Quorum Sensing and Bacterial Social Interactions in Biofilms
PERSPECTIVES OF CELL-CELL SIGNALING
The field of cell-cell signaling and coordinated microbial group behavior arose from two independent discoveries reported about 40 years ago. Tomasz stated in 1965 that a hormone-like extracellular product helped regulate competence in Streptococcus pneumoniae (94). The signal was later identified as a peptide—indeed, peptides have emerged as common molecular signals among gram-positive bacteria (31, 56). In 1970, Nealson and colleagues (70) reported that luminescence in the marine bacterium Vibrio fischeri (formerly Photobacterium fischeri) was produced only at high cell density but could be induced at low density by growth in the spent medium of a high-density culture. They referred to this phenomenon as “autoinduction” (69). The signal factor termed “autoinducer” was later identified as an acylated homoserine lactone (acyl-HSL) molecule. This class of signaling molecules predominates within the proteobacteria (97). The term “quorum sensing” (QS) was introduced to specifically refer to the cell-density-linked, coordinated gene expression in populations that experience threshold signal concentrations to induce a synchronized population response (38). Until then, the community was reluctant to accept the concept that bacterial social behaviors known for Myxococcus are the rule, rather than the exception, in the microbial world (84-86). The transformative discoveries that bacteria communicate and exist in nature predominantly as sessile biofilm communities have brought the concept of bacterial multicellularity to the forefront of microbiology.
SOCIAL EVOLUTION AND CELL-TO-CELL COMMUNICATION
Social behaviors and the dilemma of cooperation. As our understanding of the molecular mechanisms that govern social behaviors of many different microbes increases, the opportunity arises to view these systems from a social perspective.
Social behaviors are those that have fitness consequences for both the individual that performs the behavior and a recipient. Cooperation has generally been studied in animals; however, the same problems exist at all levels of biological organization . Cooperation among microbes often takes the form of a shared investment in a group resource (public good), which is costly for an individual to produce, yet provides a benefit to all the individuals in the local group and population. It is often assumed that cooperative behaviors between microbes are favored because the population benefits as a whole. However, selection for cooperation is generally not at the population level; rather, selection occurs at the level of the gene or genes responsible for the relevant social behavior. Any cooperative behavior is at the risk of invasion by selfish individuals (cheaters), who pay little (or none) of the costs of cooperation but gain all the benefits. When public goods are beneficial, the population grows faster when it consists purely of cooperators. However, in a mixed population social cheaters can outcompete cooperators, thereby gaining a fitness benefit within the population. Cooperation can then break down due to social conflict and can even lead to a population collapse or extinction—natural selection does not act with foresight (103).
Classification of social behaviors
Explanations for the evolution of cooperation.Cooperation is widespread in the natural world; thus, mechanisms must exist for its maintenance especially because of the potential spread of social cheaters. For the individual that performs the behavior, cooperation provides a direct fitness benefit that outweighs the cost of performing the behavior (81). In addition, cooperation provides indirect fitness benefits to other individuals who carry the cooperative gene. The most common reason for two individuals to share genes in common is for them to be genealogical relatives (kin), which is often termed kin selection (41). By helping a close relative reproduce, an individual transmits genes to the next generation, albeit indirectly; this class of cooperation is altruistic. Kin selection can work in two ways. First, an individual can distinguish kin from nonkin and therefore preferentially direct aid toward them. Second, the population can experience limited dispersal. Here relatives are kept close together, favoring indiscriminate altruism toward neighbors. The limited dispersal mechanism does not require complex cognition, so it could be important in a broad range of organisms, especially microbes. Indirect fitness benefits can also be obtained when cooperation is directed to nonrelatives who share the same cooperative gene (26, 41).
