Pseudomonas aeruginosa Quorum Sensing.

Pseudomonas aeruginosa, like many bacteria, uses chemical signals to communicate between cells in a process called quorum sensing (QS). QS allows groups of bacteria to sense population density and, in response to changing cell densities, to coordinate behaviors. The P. aeruginosa QS system consists of two complete circuits that involve acyl-homoserine lactone signals and a third system that uses quinolone signals. Together, these three QS circuits regulate the expression of hundreds of genes, many of which code for virulence factors. P. aeruginosa has become a model for studying the molecular biology of QS and the ecology and evolution of group behaviors in bacteria. In this chapter, we recount the history of discovery of QS systems in P. aeruginosa, discuss how QS relates to virulence and the ecology of this bacterium, and explore strategies to inhibit QS. Finally, we discuss future directions for research in P. aeruginosa QS.

A new class of homoserine lactone quorum-sensing signals.

Quorum sensing is a term used to describe cell-to-cell communication that allows cell-density-dependent gene expression. Many bacteria use acyl-homoserine lactone (acyl-HSL) synthases to generate fatty acyl-HSL quorum-sensing signals, which function with signal receptors to control expression of specific genes. The fatty acyl group is derived from fatty acid biosynthesis and provides signal specificity, but the variety of signals is limited. Here we show that the photosynthetic bacterium Rhodopseudomonas palustris uses an acyl-HSL synthase to produce p-coumaroyl-HSL by using environmental p-coumaric acid rather than fatty acids from cellular pools. The bacterium has a signal receptor with homology to fatty acyl-HSL receptors that responds to p-coumaroyl-HSL to regulate global gene expression. We also found that p-coumaroyl-HSL is made by other bacteria including Bradyrhizobium sp. and Silicibacter pomeroyi. This discovery extends the range of possibilities for acyl-HSL quorum sensing and raises fundamental questions about quorum sensing within the context of environmental signalling.

Bacterial biofilms: a common cause of persistent infections.

Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. Formation of these sessile communities and their inherent resistance to antimicrobial agents are at the root of many persistent and chronic bacterial infections. Studies of biofilms have revealed differentiated, structured groups of cells with community properties. Recent advances in our understanding of the genetic and molecular basis of bacterial community behavior point to therapeutic targets that may provide a means for the control of biofilm infections.

The involvement of cell-to-cell signals in the development of a bacterial biofilm.

Bacteria in nature often exist as sessile communities called biofilms. These communities develop structures that are morphologically and physiologically differentiated from free-living bacteria. A cell-to-cell signal is involved in the development of Pseudomonas aeruginosa biofilms. A specific signaling mutant, a lasI mutant, forms flat, undifferentiated biofilms that unlike wild-type biofilms are sensitive to the biocide sodium dodecyl sulfate. Mutant biofilms appeared normal when grown in the presence of a synthetic signal molecule. The involvement of an intercellular signal molecule in the development of P. aeruginosa biofilms suggests possible targets to control biofilm growth on catheters, in cystic fibrosis, and in other environments where P. aeruginosa biofilms are a persistent problem.

Sociomicrobiology: the connections between quorum sensing and biofilms.

In the past decade, significant debate has surrounded the relative contributions of genetic determinants versus environmental conditions to certain types of human behavior. While this debate goes on, it is with a certain degree of irony that microbiologists studying aspects of bacterial community behavior face the same questions. Information regarding two social phenomena exhibited by bacteria, quorum sensing and biofilm development, is reviewed here. These two topics have been inextricably linked, possibly because biofilms and quorum sensing represent two areas in which microbiologists focus on social aspects of bacteria. We will examine what is known about this linkage and discuss areas that might be developed. In addition, we believe that these two aspects of bacterial behavior represent a small part of the social repertoire of bacteria. Bacteria exhibit many social activities and they represent a model for dissecting social behavior at the genetic level. Therefore, we introduce the term 'sociomicrobiology'.

Self perception in bacteria: quorum sensing with acylated homoserine lactones.

A variety of Gram-negative bacteria produce membrane permeant, acylated homoserine lactone (HL) pheromones that act as cell density cues. Synthesis and response to these factors requires proteins homologous to the Luxl acylhomoserine lactone synthase and the LuxR transcription factor from Vibrio fischeri. Recent genetic and biochemical studies have begun to provide a mechanistic understanding of acyl HL dependent gene regulation. Examination of the role of acyl HLs in diverse bacteria positions LuxR-Luxl type systems within an increasingly broad regulatory context and suggests that, in some bacteria, they comprise a global regulatory circuit.

Acylated homoserine lactone detection in Pseudomonas aeruginosa biofilms by radiolabel assay.

