Chemical Communication among Bacteria

This chapter talks about the two cells that comprise the sporangium during spore formation in Bacillus subtilis. Spore formation in B. subtilis has served as an important model for cell-cell signaling in bacteria. The conversation between the forespore and mother cell provides insight into how and why cells communicate and highlights the diversity of ways in which organisms transduce information across their membranes. It is clear that there are three signal transduction pathways between the mother cell and forespore that ensure that gene expression in one compartment is linked to gene expression in the other throughout the sporulation process. The first signal transduction pathway between the forespore and mother cell has as input σF activity in the forespore and as output the activation of σE in the mother cell. Dissection of the molecular mechanisms of R-mediated GA activation and GA-mediated pro-σE processing promises to reveal general principles of how information can be transduced across a lipid bilayer. The second signal transduction pathway, the activation of σG in the forespore under the control of σE in the mother cell, has been the most refractory to genetic and molecular dissection. The third and final signal transduction pathway, the activation of σK in the mother cell under the control of σG in the forespore, is the most well understood of the three. All three of these signaling pathways have served as powerful models for studying cell-cell signaling in bacteria.

This chapter focuses on the mechanisms of extracellular peptide signaling that Bacillus subtilis utilizes to control gene expression involved in sporulation, the ComA-mediated general quorum response, and horizontal gene transfer. This signaling provides mechanisms for the cell to monitor population density and to distinguish whether neighboring cells are similar or different. All the responses described are stimulated by high population densities. The focus of the chapter is cell-cell signaling mediated by peptides. Horizontal gene transfer plays an important role in bacterial evolution. It is often regulated by cell-cell signaling, including the pheromone-responsive conjugal plasmids of Entercoccus faecalis, the autoinducer-sensing conjugal plasmids of Agrobacterium tumefaciens, and competence development in Streptococcus and Bacillus species. Self-recognition during B. subtilis competence development likely serves to limit competence development to conditions when DNA from closely related bacteria will be present. Cell-cell signaling influences the activity of integrative and conjugative element (ICEBs1) in two ways. First, signaling via host-encoded regulators indicates a high population density and the proximity of potential mating partners. Second, the signaling pentapeptide PhrI is produced by ICEBs1-containing cells and is used to inhibit transfer to potential partners that already contain the element. B. subtilis competence and sporulation are also regulated by two regulators, ComK and Spo0A, that are parts of multiple autoregulatory loops. They involve both positive and negative feedback regulation and help establish and maintain stable subpopulations of cells that exhibit specific patterns of gene expression and follow specific developmental fates.

One of the best-studied forms of microbial behavior controlled by intercellular signaling is pheromone-inducible conjugation in Enterococcus faecalis. This chapter emphasizes significant results of research on the pheromone systems during the past several years, particularly the pCF10 system. The chapter focuses on new insights into the control of pheromone activity in donor and recipient cells, on the molecular mechanism of the pheromone response, and on the evolution of pCF10 plasmids. The majority of research on pCF10 plasmids has focused on pAD1 and pCF10. The genes encoded on the approximately 70-kb pCF10 plasmid are involved in pheromone-inducible conjugation. Specific outcomes of pheromone induction are discussed in the chapter. The authors describe the synthesis of cCF10 to exemplify a process that occurs in a similar fashion for several pheromones. The genetic screens and biochemical evidence demonstrating two functions for PrgX resulted in the development of a model of how PrgX negatively regulates from the prgQ promoter as well as positively regulates its own expression by a single mechanism. The X-ray crystal structure of PrgX is mainly helical with 17 alpha-helices. The authors propose that the pCF10 system described in the chapter has been appropriated by the plasmid for the purpose of increasing the sensitivity and adaptability to multiple situations.

Since 1970, the author's research group has been trying to decipher the instructions used by Myxococcus xanthus to build its fruiting body. Fruiting body development requires a solid surface because the structure is built by cell movement. Two molecular motors, retractile type IV pili at their leading end (S motility), and nozzles for secreting a slime gel at the trailing end (A motility) provide the adhesion and thrust necessary for moving on surfaces. Outward spreading stops when M. xanthus senses that it has begun to starve; instead it moves inward to the swarm center to establish centers for fruiting body aggregation. By tradition, the aggregation of cells has been considered to arise from chemotaxis, and this view was encouraged by the discovery of many "chemotaxis genes" in M. xanthus. Aggregation by local cell contact signaling has been tested by mathematical simulation. At the beginning of sporulation the cell density in the center is about one-third the density in the outer region. Temporal changes in gene expression are necessary to adjust to starvation, to aggregate, and finally to sporulate. All the defects in aggregation and sporulation could be traced to a particular segment of the C-signal transduction pathway namely the branch from the C-signal receptor to FruA~P. All aspects of the phenotype of either deletion mutant were accounted for by the hypothesis that mutant MXAN4899 severely restricts the rise in the level of phosphorylated FruA.

