Gold nanoparticles activate hydrogenase synthesis and improve heterotrophic growth of Ralstonia eutropha H16.

Ralstonia eutropha is a facultative chemolithoautotrophic aerobic bacterium that grows using organic substrates or H2 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 H2-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. H2-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 H2 production of Escherichia coli. The findings of this study offer new perspectives for the production of oxygen-tolerant Hyds and the development of EFCs.

Role of DNA modifications in Mycoplasma gallisepticum.

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.

Molecular Basis of the Slow Growth of Mycoplasma hominis on Different Energy Sources.

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.

Thymidine utilisation pathway is a novel phenotypic switch of Mycoplasma hominis.

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.

The Dynamics of Mycoplasma gallisepticum Nucleoid Structure at the Exponential and Stationary Growth Phases.

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.

Gene Networks Underlying the Resistance of Bifidobacterium longum to Inflammatory Factors.

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.

Data on proteome of Mycoplasma hominis cultivated with arginine or thymidine as a carbon source.

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).

Data on nucleoid-associated proteins isolated from Mycoplasma Gallisepticum in different growth phases.

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.

Application of organic waste glycerol to produce crude extracts of bacterial cells and microbial hydrogenase-the anode enzymes of bio-electrochemical systems.

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.

Influence of the spacer region between the Shine-Dalgarno box and the start codon for fine-tuning of the translation efficiency in Escherichia coli.

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.

Proteomic dataset: Profiling of cultivated Echerichia coli isolates from Crohn's disease patients and healthy individuals.

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.

Direct detection of cysteine peptidases for MALDI-TOF MS analysis using fluorogenic substrates.

A method is described for the direct detection of unstable cysteine peptidase activity in polyacrylamide gels after native electrophoresis using new selective fluorogenic peptide substrates, pyroglutamyl-phenylalanyl-alanyl-4-amino-7-methylcoumaride (Glp-Phe-Ala-AMC) and pyroglutamyl-phenylalanyl-alanyl-4-amino-7-trifluoromethyl-coumaride (Glp-Phe-Ala-AFC). The detection limit of the model enzyme papain was 17 pmol (0.29 µg) for Glp-Phe-Ala-AMC and 43 pmol (0.74 µg) for Glp-Phe-Ala-AFC, with increased sensitivity and selectivity compared to the traditional method of protein determination with Coomassie G-250 staining or detection of activity using chromogenic substrates. Using this method, we easily identified the target digestive peptidases of Tenebrio molitor larvae by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. The method offers simplicity, high sensitivity, and selectivity compared to traditional methods for improved identification of unstable cysteine peptidases in multi-component biological samples.

The Cyanotoxin BMAA Induces Heterocyst Specific Gene Expression in Anabaena sp. PCC 7120 under Repressive Conditions.

Cyanobacteria synthesize neurotoxic ß-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.

Stress effects of cyanotoxin ß-methylamino-L-alanine (BMAA) on cyanobacterial heterocyst formation and functionality.

Various species of cyanobacteria, diatoms and dinoflagellates are capable of synthesizing the non-proteinogenic neurotoxic amino acid ß-N-methylamino-L-alanine (BMAA), which is known to be a causative agent of human neurodegeneration. Similar to most cyanotoxins, the biological and ecological functions of BMAA in cyanobacteria are unknown. In this study, we show for the first time that BMAA, in micromolar amounts, inhibits the formation of heterocysts (specialized nitrogen-fixing cells) in heterocystous, diazotrophic cyanobacteria [Anabaena sp. PCC 7120, Nostoc punctiforme PCC 73102 (ATCC 29133), Nostoc sp. strain 8963] under conditions of nitrogen starvation. The inhibitory effect of BMAA is abolished by the addition of glutamate. To understand the genetic reason for the observed phenomenon, we used qPCR to study the expression of key genes involved in cell differentiation and nitrogen metabolism in the model cyanobacterium Anabaena sp. PCC 7120. We observed that in the presence of BMAA, Anabaena sp. PCC 7120 does not express two essential genes associated with heterocyst differentiation, namely, hetR and hepA. We also found that addition of BMAA to cyanobacterial cultures with mature heterocysts inhibits nifH gene expression and nitrogenase activity.