We describe the development of a new radioactive assay for acyl-HSL production by bacterial cultures. The assay is based on the uptake of radiolabeled methionine and conversion of the radiolabel into SAM. The radiolabeled SAM is then incorporated into acyl-HSL by an acyl-HSL synthase. This assay is faster than previously used bioassays and shows no bias for the detection of acyl-HSLs of a particular length or side chain substitution. Acyl-HSL production can be monitored over a wide range of growth conditions in liquid culture. This assay can also be used in conjunction with a tube biofilm reactor to monitor acyl-HSL production by biofilm cultures. Ultimately this assay will allow comparison of acyl-HSL production by cells subjected to a variety of physiological conditions.

Spirochete chemotaxis, motility, and the structure of the spirochetal periplasmic flagella.

Spirochetes have a unique motility system that is characterized by flagellar filaments contained within the outer membrane sheath. Direct evidence using video microscopy has recently been obtained which indicates that these periplasmic flagella (PF) rotate in several spirochetal species. This rotation generates thrust. As shown for one spirochete, Spirochaeta aurantia, motility is driven by a proton motive force. Spirochete chemotaxis has been most thoroughly studied in S. aurantia. This spirochete exhibits three distinct behaviours, runs of smooth swimming, reversals and flexing. These behaviours are modulated by addition of attractants such that S. aurantia swims towards higher concentrations of attractants in a spatial gradient. Unlike the prototypical bacterium, Escherichia coli, chemotaxis in S. aurantia involves fluctuations in membrane potential. The PF of a number of spirochetes have been examined in considerable detail. For most species, the PF filaments are complex, consisting of an assembly of several different polypeptides. There are several antigenically related core polypeptides surrounded by an outer layer consisting of a different polypeptide. Borrelia burgdorferi and Spirochaeta zuelzerae represent exceptions where the filaments are composed of a single major polypeptide species. The genes encoding the filament polypeptides from several spirochete species have been cloned and analysed. Apparently, the outer layer polypeptides of S. aurantia, Treponema pallidum and Serpulina hyodysenteriae are transcribed from sigma-70-like promoters, whereas the core polypeptide genes are transcribed from sigma-28-like promoters. A gene encoding the hook polypeptide in Treponema phagedenis has been cloned and analysed. The product of this gene shows significant similarity to the E. coli hook protein, FlgE, and homologs have been identified in T. pallidum and B. burgdorferi.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the Spirochaeta aurantia flaA gene and transcript.

The flaA gene, which codes for the Spirochaeta aurantia flagellar filament outer layer polypeptide, FlaA, was cloned, sequenced and analysed. The gene appears to be transcribed into a monocistronic mRNA from a sigma 70-like promoter. The translational start is 31 base pairs after the start of the transcript, the open reading frame is 1011 base pairs, and a rho-independent-like transcription terminator sequence begins about 19 base pairs after the translational stop codon. The deduced amino acid sequence of the S. aurantia FlaA showed 40% identity with the Treponema pallidum FlaA, but these polypeptides did not show a significant similarity to other polypeptides for which sequence information was available.

Intragenic suppression of a luxR mutation: characterization of an autoinducer-independent LuxR.

The Vibrio fischeri luminescence genes are activated by the LuxR protein and a diffusible signal termed the autoinducer. LuxR consists of two domains, a C-terminal transcriptional activator domain, and an N-terminal autoinducer-binding domain, which serves to regulate the function of the C-terminal domain. We have isolated and characterized an intragenic suppressor of a mutation that maps to the N-terminal domain and blocks autoinducer binding. The suppressor changes an alanine residue at position-221 in the C-terminal domain to a valine. In Escherichia coli, the suppressor allows partial activation of the V. fischeri luminescence genes although E. coli containing this protein remains unable to bind autoinducer. To further analyze the influence of the second-site mutation on luxR function, we constructed a luxR gene that coded for a protein with a wild-type N-terminal domain and with the ala-221 to val substitution in the C-terminal domain. This protein activated the luminescence genes in the presence or absence of autoinducer, and it bound autoinducer at levels comparable to the wild-type LuxR protein. Apparently, the alanine to valine substitution at position-221 allows activity of the C-terminal domain in a fashion independent of whether autoinducer is bound to the N-terminal domain.

The influence of iron on Pseudomonas aeruginosa physiology: a regulatory link between iron and quorum sensing.

