Mapping Stress-Induced Changes in Autoinducer AI-2 Production in Chemostat-Cultivated Escherichia coli K-12

Abstract

Numerous gram-negative bacteria employ a cell-to-cell signaling mechanism, termed quorum sensing, for controlling gene expression in response to population density. Recently, this phenomenon has been discovered in Escherichia coli, and while pathogenic E. coli utilize quorum sensing to regulate pathogenesis (i.e., expression of virulence genes), the role of quorum sensing in nonpathogenic E. coli is less clear, and in particular, there is no information regarding the role of quorum sensing during the overexpression of recombinant proteins. The production of autoinducer AI-2, a signaling molecule employed by E. coli for intercellular communication, was studied in E. coli W3110 chemostat cultures using a Vibrio harveyi AI-2 reporter assay (M. G. Surrette and B. L. Bassler, Proc. Natl. Acad. Sci. USA 95:7046-7050, 1998). Chemostat cultures enabled a study of AI-2 regulation through steady-state and transient responses to a variety of environmental stimuli. Results demonstrated that AI-2 levels increased with the steady-state culture growth rate. In addition, AI-2 increased following pulsed addition of glucose, Fe(III), NaCl, and dithiothreitol and decreased following aerobiosis, amino acid starvation, and isopropyl-␤-D-thiogalactopyranoside-induced expression of human interleukin-2 (hIL-2). In general, the AI-2 responses to several perturbations were indicative of a shift in metabolic activity or state of the cells induced by the individual stress. Because of our interest in the expression of heterologous proteins in E. coli, the transcription of four quorum-regulated genes and 20 stress genes was mapped during the transient response to induced expression of hIL-2. Significant regulatory overlap was revealed among several stress and starvation genes and known quorum-sensing genes.Synthesis and perception of a self-produced, freely diffusible signal molecule, termed autoinducer AI-2, by the gram-negative bacterium Escherichia coli is thought to regulate the expression of a variety of genes in response to population density. This process, termed autoinduction or quorum sensing, was first described in Vibrio fischeri (39), and similar autoregulatory mechanisms have since been reported in a wide range of bacteria, including Pseudomonas aeruginosa (34), Erwinia caratovora (5), and Agrobacterium tumefaciens (40), as well as E. coli (18,44,57). Although the existence of such a mechanism in E. coli has been uncovered, the genetic, physiological, and environmental factors that contribute to and regulate the quorum circuitry remain poorly defined.Evidence of intercellular communication in E. coli came with the discovery of a quorum-regulated transcriptional transactivator (SdiA) of the cell division genes in the ftsQAZ locus homologous to LuxR of V. fischeri (18, 44, 57) and a synthase protein (LuxS E.c. ) responsible for AI-2 signal molecule production (49). Further elucidation of native E. coli quorum circuit architecture resulted from the Vibrio harveyi cross-species activity assay of Surette and Bassler (47), which has g...

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