Transformation of cellobiose during the interaction of cellobiose dehydrogenase and ß-glucosidase of Cerrena unicolor.

This work investigated the regulatory role of the interaction between cellobiose dehydrogenase (CDH) and ß-glucosidase (ß-GLU) in the conversion of cellobiose into cellobionolactone or glucose in vitro. To study the regulation, the two enzymes were isolated from the culture medium of the fungus Cerrena unicolor grown on a medium with microcrystalline cellulose. The enzymes were obtained in an electrophoretically homogeneous state. Their properties were studied. Both enzymes had acidic pH optima and were more stable in the acidic pH range. CDH was moderately thermostable, while ß-GLU had a low thermostability. Both enzymes efficiently catalyzed the transformation of cellobiose. A mixture of CDH and ß-GLU transformed cellobiose to glucose or cellobionolactone in the presence of various concentrations of laccase and hydroquinone. Formation of glucose and cellobionolactone in vitro during the competition between CDH and ß-GLU for cellobiose depended on the availability of quinones, formed as a result of the interaction of laccase and hydroquinone, for CDH. At low laccase and hydroquinone concentrations, the formation of glucose was found to predominate over that of cellobionolactone. The possible physiological role of the enzymes' interaction is discussed.

Genome analysis of E. coli isolated from Crohn's disease patients.

BACKGROUND: Escherichia coli (E. coli) has been increasingly implicated in the pathogenesis of Crohn's disease (CD). The phylogeny of E. coli isolated from Crohn's disease patients (CDEC) was controversial, and while genotyping results suggested heterogeneity, the sequenced strains of E. coli from CD patients were closely related. RESULTS: We performed the shotgun genome sequencing of 28 E. coli isolates from ten CD patients and compared genomes from these isolates with already published genomes of CD strains and other pathogenic and non-pathogenic strains. CDEC was shown to belong to A, B1, B2 and D phylogenetic groups. The plasmid and several operons from the reference CD-associated E. coli strain LF82 were demonstrated to be more often present in CDEC genomes belonging to different phylogenetic groups than in genomes of commensal strains. The operons include carbon-source induced invasion GimA island, prophage I, iron uptake operons I and II, capsular assembly pathogenetic island IV and propanediol and galactitol utilization operons. CONCLUSIONS: Our findings suggest that CDEC are phylogenetically diverse. However, some strains isolated from independent sources possess highly similar chromosome or plasmids. Though no CD-specific genes or functional domains were present in all CD-associated strains, some genes and operons are more often found in the genomes of CDEC than in commensal E. coli. They are principally linked to gut colonization and utilization of propanediol and other sugar alcohols.

Transcription profiling data set of different states of Mycoplasma gallisepticum.

Mycoplasma gallisepticum belongs to class Mollicutes and causes chronic respiratory disease in birds. It has a reduced genome, lack of cell wall and many metabolic pathways, and also easy to culture and non-pathogenic to humans. Aforementioned made it is a convenient model for studying of systems biology of minimal cell. Studying the transcriptomic level of M. gallisepticum is interesting for both understanding of common principles of transcription regulation of minimal cell and response to definite influence for pathogen bacteria. For rapid investigation of gene expression we developed microarray design including 3366 probes for 678 genes. They included 665 protein coding sequences and 13 antisense RNAs from 816 genes and 17 ncRNAs present in Mycoplasma gallisepticum. The study was performed on Agilent one-color microarray with custom design and random-T7 polymerase primer for cDNA synthesis. Here we present the data for transcription profiling of M. gallisepticum under different types of exposures: genetic knock-out mutants, cell culture exposed to sublethal concentrations of antibiotics and well-characterized heat stress effect. Mutants have transposon insertion to hypothetical membrane protein, lactate dehydrogenase, helicase with unknown function, 1-deoxy-d-xylulose 5-phosphate reductoisomerase or potential sigma factor. For inhibition of important cell systems, treatment with carbonyl cyanide m-chlorophenylhydrazone (CCCP), novobiocin or tetracycline were chosen. Data are available via NCBI Gene Expression Omnibus (GEO) with the accession number GSE85777 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE85777).

