Currently, synthetic genomics is a rapidly developing field. Its main tasks, such as the design of synthetic sequences and the assembly of DNA sequences from synthetic oligonucleotides, require specialized software. In this article, we present a program with a graphical interface that allows non-bioinformatics to perform the tasks needed in synthetic genomics. We developed BAC-browser v.2.1. It helps to design nucleotide sequences and features the following tools: generate nucleotide sequence from amino acid sequences using a codon frequency table for a specific organism, as well as visualization of restriction sites, GC composition, GC skew and secondary structure. To assemble DNA sequences, a fragmentation tool was created: regular breakdown into oligonucleotides of a certain length and breakdown into oligonucleotides with thermodynamic alignment. We demonstrate the possibility of DNA fragments assemblies designed in different modes of BAC-browser. The BAC-browser has a large number of tools for working in the field of systemic genomics and is freely available at the link with a direct link https://sysbiomed.ru/upload/BAC-browser-2.1.zip .

Ralstonia eutropha is a facultative chemolithoautotrophic aerobic bacterium that grows using organic substrates or H-2 and CO2. Hydrogenases (Hyds) are synthesized under lithoautotrophic, or energy-limited heterotrophic conditions and are used in enzyme fuel cells (EFC) as anodic catalysts. The effects of chemically synthesized gold nanoparticles (Au-NPs) on R. eutropha H16 growth, oxidation-reduction potential (ORP) kinetics, and H-2-oxidizing Hyd activity were investigated in this study. Atomic force microscopy showed that thin, plate-shaped Au-NPs were in the nanoscale range with an average size of 5.68 nm. Compared with growth in medium without Au-NPs (control), the presence of Au-NPs stimulated growth, and resulted in a decrease in ORP to negative values. H-2-oxidizing activity was not detected in the absence of Au-NPs, but activity was significantly induced (12 U/g CDW) after 24 h of growth with 18 ng/ml, increasing a further 4-fold after 72 h of growth. The results demonstrate that Au-NPs primarily influence the membrane-bound Hyd. In contrast to R. eutropha, Au-NPs had a negligible or negative effect on the growth, Hyd activity, and H-2 production of Escherichia coli. The findings of this study offer new perspectives for the production of oxygen-tolerant Hyds and the development of EFCs.

IntroductionWhiA is a conserved protein found in numerous bacteria. It consists of an HTH DNA-binding domain linked with a homing endonuclease (HEN) domain. WhiA is one of the most conserved transcription factors in reduced bacteria of the class Mollicutes. Its function in Mollicutes is unknown, while it is well-characterized in Streptomyces. Here, we focused on WhiA protein from Mycoplasma gallisepticum.MethodsWe used a combination molecular dynamics, EMSA, MST and AFM to study the DNA-binding and ATP-binding properties of WhiA from M. gallisepticum. The transcriptional repressor function of WhiA was demonstrated using gene knockdown, reporter constructs and proteome analysis.ResultsWe demonstrate that WhiA homolog from M. gallisepticum binds a conserved sequence of the GAYACRCY core (Y = C or T, R = A or G), which is located in the promoter of an operon coding for ribosomal proteins and adenylate kinase (rpsJ operon). We show that WhiA in M. gallisepticum is a repressor of rpsJ operon and a sensor of ATP. HTH domain binds to the core motif and HEN domain binds to the auxiliary motif GTTGT that is located downstream to the core motif. We show that binding by both domains to DNA is required to fulfill the transcription repressor function. Knockdown of whiA does not affect actively growing M. gallisepticum, but leads to the growth retardation after freezing.DiscussionWe propose the following model for M. gallisepticum WhiA function. WhiA remains bound to the core motif at any conditions. At low ATP concentrations (starvation) HEN domain binds auxiliary motif and represses rpsJ operon transcription. At high ATP concentrations (nutrient-rich conditions) HEN domain binds ATP and releases auxiliary motif. It leads to the de-repression of rpsJ operon and increased production of ribosomal proteins.