In iron-replete environments, the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein represses expression of two small regulatory RNAs encoded by prrF1 and prrF2. Here we describe the effects of iron and PrrF regulation on P. aeruginosa physiology. We show that PrrF represses genes encoding enzymes for the degradation of anthranilate (i.e. antABC), a precursor of the Pseudomonas quinolone signal (PQS). Under iron-limiting conditions, PQS production was greatly decreased in a DeltaprrF1,2 mutant as compared with wild type. The addition of anthranilate to the growth medium restored PQS production to the DeltaprrF1,2 mutant, indicating that its defect in PQS production is a consequence of anthranilate degradation. PA2511 was shown to encode an anthranilate-dependent activator of the ant genes and was subsequently renamed antR. AntR was not required for regulation of antA by PrrF but was required for optimal iron activation of antA. Furthermore, iron was capable of activating both antA and antR in a DeltaprrF1,2 mutant, indicating the presence of two distinct yet overlapping pathways for iron activation of antA (AntR-dependent and PrrF-dependent). Additionally, several quorum-sensing regulators, including PqsR, influenced antA expression, demonstrating that regulation of anthranilate metabolism is intimately woven into the quorum-sensing network of P. aeruginosa. Overall, our data illustrate the extensive control that both iron regulation and quorum sensing exercise in basic cellular physiology, underlining how intermediary metabolism can affect the regulation of virulence factors in P. aeruginosa.

The influence of human respiratory epithelia on Pseudomonas aeruginosa gene expression.

The opportunistic pathogen Pseudomonas aeruginosa can cause acute or chronic infections in humans. Little is known about the initial adaptation of P. aeruginosa to host tissues and the factors that determine whether a P. aeruginosa-epithelial cell interaction will manifest as an acute or a chronic infection. To gain insights into the initial phases of P. aeruginosa infections and to identify P. aeruginosa genes regulated in response to respiratory epithelia, we exposed P. aeruginosa to cultured primary differentiated human airway epithelia. We used a P. aeruginosa strain that causes acute damage to the epithelia and a mutant with defects in Type III secretion and in rhamnolipid synthesis. The mutant did not cause rapid damage to epithelia as did the wildtype. We compared the transcriptomes of the P. aeruginosa wildtype and the mutant to each other and to P. aeruginosa grown under other conditions, and we discovered overlapping sets of differentially expressed genes in the wildtype and mutant exposed to epithelia. A recent study reported that exposure of P. aeruginosa to epithelia is characterized by a repression of the bacterial iron-responsive genes. These findings were suggestive of ample iron availability during infection. In contrast, we found that P. aeruginosa shows an iron-starvation response upon exposure to epithelial cells. This observation highlights the importance of the iron starvation response in both acute and chronic infections and suggests opportunities for therapy.

Timing and localization of rhamnolipid synthesis gene expression in Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa biofilms can develop mushroom-like structures with stalks and caps consisting of discrete subpopulations of cells. Self-produced rhamnolipid surfactants have been shown to be important in development of the mushroom-like structures. The quorum-sensing-controlled rhlAB operon is required for rhamnolipid synthesis. We have introduced an rhlA-gfp fusion into a neutral site in the P. aeruginosa genome to study rhlAB promoter activity in rhamnolipid-producing biofilms. Expression of the rhlA-gfp fusion in biofilms requires the quorum-sensing signal butanoyl-homoserine lactone, but other factors are also required for expression. Early in biofilm development rhlA-gfp expression is low, even in the presence of added butanoyl-homoserine lactone. Expression of the fusion becomes apparent after microcolonies with a depth of >20 mum have formed and, as shown by differential labeling with rfp or fluorescent dyes, rhlA-gfp is preferentially expressed in the stalks rather than the caps of mature mushrooms. The rhlA-gfp expression pattern is not greatly influenced by addition of butanoyl-homoserine lactone to the biofilm growth medium. We propose that rhamnolipid synthesis occurs in biofilms after stalks have formed but prior to capping in the mushroom-like structures. The differential expression of rhlAB may play a role in the development of normal biofilm architecture.

Complete genome sequence of Vibrio fischeri: a symbiotic bacterium with pathogenic congeners.

Vibrio fischeri belongs to the Vibrionaceae, a large family of marine gamma-proteobacteria that includes several dozen species known to engage in a diversity of beneficial or pathogenic interactions with animal tissue. Among the small number of pathogenic Vibrio species that cause human diseases are Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus, the only members of the Vibrionaceae that have had their genome sequences reported. Nonpathogenic members of the genus Vibrio, including a number of beneficial symbionts, make up the majority of the Vibrionaceae, but none of these species has been similarly examined. Here we report the genome sequence of V. fischeri ES114, which enters into a mutualistic symbiosis in the light organ of the bobtail squid, Euprymna scolopes. Analysis of this sequence has revealed surprising parallels with V. cholerae and other pathogens.

Overproduction and purification of the luxR gene product: Transcriptional activator of the Vibrio fischeri luminescence system.