The definition of genetic relatedness (r) is the relatedness at the locus/loci of the behavior being considered, not the entire genome. At the Austin conference, Greg Velicer, Indiana University, described the importance of relatedness in Myxococcus xanthus at the locus of the social trait when he revealed that a mutation in a single gene restores sociality in a population that is under the threat of extinction. M. xanthus is a soil-dwelling bacterium that undergoes multicellular development during periods of starvation, which leads to the development of a wide range of fruiting body types. A cheater genotype termed the “obligate cheater” was identified. In isolation, this strain fails to produce any spores; hence, it is dependent on a social host (34). In competition experiments with cooperators, the cheaters dramatically spread in the population, resulting in population extinction. However, during one such experiment, an obligate cheater reevolved the ability to sporulate in the absence of cooperators, but unlike the wild type, it resisted the future invasion by cheater cells (34). Remarkably, this strain, termed Phoenix after the mythical burning bird that can arise from its own ashes, emerged from just a single mutation that increased the levels of an acetyltransferase (34). Thus, changes in a single genetic locus can enable populations to recover from near-extinction. It also suggests a molecular mechanism whereby cheater cells are suppressed or inhibited so that they cannot take over a population (35).
The complexity of QS.QS is generally assumed to coordinate cooperative behaviors in bacteria. Two complementary talks demonstrated that QS in Pseudomonas aeruginosa is a social trait that is exploitable by cheaters (29, 53). Both studies used minimal medium containing carbon sources that required the secretion of QS-dependent proteases (public goods) to support growth. Martin Schuster, Oregon State University (M. Schuster, CCCB-07, abstr. S5:3), demonstrated that after 100 generations, a subpopulation of lasR mutants, incapable of responding to QS, developed from the wild type (83). In mixed populations, as the relative size of the lasR mutant population grew, population fitness declined, demonstrating that the cheater load can have serious consequences for a population. Interestingly, this reduction in fitness did not occur in the long-term evolution experiments, suggesting that over time populations can adapt to the presence of cheaters (83). Steve Diggle, University of Nottingham, United Kingdom (S. Diggle, CCCB-07, abstr. S5:1), pointed out that the real cost of QS is in the response to the signal, not signal production itself.
Given the cost of QS and the opportunities for cheaters to spread, how can QS be maintained? Brown and Johnstone first proposed a theoretical kin selection model as a mechanism of maintaining QS (14). In support of the Brown and Johnstone model, Jan Kreft, University of Birmingham, United Kingdom (J. U. Kreft et al., CCCB-07, abstr. S6:1), used mathematical modeling to show that clustering of cells has a strong impact on the autoinducer concentration that a cell may sense. Cells within the same cluster can efficiently perceive signals from adjacent cells within that cluster. Importantly, cells within clusters tend to be clonal and so communication among these cells tends to be with kin. This high relatedness within clusters suppresses cheating and may also be a mechanism to prevent confusion from cross talk with neighboring bacterial species. Steve Diggle (S. Diggle, CCCB-07, abstr. S5:1) provided empirical data to show that QS may be maintained by kin selection. In both high-relatedness and low-relatedness treatments, high relatedness favored QS. Perhaps more importantly, QS was not favored in conditions of low relatedness when the wild type and QS lasR mutants were mixed (28). It is interesting that many P. aeruginosa clinical isolates sampled from the cystic fibrosis (CF) lung are signal-blind lasR mutants (82, 90). Cheating provides one explanation for this, and the CF lung may be an environment that is particularly susceptible to this type of behavior. There are, however, alternative explanations. It could be that QS is simply not important for survival in the lung and, as a consequence, is naturally lost. A third possibility is that a mutation in lasR confers a growth advantage over a QS-positive strain in the CF environment (25).
