Rathayibacter tanaceti sp. nov., a Novel Actinobacterium from Tanacetum vulgare Infested by Foliar Nematode Aphelenchoides sp.

Two novel yellow-pigmented, rod-shaped and non-motile coryneform actinobacteria, strains VKM Ac-2596T and VKM Ac-2761, were isolated from a plant Tanacetum vulgare (Asteraceae) infested by foliar nematode Aphelenchoides sp. The strains exhibited the highest 16S rRNA gene sequence similarities to Rathayibacter agropyri CA4T (99.71%), Rathayibacter rathayi DSM 7485T (99.65%) and Rathayibacter iranicus VKM Ac-1602T (99.65%). The pairwise average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between VKM Ac-2596T and VKM Ac-2671 towards the type strains of Rathayibacter species did not exceed 85.24% and 29.40%, respectively, that are well below the thresholds for species delineation. The target strains had key chemotaxonomic properties typical of the genus Rathayibacter, namely, the DAB-based peptidoglycan, rhamnose and mannose as the predominant sugars and a rhamnomannan in the cell, the major menaquinone MK-10 and fatty acids of iso-anteiso type, with a large proportion of anteiso-15:0. The strains showed clear differences from the recognized Rathayibacter species in several phenotypic characteristics, including the difference in the composition of cell wall glycopolymers. Based on the results obtained in this study and the data published previously, we provide a description of a new species, Rathayibacter tanaceti sp. nov., with DL-642T (= VKM Ac-2596T = LMG 33114T) as the type strain.

Lysobacter gummosus 10.1.1, a Producer of Antimicrobial Agents.

This work investigated the antimicrobial potential of Lysobacter gummosus 10.1.1. The culture fluid of the strain was found to contain antimicrobial agents active against Staphylococcus aureus, Micrococcus luteus, and Bacillus cereus. L. gummosus was first shown to be capable of forming outer membrane vesicles, which have a bacteriolytic effect against not only Gram-positive bacteria but also against the Gram-negative pathogen Pseudomonas aeruginosa. Transcriptomic analysis revealed the genes of almost all known bacteriolytic enzymes of Lysobacter, as well as the genes of enzymes with putative bacteriolytic activity. Also identified were genes involved in the biosynthesis of a number of secondary metabolites for which antimicrobial activities are known. This research is indicative of the relevance of isolating and studying L. gummosus antimicrobial agents.

Identification of a Gene Encoding a New Fungal Steroid 7-Hydroxylase and Its Functional Characterization in Pichia pastoris Yeast.

The hydroxylation of steroids in the C7ß position is one of the rare reactions that allow the production of value-added precursors in the synthesis of ursodeoxycholic acid and other pharmaceuticals. Recently, we discovered this activity in the ascomycete Curvularia sp. VKM F-3040. In this study, the novel gene of 7-hydroxylase (P450cur) was identified as being heterologously expressed and functionally characterized in Pichia pastoris. Transcriptome data mining and differential expression analysis revealed that 12 putative genes in Curvularia sp. mycelia significantly increased their expression in response to dehydroepiandrosterone (DHEA). The transcriptional level of the most up-regulated cytochrome P450cur gene was increased more than 300-fold. A two-gene construct with a candidate P450cur gene and the gene of its natural redox partner, NADPH-cytochrome P450 reductase (CPR), which is interconnected by a T2A element, was created. Using this construct, recombinant P. pastoris strains co-expressing fungal P450cur and CPR genes were obtained. The functional activity of the recombinant P450cur was studied in vivo during the bioconversion of androstane steroids. The fungal 7-monooxygenase predominantly catalyzed the 7ß-hydroxylation of androstadienedione (ADD), DHEA, and androstenediol, whereas 1-dehydrotestosterone was hydroxylated by P450cur mainly at the C7-Hα position. To our knowledge, this is the first report of a recombinant yeast capable of catalyzing the 7α/ß-hydroxylation of ADD and DHEA.

De novo transcriptome assembly of Curvularia sp. VKM F-3040, a promising steroid-modifying ascomycete.

This research presents de novo transcriptome shotgun assembly for Curvularia sp. VKM F-3040, which is a putative fungal strain able to modify androstane steroids with production of 7-hydroxy and 17-hydroxylated derivatives-key intermediates in the synthesis of pharmaceutical ingredients. The data are of importance for creating novel microbial biocatalysts.

Transcriptomic Analysis Followed by the Isolation of Extracellular Bacteriolytic Proteases from Lysobacter capsici VKM B-2533T.