This chapter focuses on the Dif chemosensory pathway, which regulates movement and development in different ways. The dif genes encode components of a chemosensory pathway known to be essential for S motility, biofilm formation, lipid chemotaxis, and fruiting body development. Myxococcus xanthus does not respond chemotactically to a variety of molecules that are chemoeffectors in other organisms. Several lines of evidence suggest that the M. xanthus phosphatidylethanolamine (PE) response is analogous to chemotaxis of flagellar bacteria. The Dif chemosensory pathway plays at least three roles in motility. The short diffusion range of the attractant suggests that Myxococcus chemotaxis is contact based. One of the most intriguing problems about Dif-mediated lipid chemotaxis is the need for stimulation and adaptation rates consistent with the slow rate of movement. The mechanism of signal perception was examined by making a variety of artificial DifA constructs. M. xanthus cells expressing a chimeric chemoreceptor (NafA) in which the transmembrane, periplasmic, and histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase (HAMP) linker domains of DifA are replaced by the corresponding domains of the enteric nitrate sensor NarX are defective in extracellular matrix (ECM) production. The DifA cytoplasmic region closely follows the Escherichia coli consensus and contains two methylation domains (MD) separated by the highly conserved domain (HCD) that binds CheW.

Most of the information about heterocyst development to date is based on the study of three species of heterocyst-forming filamentous cyanobacteria: Anabaena (also Nostoc) sp. strain PCC 7120, A. variabilis ATCC 29413, and Nostoc punctiforme ATCC 29133. This chapter focuses on those genes involved in signaling and regulation. HetR plays a central role in heterocyst development and pattern formation. In bacteria, calcium ions play important roles in various cellular processes such as pathogenesis, sporulation in Bacillus, chemotaxis in Escherichia coli, and heterocyst development in cyanobacteria. PatA may influence heterocyst development by attenuating the negative effects of the main inhibitory signals of heterocyst pattern formation, PatS and HetN. Late stages of heterocyst development are characterized by structural changes that include the deposition of three cell layers: an outermost fibrous layer, an envelope polysaccharide layer, and an innermost glycolipid layer. During nitrogen fixation, nitrogenase reduces atmospheric nitrogen to ammonia, which is then assimilated into amino acids. A recent epistasis analysis of four genes involved in pattern formation in Anabaena Strain PCC 7120 suggests that PatA has two distinct activities, to promote differentiation as well as to attenuate the negative effects of PatS and HetN on differentiation. Some genes that are required for heterocyst development are also involved in akinete formation, such as hetR and hepA. In the absence of heterocysts, the akinetes seem to form at random positions along the filament, whereas the presence of heterocysts influences akinete positioning, implying the existence of cell-to-cell communication.

The Actinobacteria, including the genus Streptomyces, constitute, on average, about 13% of soil bacterial communities, making them a dominant form of life on Earth. Research has shown that many wild isolates of Streptomyces spp. are capable of intercellular communication, and in one instance, the extracellular compound is a desferrioxamine siderophore. This apparent diversity of intercellular signaling mechanisms suggests that the streptomycetes rely extensively on cell-cell communication to coordinate growth with the production of secondary metabolites and sporulation. As morphological and physiological differentiation can be visually monitored, extracellular rescue of mutant phenotypes can be quite striking. However, the streptomycetes present challenges that need to be considered when investigating cell-cell signaling. The best-understood signaling systems exhibited by the streptomycetes are those mediated by the γ-butyrolactones. In certain species, including Streptomycetes lavendulae and S. coelicolor, the roles of the γ-butyrolactones appear to be restricted to secondary metabolism. To date, three classes of secreted, hydrophobic molecules have been shown to be involved in aerial hyphae formation. In S. coelicolor, these include the chaplins and a small lanthionine-containing peptide, SapB, which has orthologues in S. griseus, S. avermitilis, and S. scabies, as well as a functional homologue, SapT, produced by S. tendae. The pamamycins, a group of macrolide antibiotics produced by S. alboniger, demonstrate that a single molecule may serve more than one distinct function. At subinhibitory concentrations, they stimulate the formation of aerial hyphae, while at higher concentrations they inhibit the growth of nonproducing streptomycetes and other gram-positive bacteria.