A few years ago, the notion of diffusion sensing (DS) was introduced. It was posited that autoinducer functions chiefly to enable individual cells to sense how rapidly secreted molecules diffuse away. Therefore, DS could allow individuals to minimize the loss of costly public goods by extracellular diffusion (78). However, it has now been empirically demonstrated that QS does have social fitness consequences, providing a benefit at the group level that can be exploited by individuals that do not produce signal (28, 83). Burkhard Hense, GSF National Research Centre, Munich, Germany (B. A. Hense et al., CCCB-07, abstr. S6:5), introduced a new concept termed efficiency sensing (ES) (44). ES assumes that low-cost autoinducers are released to test the efficiency of producing costlier exoenzymes, a concept that is similar to DS. However, ES includes the potential for cooperation because microcolonies (clusters) may help protect against interference by other species and cheaters. In this way ES unifies both QS and DS, as it enables cells to sense cell density, diffusion limitation, and cell distribution (clustering) (Fig. 1) (44).
Effect of spatial clustering on QS signaling. A mathematical model for autoinducer systems with or without positive feedback was developed. The model was used to investigate the effect of the spatial arrangement of autoinducer-producing cells on the accumulation of local autoinducer. Comparing a random (a) with a clustered (b) arrangement of the same number of cells, the threshold concentration for induction is reached only within and near the clusters. The figure shows bacterial cells that are not induced in cyan and those that are induced in purple. Comparing the same clustered pattern with (b) and without (c) positive feedback demonstrates that this characteristic of autoinducer production is critical for reaching sufficient autoinducer concentrations for cells to induce autoinducer production and autoinducer-dependent genes. The autoinducer concentration, as a percentage of the threshold concentration, is indicated by contour lines and background color, for which a linear color map from red (<16%) to white (>200%) was used. In panel b, the thick contour line separates the noninduced cells from the induced cells. The three-dimensional domain is viewed from the top, onto an impermeable surface at the bottom. As the domain is otherwise infinite, the autoinducer can diffuse away.
Clearly consideration of the evolution of microbial social behaviors is a fascinating endeavor that can stimulate everyone in the field to evaluate the utility and maintenance of intercellular communication systems in nature. A challenge for the future will be to combine both mechanistic and evolutionary approaches to further understand microorganisms in their natural habitats.
CELL-CELL SIGNALING IN HOST-MICROBE INTERACTIONS
The paradigm.The marine bacterium Vibrio fischeri cycles between a free-living existence and a mutualistic association with its host, the Hawaiian bobtail squid, Euprymna scolopes, to which it contributes lux-dependent luminescence (97, 98). Luminescence is governed by the luxI/luxR QS system, which represents the mechanistic paradigm for most acyl-HSL-dependent QS regulatory systems within gram-negative bacteria (33, 61, 65, 109). In these systems, the canonical “I” gene encodes the acyl-HSL synthase, while the “R” gene encodes the regulatory protein, usually an activator. In V. fischeri, the luxI/luxR system is at the bottom of a complex hierarchical regulatory circuit governed synergistically by the AinS N-octanoyl HSL (C8-HSL) and the LuxS autoinducer 2 (AI-2) signal synthases and their cognate sensory histidine kinases LuxN and LuxP/Q, respectively (Fig. 2). LuxN and LuxQ undergo autophosphorylation in the absence of signal input, which leads to phosphorylation of the LuxO response regulator. Phosphorylated LuxO indirectly represses LitR, the central regulator of symbiosis and luminescence, via small RNA (sRNA) negative regulatory elements. Signal input transforms LuxN and LuxQ into phosphatases, leaving LuxO in a largely unphosphorylated, inactive state. Under these conditions, LitR becomes available for the activation of luxR and other symbiosis-related functions (Fig. 2). The AinS-specific C8-HSL input is particularly important for the early establishment and persistence of symbiosis and luminescence in culture. In contrast, luminescence at very high cell densities in the host light organ requires the luxI-specific signal system (for an excellent summary of the system see the review by K. L. Visick ). Interestingly, Sarah Studer from the Ruby lab, University of Wisconsin (S. Studer and E. Ruby, CCCB-07, abstr. 123B), reported that AinS and LitR appear to control acetate metabolism, which is critical for bacterial survival within the light organ. Eric Stabb, University of Georgia (A. N. Septer et al., CCCB-07, abstr. 19B), provided evidence that the V. fischeri ArcA response regulator of the redox-monitoring ArcAB two-component system represses luminescence under reducing conditions by binding to a site upstream of and proximal to the luxI promoter, thereby blocking LuxR activation of bioluminescence. The bacteria encounter oxidative conditions during early colonization of the Euprymna scolopes light organ, and the authors hypothesize that this leads to ArcA derepression of the lux operon, accounting for the significant induction of luminescence that is seen only during symbiotic infection. These findings are congruent with the hypothesis of Visick and colleagues that bioluminescence benefits the symbiotic bacteria by decreasing ambient oxygen levels to prevent antimicrobial reactive oxygen species production by the host (11, 98). Moreover, the study showed that the autoinducer, produced by LuxI, is also regulated by ArcA and that arcA mutants induce luminescence in neighboring wild-type cells. Thus, redox stresses detected by ArcAB in a subpopulation may elicit a population-wide response. This observation has led to the proposal that V. fischeri autoinducer is a “redox-responsive alarm signal” with a broader role in V. fischeri than its established function as a census-taking molecule.
