Here, we determined the relative importance of different transcriptional mechanisms in the genome-reduced bacterium Mycoplasma pneumoniae, by employing an array of experimental techniques under multiple genetic and environmental perturbations. Of the 143 genes tested (21% of the bacterium's annotated proteins), only 55% showed an altered phenotype, highlighting the robustness of biological systems. We identified nine transcription factors (TFs) and their targets, representing 43% of the genome, and 16 regulators that indirectly affect transcription. Only 20% of transcriptional regulation is mediated by canonical TFs when responding to perturbations. Using a Random Forest, we quantified the non-redundant contribution of different mechanisms such as supercoiling, metabolic control, RNA degradation, and chromosome topology to transcriptional changes. Model-predicted gene changes correlate well with experimental data in 95% of the tested perturbations, explaining up to 70% of the total variance when also considering noise. This analysis highlights the importance of considering non-TF-mediated regulation when engineering bacteria.

Determining the gene regulatory network of an organism is fundamental for achieving a global understanding of cellular behaviour. Here, we determine the relative importance of different transcription regulators in the genome-reduced bacterium Mycoplasma pneumoniae, by integrating DNA affinity chromatography, chromatin isolation, ChIP-seq, transcriptomics, proteomics and growth curves experiments for 143 genes (20.8% of the bacteria´s NCBI annotated proteins). In 55% of the cases, gene overexpression or mutation does not result in transcriptional nor growth phenotypes, highlighting the robustness of even very simple cells. We identified nine TFs (besides the sigma factor) and their targets, representing 33% of the genome. Nevertheless, we find that the majority of transcription regulation in M. pneumoniae is not mediated by canonical TFs, but rather occurs at the level of supercoiling, metabolic control, RNA degradation, and chromosome topology. For the first time, we quantify the non-redundant contribution of each of these mechanisms in various environmental perturbations using a random forest model, showing that cells use different mechanisms to respond to distinct stresses. The gene changes predicted by our model correlate with the actual changes in 95% of the tested perturbations, and after accounting for experimental and gene expression noise, we can explain over 70% of the total variance. This comprehensive analysis highlights the importance of non-TF-mediated regulation in bacteria, an ancient type of regulation that allows adaptation to mild environmental changes.

The genome of the Gram-positive soil bacterium Bacillus subtilis encodes three potential diadenylate cyclases that may synthesize the signaling nucleotide cyclic di-AMP (c-di-AMP). These enzymes are expressed under different conditions in different cell compartments, and they localize to distinct positions in the cell. Here we demonstrate the diadenylate cyclase activity of the so far uncharacterized enzymes CdaA (previously known as YbbP) and CdaS (YojJ). Our work confirms that c-di-AMP is essential for the growth of B. subtilis and shows that an excess of the molecule is also harmful for the bacteria. Several lines of evidence suggest that the diadenylate cyclase CdaA is part of the conserved essential cda-glm module involved in cell wall metabolism. In contrast, the CdaS enzyme seems to provide c-di-AMP for spores. Accumulation of large amounts of c-di-AMP impairs the growth of B. subtilis and results in the formation of aberrant curly cells. This phenotype can be partially suppressed by elevated concentrations of magnesium. These observations suggest that c-di-AMP interferes with the peptidoglycan synthesis machinery. The activity of the diadenylate cyclases is controlled by distinct molecular mechanisms. CdaA is stimulated by a regulatory interaction with the CdaR (YbbR) protein. In contrast, the activity of CdaS seems to be intrinsically restricted, and a single amino acid substitution is sufficient to drastically increase the activity of the enzyme. Taken together, our results support the idea of an important role for c-di-AMP in B. subtilis and suggest that the levels of the nucleotide have to be tightly controlled.