This chapter describes work on new and emerging systems that describe roles for excreted cellular metabolites as intercellular signals. The therapeutic value of antibiotics is unquestionable; however, understanding the biological role of these compounds in natural settings may not be as intuitive as the definition of antibiotics would suggest. Increases in biofilm formation were correlated with a 10-fold increase in expression of the ica operon upon treatment with antibiotic. The internal concentration of polyamines is tightly regulated through a combination of biosynthesis, transport, and excretion. In Escherichia coli, putrescine is synthesized either by decarboxylation of L-ornithine or decarboxylation of Larginine followed by removal of a urea molecule. P. aeruginosa is known to make and excrete both monorhamnolipids (mono-RHLs) and dirhamnolipids (di-RHLs), which have either one or two attached rhamnose groups, respectively. In E. coli, indole is imported from the extracellular environment predominantly by the Mtr permease whereas efflux of indole out of the cell is performed by the AcrEF pump. The chapter provides evidence that supports the role of various metabolites in the regulation of community-level traits, such as biofilm formation, swarming, and filamentation.

The opportunistic pathogen Pseudomonas aeruginosa provides one of the most intensely studied examples of acylhomoserine lactone (acyl-HSL)-controlled gene expression. In P. aeruginosa, quorum-sensing gene regulation is accomplished by two complete acyl-HSL systems, LasR-LasI and RhlR-RhlI, and by an orphan receptor, QscR. One microarray study provided additional insights into the signal requirements for the activation of individual quorum-controlled genes. LuxR-type polypeptides can be subdivided into two functional domains based on sequence conservation, genetic and biochemical analysis of representative members, and the crystal structure of TraR from Agrobacterium tumefaciens. LuxR-type proteins also contain a helix-turn-helix motif (HTH) in their carboxy-terminal domain that is required for DNA binding. Based on the recent biochemical characterization of other LuxR-type proteins, general patterns of acyl-HSL/receptor interaction emerge that allow us to distinguish three separate classes. The three P. aeruginosa receptors LasR, RhlR, and QscR each represent one such class: LasR is a class 1 receptor. In contrast, if LuxR-type proteins such as LasR and QscR require their ligand for proper folding, they should induce transcription of target genes relatively slowly because protein activation through translation would be slow. Once a quorum is reached, a change in the gene expression profile is triggered that leads to the formation of a mature biofilm, reminiscent of a developmental program in more complex systems.

One of the environmental signals measured by Vibrio cholerae is its own cell density, which it achieves by a quorum-sensing mechanism. During inhabitation of aquatic environments, V. cholerae lives in association with various species of phytoplankton and zooplankton, often in the form of biofilms. Once V. cholerae has entered the host and traversed the hostile stomach environment, it must penetrate the mucous layer and adhere to and colonize the epithelial cells of the small intestine. To achieve this, V. cholerae produces a number of virulence factors, including the cholerae toxin (CT) and the toxin coregulated pilus (TCP). TCP is a type IV pilus encoded by the Vibrio pathogenicity island (VPI) whose probable function is to mediate adherence to the intestinal mucosal cells. When the quorum-sensing pathways of V. cholerae were being dissected at the molecular level, it was noted that the simultaneous mutation of both the CAI-1 and AI-2 systems did not abolish density-dependent light induction from the lux operon. The mechanisms of quorum-sensing control of biofilm formation in V. cholerae is further complicated by a recent finding that the concentration of the autoinducer CAI-1 is higher in biofilms than in planktonic cultures. To assess the significance of quorum sensing, it is important to carry out experiments under conditions that mimic as closely as possible the natural habitat of V. cholerae.

In addition to the quorum-sensing two-component system (TCS) agr, three other distinct TCSs are presently known to be involved: sae, srr and arl. This chapter begins with the well-studied agr locus, ≈3 kb in length and consisting of divergent transcription units, driven by promoters P2 and P3. Indeed, agr groupings broadly correlate with strain genotypes, and analysis of several Staphylococcus aureus strain sets revealed that genotypic class, with rare exceptions, associated with a single agr group, pointing to agr group differentiation as a primary evolutionary event that preceded genotypic divergence. The role of srrAB was recently analyzed using an antisense RNA approach to repress its expression conditionally, with which it was demonstrated that this TCS differentially regulates virulence genes such as tst and spa in aerobic and anaerobic conditions. It has been noted that the pore-forming Panton-Valentine leukocidin toxin, associated with staphylococcal necrotizing pneumonia, has recently also been demonstrated to downregulate the expression of several exoproteins at the transcriptional level, and it is predicted that yet other variable genes encoding toxins that cause toxinoses will be shown to act in the manner described above. Transcription factors feed back to agr, establishing additional feedback loops, and probably interact similarly with the other TCSs. At least two of the superantigens (SAgs), signaling through BB-4, transmit information through the transcription factors for downregulation of the various exoprotein genes.