The V. fischeri lux paradigm regulatory cascade. V. fischeri produces three QS signals: the LuxI-produced 3-oxo-hexanoyl-HSL (yellow circles), AinS-produced C8-HSL (blue circles), and a LuxS-produced signal, presumably a furanosyl borate diester (AI-2; green circles). LuxR acts as a receptor for both C8-HSL and 3-O-C6-HSL (61). C8-HSL is also a signal for the membrane-bound LuxN sensor kinase, while AI-2 interacts with the LuxP periplasmic binding protein and the LuxQ sensor kinase. At low cell density (low signal concentrations), LuxN and LuxQ autophosphorylate and transfer the phosphate to LuxU, which in turn phosphorylates the LuxO transcriptional activator. Phosphorylated LuxO is predicted to activate the transcription of sRNAs, which inhibit the production of LitR protein. Increasing population density and signal concentrations switch the kinases to phosphatases, leading to dephosphorylated LuxO and production of LitR. LitR activates luxR and flagellar genes. In turn, LuxR and inducing levels of the 3-O-C6-HSL stimulate light production. (Note that aspects of this model remain to be tested in V. fischeri and are based on the experimentally defined V. harveyi model .) OM, outer membrane; IM, inner membrane. (Reprinted from reference 96 with permission.)
Computational models presented by Andrew Goryachev, University of Edinburgh, United Kingdom (A. B. Goryachev, CCCB-07, abstr. S6:3), predict that LuxR self-amplification and the presence of sRNA regulatory intermediates contribute significantly to network fitness. In this connection, Josh Willliams of the Stevens lab, Virginia Tech (J. W. Williams et al., CCCB-07, abstr. S6:4), showed that LuxR self-amplification buffers against intrinsic acyl-HSL signal variation. This ensures that the switch to inducing conditions is not easily reversed, thereby endowing the system with hysteresis.
The bilingual nature of Vibrio cholerae.V. cholerae cell-cell communication was the focus of the conference keynote presentation by Bonnie Bassler of the Howard Hughes Medical Institute and Princeton University (B. L. Bassler, CCCB-07, S1:0 Keynote; D. A. Higgins et al., CCCB-07, abstr. 55B). V. cholerae exists principally in aquatic biofilms associated with plankton and suspended particulate matter (43). Consumption of contaminated waters provides access to an alternate life-style within the intestine of a mammalian host. QS-mediated communication plays a decisive role in the switch between the free-living and host-associated virulent existence. V. cholerae senses cell density via the V. cholerae autoinducer 1 (CAI-1) and AI-2 autoinducer signals, produced by the CsqA and LuxS autoinducer synthases, respectively (Fig. 3). This organism lacks the classic acyl-HSL signal characteristic of its two marine relatives, V. fischeri and Vibrio harveyi. The CAI-1 signal system appears to be common within the Vibrio genus (46). Bassler revealed the novel chemical structure of CAI-1 as (S)-3-hydroxytridecan-4-one (Fig. 3) (46). This molecule represents a unique class of autoinducers, and structure/function studies suggest that the C13-carbon side chain length and the S enantiomeric configuration are functionally significant. Interestingly, compounds related to CAI-1 have pheromone-like functions in certain insects (46).
