Communication, cooperation & conflict in quorum sensing bacteria

The scientific community has gathered an extremely detailed and sophisticated understanding of the genetic and molecular underpinnings of microbial communication. How these microbial communication systems arise and are maintained over evolutionary time-scales however has received relatively little attention. Some major questions remain unanswered such as; what is the function of small diffusible molecules? How does population structure affect the dynamics of social communication and what is the link between the ecology of communication and the virulence of a pathogenic population? Borrowing concepts from evolutionary theory can help to unravel these fundamental questions in the context of microbial communication as it has done in other taxa displaying social behaviours. In addition microbial model organisms in which molecular and genetic tools are abundant lend enormous power to empirical tests of evolutionary theory. This work combines both of these in an attempt to understand the evolution of bacterial communication using the model organism Pseudomonas aeruginosa and its well characterised Quorum Sensing systems. Specifically the focus is in three areas. Firstly this study reveals that the stability of bacterial signalling is vulnerable to perturbations in cost and benefit and genetic conflict. Secondly this study finds that spatial structure (biofilm vs planktonic) influences the outcome of social competition over signalling and reduces population viability. Thirdly this study finds that interspecific and intraspecific competition over public goods impose divergent selective pressures on communication

Genomic characterisation of an endometrial pathogenic <i>Escherichia coli</i> strain reveals the acquisition of genetic elements associated with extra-intestinal pathogenicity

&lt;b&gt;Background&lt;/b&gt;&lt;p&gt;&lt;/p&gt; Strains of &lt;i&gt;Escherichia coli&lt;/i&gt; cause a wide variety of intestinal and extra-intestinal diseases in both humans and animals, and are also often found in healthy individuals or the environment. Broadly, a strong phylogenetic relationship exists that distinguishes most &lt;i&gt;E. Coli&lt;/i&gt; causing intestinal disease from those that cause extra-intestinal disease, however, isolates within a recently described subclass of Extra-Intestinal Pathogenic &lt;i&gt;E. Coli&lt;/i&gt; (ExPEC), termed endometrial pathogenic &lt;i&gt;E. Coli&lt;/i&gt;, tend to be phylogenetically distant from the vast majority of characterised ExPECs, and more closely related to human intestinal pathogens. In this work, we investigate the genetic basis for ExPEC infection in the prototypic endometrial pathogenic &lt;i&gt;E. Coli&lt;/i&gt; strain MS499.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Results&lt;/b&gt;&lt;p&gt;&lt;/p&gt; By investigating the genome of MS499 in comparison with a range of other E. coli sequences, we have discovered that this bacterium has acquired substantial lengths of DNA which encode factors more usually associated with ExPECs and less frequently found in the phylogroup relatives of MS499. Many of these acquired factors, including several iron acquisition systems and a virulence plasmid similar to that found in several ExPECs such as APEC O1 and the neonatal meningitis &lt;i&gt;E. Coli&lt;/i&gt; S88, play characterised roles in a variety of typical ExPEC infections and appear to have been acquired recently by the evolutionary lineage leading to MS499.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Conclusions&lt;/b&gt;&lt;p&gt;&lt;/p&gt; Taking advantage of the phylogenetic relationship we describe between MS499 and several other closely related &lt;i&gt;E. Coli&lt;/i&gt; isolates from across the globe, we propose a step-wise evolution of a novel clade of sequence type 453 ExPECs within phylogroup B1, involving the recruitment of ExPEC virulence factors into the genome of an ancestrally non-extraintestinal &lt;i&gt;E. Coli&lt;/i&gt;, which has repurposed this lineage with the capacity to cause extraintestinal disease. These data reveal the genetic components which may be involved in this phenotype switching, and argue that horizontal gene exchange may be a key factor in the emergence of novel lineages of ExPECs.&lt;p&gt;&lt;/p&gt

Quorum sensing protects bacterial co-operation from exploitation by cheats

Quorum sensing (QS) is a cell–cell communication system found in many bacterial species, commonly controlling secreted co-operative traits, including extracellular digestive enzymes. We show that the canonical QS regulatory architecture allows bacteria to sense the genotypic composition of high-density populations, and limit co-operative investments to social environments enriched for co-operators. Using high-density populations of the opportunistic pathogen Pseudomonas aeruginosa we map per-capita signal and co-operative enzyme investment in the wild type as a function of the frequency of non-responder cheats. We demonstrate mathematically and experimentally that the observed response rule of ‘co-operate when surrounded by co-operators' allows bacteria to match their investment in co-operation to the composition of the group, therefore allowing the maintenance of co-operation at lower levels of population structuring (that is, lower relatedness). Similar behavioural responses have been described in vertebrates under the banner of ‘generalised reciprocity'. Our results suggest that mechanisms of reciprocity are not confined to taxa with advanced cognition, and can be implemented at the cellular level via positive feedback circuits

