Developing a cell-based sensor for the detection of Autoinducer-2

Abstract

Abstract Bacteria use an intricate set of communi cation systems for sensing and interpreting environmental cues that coordinate population-based behavior. Quorum sensing is one of thes e systems, and it involves the production, release, and detection of small chemical signaling molecules. Recent resear ch has revealed the role of quorum sensing molecules in the control of microbial activities such as biofilm formation. In this presentation we outline the development of a recombinant E. coli cell-based sensor for detection of the quorum sensing molecule Autoinducer-2 (AI-2), as well as engineering strategies to remove sugar and anoxic inhibition of the strain. Keywords: quorum sensing, Autoinducer-2 (AI-2), sensor cell, biofilm 1. Introduction The study of bacteria cooperating to organize various cellular behaviors is a rapidly expanding area of research, impacting both the environment and industry. Bacterial colonization of medical devices and food processing equipment has long been known to be a problem; however biofilms can also exist in petroleum transport, storage, and delivery systems. The presence of these organism s causes fouling of the fuel in the system, as well as corrosion and other mechanical failures resulting in increased maintenance and ope rating costs. Microbially Induced Corrosion (MIC) is an electrochemical process in which the metabolic processes of a consortium of microorganisms initiate or enhance oxidation chemistry at a metal surface. MIC is problematic for vi rtually all industries, especia lly paper, sugar, dentistry, and the airline industry which is the largest fuel consuming industry [1]. It is estimated approximately 40% of internal pipeline degradation in the petroleum industry is the result of colonization by microorganisms [2]. Water and other contaminants collect at the bottom of fuel storage containers and pipelines, providing an environment for microbes to colonize and catabolize the hydrocarbo ns at the interface between the aqueous and organic phases. Detection of bacteria prior to the onset of MIC is problematic for a number of reasons. Many organisms found forming biofilms in fuel lines and storage containers are obligate anaerobes, making culture based detection costly, time consuming, and prone to false negatives. Additionally, biofilms tend to be cohesive structures that are resistant to sloughing, so detection strategies relying on culturing the organism, or PCR based methods may produce false negatives. A reliable method for identifying bacterial contamination prior to biofilm formation in systems not amenable to traditional culturing techniques would have significant industrial usefulness. In this paper we describe strategies for overcoming the limitations of a strain of bacteria that could be utilized to detect bacterial contamination before the onset of MIC through the sensing of small prokaryotic signaling molecules. Bacteria, once thought to be independently operating units, are now known to employ a complex set of communication systems for sensing and interpreting environmental stimuli that coordinate population-based behaviors generally referred to as Quorum Sensing (QS). QS has be en found to coordinate diverse cellular processes including bioluminescence [5], virulence [6], biofilm formation [7], cell division, motility, and metabolism. It has also been engineered to regulate recombinant protein production [8-10]. QS is conducted via the production, release, and response to small chemical signaling molecules termed autoinducers. As extracellular concentration of autoinducer increases, a threshold is reached which stimulates a signal transduction mechanism causing a coordinated change in the cellular processes of the population of bacteria [3, 4]. One signaling molecule, au toinducer-2 (AI-2), has recently been implicated as an interspecies communicati on molecule suggesting a role as a ‘unive rsal’ signal, for a variety of bacterial behaviors including the formation of biofilms [11-13]. In a recent study identifyi ng bacterial consortia isolated from pe troleum-product-transporting pipelines, the following bacteria species were identified: B. cereus ACE4, S. marcescens ACE2, B. subtilis AR12, P. aeruginosa AI1, K. oxytoca ACP, P. stutzeri AP2, B. litoralis AN1, and Bacillus sp. AN5 [1]. Of these species it has was determined that B. cereus , S. marcescens , and B. subtilis produce AI-2 [13-15]. The presence of AI-2 has historically been detected through a qualitative Vibrio harveyi based bioassay, which has several limitations. The assay utilizes the mutant strain