Application of sorting and next generation sequencing to study 5΄-UTR influence on translation efficiency in Escherichia coli.

Yield of protein per translated mRNA may vary by four orders of magnitude. Many studies analyzed the influence of mRNA features on the translation yield. However, a detailed understanding of how mRNA sequence determines its propensity to be translated is still missing. Here, we constructed a set of reporter plasmid libraries encoding CER fluorescent protein preceded by randomized 5΄ untranslated regions (5΄-UTR) and Red fluorescent protein (RFP) used as an internal control. Each library was transformed into Escherchia coli cells, separated by efficiency of CER mRNA translation by a cell sorter and subjected to next generation sequencing. We tested efficiency of translation of the CER gene preceded by each of 48 natural 5΄-UTR sequences and introduced random and designed mutations into natural and artificially selected 5΄-UTRs. Several distinct properties could be ascribed to a group of 5΄-UTRs most efficient in translation. In addition to known ones, several previously unrecognized features that contribute to the translation enhancement were found, such as low proportion of cytidine residues, multiple SD sequences and AG repeats. The latter could be identified as translation enhancer, albeit less efficient than SD sequence in several natural 5΄-UTRs.

Reconstruction of Transcription Control Networks in Mollicutes by High-Throughput Identification of Promoters.

Bacteria of the class Mollicutes have significantly reduced genomes and gene expression control systems. They are also efficient pathogens that can colonize a broad range of hosts including plants and animals. Despite their simplicity, Mollicutes demonstrate complex transcriptional responses to various conditions, which contradicts their reduction in gene expression regulation mechanisms. We analyzed the conservation and distribution of transcription regulators across the 50 Mollicutes species. The majority of the transcription factors regulate transport and metabolism, and there are four transcription factors that demonstrate significant conservation across the analyzed bacteria. These factors include repressors of chaperone HrcA, cell cycle regulator MraZ and two regulators with unclear function from the WhiA and YebC/PmpR families. We then used three representative species of the major clades of Mollicutes (Acholeplasma laidlawii, Spiroplasma melliferum, and Mycoplasma gallisepticum) to perform promoter mapping and activity quantitation. We revealed that Mollicutes evolved towards a promoter architecture simplification that correlates with a diminishing role of transcription regulation and an increase in transcriptional noise. Using the identified operons structure and a comparative genomics approach, we reconstructed the transcription control networks for these three species. The organization of the networks reflects the adaptation of bacteria to specific conditions and hosts.

Phase Transition of the Bacterium upon Invasion of a Host Cell as a Mechanism of Adaptation: a Mycoplasma gallisepticum Model.

What strategies do bacteria employ for adaptation to their hosts and are these strategies different for varied hosts? To date, many studies on the interaction of the bacterium and its host have been published. However, global changes in the bacterial cell in the process of invasion and persistence, remain poorly understood. In this study, we demonstrated phase transition of the avian pathogen Mycoplasma gallisepticum upon invasion of the various types of eukaryotic cells (human, chicken, and mouse) which was stable during several passages after isolation of intracellular clones and recultivation in a culture medium. It was shown that this phase transition is manifested in changes at the proteomic, genomic and metabolomic levels. Eukaryotic cells induced similar proteome reorganization of M. gallisepticum during infection, despite different origins of the host cell lines. Proteomic changes affected a broad range of processes including metabolism, translation and oxidative stress response. We determined that the activation of glycerol utilization, overproduction of hydrogen peroxide and the upregulation of the SpxA regulatory protein occurred during intracellular infection. We propose SpxA as an important regulator for the adaptation of M. gallisepticum to an intracellular environment.