Bacterial autoinducer 2 (AI-2) is proposed to be an interspecies mediator of cell-cell communication that enables cells to operate at the multicellular level. Many environmental stimuli have been shown to affect the extracellular AI-2 levels, carbon sources being among the most important. In this report, we show that both AI-2 synthesis and uptake in Escherichia coli are subject to catabolite repression through the cyclic AMP (cAMP)-CRP complex, which directly stimulates transcription of the lsr (for "luxS regulated") operon and indirectly represses luxS expression. Specifically, cAMP-CRP is shown to bind to a CRP binding site located in the upstream region of the lsr promoter and works with the LsrR repressor to regulate AI-2 uptake. The functions of the lsr operon and its regulators, LsrR and LsrK, previously reported in Salmonella enterica serovar Typhimurium, are confirmed here for E. coli. The elucidation of cAMP-CRP involvement in E. coli autoinduction impacts many areas, including the growth of E. coli in fermentation processes.Bacteria have evolved complex genetic circuits to modulate their physiological states and behaviors in response to a variety of extracellular signals. In a process termed quorum sensing, or density-dependent gene regulation, bacteria produce, release, and respond to signaling molecules (autoinducers), which accumulate as a function of cell density. Quorum sensing allows bacteria to communicate with each other and coordinate their activities at a multicellular level. The autoinducers of many gram-positive bacteria are secreted peptides (30, 42), while gram-negative bacteria use small chemical molecules (60). Among gram-negative bacteria, the LuxI/LuxR signal synthase-signal receptor system is the most studied at the molecular level, with the signaling species being a family of N-acylhomoserine lactones. However, the cross-species autoinducer, autoinducer 2 (AI-2), has received intense interest recently because the gene for its terminal synthase, luxS, is present in over 55 bacteria and its activity can be readily assayed biologically (61). It is known that quorum sensing regulates diverse cellular processes, including bioluminescence (19, 34), spore formation (33), motility (18, 22), competence (35), conjugation (20), antibiotic synthesis (2, 17), virulence (38,44,50), and biofilm maturation (13, 45).Our laboratory is interested in understanding and controlling microbial behavior in bioreactors in order to enhance recombinant protein synthesis and yield. Since quorum sensing is emerging as a global regulator of many intracellular processes, including those that influence protein synthesis, efforts to understand this "tunable" controller are essential. In our previous work using chemostat cultures (14), many stimuli were found to affect the level of AI-2. Among these, the pulsed addition of glucose, a common carbon source for recombinant Escherichia coli fermentations, resulted in increased AI-2 levels, but with the dynamic response dependent on the steadystate growth rate (e.g., dil...

The regulatory network for the uptake of Escherichia coli autoinducer 2 (AI-2) is comprised of a transporter complex, LsrABCD; its repressor, LsrR; and a cognate signal kinase, LsrK. This network is an integral part of the AI-2 quorum-sensing (QS) system. Because LsrR and LsrK directly regulate AI-2 uptake, we hypothesized that they might play a wider role in regulating other QS-related cellular functions. In this study, we characterized physiological changes due to the genomic deletion of lsrR and lsrK. We discovered that many genes were coregulated by lsrK and lsrR but in a distinctly different manner than that for the lsr operon (where LsrR serves as a repressor that is derepressed by the binding of phospho-AI-2 to the LsrR protein). An extended model for AI-2 signaling that is consistent with all current data on AI-2, LuxS, and the LuxS regulon is proposed. Additionally, we found that both the quantity and architecture of biofilms were regulated by this distinct mechanism, as lsrK and lsrR knockouts behaved identically. Similar biofilm architectures probably resulted from the concerted response of a set of genes including flu and wza, the expression of which is influenced by lsrRK. We also found for the first time that the generation of several small RNAs (including DsrA, which was previously linked to QS systems in Vibrio harveyi) was affected by LsrR. Our results suggest that AI-2 is indeed a QS signal in E. coli, especially when it acts through the transcriptional regulator LsrR.Bacteria communicate with each other through small "hormone-like" organic molecules referred to as autoinducers. Autoinducer-based bacterial cell-to-cell communication, enabling population-based multicellularity, has been termed quorum sensing (QS) (27). Cellular functions controlled by QS are varied and reflect the needs of a particular bacterial species for inhabiting a given niche (10,38,65).QS among Escherichia coli and Salmonella strains has been a topic of great interest, and different intercellular signaling systems have been identified: that mediated by the LuxR homolog SdiA; the LuxS/autoinducer 2 (AI-2) system, an AI-3 system, and a signaling system mediated by indole (2,19,36,57,61,68). Among these systems, the LuxS/AI-2 system possesses the unique feature of endowing cell population-dependent behavior while interacting with central metabolism through the intracellular activated methyl cycle (20,21,45,65,73). Therefore, it has the potential to influence both gene regulation and bacterial fitness.AI-2's function has been studied using luxS mutants and by adding either conditioned medium or in vitro-synthesized AI-2 to bacterial cultures. It is noteworthy that the luxS transcription profile is not synchronous with the accumulation profile of extracellular AI-2 in bacterial supernatants (5,31,75). In E.coli, extracellular AI-2 activity peaks during the mid-to lateexponential phase and rapidly decreases during entry into the stationary phase. A corresponding decrease in LuxS protein levels is not observed (31,75). The disap...

