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.

Biogenic Ferrihydrite Nanoparticles Produced by Klebsiella oxytoca: Characterization, Physicochemical Properties and Bovine Serum Albumin Interactions.

The synthesis of nanoparticles inside microorganisms is an economical alternative to chemical and physical methods of nanoparticle synthesis. In this study, ferrihydrite nanoparticles synthesized by Klebsiella oxytoca bacterium in special conditions were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDS), small-angle X-ray (SAXS), UV-Vis spectroscopy, fluorescence, fluorescence resonance energy transfer (FRET), and molecular docking. The morphology and the structure of the particles were characterized by means of SEM and SAXS. The elemental content was determined by means of the EDS method. The absorption properties of the ferrihydrite nanoparticles were investigated by UV-Vis spectroscopy. The binding mechanism of the biogenic ferrihydrite nanoparticles to Bovine Serum Albumin (BSA) protein, studied by fluorescence, showed a static and weak process, combined with FRET. Protein denaturation by temperature and urea in the presence of the ferrihydrite nanoparticles demonstrated their influence on the unfolding process. The AutoDock Vina and UCSF Chimera programs were used to predict the optimal binding site of the ferrihydrite to BSA and to find the location of the hydrophobic cavities in the sub-domain IIA of the BSA structure.

Biogenic Ferrihydrite Nanoparticles: Synthesis, Properties In Vitro and In Vivo Testing and the Concentration Effect.

Biogenic ferrihydrite nanoparticles were synthesized as a result of the cultivation of Klebsiella oxytoca microorganisms. The distribution of nanoparticles in the body of laboratory animals and the physical properties of the nanoparticles were studied. The synthesized ferrihydrite nanoparticles are superparamagnetic at room temperature, and the characteristic blocking temperature is 23-25 K. The uncompensated moment of ferrihydrite particles was determined to be approximately 200 Bohr magnetons. In vitro testing of different concentrations of ferrihydrite nanoparticles for the functional activity of neutrophilic granulocytes by the chemiluminescence method showed an increase in the release of primary oxygen radicals by blood phagocytes when exposed to a minimum concentration and a decrease in secondary radicals when exposed to a maximum concentration. In vivo testing of ferrihydrite nanoparticles on Wister rats showed that a suspension of ferrihydrite nanoparticles has chronic toxicity, since it causes morphological changes in organs, mainly in the spleen, which are characterized by the accumulation of hemosiderin nanoparticles (stained blue according to Perls). Ferrihydrite can also directly or indirectly stimulate the proliferation and intracellular regeneration of hepatocytes. The partial detection of Perls-positive cells in the liver and kidneys can be explained by the rapid elimination from organs and the high dispersion of the nanomaterial. Thus, it is necessary to carry out studies of these processes at the systemic level, since the introduction of nanoparticles into the body is characterized by adaptive-proliferative processes, accompanied by the development of cell dystrophy and tension of the phagocytic system.

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).

Propionate Induces Virulent Properties of Crohn's Disease-Associated Escherichia coli.

Crohn's disease (CD) is a severe chronic immune-mediated granulomatous inflammatory disease of the gastrointestinal tract. The mechanisms of CD pathogenesis remain obscure. Metagenomic analysis of samples from CD patients revealed that several of them have the elevated level of Escherichia coli with adhesive-invasive phenotype (AIEC). Previously, we isolated an E. coli strain CD isolate ZvL2 from a patient with CD, which features AIEC phenotype. Here, we demonstrate that prolonged growth on propionate containing medium stimulates virulent properties of CD isolate ZvL2, while prolonged growth on glucose reduces these properties to levels indistinguishable from laboratory strain K-12 MG1655. Propionate presence also boosts the ability of CD isolate ZvL2 to penetrate and colonize macrophages. The effect of propionate is reversible, re-passaging of CD isolate on M9 medium supplemented with glucose leads to the loss of its virulent properties. Proteome analysis of CD isolate ZvL2 growth in medium supplemented with propionate or glucose revealed that propionate induces expression porins OmpA and OmpW, transcription factors PhoP and OmpR, and universal stress protein UspE, which were previously found to be important for macrophage colonization by enteropathogenic bacteria.

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.

Genetic diversity of Escherichia coli in gut microbiota of patients with Crohn's disease discovered using metagenomic and genomic analyses.

