Targeted Genome Editing of Virulent Pseudomonas Phages Using CRISPR-Cas3.

The vast number of unknown phage-encoded ORFan genes and limited insights into the genome organization of phages illustrate the need for efficient genome engineering tools to study bacteriophage genes in their natural context. In addition, there is an application-driven desire to alter phage properties, which is hampered by time constraints for phage genome engineering in the bacterial host. We here describe an optimized CRISPR-Cas3 system in Pseudomonas for straightforward editing of the genome of virulent bacteriophages. The two-vector system combines a broad host range CRISPR-Cas3 targeting plasmid with a SEVA plasmid for homologous directed repair, which enables the creation of clean deletions, insertions, or substitutions in the phage genome within a week. After creating the two plasmids separately, a co-transformation to P. aeruginosa cells is performed. A subsequent infection with the targeted phage allows the CRISPR-Cas3 system to cut the DNA specifically and facilitate or select for homologous recombination. This system has also been successfully applied for P. aeruginosa and Pseudomonas putida genome engineering. The method is straightforward, efficient, and universal, enabling to extrapolate the system to other phage-host pairs.

FLAMS: Find Lysine Acylations and other Modification Sites.

SUMMARY: Today, hundreds of post-translational modification (PTM) sites are routinely identified at once, but the comparison of new experimental datasets to already existing ones is hampered by the current inability to search most PTM databases at the protein residue level. We present FLAMS (Find Lysine Acylations and other Modification Sites), a Python3-based command line and web-tool that enables researchers to compare their PTM sites to the contents of the CPLM, the largest dedicated protein lysine modification database, and dbPTM, the most comprehensive general PTM database, at the residue level. FLAMS can be integrated into PTM analysis pipelines, allowing researchers to quickly assess the novelty and conservation of PTM sites across species in newly generated datasets, aiding in the functional assessment of sites and the prioritization of sites for further experimental characterization. AVAILABILITY AND IMPLEMENTATION: FLAMS is implemented in Python3, and freely available under an MIT license. It can be found as a command line tool at https://github.com/hannelorelongin/FLAMS, pip and conda; and as a web service at https://www.biw.kuleuven.be/m2s/cmpg/research/CSB/tools/flams/.

Posttranslational modifications in bacteria during phage infection.

During phage infection, both virus and bacteria attempt to gain and/or maintain control over critical bacterial functions, through a plethora of strategies. These strategies include posttranslational modifications (PTMs, including phosphorylation, ribosylation, and acetylation), as rapid and dynamic regulators of protein behavior. However, to date, knowledge on the topic remains scarce and fragmented, while a more systematic investigation lies within reach. The release of AlphaFold, which advances PTM enzyme discovery and functional elucidation, and the increasing inclusivity and scale of mass spectrometry applications to new PTM types, could significantly accelerate research in the field. In this review, we highlight the current knowledge on PTMs during phage infection, and conceive a possible pipeline for future research, following an enzyme-target-function scheme.

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 protein PIT2 impacts Pseudomonas aeruginosa quorum sensing by direct interaction with LasR.

In recent decades, there has been a notable increase in antibiotic-resistant Pseudomonas aeruginosa isolates, necessitating the development of innovative treatments to combat this pathogen. This manuscript explores the potential of different phage proteins to attenuate virulence factors of P. aeruginosa, particularly the type II secretion system (T2SS). PIT2, a protein derived from the lytic Pseudomonas phage LMA2 inhibits the T2SS effectors PrpL and LasA and attenuates the bacterial virulence toward HeLa cells and Galleria mellonella. Using RNAseq-based differential gene expression analysis, PIT2's impact on the LasR regulatory network is revealed, which plays a key role in bacterial quorum sensing. This discovery expands our knowledge on phage-encoded modulators of the bacterial metabolism and offers a promising anti-virulence target in P. aeruginosa. As such, it lays the foundation for a new phage-inspired anti-virulence strategy to combat multidrug resistant pathogens and opens the door for SynBio applications.