The aim of the study was to search for, isolate and characterize new bacteriolytic enzymes that show promising potential for their use in medicine, agriculture and veterinary. Using a transcriptomic analysis, we annotated in Lysobacter capsici VKM B-2533T the genes of known bacteriolytic and antifungal enzymes, as well as of antibiotics, whose expression levels increased when cultivated on media conducive to the production of antimicrobial agents. The genes of the secreted putative bacteriolytic proteases were also annotated. Two new bacteriolytic proteases, Serp and Serp3, were isolated and characterized. The maximum bacteriolytic activities of Serp and Serp3 were exhibited at low ionic strength of 10 mM Tris-HCl, and high temperatures of, respectively, 80 °C and 70 °C. The pH optimum for Serp was 8.0; for Serp3, it was slightly acidic, at 6.0. Both enzymes hydrolyzed autoclaved cells of Micrococcus luteus Ac-2230T, Proteus vulgaris H-19, Pseudomonas aeruginosa and Staphylococcus aureus 209P. Serp also digested cells of Bacillus cereus 217. Both enzymes hydrolyzed casein and azofibrin. The newly discovered enzymes are promising for developing proteolytic antimicrobial drugs on their basis.

Ancylobacter radicis sp. nov., a novel aerobic methylotrophic bacteria associated with plants.

The two novel bacterial strains, designated as VTT and ML, were isolated from roots of cinquefoil (Potentilla sp.) and leaves of meadow-grass (Poa sp.) on the flooded bank of lake, respectively. These isolates were Gram-negative, non-spore-forming, non-motile, rod-shaped cells, utilized methanol, methylamine, and polycarbon compounds as carbon and energy sources. In the whole-cell fatty acid pattern of strains prevailed C18:1ω7c and C19:0cyc. Based on the phylogenetic analysis of 16S rRNA gene sequences, strains VTT and ML were closely related to the representatives of the genus Ancylobacter (98.3-98.5%). The assembled genome of strain VTT has a total length of 4.22 Mbp, and a G + C content is 67.3%. The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values between strain VTT and closely related type strains of genus Ancylobacter were 78.0-80.6%, 73.8-78.3% and 22.1-24.0%, respectively, that clearly lower than proposed thresholds for species. On the basis of the phylogenetic, phenotypic, and chemotaxonomic analysis, isolates VTT and ML represent a novel species of the genus Ancylobacter, for which the name Ancylobacter radicis sp. nov. is proposed. The type strain is VTT (= VKM B-3255T = CCUG 72400T). In addition, novel strains were able to dissolve insoluble phosphates, to produce siderophores and plant hormones (auxin biosynthesis). According to genome analysis genes involved in the biosynthesis of siderophores, polyhydroxybutyrate, exopolysaccharides and phosphorus metabolism, as well as the genes involved in the assimilation of C1-compounds (natural products of plant metabolism) were found in the genome of type strain VTT.

Draft Genome Sequences of 11 Rhodopsin Gene-Containing Actinobacteria (Geodermatophilaceae) from Saline Arid Habitats in the Central Asian Deserts.

Draft genome sequences of 11 strains of putative new species of Geodermatophilaceae were generated using Illumina technology. The genome sizes ranged from 4.19 to 4.99 Mb, with G+C contents of 73.5% to 74.6%, and contained genes for microbial rhodopsins. This study will contribute to our knowledge of the ecology and diversity of members of the family Geodermatophilaceae.

Distribution of Bacterial Endosymbionts of the Cardinium Clade in Plant-Parasitic Nematodes.

Bacteria of the genus "Candidatus Cardinium" and related organisms composing the Cardinium clade are intracellular endosymbionts frequently occurring in several arthropod groups, freshwater mussels and plant-parasitic nematodes. Phylogenetic analyses based on two gene sequences (16S rRNA and gyrB) showed that the Cardinium clade comprised at least five groups: A, B, C, D and E. In this study, a screening of 142 samples of plant-parasitic nematodes belonging to 93 species from 12 families and two orders using PCR with specific primers and sequencing, revealed bacteria of Cardinium clade in 14 nematode samples belonging to 12 species of cyst nematodes of the family Heteroderidae. Furthermore, in this study, the genome of the Cardinium cHhum from the hop cyst nematode, Heterodera humuli, was also amplified, sequenced and analyzed. The comparisons of the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values for the strain Cardinium cHhum with regard to related organisms with available genomes, combined with the data on 16S rRNA and gyrB gene sequence identities, showed that this strain represents a new candidate species within the genus "Candidatus Paenicardinium". The phylogenetic position of endosymbionts of the Cardinium clade detected in nematode hosts was also compared to known representatives of this clade from other metazoans. Phylogenetic reconstructions based on analysis of 16S rRNA, gyrB, sufB, gloEL, fusA, infB genes and genomes and estimates of genetic distances both indicate that the endosymbiont of the root-lesion nematode Pratylenchus penetrans represented a separate lineage and is designated herein as a new group F. The phylogenetic analysis also confirmed that endosymbionts of ostracods represent the novel group G. Evolutionary relationships of bacterial endosymbionts of the Cardinium clade within invertebrates are presented and discussed.