Quorum-sensing and cheating in bacterial biofilms

The idea from human societies that self-interest can lead to a breakdown of cooperation at the group level is sometimes termed the public goods dilemma. We tested this idea in the opportunistic bacterial pathogen, Pseudomonas aeruginosa, by examining the influence of putative cheats that do not cooperate via cell-to-cell signalling (quorum-sensing, QS).We found that: (i) QS cheating occurs in biofilm populations owing to exploitation of QS-regulated public goods; (ii) the thickness and density of biofilms was reduced by the presence of non-cooperative cheats; (iii) population growth was reduced by the presence of cheats, and this reduction was greater in biofilms than in planktonic populations; (iv) the susceptibility of biofilms to antibiotics was increased by the presence of cheats; and (v) coercing cooperator cells to increase their level of cooperation decreases the extent to which the presence of cheats reduces population productivity. Our results provide clear support that conflict over public goods reduces population fitness in bacterial biofilms, and that this effect is greater than in planktonic populations. Finally, we discuss the clinical implications that arise from altering the susceptibility to antibiotics

Characterisation of two quorum sensing systems in the endophytic Serratia plymuthica strain G3: differential control of motility and biofilm formation according to life-style

<p>Abstract</p> <p>Background</p> <p><it>N</it>-acylhomoserine lactone (AHL)-based quorum sensing (QS) systems have been described in many plant-associated Gram-negative bacteria to control certain beneficial phenotypic traits, such as production of biocontrol factors and plant growth promotion. However, the role of AHL-mediated signalling in the endophytic strains of plant-associated <it>Serratia </it>is still poorly understood. An endophytic <it>Serratia </it>sp. G3 with biocontrol potential and high levels of AHL signal production was isolated from the stems of wheat and the role of QS in this isolate was determined.</p> <p>Results</p> <p>Strain G3 classified as <it>Serratia plymuthica </it>based on 16S rRNA was subjected to phylogenetic analysis. Using primers to conserved sequences of <it>luxIR </it>homologues from the <it>Serratia </it>genus, <it>splIR </it>and <it>spsIR </it>from the chromosome of strain G3 were cloned and sequenced. AHL profiles from strain G3 and <it>Escherichia coli </it>DH5α expressing <it>splI </it>or <it>spsI </it>from recombinant plasmids were identified by liquid chromatography-tandem mass spectrometry. This revealed that the most abundant AHL signals produced by SplI in <it>E. coli </it>were <it>N</it>-3-oxo-hexanoylhomoserine lactone (3-oxo-C6-HSL), <it>N</it>-3-oxo-heptanoylhomoserine lactone (3-oxo-C7-HSL), <it>N</it>-3-hydroxy-hexanoylhomoserine lactone (3-hydroxy-C6-HSL), <it>N</it>-hexanoylhomoserine lactone (C6-HSL), and <it>N</it>-heptanoyl homoserine lactone (C7-HSL); whereas SpsI was primarily responsible for the synthesis of <it>N</it>-butyrylhomoserine lactone (C4-HSL) and <it>N</it>-pentanoylhomoserine lactone (C5-HSL). Furthermore, a quorum quenching analysis by heterologous expression of the <it>Bacillus </it>A24 AiiA lactonase in strain G3 enabled the identification of the AHL-regulated biocontrol-related traits. Depletion of AHLs with this lactonase resulted in altered adhesion and biofilm formation using a microtiter plate assay and flow cells coupled with confocal laser scanning microscopy respectively. This was different from the closely related <it>S. plymuthica </it>strains HRO-C48 and RVH1, where biofilm formation for both strains is AHL-independent. In addition, QS in G3 positively regulated antifungal activity, production of exoenzymes, but negatively regulated production of indol-3-acetic acid (IAA), which is in agreement with previous reports in strain HRO-C48. However, in contrast to HRO-C48, swimming motility was not controlled by AHL-mediated QS.</p> <p>Conclusions</p> <p>This is the first report of the characterisation of two AHL-based quorum sensing systems in the same isolate of the genus <it>Serratia</it>. Our results show that the QS network is involved in the global regulation of biocontrol-related traits in the endophytic strain G3. However, although free-living and endophytic <it>S. plymuthica </it>share some conservation on QS phenotypic regulation, the control of motility and biofilm formation seems to be strain-specific and possible linked to the life-style of this organism.</p

Loss of Social Behaviours in Populations of Pseudomonas aeruginosa Infecting Lungs of Patients with Cystic Fibrosis

Pseudomonas aeruginosa, is an opportunistic, bacterial pathogen causing persistent and frequently fatal infections of the lung in patients with cystic fibrosis. Isolates from chronic infections differ from laboratory and environmental strains in a range of traits and this is widely interpreted as the result of adaptation to the lung environment. Typically, chronic strains carry mutations in global regulation factors that could effect reduced expression of social traits, raising the possibility that competitive dynamics between cooperative and selfish, cheating strains could also drive changes in P. aeruginosa infections. We compared the expression of cooperative traits - biofilm formation, secretion of exo-products and quorum sensing (QS) - in P. aeruginosa isolates that were estimated to have spent different lengths of time in the lung based on clinical information. All three exo-products involved in nutrient acquisition were produced in significantly smaller quantities with increased duration of infection, and patterns across four QS signal molecules were consistent with accumulation over time of mutations in lasR, which are known to disrupt the ability of cells to respond to QS signal. Pyocyanin production, and the proportion of cells in biofilm relative to motile, free-living cells in liquid culture, did not change. Overall, our results confirm that the loss of social behaviour is a consistent trend with time spent in the lung and suggest that social dynamics are potentially relevant to understanding the behaviour of P. aeruginosa in lung infections