The Vibrio cholerae autoinducer signaling network. The signal synthases CqsA and LuxS produce autoinducers CAI-1 [(S)-3-hydroxytridecan-4-one] and AI-2 [(2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran borate], respectively. Signal inputs are transduced via LuxO to control levels of HapR. At low cell density, in the absence of autoinducers, hapR expression is repressed, thereby permitting the expression of virulence factors and biofilm formation. At high cell density and in the presence of autoinducers, LuxO is inactive, permitting HapR production. HapR represses virulence and biofilm formation while activating hap protease expression. (Reprinted from reference 42 with permission of the publisher.)
The CAI-1 and AI-2 signals are transduced by the sensor kinases CqsS and LuxP/LuxQ complex, respectively (Fig. 3). CqsS and LuxQ act as kinases in the unliganded state at low cell density. Both signal inputs are channeled to the common LuxO response regulator via LuxU. LuxO phosphorylation results in the activation of genes encoding the regulatory RNAs Qrr1 to Qrr4. The Qrr RNAs interact with Hfq, the RNA chaperone, and occlude the ribosome binding site of the mRNA encoding the master regulatory factor HapR (a LitR homolog). Low-cell-density conditions are generally experienced by free-living V. cholerae in the natural aquatic habitat, where the repression of hapR promotes biofilm growth and the expression of virulence factors including the toxin-coregulated pilus. These conditions are thought to prepare free-living bacterial populations for colonization of the mammalian host. Once V. cholerae populations in the host gut reach a high density, signal accumulates, and LuxO is dephosphorylated. HapR now represses genes needed for initial host-associated colonization and activates the production of the hemagglutinin protease. This protease promotes bacterial detachment from the intestinal epithelium, thereby stimulating bacterial dissemination so that new infection foci in the intestine can be established or the pathogen can escape into the environment, where it may find a new host (113). Jun Zhu, University of Pennsylvania (Z. Liu et al., CCCB-07, abstr. S4:6), showed that hapR repression by σ28 plays a critical role in the infection cycle. Specifically, polar flagellum-mediated motility enables bacteria to seek and penetrate the mucosal layer to access the epithelial cells and initiate pathogenesis. Intriguingly, flagella tend to break as the bacteria encounter the viscous intestinal mucosal matrix. This allows the secretion of the FlgM anti-σ28 factor, releasing σ28 to repress hapR expression, thereby priming V. cholerae cells for intestinal colonization.
Bonnie Bassler also presented data showing that the Qrr1 to Qrr4 sRNAs regulate the expression of virulence functions in a HapR-independent manner. The rapid synthesis of sRNAs without the need for translation and their inherent lability offer a highly responsive regulatory strategy for the rapid transition between two states. In addition the redundancy of sRNAs exhibited by V. cholerae may contribute to the fine-tuning of this regulatory switch. The HapR-independent, sRNA-mediated control of gene expression provides a logical explanation for why classical strains of V. cholerae have frameshift mutations in their respective hapR genes yet remain toxigenic (43). On the practical side, the suppression of virulence by CAI-1 QS offers a unique opportunity for developing pharmaceuticals based on CAI-1 signal chemistries that may be effective in preventing widespread cholera outbreaks.
