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 promoters 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

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

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

Cyanobacteria synthesize neurotoxic &#946;-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&#8315;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

Draft genome of the nitrogen-fixing bacterium Pseudomonas stutzeri strain KOS6 isolated from industrial hydrocarbon sludge

© 2013 Grigoryeva et al. Here we present a draft genome of Pseudomonas stutzeri strain KOS6. This strain was isolated from industrial hydrocarbon sludge as a diazotrophic microorganism. It represents one of the major parts of the culturable community of the waste and has potential importance for phytoremediation technology.Ministry of Science and Education of the Russian Federatio

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

Summary 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

Characteristics of the modification motif ANCNNNNCCT.

Mean Score–mean Modification QV of instances of this motif that were detected as modified; Mean IPD Ratio–mean interpulse duration (IPD) ratio of instances of this motif that were detected as modified; Mean Coverage–mean coverage of instances of this motif that were detected as modified.</p

Fig 8 -

A–Difference between the expression levels of paired genes for mutants with EGFP genes under synthetic promoters, with or without methylation sites. For each pair, the difference for EGFP gene in comparison with control genes eno, gadp, tuf, ligA, and gyrB is shown. Genes with significant difference between promoters with or without the methylation site (Student’s t-test, Benjamini–Hochberg correction, PEGFP promoters with or without methylated sites in -10 box, -35 box, between -35 and -10 boxes, and in the TSS. Methylation sites are underlined; methylated adenines in forward and reverse strands are in bold; -10 boxes are in blue and highlighted with blue boxes; -35 boxes are highlighted with green boxes and the sequence with strong consensus to the -35 box is in green; TSSs are in red and marked with red arrows; sequence differences between paired methylated or non-methylated promoters are in pink.</p

Methylated fraction of each methylation site.

A–Number of sites with different fractions of methylation. Colors represent sites located in one of the four parts of the genome: between the origin and midpoint of the chromosome, on the plus or minus strand. The panel on the right schematically shows the methylation sites that were counted in each group. B–Number of sites with different fractions of methylation. In this figure, the location of sites in the genome has not been considered. Colors represent the sites on the ANCNNNNCCT or AGGNNNNGNT strands in the double-stranded methylation motif. The panel on the right schematically shows the methylation sites that were counted in each group.</p

Genome alignment of Mycoplasma gallisepticum strains obtained using the program Mauve.

RM system MgaS6I is marked in orange, variable lipoproteins vlhA clusters in green, the CRISPR system in blue. M. gallisepticum S6 genome is on the top, below the genomes of the other strains. A–whole genome representation, B–fragment of genomes near MgaS6I genomic context. (PDF)</p