tRNA epitranscriptome determines pathogenicity of the opportunistic pathogen Pseudomonas aeruginosa.

The success of bacterial pathogens depends on the coordinated expression of virulence determinants. Regulatory circuits that drive pathogenesis are complex, multilayered, and incompletely understood. Here, we reveal that alterations in tRNA modifications define pathogenic phenotypes in the opportunistic pathogen Pseudomonas aeruginosa. We demonstrate that the enzymatic activity of GidA leads to the introduction of a carboxymethylaminomethyl modification in selected tRNAs. Modifications at the wobble uridine base (cmnm5U34) of the anticodon drives translation of transcripts containing rare codons. Specifically, in P. aeruginosa the presence of GidA-dependent tRNA modifications modulates expression of genes encoding virulence regulators, leading to a cellular proteomic shift toward pathogenic and well-adapted physiological states. Our approach of profiling the consequences of chemical tRNA modifications is general in concept. It provides a paradigm of how environmentally driven tRNA modifications govern gene expression programs and regulate phenotypic outcomes responsible for bacterial adaption to challenging habitats prevailing in the host niche.

Distinct Long- and Short-Term Adaptive Mechanisms in Pseudomonas aeruginosa.

Heterogeneous environments such as the chronically infected cystic fibrosis lung drive the diversification of Pseudomonas aeruginosa populations into, e.g., mucoid, alginate-overproducing isolates or small-colony variants (SCVs). In this study, we performed extensive genome and transcriptome profiling on a clinical SCV isolate that exhibited high cyclic diguanylate (c-di-GMP) levels and a mucoid phenotype. We observed a delayed, stepwise decrease of the high levels of c-di-GMP as well as alginate gene expression upon passaging the SCV under noninducing, rich medium growth conditions over 7 days. Upon prolonged passaging, this lagging reduction of the high c-di-GMP levels under noninducing planktonic conditions (reminiscent of a hysteretic response) was followed by a phenotypic switch to a large-colony morphology, which could be linked to mutations in the Gac/Rsm signaling pathway. Complementation of the Gac/Rsm signaling-negative large-colony variants with a functional GacSA system restored the SCV colony morphotype but was not able to restore the high c-di-GMP levels of the SCV. Our data thus suggest that expression of the SCV colony morphotype and modulation of c-di-GMP levels are genetically separable and follow different evolutionary paths. The delayed switching of c-di-GMP levels in response to fluctuating environmental conditions might provide a unique opportunity to include a time dimension to close the gap between short-term phenotypic and long-term genetic adaptation to biofilm-associated growth conditions. IMPORTANCE Extreme environments, such as those encountered during an infection process in the human host, make effective bacterial adaptation inevitable. While bacteria adapt individually by activating stress responses, long-term adaptation of bacterial communities to challenging conditions can be achieved via genetic fixation of favorable traits. In this study, we describe a two-pronged bacterial stress resistance strategy in the opportunistic pathogen Pseudomonas aeruginosa. We show that the production of adjusted elevated c-di-GMP levels, which drive protected biofilm-associated phenotypes in vivo, resembles a stable hysteretic response which prevents unwanted frequent switching. Cellular hysteresis might provide a link between individual adaptability and evolutionary adaptation to ensure the evolutionary persistence of host-adapted stress response strategies.

Evolution of Pseudomonas aeruginosa toward higher fitness under standard laboratory conditions.

Identifying genetic factors that contribute to the evolution of adaptive phenotypes in pathogenic bacteria is key to understanding the establishment of infectious diseases. In this study, we performed mutation accumulation experiments to record the frequency of mutations and their effect on fitness in hypermutator strains of the environmental bacterium Pseudomonas aeruginosa in comparison to the host-niche-adapted Salmonella enterica. We demonstrate that P. aeruginosa, but not S. enterica, hypermutators evolve toward higher fitness under planktonic conditions. Adaptation to increased growth performance was accompanied by a reversible perturbing of the local genetic context of membrane and cell wall biosynthesis genes. Furthermore, we observed a fine-tuning of complex regulatory circuits involving multiple di-guanylate modulating enzymes that regulate the transition between fast growing planktonic and sessile biofilm-associated lifestyles. The redundancy and local specificity of the di-guanylate signaling pathways seem to allow a convergent shift toward increased growth performance across niche-adapted clonal P. aeruginosa lineages, which is accompanied by a pronounced heterogeneity of their motility, virulence, and biofilm phenotypes.

