Genetic and environmental determinants of swarming in Pseudomonas aeruginosa
Swarming, in bacteria, is a type of group translocation phenomenon observed in several flagellated species characterized by their ability to spread across solid or semi-solid surfaces rapidly – often with a distinct colony pattern. For instance, swarming observed in Pseudomonas aeruginosa, an opportunistic human pathogen widely known for its ability to engage in various forms of bacterial sociality, entails a characteristic pattern composed of five to eight branches called tendrils or dendrites, which radiate outwards from the point of inoculum. While some species can swarm over a wide range of surface conditions, swarming in many species, like P. aeruginosa, is observed exclusively under a narrow range of semi-solid agar (0.4% – 0.7%) surface conditions. Interestingly, as described in species like E. coli, whether swarming is a chemotactically driven motility is often contested with a few lines of opposing evidence. Nonetheless, in P. aeruginosa, as well as in other species, swarming is strictly observed under a limited set of conditions suggesting specific extrinsic cues could influence swarming. Even though swarming has been reported in several bacterial lineages, including numerous Gram-positive and Gram-negative species, the ecological and evolutionary relevance of the phenomenon is still unclear. In this study, using PA14, a laboratory strain of P. aeruginosa as a model, we investigate the relevance of swarming as an important social attribute of bacteria through a reductionist approach. This study is designed to experiment, at least partially, with the following running hypotheses in the lab: • Swarming is a population-wide response to ecologically relevant external cues. • Multiple, yet specific, external cues can influence swarming either independently or in a combinatorial fashion. • The bacterium must encode and utilize dedicated signal perception and response units to perceive and differentiate such varied signals. In the first part of my thesis, I describe the details of the experiments we performed to understand the swarming attributes of PA14 across four swarming-supporting formulations. This chapter of my work primarily aims to uncover various genetic determinants which might be critical in modulating this behaviour. To address this, we set out to answer whether molecular sensors of the bacterium are essential for swarming. By screening a targeted subset of a transposon mutant library of P. aeruginosa, we found that multiple genes encoding sensor kinases (SKs), or response regulators (RRs), of the two-component signalling systems (TCSs) are required in a nutrient-specific manner. In the second part of the work, we attempted to identify cues sensed by the TCS system by a transcriptomic approach. For this, we performed an RNA-seq analysis by comparing the transcriptome of PA14 in two distinct phases of swarming, i.e., early swarm lag (3 hours) and late swarm lag (12 hours). In addition to numerous stationary phase and quorum sensing associated transcripts, we found that a cluster of contiguously placed seventeen genes, including those encoding a periplasmic ethanol dehydrogenase (ExaA) and its regulators, are significantly upregulated in late swarm-lag compared to early swarm-lag. Consistently, we found that ethanol, when provided in low concentrations, can serve as an extrinsic cue for swarming in P. aeruginosa. Subsequently, we further confirmed that this ethanol oxidation cluster is downstream of ErdR, an RR identified as a regulator of swarming in the screen described in the first part of the thesis. In the third part of the work, we further examined functional aspects of ErdR through molecular biology approaches. ErdR belongs to the NarL family of RRs without a known SK partner. To understand whether ErdR is activated via phosphorylation, a common feature of all canonical RRs, we mutated a conserved phosphorylatable-Asp residue to alanine (D58A) in ErdR. This mutation abolished ethanol-induced swarming in P. aeruginosa. We found that the DNA binding domain of ErdR is also essential for its swarm-promoting function. To identify sensor kinases, we screened a deletion mutant library comprising all the 62 SKs and found that two SK loci (ercS and PA14_21700) were necessary for ethanol-induced swarming. Although the location of both ercS and PA14_21700 are outside the erdR operon, their phenotypes were similar to erdR. To confirm this further, we created a transcriptional reporter of ethanol dehydrogenase (ExaA), which is downstream to ErdR. We found that both ErcS and PA14_21700 regulate ExaA expression. Thus, these two SKs appear to work in the same pathway as ErdR in promoting swarming and utilization of ethanol as a carbon source. Overall, this work has shown that P. aeruginosa utilizes ethanol, an ecologically relevant cue, to facilitate swarming. The bacterium uses a cluster of seventeen genes, well conserved in the Pseudomonas genus, to utilize ethanol as a signal for swarming. Our study provides a framework for the identification of other environmental triggers which may regulate swarming in P. aeruginosa and other bacteria.