Evolution of Quorum Sensing in Pseudomonas aeruginosa Can Occur via Loss of Function and Regulon Modulation.

Pseudomonas aeruginosa populations evolving in cystic fibrosis lungs, animal hosts, natural environments and in vitro undergo extensive genetic adaption and diversification. A common mutational target is the quorum sensing (QS) system, a three-unit regulatory system that controls the expression of virulence factors and secreted public goods. Three evolutionary scenarios have been advocated to explain selection for QS mutants: (i) disuse of the regulon, (ii) cheating through the exploitation of public goods, or (ii) modulation of the QS regulon. Here, we examine these scenarios by studying a set of 61 QS mutants from an experimental evolution study. We observed nonsynonymous mutations in all three QS systems: Las, Rhl, and Pseudomonas Quinolone Signal (PQS). The majority of the Las mutants had large deletions of the Las regulon, resulting in loss of QS function and the inability to produce QS-regulated traits, thus supporting the first or second scenarios. Conversely, phenotypic and gene expression analyses of Rhl mutants support network modulation (third scenario), as these mutants overexpressed the Las and Rhl receptors and showed an altered QS-regulated trait production profile. PQS mutants also showed patterns of regulon modulation leading to strain diversification and phenotypic tradeoffs, where the upregulation of certain QS traits is associated with the downregulation of others. Overall, our results indicate that mutations in the different QS systems lead to diverging effects on the QS trait profile in P. aeruginosa populations. These mutations might not only affect the plasticity and diversity of evolved populations but could also impact bacterial fitness and virulence in infections. IMPORTANCE Pseudomonas aeruginosa uses quorum sensing (QS), a three-unit multilayered network, to coordinate expression of traits required for growth and virulence in the context of infections. Despite its importance for bacterial fitness, the QS regulon appears to be a common mutational target during long-term adaptation of P. aeruginosa in the host, natural environments, and experimental evolutions. This raises questions of why such an important regulatory system is under selection and how mutations change the profile of QS-regulated traits. Here, we examine a set of 61 experimentally evolved QS mutants to address these questions. We found that mutations involving the master regulator, LasR, resulted in an almost complete breakdown of QS, while mutations in RhlR and PqsR resulted in modulations of the regulon, where both the regulon structure and the QS-regulated trait profile changed. Our work reveals that natural selection drives diversification in QS activity patterns in evolving populations.

Siderophores drive invasion dynamics in bacterial communities through their dual role as public good versus public bad.

Microbial invasions can compromise ecosystem services and spur dysbiosis and disease in hosts. Nevertheless, the mechanisms determining invasion outcomes often remain unclear. Here, we examine the role of iron-scavenging siderophores in driving invasions of Pseudomonas aeruginosa into resident communities of environmental pseudomonads. Siderophores can be 'public goods' by delivering iron to individuals possessing matching receptors; but they can also be 'public bads' by withholding iron from competitors lacking these receptors. Accordingly, siderophores should either promote or impede invasion, depending on their effects on invader and resident growth. Using supernatant feeding and invasion assays, we show that invasion success indeed increased when the invader could use its siderophores to inhibit (public bad) rather than stimulate (public good) resident growth. Conversely, invasion success decreased the more the invader was inhibited by the residents' siderophores. Our findings identify siderophores as a major driver of invasion dynamics in bacterial communities under iron-limited conditions.

Ecology drives the evolution of diverse social strategies in Pseudomonas aeruginosa.

Bacteria often cooperate by secreting molecules that can be shared as public goods between cells. Because the production of public goods is subject to cheating by mutants that exploit the good without contributing to it, there has been great interest in elucidating the evolutionary forces that maintain cooperation. However, little is known about how bacterial cooperation evolves under conditions where cheating is unlikely to be of importance. Here we use experimental evolution to follow changes in the production of a model public good, the iron-scavenging siderophore pyoverdine, of the bacterium Pseudomonas aeruginosa. After 1200 generations of evolution in nine different environments, we observed that cheaters only reached high frequency in liquid medium with low iron availability. Conversely, when adding iron to reduce the cost of producing pyoverdine, we observed selection for pyoverdine hyperproducers. Similarly, hyperproducers also spread in populations evolved in highly viscous media, where relatedness between interacting individuals is increased. Whole-genome sequencing of evolved clones revealed that hyperproduction is associated with mutations involving genes encoding quorum-sensing communication systems, while cheater clones had mutations in the iron-starvation sigma factor or in pyoverdine biosynthesis genes. Our findings demonstrate that bacterial social traits can evolve rapidly in divergent directions, with particularly strong selection for increased levels of cooperation occurring in environments where individual dispersal is reduced, as predicted by social evolution theory. Moreover, we establish a regulatory link between pyoverdine production and quorum-sensing, showing that increased cooperation with respect to one trait (pyoverdine) can be associated with the loss (quorum-sensing) of another social trait.

