Microbial Evolution

The observed mutational spectrum of adaptive outcomes can be constrained by many factors. For example, mutational biases can narrow the observed spectrum by increasing the rate of mutation at isolated sites in the genome. In contrast, complex environments can shift the observed spectrum by defining fitness consequences of mutational routes. We investigate the impact of different nutrient environments on the evolution of motility in   Pseudomonas fluorescens   Pf0-2x (an engineered non-motile derivative of Pf0-1) in the presence and absence of a strong mutational hotspot. Previous work has shown that this mutational hotspot can be built and broken via six silent mutations, which provide rapid access to a mutation that rescues swimming motility and confers the strongest swimming phenotype in specific environments. Here, we evolved a hotspot and non-hotspot variant strain of Pf0-2x for motility under nutrient-rich (LB) and nutrient-limiting (M9) environmental conditions. We observed the hotspot strain consistently evolved faster across all environmental conditions and its mutational spectrum was robust to environmental differences. However, the non-hotspot strain had a distinct mutational spectrum that changed depending on the nutrient environment. Interestingly, while alternative adaptive mutations in nutrient-rich environments were equal to, or less effective than, the hotspot mutation, the majority of these mutations in nutrient-limited conditions produced superior swimmers. Our competition experiments mirrored these findings, underscoring the role of environment in defining both the mutational spectrum and the associated phenotype strength. This indicates that while mutational hotspots working in concert with natural selection can speed up access to robust adaptive mutations (which can provide a competitive advantage in evolving populations), they can limit exploration of the mutational landscape, restricting access to potentially stronger phenotypes in specific environments.

Phages are ubiquitous in nature, and bacteria with very different genomics, metabolisms, and lifestyles are subjected to their predation. Yet, the defence systems that allow bacteria to resist their phages have rarely been explored experimentally outside a very limited number of model organisms. Actinobacteria (Actinomycetota) are a phylum of GC-rich Gram-positive bacteria, which often produce an important diversity of secondary metabolites. Despite being ubiquitous in a wide range of environments, from soil to fresh and sea water but also the gut microbiome, relatively little is known about the anti-phage arsenal of Actinobacteria. In this work, we used DefenseFinder to systematically detect 131 anti-phage defence systems in 22803 fully sequenced prokaryotic genomes, among which are 2253 Actinobacteria of more than 700 species. We show that, like other bacteria, Actinobacteria encode many diverse anti-phage systems that are often encoded on mobile genetic elements. We further demonstrate that most detected defence systems are absent or rarer in Actinobacteria than in other bacteria, while a few rare systems are enriched (notably gp29-gp30 and Wadjet). We characterize the spatial distribution of anti-phage systems on   Streptomyces   chromosomes and show that some defence systems (e.g. RM systems) tend to be encoded in the core region, while others (e.g. Lamassu and Wadjet) are enriched towards the extremities. Overall, our results suggest that Actinobacteria might be a source of novel anti-phage systems and provide clues to characterize mechanistic aspects of known anti-phage systems.

Evolutionary innovation of transcription factors frequently drives phenotypic diversification and adaptation to environmental change. Transcription factors can gain or lose connections to target genes, resulting in novel regulatory responses and phenotypes. However the frequency of functional adaptation varies between different regulators, even when they are closely related. To identify factors influencing propensity for innovation, we utilise a   Pseudomonas fluorescens   SBW25 strain rendered incapable of flagellar mediated motility in soft-agar plates via deletion of the flagellar master regulator (fleQ). This bacterium can evolve to rescue flagellar motility via gene regulatory network rewiring of an alternative transcription factor to rescue activity of FleQ. Previously, we have identified two members (out of 22) of the RpoN-dependent enhancer binding protein (RpoN-EBP) family of transcription factors (NtrC and PFLU1132) that are capable of innovating in this way. These two transcription factors rescue motility repeatably and reliably in a strict hierarchy – with NtrC the only route in a ∆fleQ background, and PFLU1132 the only route in a ∆fleQ∆ntrC background. However, why other members in the same transcription factor family have not been observed to rescue flagellar activity is unclear. Previous work shows that protein homology cannot explain this pattern within the protein family (RpoN-EBPs), and mutations in strains that rescued motility suggested high levels of transcription factor expression and activation drive innovation. We predict that mutations that increase expression of the transcription factor are vital to unlock evolutionary potential for innovation. Here, we construct titratable expression mutant lines for 11 of the RpoN-EBPs in   P. fluorescens  . We show that in five additional RpoN-EBPs (FleR, HbcR, GcsR, DctD, AauR and PFLU2209), high expression levels result in different mutations conferring motility rescue, suggesting alternative rewiring pathways. Our results indicate that expression levels (and not protein homology) of RpoN-EBPs are a key constraining factor in determining evolutionary potential for innovation. This suggests that transcription factors that can achieve high expression through few mutational changes, or transcription factors that are active in the selective environment, are more likely to innovate and contribute to adaptive gene regulatory network evolution.

