Data-independent proteome profile of Mycoplasma gallisepticum under normal conditions and heat stress.
ABSTRACT: The data reported is a large-scale untargeted proteome profile for Mycoplasma gallisepticum - a model organism for studying both regulation in genome-reduced bacteria and intracellular infection (Mazin et al., 2014) [1,2]. While seminal whole-proteome studies were performed on Mycoplasma genitalium  and a few proteome datasets are available for Mycoplasma pneumoniae, no data-independent (DIA) proteome profiling has been published for bacteria of Mycoplasma genus. Since DIA-based proteome profiling allows to extract evidence on presence and quantity of any protein of interest in a post-acquisition manner and the data presented is describing a model which is suitable to study both proteome regulation in general and details of mycoplasma infection process , the proteome profiling data presented here is of value for deep annotation. The data was deposited to the PRIDE repository (PXD008198).
Project description:Mycoplasma gallisepticum is a bacterium of class Mollicutes which encompasses wall-less bacteria with significantly reduced genomes. Due to their overall reduction and simplicity mycoplasmas serve as a model of minimal cell and are used for systems biology studies. Here we present raw data on translatome (ribosome-bound mRNA) analysis of Mycoplasma gallisepticum under logarithm growth and heat stress. The data supports the publication of "Ribosomal profiling of Mycoplasma gallisepticum" (G. Y. Fisunov, D. V Evsyutina, A. A. Arzamasov, I. O. Butenko, V. M. Govorun, 2015) .
Project description:Mycoplasma gallisepticum, an avian-pathogenic bacterium, glides on host tissue surfaces by using a common motility system with Mycoplasma pneumoniae In the present study, we observed and analyzed the gliding behaviors of M. gallisepticum in detail by using optical microscopes. M. gallisepticum glided at a speed of 0.27?±?0.09??m/s with directional changes relative to the cell axis of 0.6 degree?±?44.6 degrees/5 s without the rolling of the cell body. To examine the effects of viscosity on gliding, we analyzed the gliding behaviors under viscous environments. The gliding speed was constant in various concentrations of methylcellulose but was affected by Ficoll. To investigate the relationship between binding and gliding, we analyzed the inhibitory effects of sialyllactose on binding and gliding. The binding and gliding speed sigmoidally decreased with sialyllactose concentration, indicating the cooperative binding of the cell. To determine the direct energy source of gliding, we used a membrane-permeabilized ghost model. We permeabilized M. gallisepticum cells with Triton X-100 or Triton X-100 containing ATP and analyzed the gliding of permeabilized cells. The cells permeabilized with Triton X-100 did not show gliding; in contrast, the cells permeabilized with Triton X-100 containing ATP showed gliding at a speed of 0.014?±?0.007??m/s. These results indicate that the direct energy source for the gliding motility of M. gallisepticum is ATP.IMPORTANCE Mycoplasmas, the smallest bacteria, are parasitic and occasionally commensal. Mycoplasma gallisepticum is related to human-pathogenic mycoplasmas-Mycoplasma pneumoniae and Mycoplasma genitalium-which cause so-called "walking pneumonia" and nongonococcal urethritis, respectively. These mycoplasmas trap sialylated oligosaccharides, which are common targets among in?uenza viruses, on host trachea or urinary tract surfaces and glide to enlarge the infected areas. Interestingly, this gliding motility is not related to other bacterial motilities or eukaryotic motilities. Here, we quantitatively analyze cell behaviors in gliding and clarify the direct energy source. The results provide clues for elucidating this unique motility mechanism.
Project description: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.