One of the first bacterial species for which N-acylhomoserine lactone (AHL) quorum sensing (QS) was described was Erwinia carotovora. Since then, QS has been well studied in the soft-rot erwinias, where, as described in this chapter, QS plays a key role in the regulation of secreted plant cell wall-degrading enzymes (PCWDEs) production and hence in virulence. In certain strains, a well-defined AHL QS system also controls production of β-lactam antibiotic, carbapenem. In addition, it must be emphasized that QS is only one of many regulatory inputs into virulence factor production in Erwinia. The majority of the key secreted virulence factors of E. carotovora and E. chrysanthemi, including multiple Pels, Peh, Cel, and Svx, are secreted by a type II secretion system known as the Out system. There have been two reports describing the existence of AHL QS in E. amylovora. First, production of a single AHL, most likely 3-oxo-C6-HSL, was described for several Italian strains of E. amylovora; for one strain, production of AHL was observed in planta. Second, AHL activity was detected in the culture supernatant of a Swiss strain of E. amylovora. Both reports describe the detection and partial sequencing of pairs of convergent luxIR homologues, named eamIR.

A glimpse into the disease biology of Pantoea stewartii comes from early biochemical and ultra-structural studies of the organism in culture and in the infected maize tissues. The promoter region of esaR features a wellconserved lux box-like element, the esaR box, which spans a putative -10 hexameric σ70 promoter element. The authors focused initially on this promoter to define the functional role of EsaR as a transcription factor and DNA-binding protein. EsaR showed functional attributes that were essentially the reverse from that of the LuxR and TraR functional paradigms and other orthologous proteins that are acyl homoserine lactone (AHL)-dependent quorum-sensing activators. The observation that P. stewartii expresses the major EPSST virulence factor in a cell-density-dependent manner suggests a key role for the EsaI/EsaR quorum-sensing system in managing the transition between distinct phases of bacterial/biofilm development, which may be key to pathogen fitness during host colonization. In addition, other surface localized functions will be characterized to identify components that initiate the contact between the bacterium and the xylem wall, as seen in infections with the AHL mutant strain ESN51. Finally, it might be of interest to know that P. stewartii was initially selected as an experimental organism for quorum-sensing control because of its capacity to synthesize high amounts of AHL.

This chapter briefly describes the nodulation process and some of the quorum-sensing regulatory systems that rhizobia use to monitor their population density. Conjugation is common among Rhizobiaceae, and there are very strong selection pressures to optimize growth in the rhizosphere and nodulation competitiveness. The regulation of gene transfer by quorum-sensing regulation is common among rhizobia. Individual rhizobial strains can contain up to four different LuxI-type acyl-homoserine lactone (AHL) synthases and associated regulators plus several other LuxR-type regulators lacking dedicated AHL synthases. Mutations in cinI and cinR delayed and decreased the growth rate of Rhizobium etli; because such altered growth was not observed in R. leguminosarum cinI or cinR mutants, this suggests that different sets of genes are induced via this quorum-sensing regulon in these two Rhizobium species. Bradyoxetin activity was found in extracts of all α-proteobacteria tested. This suggests that compounds similar to bradyoxetin may play an important role, not only in rhizobial symbiosis, but also in other plant- and animal-bacterial interactions. Elegant studies by researchers suggest that quorum sensing modulates both intra- and inter-species cell-cell communications. It was demonstrated that the halogenated furanones modulate LuxR activity through accelerated degradation of the transcriptional activator, rather than by blocking or displacing the binding of the AHL signal. Another potential way to interfere with quorum sensing is through the degradation or inactivation of the AHL signal molecules.

This chapter reviews the role of quorum sensing in Vibrio fischeri, focusing on recent developments in one's understanding of the genetics and physiology of cell-cell signaling by populations of these bacteria, both in culture and in their light-organ symbioses. Particular emphasis is placed on outlining the regulatory factors and pathways by which quorum sensing coordinates the biological activities of this bioluminescent microbe. Early work showed that V. fischeri strains native to Euprymna scolopes were especially well adapted to this host and that nonnative strains such as MJ1 did not colonize juvenile squid well; thus, MJ1 was not an appropriate strain for studying the symbiosis between V. fischeri and E. scolopes. Production of N-octanoyl-homoserine lactone (C8-HSL) initiates stimulation of the lux operon at moderate cell densities; if ES114 had relatively low expression of AinS and low C8-HSL output, this deficiency could potentially explain why ES114 is so much dimmer than MJ1. Most likely, then, the large difference in the levels of N-3-oxo-hexanoyl-homoserine lactone (3-O-C6-HSL) production and luminescence seen between cultures of ES114 and MJ1 is due to external regulatory influences on the autoinducer synthase genes, and such regulation may be multifactorial. Biochemical and genetic studies of Acyl-homoserine lactone (AHL) signaling in Vibrio harveyi have shown that, in the presence of an inducing concentration of the LuxM-synthesized AHL, the receptor, LuxN,participates in a phosphorelay cascade by stimulating the relative dephosphorylation of LuxU.

