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.

BAC-BROWSER: The Tool for Visualization and Analysis of Prokaryotic Genomes.

Prokaryotes are actively studied objects in the scope of genomic regulation. Microbiologists need special tools for complex analysis of data to study and identification of regulatory mechanism in bacteria and archaea. We developed a tool BAC-BROWSER, specifically for visualization and analysis of small prokaryotic genomes. BAC-BROWSER provides tools for different types of analysis to study a wide set of regulatory mechanisms of prokaryotes: -transcriptional regulation by transcription factors (TFs), analysis of TFs, their targets, and binding sites.-other regulatory motifs, promoters, terminators and ribosome binding sites-transcriptional regulation by variation of operon structure, alternative starts or ends of transcription.-non-coding RNAs, antisense RNAs-RNA secondary structure, riboswitches-GC content, GC skew, codon usage BAC-browser incorporated free programs accelerating the verification of obtained results: primer design and oligocalculator, vector visualization, the tool for synthetic gene construction. The program is designed for Windows operating system and freely available for download in http://smdb.rcpcm.org/tools/index.html.

Ribosome profiling reveals an adaptation strategy of reduced bacterium to acute stress.

Bacteria of class Mollicutes (mycoplasmas) feature significant genome reduction which makes them good model organisms for systems biology studies. Previously we demonstrated, that drastic transcriptional response of mycoplasmas to stress results in a very limited response on the level of protein. In this study we used heat stress model of M. gallisepticum and ribosome profiling to elucidate the process of genetic information transfer under stress. We found that under heat stress ribosomes demonstrate selectivity towards mRNA binding. We identified that heat stress response may be divided into two groups on the basis of absolute transcript abundance and fold-change in the translatome. One represents a noise-like response and another is likely an adaptive one. The latter include ClpB chaperone, cell division cluster, homologs of immunoblocking proteins and short ORFs with unknown function. We found that previously identified read-through of terminators contributes to the upregulation of transcripts in the translatome as well. In addition we identified that ribosomes of M. gallisepticum undergo reorganization under the heat stress. The most notable event is decrease of the amount of associated HU protein. In conclusion, only changes of few adaptive transcripts significantly impact translatome, while widespread noise-like transcription plays insignificant role in translation during stress.

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.