Project description:A 150-kDa cytadhesin-like protein from Mycoplasma gallisepticum has been identified. A previously described 583-bp fragment (J.E. Dohms, L.L. Hnatow, P. Whetzel, R. Morgan and C.L. Keeler, Jr., Avian Dis. 37:380-388, 1993) was used to probe a genomic library of M. gallisepticum DNA. An 8.0-kb SacI fragment was identified, cloned, and partially sequenced. Analysis of the resulting 3,750-bp sequence revealed the presence of a 3,366-nucleotide open reading frame, mgc1. The 1,122-amino-acid protein encoded by this open reading frame, MGC1, has characteristics of a class I membrane protein and has homology with the MgPa cytadhesin of Mycoplasma genitalium (26.3%) and the P1 cytadhesin of Mycoplasma pneumoniae (28.7%). A portion of MGC1 was expressed as a glutathione S-transferase fusion protein and used to produce antiserum in rabbits. The antiserum recognizes a 150-kDa protein from M. gallisepticum. The protein is sensitive to trypsin, confirming that it is surface exposed. Primer extension analysis indicates that the mgc1 RNA starts within an upstream open reading frame, suggesting complex control of its expression. This is the first description of a functional gene from M. gallisepticum showing homology to cytadhesin genes from human mycoplasmas.
Project description:Despite the fact the term "proteome" was proposed to characterize a set of proteins in one of mycoplasma species, proteome response to various exposures in this bacteria are still obscure. Commonly, authors studying proteomic response on perturbation models in mycoplasmas use single approach and do not confirm their findings by alternative methods. Consequently, the results of proteomic analysis should be validated by complementary techniques. In this study we utilized three complementary approaches (SWATH, MRM, 2D-DIGE) to assess response of Mycoplasma gallisepticum under heat stress on proteomic level and combined these findings with metabolic response and the results of transcriptional profiling. We divide response into two modes - one is directly related to heat stress and other is triggered during heat stress, but not directly relevant to it. The latter includes accumulation of ATP and shedding of antigens. Both of these phenomena may be relevant to evasion of host's immune system and dissemination during mycoplasmosis in vivo.
Project description: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).
Project description:A putative cytadhesin-related protein (PvpA) undergoing variation in its expression was identified in the avian pathogen Mycoplasma gallisepticum. The pvpA gene was cloned, expressed in Escherichia coli, and sequenced. It exhibits 54 and 52% homology with the P30 and P32 cytadhesin proteins of the human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium, respectively. In addition, 50% homology was found with the MGC2 cytadhesin of M. gallisepticum and 49% homology was found with a stretch of 205 amino acids of the cytadherence accessory protein HMW3 of M. pneumoniae. The PvpA molecule possesses a proline-rich carboxy-terminal region (28%) containing two identical directly repeated sequences of 52 amino acids and a tetrapeptide motif (Pro-Arg-Pro-X) which is repeated 14 times. Genetic analysis of several clonal isolates representing different expression states of the PvpA product ruled out chromosomal rearrangement as the mechanism for PvpA phase variation. The molecular basis of PvpA variation was revealed in a short tract of repeated GAA codons, encoding five successive glutamate resides, located in the N-terminal region and subject to frequent mutation generating an in-frame UAA stop codon. Size variation of the PvpA protein was observed among M. gallisepticum strains, ranging from 48 to 55 kDa and caused by several types of deletions occurring at the PvpA C-terminal end and within the two directly repeated sequences. By immunoelectron microscopy, the PvpA protein was localized on the mycoplasma cell surface, in particular on the terminal tip structure. Collectively, these findings suggest that PvpA is a newly identified variable surface cytadhesin protein of M. gallisepticum.