BACKGROUND: Crohn's disease is associated with gut dysbiosis. Independent studies have shown an increase in the abundance of certain bacterial species, particularly Escherichia coli with the adherent-invasive pathotype, in the gut. The role of these species in this disease needs to be elucidated. METHODS: We performed a metagenomic study investigating the gut microbiota of patients with Crohn's disease. A metagenomic reconstruction of the consensus genome content of the species was used to assess the genetic variability. RESULTS: The abnormal shifts in the microbial community structures in Crohn's disease were heterogeneous among the patients. The metagenomic data suggested the existence of multiple E. coli strains within individual patients. We discovered that the genetic diversity of the species was high and that only a few samples manifested similarity to the adherent-invasive varieties. The other species demonstrated genetic diversity comparable to that observed in the healthy subjects. Our results were supported by a comparison of the sequenced genomes of isolates from the same microbiota samples and a meta-analysis of published gut metagenomes. CONCLUSIONS: The genomic diversity of Crohn's disease-associated E. coli within and among the patients paves the way towards an understanding of the microbial mechanisms underlying the onset and progression of the Crohn's disease and the development of new strategies for the prevention and treatment of this disease.

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.

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.

Application of Spiroplasma melliferum proteogenomic profiling for the discovery of virulence factors and pathogenicity mechanisms in host-associated spiroplasmas.

To date, no genome of any of the species from the genus Spiroplasma has been completely sequenced. Long repetitive sequences similar to mobile units present a major obstacle for current genome sequencing technologies. Here, we report the assembly of the Spiroplasma melliferum KC3 genome into 4 contigs, followed by proteogenomic annotation and metabolic reconstruction based on the discovery of 521 expressed proteins and comprehensive metabolomic profiling. A systems approach allowed us to elucidate putative pathogenicity mechanisms and to discover major virulence factors, such as Chitinase utilization enzymes and toxins never before reported for insect pathogenic spiroplasmas.

Core proteome of the minimal cell: comparative proteomics of three mollicute species.

Mollicutes (mycoplasmas) have been recognized as highly evolved prokaryotes with an extremely small genome size and very limited coding capacity. Thus, they may serve as a model of a 'minimal cell': a cell with the lowest possible number of genes yet capable of autonomous self-replication. We present the results of a comparative analysis of proteomes of three mycoplasma species: A. laidlawii, M. gallisepticum, and M. mobile. The core proteome components found in the three mycoplasma species are involved in fundamental cellular processes which are necessary for the free living of cells. They include replication, transcription, translation, and minimal metabolism. The members of the proteome core seem to be tightly interconnected with a number of interactions forming core interactome whether or not additional species-specific proteins are located on the periphery. We also obtained a genome core of the respective organisms and compared it with the proteome core. It was found that the genome core encodes 73 more proteins than the proteome core. Apart of proteins which may not be identified due to technical limitations, there are 24 proteins that seem to not be expressed under the optimal conditions.

The acylation state of surface lipoproteins of mollicute Acholeplasma laidlawii.

Acylation of the N-terminal Cys residue is an essential, ubiquitous, and uniquely bacterial posttranslational modification that allows anchoring of proteins to the lipid membrane. In gram-negative bacteria, acylation proceeds through three sequential steps requiring lipoprotein diacylglyceryltransferase, lipoprotein signal peptidase, and finally lipoprotein N-acyltransferase. The apparent lack of genes coding for recognizable homologs of lipoprotein N-acyltransferase in gram-positive bacteria and Mollicutes suggests that the final step of the protein acylation process may be absent in these organisms. In this work, we monitored the acylation state of eight major lipoproteins of the mollicute Acholeplasma laidlawii using a combination of standard two-dimensional gel electrophoresis protein separation, blotting to nitrocellulose membranes, and MALDI-MS identification of modified N-terminal tryptic peptides. We show that for each A. laidlawii lipoprotein studied a third fatty acid in an amide linkage on the N-terminal Cys residue is present, whereas diacylated species were not detected. The result thus proves that A. laidlawii encodes a lipoprotein N-acyltransferase activity. We hypothesize that N-acyltransferases encoded by genes non-homologous to N-acyltransferases of gram-negative bacteria are also present in other mollicutes and gram-positive bacteria.