Natural variation in bacterial gene regulation : a thesis presented in partial fulfilment of the requirements for the degree of Ph.D in Microbiology & Genetics, Massey University College of Science, School of Natural and Computational Sciences

Abstract

It has been over 160 years since Charles Darwin set out the theory of evolution by natural selection. This theory is broadly accepted these days. However, it is still not completely understood how natural selection shapes particular cell mechanisms and behaviours. There is a limited research about selection acting on gene regulation.

To address the questions about how selection shapes gene regulation we used a collection of environmental E. coli isolates. We quantified the genetic variability of 605 promoters within this collection of highly diverged strains. We then selected ten promoters (𝘢𝘭𝘥𝘈, 𝘺𝘩𝘫𝘟, 𝘭𝘢𝘤𝘡, 𝘢𝘤𝘦𝘉, 𝘮𝘵𝘳, 𝘤𝘥𝘥, 𝘥𝘤𝘵𝘈, 𝘱𝘵𝘴𝘎, 𝘱𝘶𝘳𝘈, and 𝘵𝘱𝘪𝘈) which highly differ in their genetic variability to analyse their phenotypic variability. We used fluorescent reporter assays with flow cytometry to measure changes in gene expression with high-throughput and at single cell resolution. In order to discern natural selection acting on gene regulation we compared phenotypes from segregating promoter variants, which have been subject to natural selection and random promoter variants that have never been subject to natural selection. We generated the random variants using PCR random mutagenesis. Beside focusing on the changes in the overall expression (i.e., transcription and translation), we examine selection acting on transcription only. This we achieved be implementing self-cleaving ribozyme insulation.

In this thesis we showed that natural selection towards high plasticity and low noise is common among regulated 𝘌. 𝘤𝘰𝘭𝘪 promoters. We also verify that the self-cleaving ribozyme RiboJ activity is highly effective and that this genetic tool can be used to detect changes in transcription alone. Utilizing the RiboJ we were then able to detect both directional and diversifying selection acting on 𝙡𝙖𝙘𝙕 promoter.

This thesis thus broadens the knowledge about natural selection acting on gene regulation and provides a new insights into how promoters are shaped in nature by selection, including some most well-characterized bacterial promoters. This work also demonstrates a new application of RiboJ ribozyme that has not been published before.