This chapter reviews the current understanding of acylated homoserine lactone (AHL) signaling in marine bacterial systems outside of the well-described Vibrio fischeriand Vibrio harveyi models. AHL production has far only been documented in proteobacterial groups. Representatives of these taxa, however, constitute one of the most numerous and functionally diverse classes of microorganisms, and they are particularly abundant in marine environments. Direct chemical analyses, such as those employed by Wagner- Dobler et al., do not have intrinsic biases but are currently at least 10-fold less sensitive than the best biosensors. AHL synthesis is often strongly regulated by other environmental conditions, and these activating conditions may not be recapitulated in standard laboratory culture. The surveys discussed in the chapter should be considered as conservative estimates of AHL production capacity among cultivatable marine bacteria and should be integrated with emerging genomic information on marine bacteria. The Roseobacteria are one of the dominant microbial groups in the ocean, and several subgroups are also the most common AHL signal producers. Ulva zoospores preferentially settled on top of bacteria, suggesting a direct interaction between the bacteria and zoospores and providing evidence that attachment is not a random process. This work led to the discovery that bacterial quorum-sensing molecules, specifically AHLs, are involved in zoospore settlement. The marine environment is a source of abundant materials and resources across the globe. The oceans are sources of diverse and complex quorum-sensing signal molecules produced by many of their endogenous proteobacteria.

The first quorum-sensing system to be discovered was the Lux system of Vibrio fischeri, which regulates light production in the light organ of deep sea fish and squid via an acyl-homoserine lactone (AHL) signaling molecule. The LuxI-type AHL synthases catalyze the formation of the AHL from the substrates S-adenosyl-L-methionine (SAM) and acyl-acyl carrier protein (acyl-ACP). Enzymatic synthesis of AHLs using purified substrates for TraI (from Agrobacterium tumefaciens) verified that both SAM and acyl-ACP are substrates for AHL synthesis in vitro. X-ray crystallographic structural analyses of two LuxI-type AHL synthases form the basis of the current molecular understanding of AHL synthesis. The structure of the EsaI enzyme from Pantoea stewartii was determined from the native sequence. The structure of the LasI enzyme from Pseudomonas aeruginosa was determined from an active form that had been engineered to improve solubility and crystallization properties. The LasI structure provided a less clear explanation for the selectivity of AHL synthases for acyl-ACPs with long acyl chains. The LasI acyl-chain binding pocket is actually an elongated tunnel through the enzyme that is formed by hydrophobic residues at similar positions in the enzyme as those in the EsaI pocket. The residues in EsaI that occlude the pocket are larger than those in the same position in LasI, which limits the acyl chain size to a C6 acyl-chain.

It has been 100 years since Agrobacterium tumefaciens was demonstrated to cause crown gall tumors at plant wound sites. This chapter first presents background information relevant to quorum sensing in A. tumefaciens and then focuses on our current knowledge of the molecular biology of the TraR-TraI system. Regulation of TraR activity is complex and occurs at the levels of transcription, protein folding, resistance to proteolysis, and the formation of quaternary complexes with other TraR subunits or with two different antiactivators of TraR. Control of traR expression by opines therefore has evolved independently in these two types of Ti plasmids. The genes of the arc operon and occ operon are not similar, except for traR. LysR and AccR are also dissimilar, as OccR is a LysR-type transcriptional activator that binds to promoter DNA both in the presence and absence of the inducing signal. Overproduction of TraR can fully overcome inhibition, suggesting that TraM acts by making stoichiometric contacts with TraR. Direct interactions were confirmed by yeast two-hybrid assays and by far Western immunoblots. The author recently used gene arrays to profile the TraR transcriptome and found that all induced genes were Ti plasmid-encoded. These genes include the tra and trb genes, which are involved in conjugal transfer; the rep genes, which are required for vegetative replication and plasmid partitioning into daughter cells; and traM.