Bacterial cell-to-cell communication facilitates coordinated expression of specific genes in a growth rate-II and cell density-dependent manner, a process known as quorum sensing. While the discovery of a diffusible Escherichia coli signaling pheromone, termed autoinducer 2 (AI-2), has been made along with several quorum sensing genes, the overall number and coordination of genes controlled by quorum sensing through the AI-2 signal has not been studied systematically. We investigated global changes in mRNA abundance elicited by the AI-2 signaling molecule through the use of a luxS mutant that was unable to synthesize AI-2. Remarkably, 242 genes, comprising ca. 5.6% of the E. coli genome, exhibited significant transcriptional changes (either induction or repression) in response to a 300-fold AI-2 signaling differential, with many of the identified genes displaying high induction levels (more than fivefold). Significant induction of ygeV, a putative 54 -dependent transcriptional activator, and yhbH, a 54 modulating protein, suggests 54 may be involved in E. coli quorum sensing.Many bacteria have evolved the ability to condition culture medium by secreting low-molecular-weight signaling pheromones in association with growth phase to control expression of specific genes, a process termed quorum sensing (19). Physiological processes controlled by quorum sensing occur in diverse species of bacteria and include bioluminescence (17), antibiotic biosynthesis (4), pathogenicity (34), and plasmid conjugal transfer (18). While acyl-homoserine lactones (HSL) appear to be the predominant quorum signal (or autoinducer [AI]) used by host-associated gram-negative bacteria, discovery of a second signaling pathway in the marine bacterium Vibrio harveyi (6,8,41) revealed an alternate AI, termed AI-2, which regulates bioluminescence in conjunction with AI-1 (N-(3-hydroxybutanoyl)-L-homoserine lactone) (7).Importantly, AI-2 (or AI-2-like) activity has been observed in virtually all strains of pathogenic and nonpathogenic Escherichia coli and Salmonella enterica serovar Typhimurium (16,(40)(41)(42), requiring the luxS gene for synthesis (43). The physiological role of AI-2 in E. coli has not been clearly elucidated, but initial findings indicate that inhibition of chromosomal replication was subject to a quorum sensing mechanism (52). More recently, quorum sensing in E. coli has been implicated in regulating the expression and activity of SdiA, a LuxR-type transcriptional activator of the cell division genes ftsQAZ, through AI-2 (15, 39). In addition, extracellular factors which accumulate in enterohemorrhagic E. coli O157:H7 culture supernatants bind to the N-terminal region of SdiA for controlling the expression of virulence factors in a quorum-dependent fashion (25). Besides possible roles in cell division and pathogenesis, quorum sensing in E. coli was postulated to play a role in stationary phase gene expression (23, 27, 39), perhaps in a bimodal fashion with the stationary phase sigma factor rpoS or with other yet-to-be-determin...

No abstract

The bacterial quorum-sensing autoinducer 2 (AI-2) has received intense interest because the gene for its synthase, luxS, is common among a large number of bacterial species. We have identified luxS-controlled genes in Escherichia coli under two different growth conditions using DNA microarrays. Twenty-three genes were affected by luxS deletion in the presence of glucose, and 63 genes were influenced by luxS deletion in the absence of glucose. Minimal overlap among these gene sets suggests the role of luxS is condition dependent. Under the latter condition, the metE gene, the lsrACDBFG operon, and the flanking genes of the lsr operon (lsrR, lsrK, tam, and yneE) were among the most significantly induced genes by luxS. The E. coli lsr operon includes an additional gene, tam, encoding an S-adenosyl-L-methionine-dependent methyltransferase. Also, lsrR and lsrK belong to the same operon, lsrRK, which is positively regulated by the cyclic AMP receptor protein and negatively regulated by LsrR. lsrK is additionally transcribed by a promoter between lsrR and lsrK. Deletion of luxS was also shown to affect genes involved in methionine biosynthesis, methyl transfer reactions, iron uptake, and utilization of carbon. It was surprising, however, that so few genes were affected by luxS deletion in this E. coli K-12 strain under these conditions. Most of the highly induced genes are related to AI-2 production and transport. These data are consistent with the function of LuxS as an important metabolic enzyme but appear not to support the role of AI-2 as a true signal molecule for E. coli W3110 under the investigated conditions.

No 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...

The obligate intracellular pathogen, Anaplasma phagocytophilum, is the causative agent of human, equine, and canine granulocytic anaplasmosis and tick-borne fever (TBF) in ruminants. A. phagocytophilum has become an emerging tick-borne pathogen in the United States, Europe, Africa, and Asia, with increasing numbers of infected people and animals every year. It has been recognized that intracellular pathogens manipulate host cell metabolic pathways to increase infection and transmission in both vertebrate and invertebrate hosts. However, our current knowledge on how A. phagocytophilum affect these processes in the tick vector, Ixodes scapularis is limited. In this study, a genome-wide search for components of major carbohydrate metabolic pathways was performed in I. scapularis ticks for which the genome was recently published. The enzymes involved in the seven major carbohydrate metabolic pathways glycolysis, gluconeogenesis, pentose phosphate, tricarboxylic acid cycle (TCA), glyceroneogenesis, and mitochondrial oxidative phosphorylation and β-oxidation were identified. Then, the available transcriptomics and proteomics data was used to characterize the mRNA and protein levels of I. scapularis major carbohydrate metabolic pathway components in response to A. phagocytophilum infection of tick tissues and cultured cells. The results showed that major carbohydrate metabolic pathways are conserved in ticks. A. phagocytophilum infection inhibits gluconeogenesis and mitochondrial metabolism, but increases the expression of glycolytic genes. A model was proposed to explain how A. phagocytophilum could simultaneously control tick cell glucose metabolism and cytoskeleton organization, which may be achieved in part by up-regulating and stabilizing hypoxia inducible factor 1 alpha in a hypoxia-independent manner. The present work provides a more comprehensive view of the major carbohydrate metabolic pathways involved in the response to A. phagocytophilum infection in ticks, and provides the basis for further studies to develop novel strategies for the control of granulocytic anaplasmosis.