Microbial Mutualism: Will You Still Need Me, Will You Still Feed Me?

How might costly cooperation evolve from scratch? A new study using cross-feeding in a bacterial system suggests that spatial proximity between partners and reciprocal fitness feedbacks between them are essential drivers of stable cooperative partnerships.

Costs and benefits of multiple mating in a species with first-male sperm precedence.

Different patterns of sperm precedence are expected to entail different costs and benefits of mating for each sex that translate into distinct predictions regarding mating system evolution. Still, most studies addressing these costs and benefits have focused on species with mixed paternity or last male precedence, neglecting first-male sperm precedence. We attempted to understand whether this latter pattern of sperm precedence translates into different costs and benefits for each sex in the haplodiploid spider mite Tetranychus urticae, a species in which female multiple mating is prevalent but most offspring are sired by first males. First, we assessed the stability of the sperm precedence pattern. To do so, we measured offspring paternity after exposing females to a different number of matings and mating intervals. Next, to determine the potential costs or benefits of multiple mating for females under different contexts, we measured the fecundity and survival of females that re-mated at different time points. To measure the potential costs of multiple mating for males, we analysed male survival in the presence of different numbers of virgin or mated females. We also tested whether males can reduce offspring production of their competitors, by reducing the production of fertilized offspring of mated females. We found no change in the pattern of sperm precedence, independently of the mating interval between matings and the number of matings. Females paid a cost of mating, as multiply-mated females laid fewer eggs than once-mated females. However, while males had reduced survival when exposed to an intermediate number of virgin females, they paid no additional costs of mating with mated females. Moreover, females that mated multiple times produced fewer fertilized offspring than females that mated once. Thus, males that copulated with mated females reduced the fitness of other males, potentially leading to a relative fitness benefit for themselves. Our results show that complex costs and benefits may arise in males in species with first-male sperm precedence. How these costs and benefits affect the maintenance of selection for polyandry remains an open question.

Spider mites escape bacterial infection by avoiding contaminated food.

To fight infection, arthropods rely on the deployment of an innate immune response but also upon physical/chemical barriers and avoidance behaviours. However, most studies focus on immunity, with other defensive mechanisms being relatively overlooked. We have previously shown that the spider mite Tetranychus urticae does not mount an induced immune response towards systemic bacterial infections, entailing very high mortality rates. Therefore, we hypothesized that other defence mechanisms may be operating to minimize infection risk. Here, we test (a) if spider mites are also highly susceptible to other infection routes-spraying and feeding-and (b) if they display avoidance behaviours towards infected food. Individuals sprayed with or fed on Escherichia coli or Pseudomonas putida survived less than the control, pointing to a deficient capacity of the gut epithelium, and possibly of the cuticle, to contain bacteria. Additionally, we found that spider mites prefer uninfected food to food contaminated with bacteria, a choice that probably does not rely on olfactory cues. Our results suggest that spider mites may rely mostly on avoidance behaviours to minimize bacterial infection and highlight the multi-layered nature of immune strategies present in arthropods.

Male spider mites use chemical cues, but not the female mating interval, to choose between mates.

The choice of the partner an individual will mate with is expected to strongly impact its fitness. Hence, natural selection has favoured the evolution of cues to distinguish among mates that will provide different fitness benefits to the individual that is choosing. In species with first-male sperm precedence, this is particularly important for males, as mating with mated females will result in no offspring. In the spider mite Tetranychus urticae only the first mating is effective, except if the interval between first and second copulations is shorter than 24 h. In line with this, males prefer to mate with virgin over mated females. They do not, however, choose between females that have mated at different time intervals. Here, we tested which type of cues males use to distinguish between females with different mating status (virgin versus mated). To do so, we firstly confirmed that males prefer virgins over mated females and that they do not select females on the basis of their age or mating interval. Next, we tested whether contact and volatile compounds or chemical trails affected male discrimination between mated and virgin females, by systematically varying the exposure of males to these cues. We found that volatile compounds and chemical trails were sufficient to induce discrimination between virgin and mated females in males. Direct contact with females, however, does not seem to play a role in this discrimination. The composition of such chemical cues (trails and volatiles) remains to be identified.