  Ralstonia solanacearum   species complex (RSSC) is a destructive group of plant pathogenic bacteria and the causative agent of bacterial wilt disease. Experimental studies have attributed RSSC virulence to insertion sequences (IS), transposable genetic elements which can both disrupt and activate host genes. Yet, the global diversity and distribution of RSSC IS are unknown. In this study, IS were bioinformatically identified in a diverse collection of 356 RSSC isolates representing five phylogenetic lineages and their diversity investigated based on genetic distance measures and comparisons with the ISFinder database. IS phylogenetic associations were determined based on their distribution across the RSSC phylogeny. Moreover, IS positions within genomes were characterised and their potential gene disruptions determined based on IS proximity to coding sequences. In total, we found 24732 IS belonging to eleven IS families and 26 IS subgroups with over half of the IS found in the megaplasmid. While IS families were generally widespread across the RSSC phylogeny, IS subgroups showed strong lineage-specific distributions and genetically similar bacterial isolates had similar IS contents. Similar associations with bacterial host genetic background were also observed with IS insertion positions which were highly conserved in closely related bacterial isolates. Finally, IS were found to disrupt genes with predicted functions in virulence, stress tolerance, and metabolism suggesting that they might be adaptive. This study highlights that RSSC insertion sequences track the evolution of their bacterial hosts potentially contributing to both intra- and inter-lineage genetic diversity.

Pharmacokinetic–pharmacodynamic (PKPD) models, which describe how drug concentrations change over time and how that affects pathogen growth, have proven highly valuable in designing optimal drug treatments aimed at bacterial eradication. However, the fast rise of antimicrobial resistance calls for increased focus on an additional treatment optimization criterion: avoidance of resistance evolution. We demonstrate here how coupling PKPD and population genetics models can be used to determine treatment regimens that minimize the potential for antimicrobial resistance evolution. Importantly, the resulting modelling framework enables the assessment of resistance evolution in response to dynamic selection pressures, including changes in antimicrobial concentration and the emergence of adaptive phenotypes. Using antibiotics and antimicrobial peptides as an example, we discuss the empirical evidence and intuition behind individual model parameters. We further suggest several extensions of this framework that allow a more comprehensive and realistic prediction of bacterial escape from antimicrobials through various phenotypic and genetic mechanisms.

Burkholderia cenocepacia infections are difficult to treat and there is an urgent need for alternative (combination) treatments. The use of anti-virulence therapies in combination with antibiotics is a possible strategy to increase the antimicrobial susceptibility of the pathogen and to slow down the development of resistance. In the present study we evaluated the β-lactam and colistin-potentiating activity, and anti-virulence effect of the non-mevalonate pathway inhibitor FR900098 against B. cenocepacia in various in vitro and in vivo models. In addition, we evaluated whether repeated exposure to FR900098 alone or when combined with ceftazidime leads to increased resistance. FR900098 potentiated the activity of colistin and several β-lactam antibiotics (aztreonam, cefepime, cefotaxime, ceftazidime, mecillinam and piperacillin) but not of imipenem and meropenem. When used alone or in combination with ceftazidime, FR900098 increased the survival of infected Galleria mellonella and Caenorhabditis elegans. Furthermore, combining ceftazidime with FR900098 resulted in a significant inhibition of the biofilm formation of B. cenocepacia . Repeated exposure to FR900098 in the C. elegans infection model did not lead to decreased activity, and the susceptibility of the evolved B. cenocepacia HI2424 lineages to ceftazidime, FR900098 and the combination of both remained unchanged. In conclusion, FR900098 reduces B. cenocepacia virulence and potentiates ceftazidime in an in vivo C. elegans model, and this activity is not lost during the experimental evolution experiment carried out in the present study.