IntroductionSmall mammals, especially rodents and bats, are known reservoirs of zoonotic viruses, but little is known about the viromes of insectivorous species including hedgehogs (order Eulipotyphla), which often live near human settlements and come into contact with humans.MethodsWe used high-throughput sequencing and metaviromic analysis to describe the viromes of 21 hedgehogs (Erinaceus sp.) sampled from summer 2022 to spring 2023. We captured 14 active animals from the wild (seven in European Russia and the other seven in Central Siberia). The remaining 7 animals were hibernating in captivity (captured in European Russia before the experiment).Results and discussionThe diversity of identified viral taxa as well as the total number of reads classified as viral was high in all active animals (up to eight different viral families per animal), but significantly lower in hibernating animals (zero or no more than three different viral families per animal). The present study reports, for the first time, betacoronaviruses and mammasrenaviruses in hedgehogs from Russia. Erinaceus coronaviruses (EriCoVs) were found in 4 of 7 active animals captured in the wild, in European Russia, making it is the easiest finding of EriCoVs in Europe. One animal was found to carry of two different EriCoVs. Both strains belong to the same phylogenetic clade as other coronaviruses from European hedgehogs. Pairwise comparative analysis suggested that one of these two strains arose by recombination with an unknown coronavirus, since all of identified SNPs (n = 288) were found only in the local genome region (the part of ORF1b and S gene). The novel mammarenaviruses (EriAreVs) were detected in 2 out of 7 active and in 2 out of 7 hibernating animals from the European Russia. Several complete L and S segments of EriAreVs were assembled. All identified EriAreVs belonged to the same clade as the recently described MEMV virus from Hungarian hedgehogs. As the hibernating hedgehogs were positive for EriAreVs when kept in controlled conditions without contact with each other, we suggest the possibility of persistent arenavirus infection in hedgehogs, but further experiments are needed to prove this.

At present, bacteriophages are considered as an alternative to antibiotics in prevention and treatment of bacterial infections, in particular cholera.The aim of the work was to demonstrate a method to obtain synthetic bacteriophage against Vibrio cholerae. Vibriophage N4 was selected as a subject for the study.Materials and methods. The genome sequence of vibriophage N4 (38.5 kb) was taken from the NCBI GenBank database. The sequence was divided into gene blocks of 1500–2000 bp. The gene blocks, in turn, were split into oligonucleotides. Sequence partitioning was carried out using the BAC-browser software that we have developed. Oligonucleotides were chemically synthesized; gene blocks were assembled from them. After that, the complete genome of vibriophage N4 was synthesized from the obtained gene blocks. The assembly of the synthetic genome took place in two stages. At the first stage, gene block cassettes of 5–7 pieces with sizes ranging from 7 to 10.5 thousand bp were generated via homologous recombination in yeast. The resulting cassettes were then amplified and used for in vitro assembly using 5’-3’ exonuclease and thermostable DNA polymerase. The resulting preparation was used for electroporation of V. cholerae cells.Results and discussion. The synthetic genome of vibriophage N4 was delivered into the V. cholerae strain M818 O1 biovar El Tor using electroporation. As a result, the formation of lytic plaques on the lawn of V. cholerae was observed. The range of technologies we have developed: software for assembly design, enzymes and buffers for the synthesis of gene blocks and their crosslinking by homologous recombination in vitro, the method for producing large-sized assemblies in yeast can be used to obtain artificial bacteriophages with a rational genome design.

The standardization of DNA fragment assembly methods for many tasks of synthetic biology is crucial. This is necessary for synthesizing a wider repertoire of sequences, as well as for further automation and miniaturization of such reactions. In this work, we proposed conditions for the assembly of DNA fragments from chemically synthesized oligonucleotides and we identified the errors occurring in the sequence under these conditions. Additionally, we proposed conditions for further combining synthetic fragments into larger DNA fragments. We showed that the optimized conditions are suitable for the assembly of a wide range of sequences.