The human pathogen Mycoplasma pneumoniae has maintained only a small repertoire of genes putatively involved in transcription regulation. This reflects its adaption to a nutrient rich and rather constant habitat, the human lung epithelium. To date, the heat shock response regulator HrcA is the only characterized protein involved in transcription regulation in this organism. However, several transcription regulation events have been described, leading to the question which mechanisms or which regulators are responsible for these events. This work aimed to study the implication of the putative, so far uncharacterized regulators in transcription in M. pneumoniae. Screening of a mutant library showed that the majority of the genes encoding these regulators are essential for viability. Only mutants affected in genes relA and whiA, both widely distributed and highly conserved in bacteria, could be isolated from the library. In addition, interaction of protein Mpn266 with RpoA could be shown, providing evidence that this protein is an orthologue of transcription regulator Spx in the Firmicutes. However, this regulator is essential, too. Several experiments have been performed to characterize the role of RelA and the alarmone (p)ppGpp in M. pneumoniae. Whereas it could be shown that mechanisms leading to (p)ppGpp formation in this organism are conserved, no additional phenotype could be identified in a relA mutant despite an inability to synthesize these alarmones. An effect on transcription regulation could not be shown by microarray analysis. However, evidence was provided that (p)ppGpp formation is not involved in short-term regulation of transcription in M. pneumoniae. Experimental data indicate that the phosphorylation state of HPr is involved in regulating the uptake of glucose and other PTS sugars in a pH dependent manner, rather then in transcription regulation. Microarray analysis of a whiA mutant showed that a large operon consisting mainly of gene s encoding ribosomal proteins is constitutively up-regulated in this strain. Both the regulator and the operon co-occur in Gram-positive bacteria, suggesting a similar function of this regulator in other bacteria. The finding of a conserved DNA sequence upstream of this operon supports this hypothesis. In summary, this work provides the first microarray analyses of M. pneumoniae mutants. Furthermore, the first M. pneumoniae mutant impaired in transcription regulation and a novel putative role of HPr phosphorylation were described. Thus, this work provides a basis for future experiments that could help to understand regulatory mechanisms in this minimal organism.

The genera Mycoplasma and Spiroplasma belong to the mollicutes, a group of Gram-positive firmicutes that lack a cell wall. These bacteria live in close association with their eukaryotic hosts and are characterized by an extreme reduction of the genome. While the members of the genus Mycoplasma infect vertebrates and humans, the Spiroplasma species infect insects and plants and cause severe plant diseases. In both genera, pathogenicity is intimately linked to carbon metabolism. Mycoplasma species affect their hosts by producing hydrogen peroxide, which is generated by two enzymes that use water instead of NAD+ as electron acceptor. Spiroplasma species degrade the sucrose that is present in the host tissues to fructose and glucose. Given that only the fructose is catabolized by the bacteria, the remaining glucose accumulates in the plant and causes damage. The cells of the mollicutes are usually pleomorphic, with a specialized terminal organelle structure. This structure is involved in cell division, movement, and attachment to the host cells. Because of their extreme genome reduction, the mollicutes are workhorses for the identification of the minimal gene set that is required for life. This is the basis for the new research field of synthetic biology.

The nucleotide sequences that control transcription initiation and regulation in Mycoplasma pneumoniae are poorly understood. Moreover, only few regulatory events have been reported for M. pneumoniae. We have studied changes in the global protein synthesis pattern in M. pneumoniae in response to the presence of glycerol. The ackA and ldh genes, encoding acetate kinase and lactate dehydrogenase, respectively, were controlled in a carbon source-dependent manner. While the ackA gene was strongly expressed in the presence of glucose, transcription of ldh was induced by glycerol. The promoters of both genes were mapped by primer extension analysis. Molecular analysis of transcription regulatory mechanisms in M. pneumoniae has so far not been possible due to the lack of appropriate reporter systems that can be used to study the activity of promoter fragments and their mutant derivatives in vivo. Recently, a reporter system has been developed which allows cloning of promoter fragments in front of a promoterless lacZ gene and inserting this construct into the genome of M. pneumoniae. To study the requirements of M. pneumoniae RNA polymerase for promoter recognition, a series of fusions of deletion and mutant variants of the ldh promoter was constructed and analyzed in vivo. While mutations affecting the -10 region strongly interfered with gene expression, the -35 region seems to be of minor importance in M. pneumoniae.