collected and analyzed. Clusters of genes encoding enzymes for the degradation of naphthalene and n-alkanes are localized on the chromosome. Based on the Average Nucleotide Identity and digital DNA-DNA Hybridization compared with type strains of the group of fluorescent pseudomonads, the bacterium studied probably belongs to a new species. Using light, fluorescent, and scanning electron microscopy, the ability of the studied bacterium to form biofilms of different architectures when cultured in liquid mineral medium with different carbon sources, including naphthalene and n-dodecane, was demonstrated. When grown on a mixture of naphthalene and n-dodecane, the strain first consumed naphthalene and then n-dodecane. Cultivation of the strain on n-dodecane was characterized by a long adaptation phase, in contrast to cultivation on naphthalene and a mixture of naphthalene and n-dodecane.

Characterization and Genomic Analysis of the Naphthalene-Degrading Delftia tsuruhatensis ULwDis3 Isolated from Seawater.

Strains of the genus Delftia are poorly studied microorganisms. In this work, the complete genome of the naphthalene-degrading Delftia tsuruhatensis strain ULwDis3 isolated from seawater of the Gulf of Finland of the Baltic Sea was assembled. For the first time, genes encoding naphthalene cleavage pathways via salicylate and gentisate were identified in a strain of the genus Delftia. The genes are part of one operon (nag genes). Three open reading frames (ORFs) were found in the genome of D. tsuruhatensis strain ULwDis3 that encode gentisate 1.2-dioxygenase. One of the ORFs is part of the nag operon. The physiological and biochemical characteristics of the strain ULwDis3 when cultured in mineral medium with naphthalene as the sole source of carbon and energy were also studied. It was found that after 22 h of growth, the strain stopped consuming naphthalene, and at the same time, naphthalene 1.2-dioxygenase and salicylate 5-hydroxylase activities were not detected. Later, a decrease in the number of living cells and the death of the culture were observed. Gentisate 1.2-dioxygenase activity was detected from the time of gentisate formation until culture death.

Diversity of Microbial Communities, PAHs, and Metals in Road and Leaf Dust of Functional Zones of Moscow and Murmansk.

The impact of geographical factors, functional zoning, and biotope type on the diversity of microbial communities and chemical components in the dust of urban ecosystems was studied. Comprehensive analyses of bacterial and fungal communities, polycyclic aromatic hydrocarbons (PAHs), and metals in road and leaf dust in three urban zones of Murmansk and Moscow with contrasting anthropogenic load were conducted. We found that the structure of bacterial communities affected the functional zoning of the city, biotope type, and geographical components. Fungal communities were instead impacted only by biotope type. Our findings revealed that the structure of fungal communities was mostly impacted by PAHs whereas bacterial communities were sensitive to metals. Bacteria of the genus Sphingomonas in road and leaf dust as indicators of the ecological state of the urban ecosystems were proposed.

Current research on simultaneous oxidation of aliphatic and aromatic hydrocarbons by bacteria of genus Pseudomonas.

One of the most frequently used methods for elimination of oil pollution is the use of biological preparations based on oil-degrading microorganisms. Such microorganisms often relate to bacteria of the genus Pseudomonas. Pseudomonads are ubiquitous microorganisms that often have the ability to oxidize various pollutants, including oil hydrocarbons. To date, individual biochemical pathways of hydrocarbon degradation and the organization of the corresponding genes have been studied in detail. Almost all studies of this kind have been performed on degraders of individual hydrocarbons belonging to a single particular class. Microorganisms capable of simultaneous degradation of aliphatic and aromatic hydrocarbons are very poorly studied. Most of the works on such objects have been devoted only to phenotype characteristic and some to genetic studies. To identify the patterns of interaction of several metabolic systems depending on the growth conditions, the most promising are such approaches as transcriptomics and proteomics, which make it possible to obtain a comprehensive assessment of changes in the expression of hundreds of genes and proteins at the same time. This review summarizes the existing data on bacteria of the genus Pseudomonas capable of the simultaneous oxidation of hydrocarbons of different classes (alkanes, monoaromatics, and polyaromatics) and presents the most important results obtained in the studies on the biodegradation of hydrocarbons by representatives of this genus using methods of transcriptomic and proteomic analyses.

Phylloplane Biodiversity and Activity in the City at Different Distances from the Traffic Pollution Source.