Introduction.Mycoplasma hominis is a bacterium belonging to the class Mollicutes. It causes acute and chronic infections of the urogenital tract. The main features of this bacterium are an absence of cell wall and a reduced genome size (517-622 protein-encoding genes). Previously, we have isolated morphologically unknown M. hominis colonies called micro-colonies (MCs) from the serum of patients with inflammatory urogenital tract infection.Hypothesis. MCs are functionally different from the typical colonies (TCs) in terms of metabolism and cell division.Aim. To determine the physiological differences between MCs and TCs of M. hominis and elucidate the pathways of formation and growth of MCs by a comparative proteomic analysis of these two morphological forms.Methodology. LC-MS proteomic analysis of TCs and MCs using an Ultimate 3000 RSLC nanoHPLC system connected to a QExactive Plus mass spectrometer.Results. The study of the proteomic profiles of M. hominis colonies allowed us to reconstruct their energy metabolism pathways. In addition to the already known pentose phosphate and arginine deamination pathways, M. hominis can utilise ribose phosphate and deoxyribose phosphate formed by nucleoside catabolism as energy sources. Comparative proteomic HPLC-MS analysis revealed that the proteomic profiles of TCs and MCs were different. We assume that MC cells preferably utilised deoxyribonucleosides, particularly thymidine, as an energy source rather than arginine or ribonucleosides. Utilisation of deoxyribonucleosides is less efficient as compared with that of ribonucleosides and arginine in terms of energy production. Thymidine phosphorylase DeoA is one of the key enzymes of deoxyribonucleosides utilisation. We obtained a DeoA overexpressing mutant that exhibited a phenotype similar to that of MCs, which confirmed our hypothesis.Conclusion. In addition to the two known pathways for energy production (arginine deamination and the pentose phosphate pathway) M. hominis can use deoxyribonucleosides and ribonucleosides. MC cells demonstrate a reorganisation of energy metabolism: unlike TC cells, they preferably utilise deoxyribonucleosides, particularly thymidine, as an energy source rather than arginine or ribonucleosides. Thus MC cells enter a state of energy starvation, which helps them to survive under stress, and in particular, to be resistant to antibiotics.

Mycoplasma hominis is an opportunistic urogenital pathogen in vertebrates. It is a non-glycolytic species that produces energy via arginine degradation. Among genital mycoplasmas, M. hominis is the most commonly reported to play a role in systemic infections and can persist in the host for a long time. However, it is unclear how M. hominis proceeds under arginine limitation. The recent metabolic reconstruction of M. hominis has demonstrated its ability to catabolize deoxyribose phosphate to produce ATP. In this study, we cultivated M. hominis on two different energy sources (arginine and thymidine) and demonstrated the differences in growth rate, antibiotic sensitivity, and biofilm formation. Using label-free quantitative proteomics, we compared the proteome of M. hominis under these conditions. A total of 466 proteins were identified from M. hominis, representing approximately 85% of the predicted proteome, while the levels of 94 proteins changed significantly. As expected, we observed changes in the levels of metabolic enzymes. The energy source strongly affects the synthesis of enzymes related to RNA modifications and ribosome assembly. The translocation of lipoproteins and other membrane-associated proteins was also impaired. Our study, the first global characterization of the proteomic switching of M. hominis in arginine-deficiency media, illustrates energy source-dependent control of pathogenicity factors and can help to determine the mechanisms underlying the interaction between the growth rate and fitness of genome-reduced bacteria.

The epigenetics of bacteria, and bacteria with a reduced genome in particular, is of great interest, but is still poorly understood. Mycoplasma gallisepticum, a representative of the class Mollicutes, is an excellent model of a minimal cell because of its reduced genome size, lack of a cell wall, and primitive cell organization. In this study we investigated DNA modifications of the model object Mycoplasma gallisepticum and their roles. We identified DNA modifications and methylation motifs in M. gallisepticum S6 at the genome level using single molecule real time (SMRT) sequencing. Only the ANCNNNNCCT methylation motif was found in the M. gallisepticum S6 genome. The studied bacteria have one functional system for DNA modifications, the Type I restriction-modification (RM) system, MgaS6I. We characterized its activity, affinity, protection and epigenetic functions. We demonstrated the protective effects of this RM system. A common epigenetic signal for bacteria is the m6A modification we found, which can cause changes in DNA-protein interactions and affect the cell phenotype. Native methylation sites are underrepresented in promoter regions and located only near the -35 box of the promoter, which does not have a significant effect on gene expression in mycoplasmas. To study the epigenetics effect of m6A for genome-reduced bacteria, we constructed a series of M. gallisepticum strains expressing EGFP under promoters with the methylation motifs in their different elements. We demonstrated that m6A modifications of the promoter located only in the -10-box affected gene expression and downregulated the expression of the corresponding gene.