The Legionella parallel.The gram-negative bacterium Legionella pneumophila, which causes a severe pneumonia known as Legionnaires' disease, also alternates between a free-living virulent and a host-associated replicative state. Hosts include protists and alveolar macrophages (68), where the bacteria are contained within a specialized endoplasmic reticulum-derived vacuole. In the replicative phase, the expression of virulence factors and motility are down-regulated, but as cells enter stationary phase, they switch to the transmissive mode in which virulence and motility functions are up-regulated. Thomas Spirig from the Hilbi lab, ETH Zürich, Switzerland (T. Spirig, CCCB-07, abstr. S1:4), reported that L. pneumophila harbors a gene system (lqs) similar to the V. cholerae cqsAS CAI-1 QS system. The functionality of lqsA was verified using a CAI-1 reporter strain and by genetic complementation of a V. cholerae cqsA-null mutant. lqsA encodes a homolog of the BioF 8-amino-7-oxononanoate synthase that catalyzes the condensation of -alanine and 6-carboxyhexanoyl coenzyme A. This enzyme has a high degree of homology to CqsA. The lqsR gene, which is not present in V. cholerae, appears to play a key role in governing the transition between the replicative and transmissive (virulent) phase (93). Interestingly, similar gene systems exist in a number of other bacteria including Burkholderia xenovorans, an organism that has adapted to complex and diverse niches.
The “ins and outs” of Yersinia pseudotuberculosis.Y. pseudotuberculosis is a mammalian enteropathogen that causes gastroenteritis. The organism alternates between a free-living aqueous or food-borne and host-associated existence. As described by Hannah Patrick of the Williams group, University of Nottingham, United Kingdom (H. L. Patrick et al., CCCB-07, abstr. S2:3), Y. pseudotuberculosis harbors two luxR/I-type systems, ypsR/I and ytbR/I, which together produce a suite of short- and long-chained acyl-HSLs. Detailed genetic studies show that YpsR/I positively activates YtbR/I. These QS systems govern the differential expression of genes related to Y. pseudotuberculosis aggregation, motility, and virulence (3). It will be interesting to explore to what degree these regulatory systems contribute to the biphasic life-style of this organism.
An orphan with a mission.The QscR regulator in P. aeruginosa is characterized as an “orphan” because it lacks a cognate acyl-HSL synthase gene (22). Pete Greenberg, University of Washington, reported that QscR is critical to the pathogenesis of P. aeruginosa and that a qscR mutant is hypervirulent due to enhanced expression of RhlR/LasR-controlled phenazine, hydrogen cyanide, and elastase virulence functions. To distinguish between a number of possible QscR regulatory mechanisms, Lequette and colleagues (58) conducted a transcriptome study and showed that the QscR regulon partially overlaps with the LasR and RhlR regulons but also regulates genes not governed by either LasR or RhlR. QscR directly activates genes in a 3-oxo-dodecanoyl-HSL (3-O-C12-HSL)-dependent manner, although the protein receptor is more responsive to decanoyl-HSL (C10-HSL) (58), suggesting a possible role for QscR in sensing coexisting, competing neighbors such as Burkholderia vietnamensis, a C10-HSL producer. This rationale led to the prediction that QscR might have less-stringent ligand binding characteristics than the highly stable, nearly irreversible ligand binding properties of LasR. Indeed, in Escherichia coli reporter assays probing the diffusibility of ligand bound to QscR and LasR, sufficient signal diffused only from QscR. Most recently, Greenberg's group working together with Helen Blackwell's group at the University of Wisconsin identified small molecule inhibitors of LasR that function as agonists for QscR (63). Such compounds could mitigate the antagonistic effects of LasR and QscR on virulence.
“Elaborate Lives,” an aria about AidA.Caenorhabditis elegans is a useful nonmammalian infection model. Leo Eberl, University of Zürich, Switzerland (J. Wopperer et al., CCCB-07, abstr. S6:2), described the utility of this system to study the role of AidA, a protein required for nematode colonization by strains of the Burkholderia cepacia complex. All strains expressing the AiiA acyl-HSL lactonase inhibited the expression of AidA, demonstrating that this protein is QS regulated. AidA is not a virulence factor per se but is required for B. cepacia complex strains to persist in C. elegans. Furthermore, active QS is essential for nematode pathogenicity by all these strains, leading to the prediction that QS is likely to control other nematocidal determinants (15).