Metabolic reprogramming of Pseudomonas aeruginosa by phage-based quorum sensing modulation.

The Pseudomonas quinolone signal (PQS) is a multifunctional quorum sensing molecule of key importance to P. aeruginosa. Here, we report that the lytic Pseudomonas bacterial virus LUZ19 targets this population density-dependent signaling system by expressing quorum sensing targeting protein (Qst) early during infection. We demonstrate that Qst interacts with PqsD, a key host quinolone signal biosynthesis pathway enzyme, resulting in decreased levels of PQS and its precursor 2-heptyl-4(1H)-quinolone. The lack of a functional PqsD enzyme impairs LUZ19 infection but is restored by external supplementation of 2-heptyl-4(1H)-quinolone, suggesting that LUZ19 exploits the PQS system for successful infection. We establish a broad functional interaction network of Qst, which includes enzymes of cofactor biosynthesis pathways (CoaC/ThiD) and a non-ribosomal peptide synthetase pathway (PA1217). Qst therefore represents an exquisite example of intricate reprogramming of the bacterium by a phage, which may be further exploited as tool to combat antibiotic resistant bacterial pathogens.

qDNase assay: A quantitative method for real-time assessment of DNase activity on coated surfaces.

DNase coatings show great potential to prevent biofilm formation in various applications of the medical implant, food and marine industry. However, straightforward and quantitative methods to characterize the enzymatic activity of these coatings are currently not available. We here introduce the qDNase assay, a quantitative, real-time method to characterize the activity of DNase coatings. The assay combines (1) the use of an oligonucleotide probe, which fluoresces upon cleavage by coated DNases, and (2) the continuous read-out of the fluorescent signal within a microplate fluorometer format. The combination of these two properties results in a real-time fluorescent signal that is used to directly quantify the activity of DNase coatings. As a proof of concept, bovine DNase I coatings were immobilized on titanium by means of chemical grafting and their activity was estimated at 3.87 × 10-4 U. To our knowledge, the qDNase assay provides the first approach to report the activity of a DNase coating in absolute DNase activity units. This assay will not only serve to compare existing DNase coating methods more accurately, but will also enable the rational design of new DNase coating methods in the future.

Phage-based target discovery and its exploitation towards novel antibacterial molecules.

The deeply intertwined evolutionary history between bacteriophages and bacteria has endowed phages with highly specific mechanisms to hijack bacterial cell metabolism for their propagation. Here, we present a comprehensive, phage-driven strategy to reveal novel antibacterial targets by the exploitation of phage-bacteria interactions. This strategy will enable the design of small molecules, which mimic the inhibitory phage proteins, and allow the subsequent hit-to-lead development of these antimicrobial compounds. This proposed small molecule approach is distinct from phage therapy and phage enzyme-based antimicrobials and may produce a more sustainable generation of new antibiotics that exploit novel bacterial targets and act in a pathogen-specific manner.

Bacteriophage-mediated interference of the c-di-GMP signalling pathway in Pseudomonas aeruginosa.

C-di-GMP is a key signalling molecule which impacts bacterial motility and biofilm formation and is formed by the condensation of two GTP molecules by a diguanylate cyclase. We here describe the identification and characterization of a family of bacteriophage-encoded peptides that directly impact c-di-GMP signalling in Pseudomonas aeruginosa. These phage proteins target Pseudomonas diguanylate cyclase YfiN by direct protein interaction (termed YIPs, YfiN Interacting Peptides). YIPs induce an increase of c-di-GMP production in the host cell, resulting in a decrease in motility and an increase in biofilm mass in P. aeruginosa. A dynamic analysis of the biofilm morphology indicates a denser biofilm structure after induction of the phage protein. This intracellular signalling interference strategy by a lytic phage constitutes an unexplored phage-based mechanism of metabolic regulation and could potentially serve as inspiration for the development of molecules that interfere with biofilm formation in P. aeruginosa and other pathogens.

