Rapid evolutionary tuning of endospore quantity versus quality trade-off via a phase-variable contingency locus.

The UV resistance of bacterial endospores is an important quality supporting their survival in inhospitable environments and therefore constitutes an essential driver of the ecological success of spore-forming bacteria. Nevertheless, the variability and evolvability of this trait are poorly understood. In this study, directed evolution and genetics approaches revealed that the Bacillus cereus pdaA gene (encoding the endospore-specific peptidoglycan-N-acetylmuramic acid deacetylase) serves as a contingency locus in which the expansion and contraction of short tandem repeats can readily compromise (PdaAOFF) or restore (PdaAON) the pdaA open reading frame. Compared with B. cereus populations in the PdaAON state, populations in the PdaAOFF state produced a lower yield of viable endospores but endowed them with vastly increased UV resistance. Moreover, selection pressures based on either quantity (i.e., yield of viable endospores) or quality (i.e., UV resistance of viable endospores) aspects could readily shift populations between PdaAON and PdaAOFF states, respectively. Bioinformatic analysis also revealed that pdaA homologs within the Bacillus and Clostridium genera are often equipped with several short tandem repeat regions, suggesting a wider implementation of the pdaA-mediated phase variability in other sporeformers as well. These results for the first time reveal (1) pdaA as a phase-variable contingency locus in the adaptive evolution of endospore properties and (2) bet-hedging between what appears to be a quantity versus quality trade-off in endospore crops.

Phage-host co-evolution has led to distinct generalized transduction strategies.

Generalized transduction is pivotal in bacterial evolution but lacks comprehensive understanding regarding the facilitating features and variations among phages. We addressed this gap by sequencing and comparing the transducing particle content of three different Salmonella Typhimurium phages (i.e. Det7, ES18 and P22) that share a headful packaging mechanism that is typically initiated from a cognate pac site within the phage chromosome. This revealed substantial disparities in both the extent and content of transducing particles among these phages. While Det7 outperformed ES18 in terms of relative number of transducing particles, both phages contrasted with P22 in terms of content. In fact, we found evidence for the presence of conserved P22 pac-like sequences in the host chromosome that direct tremendously increased packaging and transduction frequencies of downstream regions by P22. More specifically, a ca. 561 kb host region between oppositely oriented pac-like sequences in the purF and minE loci was identified as highly packaged and transduced during both P22 prophage induction and lytic infection. Our findings underscore the evolution of phage transducing capacity towards attenuation, promiscuity or directionality, and suggest that pac-like sequences in the host chromosome could become selected as sites directing high frequency of transduction.

A SEVA-based, CRISPR-Cas3-assisted genome engineering approach for Pseudomonas with efficient vector curing.

IMPORTANCE: The CRISPR-Cas3 editing system as presented here facilitates the creation of genomic alterations in Pseudomonas putida and Pseudomonas aeruginosa in a straightforward manner. By providing the Cas3 system as a vector set with Golden Gate compatibility and different antibiotic markers, as well as by employing the established Standard European Vector Architecture (SEVA) vector set to provide the homology repair template, this system is flexible and can readily be ported to a multitude of Gram-negative hosts. Besides genome editing, the Cas3 system can also be used as an effective and universal tool for vector curing. This is achieved by introducing a spacer that targets the origin-of-transfer, present on the majority of established (SEVA) vectors. Based on this, the Cas3 system efficiently removes up to three vectors in only a few days. As such, this curing approach may also benefit other genomic engineering methods or remove naturally occurring plasmids from bacteria.

The phage-encoded PIT4 protein affects multiple two-component systems of Pseudomonas aeruginosa.

IMPORTANCE: More and more Pseudomonas aeruginosa isolates have become resistant to antibiotics like carbapenem. As a consequence, P. aeruginosa ranks in the top three of pathogens for which the development of novel antibiotics is the most crucial. The pathogen causes both acute and chronic infections, especially in patients who are the most vulnerable. Therefore, efforts are urgently needed to develop alternative therapies. One path explored in this article is the use of bacteriophages and, more specifically, phage-derived proteins. In this study, a phage-derived protein was studied that impacts key virulence factors of the pathogen via interaction with multiple histidine kinases of TCSs. The fundamental insights gained for this protein can therefore serve as inspiration for the development of an anti-virulence compound that targets the bacterial TCS.

Assessing the Orthogonality of Phage-Encoded RNA Polymerases for Tailored Synthetic Biology Applications in Pseudomonas Species.

The phage T7 RNA polymerase (RNAP) and lysozyme form the basis of the widely used pET expression system for recombinant expression in the biotechnology field and as a tool in microbial synthetic biology. Attempts to transfer this genetic circuitry from Escherichia coli to non-model bacterial organisms with high potential have been restricted by the cytotoxicity of the T7 RNAP in the receiving hosts. We here explore the diversity of T7-like RNAPs mined directly from Pseudomonas phages for implementation in Pseudomonas species, thus relying on the co-evolution and natural adaptation of the system towards its host. By screening and characterizing different viral transcription machinery using a vector-based system in P. putida., we identified a set of four non-toxic phage RNAPs from phages phi15, PPPL-1, Pf-10, and 67PfluR64PP, showing a broad activity range and orthogonality to each other and the T7 RNAP. In addition, we confirmed the transcription start sites of their predicted promoters and improved the stringency of the phage RNAP expression systems by introducing and optimizing phage lysozymes for RNAP inhibition. This set of viral RNAPs expands the adaption of T7-inspired circuitry towards Pseudomonas species and highlights the potential of mining tailored genetic parts and tools from phages for their non-model host.

Preparing for the KIL: Receptor Analysis of Pseudomonas syringae pv. porri Phages and Their Impact on Bacterial Virulence.

The prevalence of Pseudomonas syringae pv. porri (Pspo) in Belgium continues to increase and sustainable treatments for this pathogen remain unavailable. A potentially attractive biocontrol strategy would be the application of bacteriophages. The ideal application strategy of phages in an agricultural setting remains unclear, especially in a field-based production such as for leek plants in Flanders. Therefore, more insight in bacteria-phage interaction is required, along with the evaluation of different application strategies. In this study, we further characterized the infection strategy of two Pspo phages, KIL3b and KIL5. We found that both phages recognize lipopolysaccharide (LPS) moieties on the surface of the bacterium. LPS is an important pathogenicity factor of Pspo. Our data also suggest that KIL5 requires an additional protein in the bacterial cytoplasmatic membrane to efficiently infect its host. Virulence tests showed that this protein also contributes to Pspo virulence. Furthermore, a cocktail of both phages was applied in a seed bioassay. A combination of KIL3b and KIL5 reduced the bacterial concentration 100-fold. However, in vitro Pspo resistance against phage infection developed quite rapidly. However, the impact of this phage resistance might be mitigated as is suggested by the fact that those resistance mutations preferably occur in genes involved in LPS metabolism, and that the virulence of those mutants is possibly reduced. Our data suggest that the phage cocktail has promising potential to lower the prevalence of Pspo and to be integrated in a pest management strategy. Targeted research is needed to further explore the applicability of the phages in combination with other disease control strategies.