Environmental modification via a quorum sensing molecule influences the social landscape of siderophore production

Bacteria produce a wide variety of exoproducts that favourably modify their environment and increase their fitness. These are often termed ‘public goods’ because they are costly for individuals to produce and can be exploited by non-producers (‘cheats’). The outcome of conflict over public goods is dependent upon the prevailing environment and the phenotype of the individuals in competition. Many bacterial species use quorum sensing (QS) signalling molecules to regulate the production of public goods. QS therefore determines the cooperative phenotype of individuals, and influences conflict over public goods. In addition to their regulatory functions, many QS molecules have additional properties that directly modify the prevailing environment. This leads to the possibility that QS molecules could influence conflict over public goods indirectly through non-signalling effects, and the impact of this on social competition has not previously been explored. The Pseudomonas aeruginosa QS signal molecule PQS is a powerful chelator of iron which can cause an iron starvation response. Here we show that PQS stimulates a concentration-dependent increase in the cooperative production of iron scavenging siderophores, resulting in an increase in the relative fitness of non-producing siderophore cheats. This is likely due to an increased cost of siderophore output by producing cells and a concurrent increase in the shared benefits, which accrue to both producers and cheats. Although PQS can be a beneficial signalling molecule for P. aeruginosa, our data suggests that it can also render a siderophore-producing population vulnerable to competition from cheating strains. More generally our results indicate that the production of one social trait can indirectly affect the costs and benefits of another social trait

Conflict of interest and signal interference lead to the breakdown of honest signalling

Animals use signals to coordinate a wide range of behaviours, from feeding offspring to predator avoidance. This poses an evolutionary problem, because individuals could potentially signal dishonestly to coerce others into behaving in ways that benefit the signaller. Theory suggests that honest signalling is favoured when individuals share a common interest and signals carry reliable information. Here, we exploit the opportunities offered by bacterial signalling, to test these predictions with an experimental evolution approach. We show that: (1) a reduced relatedness leads to the relative breakdown of signalling; (2) signalling breaks down by the invasion of mutants that show both reduced signalling and reduced response to signal; (3) the genetic route to signalling breakdown is variable; (4) the addition of artificial signal, to interfere with signal information, also leads to reduced signalling. Our results provide clear support for signalling theory, but we did not find evidence for the previously predicted coercion at intermediate relatedness, suggesting that mechanistic details can alter the qualitative nature of specific predictions. Furthermore, populations evolved under low relatedness caused less mortality to insect hosts, showing how signal evolution in bacterial pathogens can drive the evolution of virulence in the opposite direction to that often predicted by theory

4,6-Bis(4-fluoro­phen­yl)-2-phenyl-1H-indazol-3(2H)-one

In the title compound, C25H16F2N2O, the pyrazole ring is almost planar (r.m.s. deviation = 0.028 Å) and makes a dihedral angle of 5.86 (11)° with the indazole benzene ring. The dihedral angle between the pyrazole ring and the unsubstituted phenyl ring is 28.19 (11)°. The dihedral angles between the unsubstituted phenyl and the two fluoro­phenyl groups are 57.69 (10) and 18.01 (10)°. In the crystal, mol­ecules are linked by inter­molecular N—H⋯O and C—H⋯F inter­actions, forming infinite chains along the b axis with graph-set motif R 3 2(19). The crystal structure is further consolidated by π–π stacking [centroid–centroid distances = 3.5916 (13) and 3.6890 (13) Å] and C—H⋯π inter­actions

Conflict of interest and signal interference lead to the breakdown of honest signalling

Animals use signals to coordinate a wide range of behaviours, from feeding offspring to predator avoidance. This poses an evolutionary problem, because individuals could potentially signal dishonestly to coerce others into behaving in ways that benefit the signaller. Theory suggests that honest signalling is favoured when individuals share a common interest and signals carry reliable information. Here, we exploit the opportunities offered by bacterial signalling, to test these predictions with an experimental evolution approach. We show that: (1) a reduced relatedness leads to the relative breakdown of signalling; (2) signalling breaks down by the invasion of mutants that show both reduced signalling and reduced response to signal; (3) the genetic route to signalling breakdown is variable; (4) the addition of artificial signal, to interfere with signal information, also leads to reduced signalling. Our results provide clear support for signalling theory, but we did not find evidence for the previously predicted coercion at intermediate relatedness, suggesting that mechanistic details can alter the qualitative nature of specific predictions. Furthermore, populations evolved under low relatedness caused less mortality to insect hosts, showing how signal evolution in bacterial pathogens can drive the evolution of virulence in the opposite direction to that often predicted by theory