The structure and dynamics of bacterial nucleoids play important roles in regulating gene expression. Bacteria of class Mollicutes and, in particular, mycoplasmas feature extremely reduced genomes. They lack multiple structural proteins of the nucleoid, as well as regulators of gene expression. We studied the organization of Mycoplasma gallisepticum nucleoids in the stationary and exponential growth phases at the structural and protein levels. The growth phase transition results in the structural reorganization of M. gallisepticum nucleoid. In particular, it undergoes condensation and changes in the protein content. The observed changes corroborate with the previously identified global rearrangement of the transcriptional landscape in this bacterium during the growth phase transition. In addition, we identified that the glycolytic enzyme enolase functions as a nucleoid structural protein in this bacterium. It is capable of non-specific DNA binding and can form fibril-like complexes with DNA.

Mycoplasma hominis is an opportunistic bacterium that can cause acute and chronic infections of the urogenital tract. This bacterium, like all other Mycoplasma species, is characterized by the reduced genome size, and, consequently, reduction of the main metabolic pathways. M. hominis cells cannot effectively use glucose as a carbon and energy source. Therefore, the main pathway of energy metabolism is the arginine dihydrolase pathway. However, several bacteria can use nucleosides as the sole energy source. Biochemical studies using Salmonella typhimurium have shown that three enzymes (thymidine phosphorylase, phosphopentose mutase and deoxyribose-phosphate aldolase) are involved in the thymidine catabolic pathway. All these enzymes are present in M. hominis. For understanding changes in the energy metabolism of M. hominis we performed shotgun proteome analysis of M. hominis cells in liquid medium with arginine or thymidine as a carbon source. LC-MS analysis was performed with an Ultimate 3000 Nano LC System (Thermo Fisher Scientific) coupled to a Q Exactive HF benchtop Orbitrap mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific). Data are available via ProteomeXchange with identifier PXD018714 (https://www.ebi.ac.uk/pride/archive/projects/PXD018714).

Summary Translation efficiency contributes several orders of magnitude difference in the overall yield of exogenous gene expression in bacteria. In diverse bacteria, the translation initiation site, whose sequence is the primary determinant of the translation performance, is comprised of the start codon and the Shine–Dalgarno box located upstream. Here, we have examined how the sequence of a spacer between these main components of the translation initiation site contributes to the yield of synthesized protein. We have created a library of reporter constructs with the randomized spacer region, performed fluorescently activated cell sorting and applied next‐generation sequencing analysis (the FlowSeq protocol). As a result, we have identified sequence motifs for the spacer region between the Shine–Dalgarno box and AUG start codon that may modulate the translation efficiency in a 100‐fold range.

Mycoplasma gallisepticum (MG) is one of the smallest free-living and self-replicating organisms, it is characterized by lack of cell wall and reduced genome size. As a result of genome reduction, MG has a limited variety of DNA-binding proteins and transcription factors. To investigate the dynamic changes of the proteomic profile of MG nucleoid, that may assist in revealing its mechanisms of functioning, regulation of chromosome organization and stress adaptation, a quantitative proteomic study was performed on MG nucleoids obtained from the cell culture in logarithmic and stationary phases of synchronous growth. MG cells were grown on a liquid medium with a 9 h starvation period. Nucleoids were obtained from the cell culture at the 26th and the 50th hour (logarithmic and stationary growth phases respectively) by sucrose density gradient centrifugation. LC-MS analysis was carried out on an Ultimate 3000 RSLCnano HPLC system connected to a Fusion Lumos mass spectrometer, controlled by XCalibur software (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific). For comprehensive peptide library generation one sample from each biological replicate was run in DDA mode. Then, all the samples were run in a single LC-MS DIA run. Identification of DDA files and DIA quantitation was performed with MaxQuant and Skyline software, correspondingly. All raw data generated from IDA and DDA acquisitions are presented in the PRIDE database with identifier PXD019077.

