Escherichia coli has evolved in environments which may commonly be acidic and thus developed adaptive mechanisms to minimise acid-induced damage. It has previously been observed that adapted bacteria to moderately acidic conditions can grow in media considerably below their optimum growth pH. To explain this phenomenon, a hypothesis which suggested that diffusible molecules (alarmones) may serve as early warning systems of acidic conditions was proposed. Alarmones are thought to be produced upon exposure to mildly-acidic conditions. They then diffuse in the environment and elicit a protective response against acid in recipient cells. The protective activity of those putative alarmones against lethal acid was investigated. The main aim of this project is to determine the mode of action of those alarmones at the molecular level. Preliminary experiments confirmed acid resistance conferred by alarmones to populations of E. coli C600. The stability of those alarmones at different temperatures and following proteinase K treatment was investigated. Moreover, investigations into whether alarmones are autoinducer-2 (AI-2) molecules and whether alarmones increase the percentage of persisters in an E. coli population were undertaken. Subsequently, microarray analyses of both alarmone-induced and non-induced cultures were performed to reveal E. coli genes induced by alarmones. Moreover, proteomic studies using two-dimensional gel electrophoresis were conducted to reveal proteins induced by alarmones. Supernatants from alarmone-induced cultures conferred statistically significant protection (p<0.01) on recipient cultures against lethal acid (pH3). Alarmones were inactivated by heat (60oC) and by proteinase K. The autoinducer-2 (AI-2) assay revealed that alarmones are not AI-2 molecules. In addition, alarmones did not increase the percentage of persisters. In order to elucidate potential mechanisms for alarmone-mediated protection, the genomic expression and protein induction of alarmone-induced cells using microarray analysis and two-dimensional gel electrophoresis, respectively, were performed. Two-dimensional gel electrophoresis of transduced cultures indicated that around 13 proteins were induced in the alarmone-protected populations of E. coli C600. Mass-spectrometric analysis revealed that these alarmone-inducible proteins include the acid stress chaperone HdeB and the DNA-binding transcriptional dual regulator, H-NS which plays an important role in stress adaptation. Microarray analyses of transduced cultures indicated that 671 open reading frames (ORFs) were significantly differentially expressed between alarmone-protected and control populations (p<0.05). 508 ORFs were upregulated in the induced cells including 10 genes related to acid-resistance and 36 different genes related to multidrug efflux system proteins whereas 163 ORFs including the autoinducer-2 system were downregulated. E. coli releases diffusible signalling compounds which mediate adaptation to acid stress of recipient cells. Microarray and proteomic data show that two acid fitness island genes (hdeB and hdeD) and three genes that encode the antiporters of the three amino-acid-dependent acid resistance mechanisms (gadC, adiC and cadB) were among the upregulated genes. This work confirms that there is communication between bacterial cells in general and warning messages amongst E. coli C600 cells in particular in the presence of stress.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:606885 |
| Date | January 2012 |
| Creators | Moumene, Souad |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://www.manchester.ac.uk/escholar/uk-ac-man-scw:162972 |
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