Signaling in microbial-plant interactions.Max Dow, National University of Ireland, Ireland (M. Dow, CCCB-07, abstr. S2-, described the role of an unusual signaling molecule, cis-11-methyl-dodecenoic acid, known as DSF (diffusible signaling factor), in the plant-pathogenic bacterium Xanthomonas campestris pathovar campestris. Biosynthesis of DSF is dependent on the putative enoyl coenzyme A hydratase RpfF (30). DSF signal perception and transduction involve the RpfC hybrid sensor kinase/response regulator and the atypical RpfG response regulator. RpfG features an HD-GYP cyclic-di-GMP phosphodiesterase domain (80) and interacts with a subset of GGDEF domain proteins (2). Thus, DSF exerts its effect through an unusual signal transduction system that blends elements of a two-component system with that of a cyclic-di-GMP-specific second messenger pathway. X. campestris encodes 37 proteins with GGDEF, EAL, or HD-GYP domains, some of which are dedicated to the expression of virulence factors while others have a role in motility. One might envisage that these proteins integrate the cell-cell signal and environmental inputs to optimize bacterial development and virulence. In a separate study, Dow and colleagues (R. P. Ryan et al., CCCB-07, abstr. 9B) studied the interaction between Stenotrophomonas maltophilia and P. aeruginosa. These bacteria frequently share a common niche in the rhizosphere, as well as in the CF lung. S. maltophilia also produces DSF and carries rpf homolog genes. In coculture, S. maltophilia has substantial effects on P. aeruginosa growth and biofilm formation (Fig. 4). When grown alone, S. maltophilia forms biofilms with a filamentous architecture, whereas P. aeruginosa forms flat biofilms. In coculture, however, P. aeruginosa develops structures with a filamentous architecture within a mixed biofilm. These effects depend on DSF production by S. maltophilia and are mediated by a gene designated PA1396 that encodes a receptor protein with a sensory domain related to RpfC. Similar signal systems are present in other plant-associated bacteria, indicating that extensive DSF-specific interspecies communication may occur in nature (36, 79).
DSF from Stenotrophomonas maltophilia influences biofilm architecture of Pseudomonas aeruginosa PAO1, which does not produce the signal. Images are of 4-day-old biofilms in flow cells. (A) P. aeruginosa PAO1; (B) S. maltophilia K279a; (C) mixed culture of P. aeruginosa PAO1 and S. maltophilia K279a; (D) mixed culture of P. aeruginosa PAO1 and S. maltophilia K279arpfF (DSF negative). Bars, 20 μm. (Courtesy of Max Dow, National University of Ireland, Cork; reproduced with permission).
The Pantoea stewartii paradigm.Pantoea stewartii subsp. stewartii is a plant pathogen that causes vascular wilt in maize. The bacterium colonizes the xylem as cell-wall-adherent, stewartan exopolysaccharide (EPS)-encased biofilms (55, 99). In this system, the unliganded apo form of EsaR, the LuxR homolog QS regulator of P. stewartii, binds DNA and represses rcsA. RcsA is a transcription factor of the Rcs phosphorelay system that together with RcsB activates EPS biosynthesis in P. stewartii (16, 66). Susanne von Bodman, University of Connecticut (S. B. von Bodman et al., CCCB-07, abstr. S4:5), described two previously unidentified genetic loci essential for stewartan EPS synthesis that are coordinately activated by RcsA/RcsB under acyl-HSL-inducing conditions. Interestingly, an EPS hydrolase is located within the primary EPS biosynthetic cluster. The function of the hydrolase appears to be a stewartan-lipopolysaccharide chain length determinant that protects P. stewartii from stewartan-
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