Project description:Measureable rates of genome evolution are well documented in human pathogens but are less well understood in bacterial pathogens in the wild, particularly during and after host switches. Mycoplasma gallisepticum (MG) is a pathogenic bacterium that has evolved predominantly in poultry and recently jumped to wild house finches (Carpodacus mexicanus), a common North American songbird. For the first time we characterize the genome and measure rates of genome evolution in House Finch isolates of MG, as well as in poultry outgroups. Using whole-genome sequences of 12 House Finch isolates across a 13-year serial sample and an additional four newly sequenced poultry strains, we estimate a nucleotide diversity in House Finch isolates of only ?2% of ancestral poultry strains and a nucleotide substitution rate of 0.8-1.2×10(-5) per site per year both in poultry and in House Finches, an exceptionally fast rate rivaling some of the highest estimates reported thus far for bacteria. We also found high diversity and complete turnover of CRISPR arrays in poultry MG strains prior to the switch to the House Finch host, but after the invasion of House Finches there is progressive loss of CRISPR repeat diversity, and recruitment of novel CRISPR repeats ceases. Recent (2007) House Finch MG strains retain only ?50% of the CRISPR repertoire founding (1994-95) strains and have lost the CRISPR-associated genes required for CRISPR function. Our results suggest that genome evolution in bacterial pathogens of wild birds can be extremely rapid and in this case is accompanied by apparent functional loss of CRISPRs.
Project description:Mycoplasma gallisepticum transcriptome comparison between in vitro grown cultures of strains Rlow and F utilizing oligo DNA microarrays.
Project description:BACKGROUND: DNA repair is essential for the maintenance of genome stability in all living beings. Genome size as well as the repertoire and abundance of DNA repair components may vary among prokaryotic species. The bacteria of the Mollicutes class feature a small genome size, absence of a cell wall, and a parasitic lifestyle. A small number of genes make Mollicutes a good model for a "minimal cell" concept. RESULTS: In this work we studied the DNA repair system of Mycoplasma gallisepticum on genomic, transcriptional, and proteomic levels. We detected 18 out of 22 members of the DNA repair system on a protein level. We found that abundance of the respective mRNAs is less than one per cell. We studied transcriptional response of DNA repair genes of M. gallisepticum at stress conditions including heat, osmotic, peroxide stresses, tetracycline and ciprofloxacin treatment, stationary phase and heat stress in stationary phase. CONCLUSIONS: Based on comparative genomic study, we determined that the DNA repair system M. gallisepticum includes a sufficient set of proteins to provide a cell with functional nucleotide and base excision repair and mismatch repair. We identified SOS-response in M. gallisepticum on ciprofloxacin, which is a known SOS-inducer, tetracycline and heat stress in the absence of established regulators. Heat stress was found to be the strongest SOS-inducer. We found that upon transition to stationary phase of culture growth transcription of DNA repair genes decreases dramatically. Heat stress does not induce SOS-response in a stationary phase.
Project description:Mycoplasma gallisepticum transcriptome comparison between in vitro grown cultures of strains Rlow and F utilizing oligo DNA microarrays. Two-condition experiment, Rlow vs. F strain cells. Biological replicates: 3. 1 technical replicate per biological replicate which includes a dye swap.
Project description:Mycoplasma gallisepticum is an intracellular parasite affecting respiratory tract of poultry that belongs to class Mollicutes. M. gallisepticum features numerous variable lipoprotein hemagglutinin genes (vlhA) that play a role in immune escape. The vlhA promoters have a set of distinct properties in comparison to promoters of the other genes. The vlhA promoters carry a variable GAA repeats region at approximately 40 nts upstream of transcription start site. The promoters have been considered active only in the presence of exactly 12 GAA repeats. The mechanisms of vlhA expression regulation and GAA number variation are not described. Here we tried to understand these mechanisms using different computational methods. We conducted a comparative analysis among several M. gallisepticum strains. Nucleotide sequences analysis showed the presence of highly conserved regions flanking repeated trinucleotides that are not linked to GAA number variation. VlhA genes with 12 GAA repeats and their orthologs in 12 M. gallisepticum strains are more conserved than other vlhA genes and have narrower GAA number distribution. We conducted comparative analysis of physicochemical profiles of M. gallisepticum vlhA and sigma-70 promoters. Stress-induced duplex destabilization (SIDD) profiles showed that sigma-70 group is characterized by the common to prokaryotic promoters sharp maxima while vlhA promoters are hardly destabilized with the region between GAA repeats and transcription start site having zero opening probability. Electrostatic potential profiles of vlhA promoters indicate the presence of the distinct patterns that appear to govern initial stages of specific DNA-protein recognition. Open state dynamics profiles of vlhA demonstrate the pattern that might facilitate transcription bubble formation. Obtained data could be the basis for experimental identification of mechanisms of phase variation in M. gallisepticum.
