To study basic principles of transcriptome organization in bacteria, we analyzed one of the smallest self-replicating organisms, Mycoplasma pneumoniae. We combined strand-specific tiling arrays, complemented by transcriptome sequencing, with more than 252 spotted arrays. We detected 117 previously undescribed, mostly noncoding transcripts, 89 of them in antisense configuration to known genes. We identified 341 operons, of which 139 are polycistronic; almost half of the latter show decaying expression in a staircase-like manner. Under various conditions, operons could be divided into 447 smaller transcriptional units, resulting in many alternative transcripts. Frequent antisense transcripts, alternative transcripts, and multiple regulators per gene imply a highly dynamic transcriptome, more similar to that of eukaryotes than previously thought.

To understand basic principles of bacterial metabolism organization and regulation, but also the impact of genome size, we systematically studied one of the smallest bacteria, Mycoplasma pneumoniae. A manually curated metabolic network of 189 reactions catalyzed by 129 enzymes allowed the design of a defined, minimal medium with 19 essential nutrients. More than 1300 growth curves were recorded in the presence of various nutrient concentrations. Measurements of biomass indicators, metabolites, and 13C-glucose experiments provided information on directionality, fluxes, and energetics; integration with transcription profiling enabled the global analysis of metabolic regulation. Compared with more complex bacteria, the M. pneumoniae metabolic network has a more linear topology and contains a higher fraction of multifunctional enzymes; general features such as metabolite concentrations, cellular energetics, adaptability, and global gene expression responses are similar, however.

Negative feedback loops have been invoked as a way to control and decrease transcriptional noise. Here, we have built three circuits to test the effect of negative feedback loops on transcriptional noise of an autoregulated gene encoding a transcription factor (TF) and a downstream gene (DG), regulated by this TF. Experimental analysis shows that self-repression decreases noise compared to expression from a non-regulated promoter. Interestingly enough, we find that noise minimization by negative feedback loop is optimal within a range of repression strength. Repression values outside this range result in noise increase producing a U-shaped behaviour. This behaviour is the result of external noise probably arising from plasmid fluctuations as shown by simulation of the network. Regarding the target gene of a self-repressed TF (sTF), we find a strong decrease of noise when repression by the sTF is strong and a higher degree of noise anti-correlation between sTF and its target. Simulations of the circuits indicate that the main source of noise in these circuits could come from plasmid variation and therefore that negative feedback loops play an important role in suppressing both external and internal noise. An important observation is that DG expression without negative feedback exhibits bimodality at intermediate TF repression values. This bimodal behaviour seems to be the result of external noise as it can only be found in those simulations that include plasmid variation.