In syntrophic interactions, metabolic pathways are integrated over different cell types. This chapter focuses on two seemingly well-defined groups of secondary metabolites, quorum-sensing signals and antibiotics, to demonstrate that their described biological activities do not necessarily define their functional roles in microbial communities. The study of the biology of living organisms and associated biochemical processes has, to date, focused primarily on the structures and functions of DNA, RNA, proteins, lipids, carbohydrates, and their macromolecular complexes. Many bacteria regulate gene expression in response to accumulation of secondary metabolites, and this behavior has been collectively referred to as quorum sensing or cell-cell communication. The generalization of quorum sensing as a density-dependent process ignores the reality that most bacteria do not exist in well-stirred reactors and the signaling will largely be a local event between small groups of cells. The dual role of quorum-sensing signal and antibiotic is not exclusive to nisin, subtilin, and mercascidin peptide antibiotics. A bactericidal activity produced by a strain of Rhizobium leguminosarum that inhibited the growth of several related strains was purified and demonstrated to be a typical acyl homoserine lactone (AHL) [N-(3-hydroxy-7-cis-tetradecenoyl)-L-homoserine lactone]. The streptomycin and chloramphenicol resistance determinants were later shown to catalyze chemical inactivation of the corresponding antibiotic. In addition to the widespread occurrence of antibiotic resistance mechanisms, it has become apparent in recent years that there are many naturally occurring systems that interfere with cell-cell signaling pathways.

The marine animal pathogen Vibrio harveyi and the human pathogen Vibrio cholerae are aquatic bacteria that engage in a process of cell-cell communication called quorum sensing (QS). Autoinducer (AI)-2 is derived from S-adenosylmethionine in three enzymatic steps. First, S-adenosylmethionine serves as a methyl donor for many biochemical processes, and these methyltransferase-dependent reactions yield S-adenosylhomocysteine. Second, S-adenosylhomocysteine is metabolized to adenine and S-ribosylhomocysteine by the enzyme Pfs, and third, S-ribosylhomocysteine is the substrate for the LuxS enzyme. In mixed species consortia, other microbes also have the potential to alter AI-2 levels, and other classes of AIs are clearly manipulated, but the authors have restricted the discussion to AI-2 and how that pertains to Vibrio QS. Appropriate and distinct responses to potentially different communities are possible because of signal integration in the Vibrio circuits. Channel proteins LsrC and LsrD mediate the delivery of the ligand across the membrane. LsrA is an ATPase that supplies the energy required for transport. Rapid Lsr-dependent transport of R-THMF into the cell occurs at high cell densities. Recent studies in V. harveyi show that it possesses five qrr genes, like its closest Vibrio relatives. Examination of their functions reveals that, in stark contrast to V. cholerae, in V. harveyi the quorum-regulatory RNAs (Qrr) sRNAs act additively to control luxR mRNA levels.

The gram-negative bacterium Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that causes infection in immunocompromised individuals, including those with the heritable disease cystic fibrosis (CF). Quorum sensing (QS) has been proposed to be important for colonization of the CF lung, and virulence studies indicate that inactivation of QS in P. aeruginosa significantly reduces virulence in mammalian, plant, and insect models. Pseudomonas quinolone signal (PQS) biosynthesis proceeds through a head-to- head condensation of anthranilic acid and β-keto-decanoic acid to form the immediate precursor of PQS, 2-heptyl-4-quinolone (HHQ). This chapter focuses on membrane vesicles (MVs), including their potential use as trafficking vehicles for a variety of cargo, including cell-cell signals. To be utilized as trafficking vehicles, MVs must (i) have the ability to deliver their cargo to other cells and (ii) possess physiologically relevant cargo, necessitating transfer between cells. MVs isolated from P. aeruginosa have significant antimicrobial activity, particularly against gram-positive bacteria. This antimicrobial activity is multifaceted, including both small molecule and protein components. Recent studies using thin sectioning and transmission electron microscopy revealed that MVs were consistently present in the biofilm EPS matrix.

This chapter outlines the up-regulation of genes in response to the competence-stimulating peptide (CSP) , as well as a number of recent discoveries indicating that the coordination of gene expression mediated among neighboring Streptococcus pneumoniae cells by CSP makes important contributions to the interaction of this pathogen with its human host, and thus has significance beyond the acquisition of new genetic information that originally drew attention to the system. The sigma factor, ComX or sigma X, is encoded by two identical genes, which are located upstream of two separate 16s rRNA operons. Due to the unusual duplication, a role for this sigma in competence regulation was discovered not by genetic approaches, but only by reverse genetics: the protein was found as a minor component of RNA polymerase uniquely present in competent cultures. Identification of the peptide pheromone, the corresponding signal transduction pathway, and several downstream regulons has revealed many elements of the competence regulatory mechanism. The importance of cell-cell signaling via the competence system in biofilm formation is common in the related oral streptococci. Some of the principal pneumococcal phenotypes related to competence gene expression in biofilm and during infection are summarized. Although expression in bacteremic sepsis was found to be very low for competence genes, no data are yet available for colonization or even the intracellular life of S. pneumoniae.