Untargeted LC-MS Metabolomics Differentiates Between Virulent and Avirulent Clinical Strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a facultative pathogen that can cause, inter alia, acute or chronic pneumonia in predisposed individuals. The gram-negative bacterium displays considerable genomic and phenotypic diversity that is also shaped by small molecule secondary metabolites. The discrimination of virulence phenotypes is highly relevant to the diagnosis and prognosis of P. aeruginosa infections. In order to discover small molecule metabolites that distinguish different virulence phenotypes of P. aeruginosa, 35 clinical strains were cultivated under standard conditions, characterized in terms of virulence and biofilm phenotype, and their metabolomes were investigated by untargeted liquid chromatography-mass spectrometry. The data was both mined for individual candidate markers as well as used to construct statistical models to infer the virulence phenotype from metabolomics data. We found that clinical strains that differed in their virulence and biofilm phenotype also had pronounced divergence in their metabolomes, as underlined by 332 features that were significantly differentially abundant with fold changes greater than 1.5 in both directions. Important virulence-associated secondary metabolites like rhamnolipids, alkyl quinolones or phenazines were found to be strongly upregulated in virulent strains. In contrast, we observed little change in primary metabolism. A hitherto novel cationic metabolite with a sum formula of C12H15N2 could be identified as a candidate biomarker. A random forest model was able to classify strains according to their virulence and biofilm phenotype with an area under the Receiver Operation Characteristics curve of 0.84. These findings demonstrate that untargeted metabolomics is a valuable tool to characterize P. aeruginosa virulence, and to explore interrelations between clinically important phenotypic traits and the bacterial metabolome.

Single-Nucleotide Polymorphism-Based Genetic Diversity Analysis of Clinical Pseudomonas aeruginosa Isolates.

Extensive use of next-generation sequencing has the potential to transform our knowledge on how genomic variation within bacterial species impacts phenotypic versatility. Because different environments have unique selection pressures, they drive divergent evolution. However, there is also parallel or convergent evolution of traits in independent bacterial isolates inhabiting similar environments. The application of tools to describe population-wide genomic diversity provides an opportunity to measure the predictability of genetic changes underlying adaptation. Here, we describe patterns of sequence variations in the core genome among 99 individual Pseudomonas aeruginosa clinical isolates and identified single-nucleotide polymorphisms that are the basis for branching of the phylogenetic tree. We also identified single-nucleotide polymorphisms that were acquired independently, in separate lineages, and not through inheritance from a common ancestor. Although our results demonstrate that the Pseudomonas aeruginosa core genome is highly conserved and in general, not subject to adaptive evolution, instances of parallel evolution will provide an opportunity to uncover genetic changes that underlie phenotypic diversity.

Genetically diverse Pseudomonas aeruginosa populations display similar transcriptomic profiles in a cystic fibrosis explanted lung.

Previous studies have demonstrated substantial genetic diversification of Pseudomonas aeruginosa across sub-compartments in cystic fibrosis (CF) lungs. Here, we isolate P. aeruginosa from five different sampling areas in the upper and lower airways of an explanted CF lung, analyze ex vivo transcriptional profiles by RNA-seq, and use colony re-sequencing and deep population sequencing to determine the genetic diversity within and across the various sub-compartments. We find that, despite genetic variation, the ex vivo transcriptional profiles of P. aeruginosa populations inhabiting different regions of the CF lung are similar. Although we cannot estimate the extent to which the transcriptional response recorded here actually reflects the in vivo transcriptomes, our results indicate that there may be a common in vivo transcriptional profile in the CF lung environment.

Establishment of an induced memory response in Pseudomonas aeruginosa during infection of a eukaryotic host.

In a given habitat, bacterial cells often experience recurrent exposures to the same environmental stimulus. The ability to memorize the past event and to adjust current behaviors can lead to efficient adaptation to the recurring stimulus. Here we demonstrate that the versatile bacterium Pseudomonas aeruginosa adopts a virulence phenotype after serial passage in the invertebrate model host Galleria mellonella. The virulence phenotype was not linked to the acquisition of genetic variations and was sustained for several generations, despite cultivation of the ex vivo virulence-adapted P. aeruginosa cells under rich medium conditions in vitro. Transcriptional reprogramming seemed to be induced by a host-specific food source, as reprogramming was also observed upon cultivation of P. aeruginosa in rich medium supplemented with polyunsaturated long-chain fatty acids. The establishment of induced memory responses adds a time dimension and seems to fill the gap between long-term evolutionary genotypic adaptation and short-term induced individual responses. Efforts to unravel the fundamental mechanisms that underlie the carry-over effect to induce such memory responses will continue to be of importance as hysteretic behavior can serve survival of bacterial populations in changing and challenging habitats.

BACTOME-a reference database to explore the sequence- and gene expression-variation landscape of Pseudomonas aeruginosa clinical isolates.

Extensive use of next-generation sequencing (NGS) for pathogen profiling has the potential to transform our understanding of how genomic plasticity contributes to phenotypic versatility. However, the storage of large amounts of NGS data and visualization tools need to evolve to offer the scientific community fast and convenient access to these data. We introduce BACTOME as a database system that links aligned DNA- and RNA-sequencing reads of clinical Pseudomonas aeruginosa isolates with clinically relevant pathogen phenotypes. The database allows data extraction for any single isolate, gene or phenotype as well as data filtering and phenotypic grouping for specific research questions. With the integration of statistical tools we illustrate the usefulness of a relational database structure for the identification of phenotype-genotype correlations as an essential part of the discovery pipeline in genomic research. Furthermore, the database provides a compilation of DNA sequences and gene expression values of a plethora of clinical isolates to give a consensus DNA sequence and consensus gene expression signature. Deviations from the consensus thereby describe the genomic landscape and the transcriptional plasticity of the species P. aeruginosa. The database is available at https://bactome.helmholtz-hzi.de.