The phylloplane is an integrated part of green infrastructure which interacts with plant health. Taxonomic characterization of the phylloplane with the aim to link it to ecosystem functioning under anthropogenic pressure is not sufficient because only active microorganisms drive biochemical processes. Activity of the phylloplane remains largely overlooked. We aimed to study the interactions among the biological characteristics of the phylloplane: taxonomic diversity, functional diversity and activity, and the pollution grade. Leaves of Betula pendula were sampled in Moscow at increasing distances from the road. For determination of phylloplane activity and functional diversity, a MicroResp tool was utilized. Taxonomic diversity of the phylloplane was assessed with a combination of microorganism cultivation and molecular techniques. Increase of anthropogenic load resulted in higher microbial respiration and lower DNA amount, which could be viewed as relative inefficiency of phylloplane functioning in comparison to less contaminated areas. Taxonomic diversity declined with road vicinity, similar to the functional diversity pattern. The content of Zn in leaf dust better explained the variation in phylloplane activity and the amount of DNA. Functional diversity was linked to variation in nutrient content. The fraction of pathogenic fungi of the phylloplane was not correlated with any of the studied elements, while it was significantly high at the roadsides. The bacterial classes Gammaproteobacteria and Cytophagia, as well as the Dothideomycetes class of fungi, are exposed to the maximal effect of distance from the highway. This study demonstrated the sensitivity of the phylloplane to road vicinity, which combines the effects of contaminants (mainly Zn according to this study) and potential stressful air microclimatic conditions (e.g., low relative air humidity, high temperature, and UV level). Microbial activity and taxonomic diversity of the phylloplane could be considered as an additional tool for bioindication.

Polycyclic Aromatic Hydrocarbon-Degrading Bacteria in Three Different Functional Zones of the Cities of Moscow and Murmansk.

We performed a comparative study of the total bacterial communities and communities of cultivable polycyclic aromatic hydrocarbons (PAH)-degrading bacteria in different functional zones of Moscow and Murmansk that were formed under the influence of the PAH composition in road and leaf dust. The PAHs were determined by high-performance liquid chromatography (HPLC); the bacterial communities' diversity was assessed by metabarcoding. The degraders were isolated by their direct plating on a medium with the PAHs. The PAH total quantity declined in the leaf dust from the traffic to the recreational zone. For the road dust, a negative gradient with pollution was observed for Rhodococcus and Acinetobacter degraders and for their relative abundance in the microbiome for the functional zones of Moscow. The opposite effect was observed in the Murmansk leaf dust for the Rothia and Pseudomonas degraders and in the Moscow road dust for Microbacterium. The PCA and linear regression analyses showed that the Micrococcus degraders in the dust were sensitive to anthropogenic pollution, so they can be used as a tool for monitoring anthropogenic changes in the biosphere. The data on the degraders' and microbial communities' diversity suggest that minor degrading strains can play a key role in PAH degradation.

The organization of naphthalene degradation genes in Pseudomonas putida strain AK5.

The Pseudomonas putida АК5 that was isolated from the slime pit of a Nizhnekamsk oil chemical factory can metabolize naphthalene via salicylate and gentisate. Catabolic genes are localized on non-conjugative IncP-7 plasmid pAK5 of about 115 kb in size. The "classical"nah-1 operon and the novel sgp-operon (salicylate-gentisate pathway) are both involved in naphthalene degradation by P. putida АК5, that was first described for Pseudomonas. The sgp-operon includes six open reading frames (ORFs) (sgpAIKGHB). The four ORFs code for the entire salicylate 5-hydroxylase - oxidoreductase component (sgpA), large and small subunits of the oxigenase component (sgpG and sgpH) and 2Fe-2S ferredoxin (sgpB). Genes for gentisate 1, 2-dioxygenase (sgpI) and fumarylpyruvate hydrolase (sgpK) are located in salicylate 5-hydroxylase genes clustering between sgpA and sgpG. The putative positive regulator for the sgp-operon (sgpR) was found upstream of the sgpA gene and oriented in the opposite direction from sgpA. The putative maleylacetoacetate isomerase gene is located apart, directly downstream from the sgp-operon. The sgp-operon organization and phylogenetic analysis of deduced amino acid sequences indicate that this operon has a mosaic structure according to the modular theory of the evolution of modern catabolic pathways.