Bacteria often evolve resistance to phage through the loss or modification of cell surface receptors. In   Escherichia coli   and phage λ, such resistance can catalyze a coevolutionary arms race focused on host and phage structures that interact at the outer membrane. Here, we analyse another facet of this arms race involving interactions at the inner membrane, whereby   E. coli   evolves mutations in mannose permease-encoding genes manY and manZ that impair λ’s ability to eject its DNA into the cytoplasm. We show that these man mutants arose concurrently with the arms race at the outer membrane. We tested the hypothesis that λ evolved an additional counter-defence that allowed them to infect bacteria with deleted man genes. The deletions severely impaired the ancestral λ, but some evolved phage grew well on the deletion mutants, indicating that they regained infectivity by evolving the ability to infect hosts independently of the mannose permease. This coevolutionary arms race fulfils the model of an inverse gene-for-gene infection network. Taken together, the interactions at both the outer and inner membranes reveal that coevolutionary arms races can be richer and more complex than is often appreciated.

In the opportunistic pathogen Pseudomonas aeruginosa, quorum sensing (QS) is a social trait that is exploitable by non-cooperating cheats. Previously it has been shown that by linking QS to the production of both public and private goods, cheats can be prevented from invading populations of cooperators and this was described by Dandekar et al. (Science 2012;338:264–266) as ‘a metabolic incentive to cooperate’. We hypothesized that P. aeruginosa could evolve novel cheating strategies to circumvent private goods metabolism by rewiring its combinatorial response to two QS signals (3O-C12-HSL and C4-HSL). We performed a selection experiment that cycled P. aeruginosa between public and private goods growth media and evolved an isolate that rewired its control of cooperative protease expression from a synergistic (AND-gate) response to dual-signal input to a 3O-C12-HSL-only response. We show that this isolate circumvents metabolic incentives to cooperate and acts as a combinatorial signalling cheat, with higher fitness in competition with its ancestor. Our results show three important principles: first, combinatorial QS allows for diverse social strategies to emerge; second, restrictions levied by private goods are not sufficient to explain the maintenance of cooperation in natural populations; and third, modifying combinatorial QS responses could result in important physiological outcomes in bacterial populations.

The acquisition of plasmids is often accompanied by fitness costs such that compensatory evolution is required to allow plasmid survival, but it is unclear whether compensatory evolution can be extensive or rapid enough to maintain plasmids when they are very costly. The mercury-resistance plasmid pQBR55 drastically reduced the growth of its host, Pseudomonas fluorescens SBW25, immediately after acquisition, causing a small colony phenotype. However, within 48 h of growth on agar plates we observed restoration of the ancestral large colony morphology, suggesting that compensatory mutations had occurred. Relative fitness of these evolved strains, in lab media and in soil microcosms, varied between replicates, indicating different mutational mechanisms. Using genome sequencing we identified that restoration was associated with chromosomal mutations in either a hypothetical DNA-binding protein PFLU4242, RNA polymerase or the GacA/S two-component system. Targeted deletions in PFLU4242, gacA or gacS recapitulated the ameliorated phenotype upon plasmid acquisition, indicating three distinct mutational pathways to compensation. Our data shows that plasmid compensatory evolution is fast enough to allow survival of a plasmid despite it imposing very high fitness costs upon its host, and indeed may regularly occur during the process of isolating and selecting individual plasmid-containing clones.

For micro-organisms cycling between free-living and host-associated stages, where reproduction occurs in both of these lifestyles, an interesting inquiry is whether evolution during the free-living stage can be positively pleiotropic to microbial fitness in a host environment. To address this topic, the squid host Euprymna tasmanica and the marine bioluminescent bacterium Vibrio fischeri were utilized. Microbial ecological diversification in static liquid microcosms was used to simulate symbiont evolution during the free-living stage. Thirteen genetically distinct V. fischeri strains from a broad diversity of ecological sources (e.g. squid light organs, fish light organs and seawater) were examined to see if the results were reproducible in many different genetic settings. Genetic backgrounds that are closely related can be predisposed to considerable differences in how they respond to similar selection pressures. For all strains examined, new mutations with striking and facilitating effects on host colonization arose quickly during microbial evolution in the free-living stage, regardless of the ecological context under consideration for a strain’s genetic background. Microbial evolution outside a host environment promoted host range expansion, improved host colonization for a micro-organism, and diminished the negative correlation between biofilm formation and motility.