Summary 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.

Bacteria present a promising delivery system for treating human diseases. Here, we engineered the genome‐reduced human lung pathogen Mycoplasma pneumoniae as a live biotherapeutic to treat biofilm‐associated bacterial infections. This strain has a unique genetic code, which hinders gene transfer to most other bacterial genera, and it lacks a cell wall, which allows it to express proteins that target peptidoglycans of pathogenic bacteria. We first determined that removal of the pathogenic factors fully attenuated the chassis strain in vivo . We then designed synthetic promoters and identified an endogenous peptide signal sequence that, when fused to heterologous proteins, promotes efficient secretion. Based on this, we equipped the chassis strain with a genetic platform designed to secrete antibiofilm and bactericidal enzymes, resulting in a strain capable of dissolving Staphylococcus aureus biofilms preformed on catheters in vitro , ex vivo , and in vivo . To our knowledge, this is the first engineered genome‐reduced bacterium that can fight against clinically relevant biofilm‐associated bacterial infections.

Summary Trans-translation is a ubiquitous bacterial mechanism of ribosome rescue mediated by a transfer-messenger RNA (tmRNA) that adds a degradation tag to the truncated nascent polypeptide. Here, we characterize this quality control system in a genome-reduced bacterium, Mycoplasma pneumoniae (MPN), and perform a comparative analysis of protein quality control components in slow and fast-growing prokaryotes. We show in vivo that in MPN the sole quality control cytoplasmic protease (Lon) degrades efficiently tmRNA-tagged proteins. Analysis of tmRNA-mutants encoding a tag resistant to proteolysis reveals extensive tagging activity under normal growth. Unlike knockout strains, these mutants are viable demonstrating the requirement of tmRNA-mediated ribosome recycling. Chaperone and Lon steady-state levels maintain proteostasis in these mutants suggesting a model in which co-evolution of Lon and their substrates offer simple mechanisms of regulation without specialized degradation machineries. Finally, comparative analysis shows relative increase in Lon/Chaperone levels in slow-growing bacteria suggesting physiological adaptation to growth demand.

In this study we explored the adaptability and robustness of glycolysis and pyruvate metabolism of Mycoplasma pneumoniae (MPN). We used a dual approach, we analysed metabolomics data collected for a large number of OE and KO mutants and perturbation samples. Furthermore, we trained a dynamic model of central carbon metabolism and tested the model capacity to predict these mutants and perturbation samples as well as identify key controlling factors in central carbon metabolism. Our analysis of metabolite data as well as our model analysis indicate MPN metabolism is inherently robust against perturbations due to its network structure. Two key control hubs of central carbon metabolism were identified.