Project description:Mycoplasma gallisepticum is a convenient model object for studying the regulation of transcription because it has a reduced genome, lack of cell wall and many metabolic pathways, and also easy to culture and non-pathogenic to humans. For rapid investigation of gene expression we developed microarray design including 3 366 probes for 678 genes. They included 665 protein coding sequences and 13 antisense RNAs from 816 genes and 17 ncRNAs present in Mycoplasma gallisepticum. This work was carried out transcriptomic profiling for different types of effects on the expression of genes of Mycoplasma gallisepticum: 1) genetic knock-out mutants; 2) cell culture exposed to sublethal concentrations of antibiotics; and 3) well-characterized heat stress effect. The study was performed on Agilent one-color microarray with custom design and random-T7 polymerase primer for cDNA synthesis. Using set of different probes for each gene or ncRNA allows to increase accuracy of gene expression quality. Overall design: Transcriptome profiling of 1) wild type Mycoplasma gallisepticum cell culture and mutants with transposone insertion to 5’ UTR of hypothetical protein GCW_03380, RBS of lactate dehydrogenase GCW_00390, helicase SNF2 GCW_03935, 1-deoxy-D-xylulose 5-phosphate reductoisomerase GCW_00495 or potential sigma factor GCW_00440 in exponential growth phase; 2) Mycoplasma gallisepticum cell culture under treatment with sublethal concentrations of carbonyl cyanide m-chlorophenylhydrazone (CCCP), novobiocin or tetracycline; and 3) Mycoplasma gallisepticum cell culture under heat stress at 46C during 15 min. All experiments were carried out in 2 biological replicates.
Project description:A second cytadhesin-like protein, MGC2, was identified in the avian respiratory pathogen Mycoplasma gallisepticum. The 912-nucleotide mgc2 gene encodes a 32.6-kDa protein with 40.9 and 31.4% identity with the M. pneumoniae P30 and M. genitalium P32 cytadhesins, respectively. Functional studies with reverse transcription-PCR, immunoblotting, double-sided immunogold labeling, and attachment inhibition assays demonstrated homology to the human mycoplasmal P30 and P32 cytadhesins. These findings suggest that there is a family of cytadhesin genes conserved among pathogenic mycoplasmas infecting widely divergent hosts.
Project description:While the genomes of a number of Mycoplasma species have been fully determined, there has been limited characterization of which genes are essential. The surface protein (p47) identified by monoclonal antibody B3 is the basis for an enzyme-linked immunosorbent assay for serological detection of Mycoplasma gallisepticum infection and appears to be constitutively expressed. Its gene was cloned, and the DNA sequence was determined. Subsequent analysis of the p47 amino acid sequence and searches of DNA databases found homologous gene sequences in the genomes of M. pneumoniae and M. genitalium and identity with a gene family in Ureaplasma urealyticum and genes in M. agalactiae and M. fermentans. The proteins encoded by these genes were found to belong to a family of basic membrane proteins (BMP) that are found in a wide range of bacteria, including a number of pathogens. Several of the BMP family members, including p47, contain selective lipoprotein-associated motifs that are found in macrophage-activating lipoprotein 404 of M. fermentans and lipoprotein P48 of M. agalactiae. The p47 gene was predicted to encode a 59-kDa peptide, but affinity-purified p47 had a molecular mass of approximately 47 kDa, as determined by polyacrylamide gel analysis. Analysis of native and recombinant p47 by mass peptide fingerprinting revealed the absence of the carboxyl end of the protein encoded by the p47 gene in native p47, which would account for the difference seen in the predicted and measured molecular weights and indicated posttranslational cleavage of the lipoprotein at its carboxyl end. A DNA construct containing the p47 gene interrupted by the gene encoding tetracycline resistance was used to transform M. gallisepticum cells. A tetracycline-resistant mycoplasma clone, P2, contained the construct inserted within the genomic p47 gene, with crossovers occurring between 73 bp upstream and 304 bp downstream of the inserted tetracycline resistance gene. The absence of p47 protein in clone P2 was determined by the lack of reactivity with rabbit anti-p47 sera or monoclonal antibody B3 in Western blots of whole-cell proteins. There was no difference between the p47(-) mutant and wild-type M. gallisepticum in pathogenicity in chicken tracheal organ cultures. Thus, p47, although homologous to genes that occur in many prokaryotes, is not essential for growth in vitro or for attachment and the initial stages of pathogenesis in chickens.