Insight into Different Stages of Steroid Degradation in Thermophilic Saccharopolyspora hirsuta VKM Ac-666T Strain.

Steroids are abundant molecules in nature, and various microorganisms evolved to utilize steroids. Thermophilic actinobacteria play an important role in such processes. However, very few thermophiles have so far been reported capable of degrading or modifying natural sterols. Recently, genes putatively involved in the sterol catabolic pathway have been revealed in the moderately thermophilic actinobacterium Saccharopolyspora hirsuta VKM Ac-666T, but peculiarities of strain activity toward sterols are still poorly understood. S. hirsuta catalyzed cholesterol bioconversion at a rate significantly inferior to that observed for mesophilic actinobacteria (mycobacteria and rhodococci). Several genes related to different stages of steroid catabolism increased their expression in response to cholesterol as was shown by transcriptomic studies and verified by RT-qPCR. Sequential activation of genes related to the initial step of cholesterol side chain oxidation (cyp125) and later steps of steroid core degradation (kstD3, kshA, ipdF, and fadE30) was demonstrated for the first time. The activation correlates with a low cholesterol conversion rate and intermediate accumulation by the strain. The transcriptomic analyses revealed that the genes involved in sterol catabolism are linked functionally, but not transcriptionally. The results contribute to the knowledge on steroid catabolism in thermophilic actinobacteria and could be used at the engineering of microbial catalysts.

Whole-Genome Sequencing of Lysobacter capsici VKM B-2533T and Lysobacter gummosus 10.1.1, Promising Producers of Lytic Agents.

Lysobacter capsici VKM B-2533T and Lysobacter gummosus 10.1.1 are promising strains for use in biomedicine as sources of new antimicrobial agents. Here, we report the whole-genome sequences of both strains (total lengths, 6,239,188 bp and 6,056,609 bp, respectively), obtained using the Illumina and Nanopore platforms.

Draft Genome Sequences of 9 Actinobacteria from the Family Microbacteriaceae Associated with Insect- and Nematode-Damaged Plants.

Draft genome sequences of 9 strains of known and putative new species of Microbacteriaceae isolated from insect- and nematode-damaged plants were generated using Illumina technology. The data obtained will contribute to the development of the genome-based prokaryote taxonomy and the knowledge on the biology of the microbial group investigated.

Expression of a Stilbene Synthase Gene from the Vitis labrusca x Vitis vinifera L. Hybrid Increases the Resistance of Transgenic Nicotiana tabacum L. Plants to Erwinia carotovora.

'Isabel' grape (Vitis labrusca x V. vinifera L. hybrid) is one of the main grape cultivars in Russia and some other countries for processing, due to its vigor, tolerance to the main fungal diseases, high yield and potential for sugar accumulation. The stilbene synthase gene VlvSTS was isolated from the hybrid grape cv. Isabel and cloned into a pSS plant transformation vector under the control of a constitutive 35S RNA double promoter of the cauliflower mosaic virus, CaMV 35SS. VlvSTS-gene containing transgenic tobacco lines were obtained and analyzed. For the first time plants expressing the VlvSTS gene were shown to have an enhanced resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora B15. Transgenic plants were tested for resistance to a number of fungal pathogens. The plants were resistant to the grey mould fungus Botrytis cinerea, but not to the fungi Fusarium oxysporum, F. sporotrichioides, or F. culmorum. According to the results of a high performance liquid chromatography-mass spectrometry analysis, the amount of trans-resveratrol in leaves of transgenic plants with the highest expression of the VlvSTS gene was in a range from 150 to 170 µg/g of raw biomass. Change in the color and a decreased anthocyanin content in the flower corollas of transgenic plants were observed in transgenic lines with the highest expression of VlvSTS. A decrease in total flavonoid content was found in the flower petals but not the leaves of these tobacco lines. High expression of the VlvSTS gene influenced pollen development and seed productivity in transgenic plants. The size of pollen grains increased, while their total number per anther decreased. A decrease in the number of fertile pollen grains resulted in a decreased average weight of a seed boll in transgenic plants.

Steroid Metabolism in Thermophilic Actinobacterium Saccharopolyspora hirsuta VKM Ac-666T.