This chapter deals with the study of the biology and chemistry of γ-butyrolactone-type autoregulators that switch on secondary metabolism and morphological differentiation in Streptomyces. The A factor and receptor system in Streptomyces griseus acts as an all-or-nothing switch for both morphological and physiological differentiation. Escherichia coli carrying afsA produces two new substances that are absent in the broth of E. coli without afsA with their m/z 241 and 213 and the same MS/MS fragmentation pattern as A factor. AfsA is thus the key enzyme for the biosynthesis of γ-butyrolactones. Interestingly, a database search predicts that afsA and its homologs are distributed only in actinomycetes. In S. griseus, A factor production is controlled directly or indirectly by adpA in a two-step regulatory feedback loop. The major streptomycin resistance determinant, aphD, located just downstream of strR, encoding streptomycin-6-phosphotransferase, is also transcribed by read-through from the A factor-dependent strR promoter. The cotranscription of strR and aphD accounts for the prompt induction of streptomycin resistance by A factor and achieves a rapid increase in self-resistance just before induction of streptomycin biosynthesis.

This chapter focuses on the impact and molecular mechanisms of quorum quenching, with emphasis on quorum-quenching enzymes and other signal disruption mechanisms. There are five key processes in quorum-sensing circuit, (i) signal generation, (ii) signal transportation, (iii) signal accumulation, (iv) signal recognition, and (v) signal autoinduction. The authors have confirmed recently that replacement of His104 with alanine in AiiA240B1 almost completely abolishes the enzyme activity. By sequence alignment, it was found that all the residues implicated in metal coordination are conserved in the reported acyl-homoserine lactone (AHL)-lactonases. In contrast to the three groups of enzymes, i.e., AHL-lactonase, PON enzymes, and AHL-acylase, which degrade AHL signals by breaking the bond in either the lactone ring or in the junction connecting fatty acid moiety and homoserine lactone component, AHL-oxidoreductase modifies the 3-oxo group of the AHL signals with the corresponding substitution to generate corresponding 3-hydroxy derivatives. The small GTPase proteins Rac2, Cdc42, and Rap1A are involved in the assembly and activation of the NADPH oxidase. This system is arranged vectorially in the phagosome membrane so that electrons pass through it from the NADPH-oxidizing site to the O2- reducing site, resulting in the production of superoxide anion and, consequently, the generation of reactive oxygen species such as H2O2, ONOO–, and HOCl. The biological importance of quorum-sensing communication in bacterial pathogens and fair understanding of the general molecular mechanisms have significantly propelled our effort in searching for effective quorum-quenching mechanisms.

As the elucidation of the molecular mechanisms behind quorum sensing (QS) gained momentum, researchers conceived several strategies to block QS systems. Indeed, QS signals can be detected in biofilms across a range of environments. For example, biofilms grown on rocks in the San Marcos River in Texas have been shown to produce acyl-homoserine lactone (AHL) signals, as have biofilm communities on marine snow particles and sponge surfaces. One of the major problems with conventional biofilm-control measures is the development of resistance. The red seaweed Delisea pulchra produces a range of halogenated furanone compounds that display antifouling and antimicrobial properties, altering the abundance and composition of the bacterial community on the surface and hence the subsequent development of a biofouling community. Transcriptomic analysis of gene expression shows that 4-nitro-pyridine-N-oxide (4-NPO) mainly affects genes that are regulated by either RhlR alone or RhlR and LasR in concert. A recent transcriptomic analysis strongly suggests that the wild-type Pseudomonas aeruginosa further activates a QS-controlled strategic defense system, which reacts upon the encounter with polymorphonuclear leukocytes (PMNs) and suppresses the powerful cellular immune response by paralyzing the PMNs. Since the transcriptomic studies of in vitro biofilms suggest the existence of multiple pathways by which a biofilm can be built, the use and administration of QS blocking technologies may undergo further development and be combined with treatments directed at targets in additional pathways instrumental for biofilm development.

This chapter defines four signaling categories that describe the different interdomain signaling mechanisms. These mechanisms are: (i) one-way sensing—one organism senses and responds to a diffusible signal produced by a second organism; (ii) co-opting of a signal—one organism uses the signal produced by another to regulate its own gene expression; (iii) modulation of a signal—one organism alters the production or stability of a signal from another organism; and (iv) two-way communication—multiple signals are exchanged between organisms. Pseudomonas aeruginosa produces two acylhomoserine lactones (AHLs) signals, 3-oxo-C12-homoserine lactone (3-O-C12- HSL) and C4-homoserine lactone (C4-HSL). The increased virulence in the presence of dynorphin is due to induction of the pqs biosynthetic operon, which leads to an increased production of hydroxy-alkylquinolones, including the P. aeruginosa quinolone signal (PQS) quorum-sensing molecule, which regulates a number of cytotoxic factors. Genetic and biochemical experiments indicated that P. aeruginosa 3-O-C12-HSL, described for its role in quorum sensing, is sufficient to suppress C. albicans hypha formation. Although the chapter has focused on signaling interactions that involve molecules whose primary role appears to be the transmission of information, there are likely many other signals, such as metabolites or pH and oxygen gradients, that may play equally important roles in the regulation of processes important during interdomain interactions. Physical interactions between organisms may also be important for communication within mixed species communities.

