Understanding bacterial heterogeneity in gene expression under specific and global regulatory control /

Includes bibliographical references

PhD;2024;Molecular Biophysics Unit

Intrinsic heterogeneity in natural bacterial populations enable them to escape sudden changes in environment and evolve. Processes like cellular crosstalk, abiotic interactors, host interactions, intracellular signalling and gene expression stochasticity leads to emergence of phenotypically distinct subpopulations within identical genetic backgrounds. Such phenotypic heterogeneities are controlled by specific gene regulation mechanisms mediated by specific stimuli and dedicated operons e.g. sporulation under nutrient deprivation or global gene expression regulation affecting multiple cellular processes like emergence of metabolically dormant cells. In this talk, I will elucidate the mechanism for emergence of bacterial heterogeneity in two well studied systems. To understand phenotypic heterogeneity in stimuli specific gene regulation, we study the arabinose inducible Ara operon of planktonic E. coli cells. We demonstrate consistent inducer concentration dependent bimodality in expression. The uninduced fraction decreases with increase in inducer concentration and over time. In a small subset of inducer concentrations, however, the cells became non-responsive to the presence of the inducer. RNA quantification revealed that this desensitization occurs at the transcriptional level for all arabinose responsive promoters except the regulator AraC. The expression kinetics was also found to be dependent on the AraE and AraFGH transporter systems with each transporter contributing in their own way. To investigate heterogeneity in global gene expression of bacteria, we investigated emergence of global phenotypic heterogeneity in bacterial biofilms. We track the global regulation of the S13 ribosomal protein in E. coli biofilms using GFP reporter and show that the S13 promoter activity is highly heterogeneous and temporally variable. These stabilizes two dominant population of cells with only fraction maintaining active S13 promoter activity. The distribution of the promoter activity was also found to be independent of the inoculum density or growth phase of the starter cells suggesting a biofilm phenotype. We further evaluated susceptibility of these subpopulations towards antibiotics and found both to have similar levels of susceptibilities therefore the changes are largely metabolic. We also examined the effect of antibiotics on the whole biofilm and found that gentamicin and ofloxacin can prevent biofilm formation but can’t remove preformed biofilms. Colistin, however could neither prevent nor remove biofilms at the concentration range used; however, colistin exposure showed an increase in biofilm growth at very high concentrations, hinting at a rapid emergence of resistance by the cells. The findings of this research underscore the significance of understanding bacterial gene expression heterogeneity, both in response to specific stimuli and within biofilm communities. These insights into the regulatory mechanisms driving phenotypic diversity can inform the development of more effective antibacterial strategies and enhance our ability to predict bacterial behaviour in varying environmental conditions, ultimately contributing to improved management of bacterial infections and resistance.