The application of thermophilic microorganisms opens new prospects in steroid biotechnology, but little is known to date on steroid catabolism by thermophilic strains. The thermophilic strain Saccharopolyspora hirsuta VKM Ac-666T has been shown to convert various steroids and to fully degrade cholesterol. Cholest-4-en-3-one, cholesta-1,4-dien-3-one, 26-hydroxycholest-4-en-3-one, 3-oxo-cholest-4-en-26-oic acid, 3-oxo-cholesta-1,4-dien-26-oic acid, 26-hydroxycholesterol, 3ß-hydroxy-cholest-5-en-26-oic acid were identified as intermediates in cholesterol oxidation. The structures were confirmed by 1H and 13C-NMR analyses. Aliphatic side chain hydroxylation at C26 and the A-ring modification at C3, which are putatively catalyzed by cytochrome P450 monooxygenase CYP125 and cholesterol oxidase, respectively, occur simultaneously in the strain and are followed by cascade reactions of aliphatic sidechain degradation and steroid core destruction via the known 9(10)-seco-pathway. The genes putatively related to the sterol and bile acid degradation pathways form three major clusters in the S. hirsuta genome. The sets of the genes include the orthologs of those involved in steroid catabolism in Mycobacterium tuberculosis H37Rv and Rhodococcus jostii RHA1 and related actinobacteria. Bioinformatics analysis of 52 publicly available genomes of thermophilic bacteria revealed only seven candidate strains that possess the key genes related to the 9(10)-seco pathway of steroid degradation, thus demonstrating that the ability to degrade steroids is not widespread among thermophilic bacteria.

Draft Genome Sequences of 28 Actinobacteria of the Family Microbacteriaceae Associated with Nematode-Infected Plants.

Draft genome sequences of 28 strains of Microbacteriaceae from plants infested by plant-parasitic nematodes were obtained using Illumina technology. The sequence data will provide useful baseline information for the development of comparative genomics and systematics of Microbacteriaceae and facilitate understanding of molecular mechanisms involved in interactions between plants and nematode-associated bacterial complexes.

Draft Genome Sequence of the Type Strain Lysobacter capsici VKM B-2533.

Lysobacter capsici VKM B-2533T is a promising strain for isolation of new lytic agents. Here, we report a draft genome sequence of this strain, consisting of 131 scaffolds with a total length of 6,196,943 bp. The results obtained will aid in the discovery and study of biologically active compounds important for biomedicine.

Draft Genome Sequences of 13 Plant-Associated Actinobacteria of the Family Microbacteriaceae.

Draft genome sequences of 13 bacterial strains from the family Microbacteriaceae were generated using Illumina technology. The genome sizes varied from 3.0 to 4.8 Mb, and the DNA G+C content was 68.1 to 72.5%. The sequences obtained will contribute to the development of genome-based taxonomy and understanding of molecular interactions between bacteria and plants.

Comparative Genomics of Beggiatoa leptomitoformis Strains D-401 and D-402T with Contrasting Physiology but Extremely High Level of Genomic Identity.

Representatives of filamentous colorless sulfur-oxidizing bacteria often dominate in sulfide biotopes, preventing the diffusion of toxic sulfide into the water column. One of the most intriguing groups is a recently described Beggiatoa leptomitoformis including strains D-401 and D-402T. Both strains have identical genes encoding enzymes which are involved in the oxidation of hydrogen sulfide and thiosulfate. Surprisingly, the B. leptomitoformis strain D-401 is not capable to grow lithotrophically in the presence of reduced sulfur compounds and to accumulate elemental sulfur inside the cells, in contrast to the D-402T strain. In general, genomes of D-401 and D-402T have an extremely high level of identity and only differ in 1 single-letter substitution, 4 single-letter indels, and 16 long inserts. Among long inserts, 14 are transposons. It was shown that in the D-401 strain, a gene coding for a sulfur globule protein was disrupted by one of the mentioned transposons. Based on comparative genomics, RT-qPCR, and HPLC-MS/MS, we can conclude that this gene plays a crucial role in the formation of the sulfur globules inside the cells, and the disruption of its function prevents lithotrophic growth of B. leptomitoformis in the presence of reduced sulfur compounds.

Complete and Draft Genome Sequences of 12 Plant-Associated Rathayibacter Strains of Known and Putative New Species.

Complete and draft genome sequences of 12 Rathayibacter strains were generated using Oxford Nanopore and Illumina technologies. The genome sizes of these strains are 3.21 to 4.61 Mb, with high G+C content (67.2% to 72.7%) genomic DNA. Genomic data will provide useful baseline information for natural taxonomy and comparative genomics of members of the genus Rathayibacter.

Draft Genome Sequence of Glyphosate-Degrading Achromobacter insolitus Strain Kg 19 (VKM B-3295), Isolated from Agricultural Soil.

The genome of an Achromobacter insolitus strain isolated from an agricultural soil polluted with the herbicide glyphosate is reported. The genome size is 6.4 Mb, with an average G+C content of 65.2%. These genomic data could contribute to a better understanding of the biochemistry and regulatory mechanisms of the microbial degradation of glyphosate and aminomethylphosphonate.