Generating a SlyA Mutation in E. Coli and Validating Its Effect on Microbial Response to Environmental Stress Factors

Abstract

Background: In the human host, environments encountered by bacteria can vary widely. Bacteria experience stress from their initial moment of contact with the host. Therefore, adaptation and survival of the bacteria hinge on their ability to probe the environment and respond appropriately. Capsule expression is one of the responses to stress. In E. coli K5 capsule expression is temperature regulated and SlyA was proven to be involved in E. coli capsule gene expression. Methodology: Using HA1 cells as a model, cells were grown under different environmental conditions using a set of chemical substances and the b-galactosidase activity was measured and used as an indicator of capsule expression. pGEM-T easy vector and a series of PCR were used in cloning and generating a tet-insertional mutation of the slyA gene. Results: Maximum activity of capsule expression was under anaerobic conditions, then microaerophilic with least activity been obtained aerobically. Maximum capsule expression was obtained in presence of 0.4M sucrose or NaCl and 2mM MgSO 4 at pH 7.5. In all conditions capsule expression was increased ~2.4 fold in the presence of multicopy slyA plasmid; an effect that disappeared with tet:: slyA. Conclusion: Intestinal environmental cues in terms of anaerobiosis, relatively high osmolarity in addition to the slightly alkaline pH, all can contribute to up-regulating capsule gene expression in E. coli intestinal flora. Many aspects of clinical host-pathogen interactions involve stress responses and adaptive responses allow different pathogenic bacteria to resist host defences. Capsule expression was significantly increased in the presence of a multicopy slyA