The rhomboid family of intramembrane serine proteases controls a variety of functions in both eukaryotes and prokaryotes. The rhomboid proteins were originally identified in Drosophila, where they are required for growth factor signal generation. However, in recent years, a number of diverse functions for the rhomboid proteins have been identified. These functions include (i) the cleavage of TatA, a membrane-bound component of the twin arginine transport system that is required for cell-cell signaling in a prokaryote; (ii) regulating mitochondrial membrane fusion in Saccharomyces; and (iii) cleavage of cell surface adhesions in apicomplexan parasites. Recent biochemical analyses combined with crystallography studies have confirmed these enzymes use a Ser-His catalytic dyad. Moreover, the active-site serine of these enzymes is embedded within the membrane bilayer, and access to water in the membrane is mediated by a hydrophilic cavity that extends from the extracellular environment to the active-site serine. This chapter expands on each of the above themes and provides some future directions for the analysis of this novel class of membrane proteases.

The most well studied of the more recently identified quorum-sensing molecules in fungi are small primary alcohols, thus chemically different from the acyl-homoserine lactones and modified peptides preferred by bacteria. These primary alcohols include farnesol and tyrosol in Candida albicans, and phenylethanol and tryptophol in Saccharomyces cerevisiae. This chapter talks about molecules after a review of the mating pheromones and a more detailed discussion of the primary alcohol quorum-sensing molecules. In S. cerevisiae, members of the same mitogen-activated protein kinase (MAPK) signaling cascade are involved in both pheromone response and agar invasion in haploid cells, and in Ustilago maydis, the loci expressed in response to mating pheromone contain genes involved in filamentous growth and pathogenicity. This coordination between mating and morphogenesis in fungi is analogous to the connection between competence and virulence in Streptococcus pneumoniae and other gram-positive bacteria, which is also regulated by quorum-sensing peptides. In addition to the mating pheromones and four primary alcohols discussed, there are various examples of quorum-sensing-like phenomena and molecules in the literature, indicating that quorum sensing may indeed be as ubiquitous among fungi as it is among bacteria.

Rotifer quorum-sensing is similar in many respects to the more intensely studied bacterial quorum-sensing processes, suggesting that chemosensory processes for assessing conspecific population density may have ancient origins. The apparent involvement of the human steroidogenesis-inducing protein (SIP) in steroid production suggests a similar role for the rotifer mixis-inducing protein (MIP), such that MIP quorum sensing could trigger steroid hormone production in female rotifers, leading to meiotic oogenesis. Population genetics approaches can be used to determine when the mixis pathways of different groups of rotifers diverge and to test hypotheses related to the importance of rotifer mixis in evolutionary processes. The trade-offs between accurate population assessment and the energy costs associated with loss of a complex signaling molecule to the environment have been recognized in bacterial quorum sensing and may apply similarly to rotifers. Quorum sensing among rotifers, therefore, would be analogous to cell-cell communication between tissues of a complex multicellular organism-both require coordination, whether of animal behavior or cell physiology. Induction of sexual reproduction in rotifers satisfies the conditions that define quorum sensing, including the production and release of an autoinducing chemical cue (MIP) by rotifers in a density-dependent manner, the response by conspecifics upon reaching a threshold concentration, and the coordination of this response, which includes both physiological and behavioral changes.

This chapter focuses on the understanding of the regulation of division of labor in honeybee colonies from the perspective of quorum sensing. A flexible system of division of labor presumably is very important to colony fitness because a bee colony must develop and produce reproductive individuals despite constant changes in external and colony conditions. Three pheromones have been identified that regulate honeybee division of labor: a worker inhibitory pheromone, queen mandibular pheromone, and brood pheromone. These pheromones act directly or indirectly on physiological factors including juvenile hormone and molecular pathways associated with the foraging, malvolio, and vitellogenin genes. Ethyl oleate (EO), a substance produced by adult forager honeybees, was recently identified as a chemical inhibitory factor, delaying the age of onset of foraging in field experiments. Bees detect molecules (pheromones) that reflect the abundance of a particularly relevant category of conspecifics and respond accordingly. With the identification of these pheromones and the ability to detect their effects on brain gene expression, it should be possible to trace the flow of information from the environment ultimately to neurons in the brain to determine whether quorum sensing in bacteria and pheromone regulation of division of labor in the honey bee share any features at the molecular level.