As permanent residents of the normal gut microbiota, bifidobacteria have evolved to adapt to the host’s immune response whose priority is to eliminate pathogenic agents. The mechanisms that ensure the survival of commensals during inflammation and maintain the stability of the core component of the normal gut microbiota in such conditions remain poorly understood. We propose a new in vitro approach to study the mechanisms of resistance to immune response factors based on high-throughput sequencing followed by transcriptome analysis. This approach allowed us to detect differentially expressed genes associated with inflammation. In this study, we demonstrated that the presence of the pro-inflammatory cytokines IL-6 and TNFα to the growth medium of the B. longum subsp. longum GT15 strain changes the latter’s growth rate insignificantly while affecting the expression of certain genes. We identified these genes and performed a COG and a KEGG pathway enrichment analysis. Using phylogenetic profiling we predicted the operons of genes whose expression was triggered by the cytokines TNFα and IL-6 in vitro. By mapping the transcription start points, we experimentally validated the predicted operons. Thus, in this study, we predicted the genes involved in a putative signaling pathway underlying the mechanisms of resistance to inflammatory factors in bifidobacteria. Since bifidobacteria are a major component of the human intestinal microbiota exhibiting pronounced anti-inflammatory properties, this study is of great practical and scientific relevance.

ABSTRACT Glycerol is an organic waste material that can be used for the production of microbial biomass, consequently providing valuable biocatalysts promoting the generation of electrical current in microbial fuel cells (MFCs). [NiFe]–Hydrogenases (Hyds) of Escherichia coli and Ralstonia eutropha may be applied as potential anode biocatalysts in MFCs. In this study, E. coli K12 whole cells or crude extracts and R. eutropha HF649 synthesizing Strep-tagged membrane-bound Hyds (MBH) were evaluated as anode enzymes in a bioelectrochemical system. The samples were immobilized on the sensors with polyvinyl acetate support. Mediators like ferrocene and its derivatives (ferrocene-carboxy-aldehyde, ferrocene-carboxylic acid, methyl-ferrocene-methanol) were employed. The maximal level of bioelectrocatalytic activity of Hyds was demonstrated at 500 mV voltage. Depending on the mediator and biocatalyst, current strength varied from 5 to 42 μA. Introduction of ferrocene-carboxylic acid enhanced current strength; moreover, the current flow was directly correlated with H2 concentration. The maximal value (up to 150 μA) of current strength was achieved with a 2-fold hydrogen supply. It may be inferred that Hyds are efficiently produced by E. coli and R. eutropha grown on glycerol, while ferrocene derivatives act as agents mediating the electrochemical activity of Hyds.

One of the dysbioses often observed in Crohn's disease (CD) patients is an increased abundance of Escherichia coli (10-100 fold compared to healthy individuals) (Gevers et al., 2014). The data reported is a large-scale proteome profile for E. coli isolates collected from CD patients and healthy individuals. 43 isolates were achieved from 30 CD patients (17 male, 12 female, median age 30) and 19 isolates from 7 healthy individuals (7 male, median age 19). Isolates were cultivated on LB medium at aerobic conditions up to medium log phase. Protein extraction was performed with sodium deoxycholate (DCNa) and urea, alcylation with tris(2-carboxyethyl) phosphine and iodacetamide. Protein trypsinolysis was performed as described in (Matyushkina et al., 2016). Total cell proteomes were analysed by shotgun proteomics with HPLC-MS/MS on a maXis qTOF mass-spectrometer. The data including HPLC-MS/MS raw files and exported Mascot search results was deposited to the PRIDE repository project accession: PXD010920, project https://doi. org/10.6019/PXD010920. (C) 2019 The Author(s). Published by Elsevier Inc.

Cyanobacteria synthesize neurotoxic β-N-methylamino-l-alanine (BMAA). The roles of this non-protein amino acid in cyanobacterial cells are insufficiently studied. During diazotrophic growth, filamentous cyanobacteria form single differentiated cells, called heterocysts, which are separated by approximately 12–15 vegetative cells. When combined nitrogen is available, heterocyst formation is blocked and cyanobacterial filaments contain only vegetative cells. In the present study, we discovered that exogenous BMAA induces the process of heterocyst formation in filamentous cyanobacteria under nitrogen-replete conditions that normally repress cell differentiation. BMAA treated cyanobacteria form heterocyst-like dark non-fluorescent non-functional cells. It was found that glutamate eliminates the BMAA mediated derepression. Quantitative polymerase chain reaction (qPCR) permitted to detect the BMAA impact on the transcriptional activity of several genes that are implicated in nitrogen assimilation and heterocyst formation in Anabaena sp. PCC 7120. We demonstrated that the expression of several essential genes increases in the BMAA presence under repressive conditions.