Expression of Vibrio fischeri luminescence genes requires an inducer, termed autoinducer, and a positive regulatory element, the luxR gene product. A plasmid containing luxR under control of a tac promoter was engineered to overproduce this gene product. The overproduced luxR gene product was active in vivo, and its apparent monomeric molecular weight was indistinguishable from that of the protein encoded by luxR under control of its own promoter (M(r) 27,000). The new tac-luxR construct directed the synthesis of large quantities of the luxR gene product in induced Escherichia coli cells lacking other lux genes. In the presence of the other lux genes, overexpression of the tac-luxR construct was not detected. The overproduced luxR gene product, which formed cytoplasmic inclusion bodies, was purified and used in subsequent studies. Nonequilibrium pH gradient electrophoresis indicated that the protein was basic, and the amino-terminal 15 amino acids were sequenced. DNA-binding activity was detected by membrane filter binding assays; under the conditions used, the binding was not lux DNA-specific. Binding of tritium-labeled autoinducer to the luxR gene product was not detected, and autoinducer enhancement of the binding of the luxR gene product to DNA could not be detected reproducibly.

Biochemical and cytological analysis of the complex periplasmic flagella from Spirochaeta aurantia.

The periplasmic flagella of Spirochaeta aurantia were isolated and were found to be ultrastructurally and biochemically complex. Generally, flagellar filaments were 18 to 20 nm in diameter and appeared to consist of an 11 to 13-nm-wide inner region and an outer layer. The hook-basal body region consisted of two closely apposed disks connected to a hook by a rod. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified flagella together with a Western blot analysis of a motility mutant that produces hooks and basal bodies but not flagellar filaments revealed that the filaments were composed of three major polypeptides of 37,500, 34,000, and 31,500 apparent molecular weight (37.5K, 34K, and 31.5K polypeptides) and three minor polypeptides of 36,000, 33,000, and 32,000 apparent molecular weight (36K, 33K, and 32K polypeptides). Purified hook-basal body preparations were greatly enriched in three polypeptides in the range of 62,000 to 66,000 apparent molecular weight. Immunogold labeling experiments with a monoclonal antibody specific for the 37.5K flagellin and one that reacts with an epitope common to the 36K, 34K, 33K, 32K, and 31.5K flagellins revealed that the 37.5K major polypeptide was a component of the outer layer, whereas one or more of the other polypeptides constituted the core.

Control of Vibrio fischeri lux gene transcription by a cyclic AMP receptor protein-luxR protein regulatory circuit.

Expression of the Vibrio fischeri luminescence genes (lux genes) requires two transcriptional activators: the V. fischeri luxR gene product with autoinducer and the cyclic AMP (cAMP) receptor protein (CRP) with cAMP. It has been established that autoinducer and the luxR gene product are required for transcriptional activation of the luxICDABE operon, which contains a gene required for autoinducer synthesis and genes required for light emission. However, the role of cAMP-CRP in the induction of luminescence is not clear. We examined transcriptional control of the lux genes in Escherichia coli, using catabolite repression mutants carrying lux DNA-containing plasmids. Transcriptional fusions between the lacZ gene on Mu dI and luxR were used to assess luxR promoter activity, and the luxAB genes (which encode the two luciferase subunits) were used as a natural reporter of luxICDABE promoter activity. A plasmid containing luxR under control of the cAMP-CRP-independent tac promoter was constructed to direct the synthesis of the luxR gene product in cells containing compatible luxR::Mu dI insertion mutant plasmids. In E. coli, cAMP-CRP activated transcription of luxR and concurrently decreased luxICDABE transcription. In the presence of relatively high levels of the luxR gene product, cAMP and CRP were not required for induction of the luxICDABE operon. The luxR gene product in the presence of autoinducer activated transcription of the luxICDABE operon, as has been shown previously, and we demonstrate that it also decreased luxR transcription. Apparently, control of the V. fischeri luminescence genes involves a regulatory circuit in which cAMP and CRP activate luxR transcription and in turn the luxR gene product activates transcription of the operon responsible for light emission (uxICDABE). Furthermore, in lux gene regulation cAMP-CRP and autoinducer-LuxR protein appear to function as transcriptional antagonists.

Motility and chemotaxis of Spirochaeta aurantia: computer-assisted motion analysis.

A computer program has been designed to study behavior in populations of Spirochaeta aurantia cells, and this program has been used to analyze changes in behavior in response to chemoattractants. Three kinds of behavior were distinguished: smooth swimming, flexing, and reversals in direction of swimming after a short pause (120 ms). Cell populations exposed to chemoattractants spent, on average, 66, 33, and 1% of the time in these modes, respectively. After the addition of a chemoattractant, behavior was modified transiently--smooth swimming increased, flexing decreased, and reversals were suppressed. After addition of D-xylose (final concentration, 10 mM), the adaptation time (the time required for the populations to return to the unmodified behavior) for S. aurantia was 1.5 to 2.0 min. A model to explain the behavior of S. aurantia and the response of cells to chemoattractants is described. This model includes a coordinating mechanism for flagellar motor operation and a motor switch synchronizing device.