Analysis of Posttranslational Activation of Alternative Oxidase Isoforms.

Mitochondrial alternative oxidase (AOX) in plants is a non-proton-motive ubiquinol oxidase that is activated by redox mechanisms and 2-oxo acids. A comparative analysis of the AOX isoenzymes AOX1A, AOX1C, and AOX1D from Arabidopsis (Arabidopsis thaliana) revealed that cysteine residues, CysI and CysII, are both involved in 2-oxo acid activation, with AOX1A activity being more increased by 2-oxo acids than that of AOX1C and AOX1D. Substitution of cysteine in AOX1A by glutamate mimicked its activation by pyruvate or glyoxylate, but not in AOX1C and AOX1D. CysIII, only present in AOX1A, is not involved in activation by reduction or metabolites, but substitutions at this position affected activity. AOX1A carrying a serine residue at position CysI was activated by succinate, while correspondingly substituted variants of AOX1C and AOX1D were insensitive. Activation by glutamate at CysI and CysII is consistent with the formation of the thiohemiacetal, while succinate activation after changing CysI to serine suggests hemiacetal formation. Surprisingly, in AOX1A, replacement of CysI by alanine, which cannot form a (thio)hemiacetal, led to even higher activities, pointing to an alternative mechanism of activation. Taken together, our results demonstrate that AOX isoforms are differentially activated and that activation at CysI and CysII is additive.

Identification of a Pseudomonas aeruginosa PAO1 DNA Methyltransferase, Its Targets, and Physiological Roles.

DNA methylation is widespread among prokaryotes, and most DNA methylation reactions are catalyzed by adenine DNA methyltransferases, which are part of restriction-modification (R-M) systems. R-M systems are known for their role in the defense against foreign DNA; however, DNA methyltransferases also play functional roles in gene regulation. In this study, we used single-molecule real-time (SMRT) sequencing to uncover the genome-wide DNA methylation pattern in the opportunistic pathogen Pseudomonas aeruginosa PAO1. We identified a conserved sequence motif targeted by an adenine methyltransferase of a type I R-M system and quantified the presence of N6-methyladenine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes in the PAO1 methylation status were dependent on growth conditions and affected P. aeruginosa pathogenicity in a Galleria mellonella infection model. Furthermore, we found that methylated motifs in promoter regions led to shifts in sense and antisense gene expression, emphasizing the role of enzymatic DNA methylation as an epigenetic control of phenotypic traits in P. aeruginosa Since the DNA methylation enzymes are not encoded in the core genome, our findings illustrate how the acquisition of accessory genes can shape the global P. aeruginosa transcriptome and thus may facilitate adaptation to new and challenging habitats.IMPORTANCE With the introduction of advanced technologies, epigenetic regulation by DNA methyltransferases in bacteria has become a subject of intense studies. Here we identified an adenosine DNA methyltransferase in the opportunistic pathogen Pseudomonas aeruginosa PAO1, which is responsible for DNA methylation of a conserved sequence motif. The methylation level of all target sequences throughout the PAO1 genome was approximated to be in the range of 65 to 85% and was dependent on growth conditions. Inactivation of the methyltransferase revealed an attenuated-virulence phenotype in the Galleria mellonella infection model. Furthermore, differential expression of more than 90 genes was detected, including the small regulatory RNA prrF1, which contributes to a global iron-sparing response via the repression of a set of gene targets. Our finding of a methylation-dependent repression of the antisense transcript of the prrF1 small regulatory RNA significantly expands our understanding of the regulatory mechanisms underlying active DNA methylation in bacteria.

Unravelling post-transcriptional PrmC-dependent regulatory mechanisms in Pseudomonas aeruginosa.

Transcriptional regulation has a central role in cellular adaptation processes and is well investigated. In contrast, the importance of the post-transcriptional regulation on these processes is less well defined. The technological advancements have been critical to precisely quantify protein and mRNA level changes and hold promise to provide more insights into how post-transcriptional regulation determines phenotypes. In Pseudomonas aeruginosa the methyltransferase PrmC methylates peptide chain release factors to facilitate translation termination. Loss of PrmC activity abolishes anaerobic growth and leads to reduced production of quorum sensing-associated virulence factors. Here, by applying SILAC technology in combination with mRNA-sequencing, they provide evidence that the P. aeruginosa phenotype can be attributed to a change in protein to mRNA ratios of selected protein groups. The UAG-dependent translation termination was more dependent on PrmC activity than the UAA- and UGA-dependent translation termination. Additionally, a bias toward UAG stop codons in global transcriptional regulators was found. The finding that this bias in stop codon usage determines the P. aeruginosa phenotype is unexpected and adds complexity to regulatory circuits. Via modulation of PrmC activity the bacterial cell can cross-regulate targets independently of transcriptional signals, a process with an underestimated impact on the bacterial phenotype.