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An RNA-based mechanism for the nutritional control of bacterial DNA replication initiation

Periodic Reporting for period 1 - NutriCoRe (An RNA-based mechanism for the nutritional control of bacterial DNA replication initiation)

Reporting period: 2018-06-01 to 2020-05-31

Bacteria form the largest part of the Earth’s biomass, playing essential roles in natural environments and performing important functions in industrial processes. Nevertheless, uncontrolled growth of bacteria remains a major threat to humankind, posing various problems in industry and public health, especially due to the constant rise of antibiotic resistance. Under optimal conditions, bacteria thrive and proliferate with an astonishing rate. On the other hand, in response to adverse conditions, including nutrient depletion or environmental stress, bacteria can reduce or completely arrest their growth and proliferation to ensure their survival. Indeed, entering a non-proliferating state has been demonstrated to enhance bacterial drug tolerance and intracellular persistence of pathogenic bacteria. Knowledge about the mechanisms that allow bacteria to adjust their own growth and proliferation in response to changing conditions is thus critically important for a better understanding of infectious diseases and antibiotic resistance. Growth arrest requires a decline in the production of cellular mass and the concomitant cessation of cell cycle processes, including DNA replication. DNA replication is the process enabling the transmission of genetic information to the progeny and it is followed by chromosome segregation and cell division. It has been observed that under nutrient-starvation conditions different bacteria arrest the cell cycle with a reduced number of fully replicated chromosomes indicating that DNA replication is blocked at the replication initiation step. However, a molecular mechanism by which DNA replication initiation is nutritionally controlled has never been reported. The initiation of DNA replication in nearly all bacteria requires the product of the gene dnaA, an enzyme that recognises and binds specific sequences present on the bacterial chromosome at the origin of replication. DnaA forms an oligomeric structure that unwinds DNA and recruits the DNA replication apparatus. Given its central role in timing the initiation of DNA replication, DnaA represents a strong candidate for transducing environmental information into the cell cycle. The NutriCoRe project aimed to elucidate the molecular mechanisms involved in the DNA replication arrest in response to nutrient-starvation in bacteria. To address this fundamental question, I used the model bacterium Caulobacter crescentus. Under carbon depletion conditions and during the transition to the stationary phase, DnaA levels rapidly drop in C. crescentus and DNA replication is arrested. By combining bioinformatics, classical biochemistry, genetics, and cutting-edge molecular biology techniques, this project led to an improved understanding of the mechanism of nutritional control of DNA replication. In particular, I discovered that under carbon-limiting conditions, DnaA translation ceases, likely through the nutrient-dependent pausing of the protein synthesis machinery (i.e. the ribosome) during the synthesis DnaA’s nascent chain (Nterm). I hypothesise that the complex formed by the ribosome and DnaA Nterm is able to sense the nutritional state of the cell, thus arresting the synthesis of new DnaA. Overall, this project sheds new light onto the mechanisms transducing nutritional information into bacterial cell cycle.
In order to understand the precise molecular mechanism underlying the post-transcriptional regulation of DnaA in response to nutrient starvation, the NutriCoRe project focused on three main areas.
First, I studied and compared Caulobacter’s cellular responses to the depletion of four different macronutrients (i.e. carbon, nitrogen, phosphate and sulfur sources). I discovered that, although in all the investigated stress conditions I observed a pronounced DNA replication arrest and a drop in DnaA levels, the pathways leading dnaA downregulation depend on the sort of nutrient starvation. Second, focusing on Caulobacter’s response to carbon-limiting conditions, I investigated the role of the coding and non-coding regions of dnaA mRNA in the regulation of DnaA synthesis. Here, I discovered that the drop of DnaA levels is due to an arrest of protein synthesis during the first stages of DnaA nascent chain elongation (Nterm). Under these conditions, the concomitant degradation by Lon, leads to the complete clearance of DnaA from the cell. Last, I generated a series of Nterm mutations to shed new light onto the mechanism of DnaA translation arrest. In the final model, I suggest that DnaA translation is downregulated due to a starvation-induced ribosome pausing event that requires the interaction between the ribosome itself and specific amino acid residues in DnaA Nterm.
The main results of the NutriCoRe project have been presented in two international conferences and will be published soon in important open access peer-reviewed journals. The improved understanding about the mechanisms of dnaA regulation in response to nutrient limitations and the newly established investigation tools will be the base for a number of follow-up research projects. In the future, we aim to elucidate the structural details of the starvation-induced elongation pausing, to explore the different regulation pathways associated with nutritional stress (e.g. nitrogen starvation) and the possible involvement of small non-coding RNAs in dnaA regulation.
Understanding the molecular mechanisms that regulate bacterial proliferation remains a fundamental challenge in biology. The NutriCoRe project provides important insights into the regulation of DnaA and DNA replication. The results of this study, interpreted in a comparative framework together with existing data from distantly related bacteria, will provide in the future key insights into the evolution and the differentiation of one of the most important biological processes: the regulation of DNA replication.
Besides a better understanding of the very fundamental mechanisms of life, the outcome of the present action, potentially will contribute to the advance of new strategies to treat bacterial infections, through the development of antibiotic drugs that directly target components of the cell cycle apparatus. Furthermore, my work could contribute to developing new strategies for bacterial growth control in industry and biotechnology, for instance by engineering bacteria with custom functions. In fact, the discovery of cis- and trans-acting elements responsible for dnaA posttranscriptional regulation could provide the synthetic biology community with new tools for the regulation of the genome copy-number in artificial cells. Importantly, as a member of the Alphaproteobacteria, Caulobacter is closely related to organisms relevant for human health, ecology and biotechnology.
Effects of carbon starvation on Caulobacter cell morphology, chromosome content and DnaA levels