Project description: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.
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.
Project description:The aim of this study was to explore the prudent use of tylosin for the treatment of chronic respiratory infectious diseases in chickens caused by Mycoplasma gallisepticum (MG) based on its clinical breakpoint (CBP) and its effect on lung microbiota. The CBP was established based on the wild-type/epidemiological cutoff value (COWT/ECV), pharmacokinetics-pharmacodynamics (PK-PD) cutoff value (COPD), and clinical cutoff value (COCL) of tylosin against MG. The minimum inhibitory concentration (MIC) of tylosin against 111 MG isolates was analyzed and the COWT was 2 μg/ml. M17 with MIC of 2 μg/ml was selected as a representative strain for the PK-PD study. The COPD of tylosin against MG was 1 μg/ml. The dosage regimen formulated by the PK-PD study was 3 days administration of tylosin at a dose of 45.88 mg/kg b.w. with a 24-h interval. Five different MIC MGs were selected for clinical trial, and the COCL of tylosin against MG was 0.5 μg/ml. According to the CLSI decision tree, the CBP of tylosin against MG was set up as 2 μg/ml. The effect of tylosin on lung microbiota of MG-infected chickens was analyzed by 16S rRNA gene sequencing. Significant change of the lung microbiota was observed in the infection group and treatment group based on the principal coordinate analysis and the Venn diagrams of the core and unique OTU. The phyla Firmicutes and Proteobacteria showed difference after MG infection and treatment. This study established the CBP of tylosin against MG. It also provided scientific data for the prudent use of tylosin based on the evaluation of MG infection and tylosin treatment on the lung microbiota.
Project description:Respiratory pathogens are a health threat for poultry. Co-infections lead to the exacerbation of clinical symptoms and lesions. Mycoplasma gallisepticum (M. gallispeticum) and Avian Metapneumovirus (AMPV) are two avian respiratory pathogens that co-circulate worldwide. The knowledge about the host-pathogen interaction of M. gallispeticum and AMPV in the chicken respiratory tract is limited. We aimed to investigate how co-infections affect the pathogenesis of the respiratory disease and whether the order of invading pathogens leads to changes in host-pathogen interaction. We used chicken tracheal organ cultures (TOC) to investigate pathogen invasion and replication, lesion development, and selected innate immune responses, such as interferon (IFN) α, inducible nitric oxide synthase (iNOS) and IFNλ mRNA expression levels. We performed mono-inoculations (AMPV or M. gallispeticum) or dual-inoculations in two orders with a 24-h interval between the first and second pathogen. Dual-inoculations compared to mono-inoculations resulted in more severe host reactions. Pre-infection with AMPV followed by M. gallispeticum resulted in prolonged viral replication, more significant innate immune responses, and lesions (p < 0.05). AMPV as the secondary pathogen impaired the bacterial attachment process. Consequently, the M. gallispeticum replication was delayed, the innate immune response was less pronounced, and lesions appeared later. Our results suggest a competing process in co-infections and offer new insights in disease processes.
Project description:The goal of this study was to identify genes important to the interaction of Mycoplasma gallisepticum with host cells in an in vitro system. An interaction time of one hour was chosen since it is less than a generation time and thus all changes can be attributed to transcriptional changes. A total of 60 transcripts were determined to be significantly up- or down-regulated under these conditions, including several hypothetical genes. Also several metabolism-related genes and ATP synthase genes were significantly down-regulated. Keywords: transcriptional response to host cells