An Investigation of the Genetic Control of Biofilm Formation in Bacteria (E. coli K-12 MG1655)

Abstract

The ability to adapt to changing environments is essential for survival. Bacteria have developed sophisticated means by which they sense and respond to stresses imposed by changes in the environment. Escherichia coli (E. coli) have served as a model organism for studies in molecular genetics and physiology since the 1960s. I have undertaken this study and address three outstanding questions. Firstly, the involvement of morphogene bolA and RNA polymerase sigma factor (rpoS) in biofilm formation. Secondly, the effect on respiratory activity of E. coli in presence and absence of these two genes and thirdly, the adherence pattern and formation of biofilm by E. coli on stainless steel, polypropylene and silicon surfaces under various stress-induced conditions.
Bacterial biofilms are structural assemblages of microbial cells that encase themselves in a protective self produced matrix and irreversibly attach to surface. Their intense resistance to antibiotic and various environmental stresses has implicated them as playing a possible role in the pathogenesis of many chronic diseases. Although, the role of rpoS and bolA genes in long term stationary phase growth conditions and their response to it is now well-known, their objective presence and importance in short term response to different environmental cues which may lead to biofilm formation remains unknown.
The rpoS gene encodes a stationary phase specific sigma factor of RNA polymerase and is a key regulator of E. coli stationary phase responses. It has been experiential under laboratory conditions that gene expression is induced by stressful environmental conditions and certain metabolic intermediates. Various stress environments were employed both in planktonic and biofilm phases to examine the sudden response of rpoS against different environmental conditions. However, it was observed that sudden rpoS response varies from stress to stress conditions. The gene bolA has been shown to trigger the formation of round cells when over expressed in stationary phase. From this research, it is concluded that bolA is not only confined to stationary phase, it also involves in biofilm formation under stress environments and essential for normal cell morphology. It also plays a major role in respiration and attachment of E. coli under diverse environmental stress surroundings.
The main objective of this study was to understand the impact of heat, cold, acid and hydrogen peroxide on E. coli K-12 MG1655 and its stress response in presence and absence of rpoS and bolA genes. E. coli cells were exposed to sub-lethal levels of each stress for 15 minutes in both planktonic and biofilm phases and post-stress response i.e. gene expression level was evaluated. A real-time reverse transcription polymerase chain reaction (RT-PCR) assay, using the Applied Biosystems 7500™ real-time cycler, was developed for the purpose of this investigation of rpoS and bolA genes transcription. The assay was used specifically to quantify rpoS and bolA mRNA levels; however the method can readily be applied to the study of other E. coli genes. The method was uniquely applied to the investigation of these two genes throughout the growth cycle of E. coli in planktonic and biofilm phase in LB broth, in order to ascertain the patterns of expression for these genes. Scanning electron microscopy (SEM) was used for direct examining the cell attachment and biofilm formation on various surfaces under different stress conditions.
In summary, this thesis embodies research investigating the role of rpoS and bolA genes in E. coli K-12 MG1655 biofilm formation and provides further evidence, that bacterial biofilms play a major role in resistance to various environmental cues.