The Phage-Encoded N-Acetyltransferase Rac Mediates Inactivation of Pseudomonas aeruginosa Transcription by Cleavage of the RNA Polymerase Alpha Subunit.

In this study, we describe the biological function of the phage-encoded protein RNA polymerase alpha subunit cleavage protein (Rac), a predicted Gcn5-related acetyltransferase encoded by phiKMV-like viruses. These phages encode a single-subunit RNA polymerase for transcription of their late (structure- and lysis-associated) genes, whereas the bacterial RNA polymerase is used at the earlier stages of infection. Rac mediates the inactivation of bacterial transcription by introducing a specific cleavage in the α subunit of the bacterial RNA polymerase. This cleavage occurs within the flexible linker sequence and disconnects the C-terminal domain, required for transcription initiation from most highly active cellular promoters. To achieve this, Rac likely taps into a novel post-translational modification (PTM) mechanism within the host Pseudomonas aeruginosa. From an evolutionary perspective, this novel phage-encoded regulation mechanism confirms the importance of PTMs in the prokaryotic metabolism and represents a new way by which phages can hijack the bacterial host metabolism.

Phage S144, A New Polyvalent Phage Infecting Salmonella spp. and Cronobacter sakazakii.

Phages are generally considered species- or even strain-specific, yet polyvalent phages are able to infect bacteria from different genera. Here, we characterize the novel polyvalent phage S144, a member of the Loughboroughvirus genus. By screening 211 Enterobacteriaceae strains, we found that phage S144 forms plaques on specific serovars of Salmonella enterica subsp. enterica and on Cronobacter sakazakii. Analysis of phage resistant mutants suggests that the O-antigen of lipopolysaccharide is the phage receptor in both bacterial genera. The S144 genome consists of 53,628 bp and encodes 80 open reading frames (ORFs), but no tRNA genes. In total, 32 ORFs coding for structural proteins were confirmed by ESI-MS/MS analysis, whereas 45 gene products were functionally annotated within DNA metabolism, packaging, nucleotide biosynthesis and phage morphogenesis. Transmission electron microscopy showed that phage S144 is a myovirus, with a prolate head and short tail fibers. The putative S144 tail fiber structure is, overall, similar to the tail fiber of phage Mu and the C-terminus shows amino acid similarity to tail fibers of otherwise unrelated phages infecting Cronobacter. Since all phages in the Loughboroughvirus genus encode tail fibers similar to S144, we suggest that phages in this genus infect Cronobacter sakazakii and are polyvalent.

Isolation and Characterization of Pectobacterium Phage vB_PatM_CB7: New Insights into the Genus Certrevirus.

To date, Certrevirus is one of two genera of bacteriophage (phage), with phages infecting Pectobacterium atrosepticum, an economically important phytopathogen that causes potato blackleg and soft rot disease. This study provides a detailed description of Pectobacterium phage CB7 (vB_PatM_CB7), which specifically infects P. atrosepticum. Host range, morphology, latent period, burst size and stability at different conditions of temperature and pH were examined. Analysis of its genome (142.8 kbp) shows that the phage forms a new species of Certrevirus, sharing sequence similarity with other members, highlighting conservation within the genus. Conserved elements include a putative early promoter like that of the Escherichia coli sigma70 promoter, which was found to be shared with other genus members. A number of dissimilarities were observed, relating to DNA methylation and nucleotide metabolism. Some members do not have homologues of a cytosine methylase and anaerobic nucleotide reductase subunits NrdD and NrdG, respectively. Furthermore, the genome of CB7 contains one of the largest numbers of homing endonucleases described in a single phage genome in the literature to date, with a total of 23 belonging to the HNH and LAGLIDADG families. Analysis by RT-PCR of the HNH homing endonuclease residing within introns of genes for the large terminase, DNA polymerase, ribonucleotide reductase subunits NrdA and NrdB show that they are splicing competent. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was also performed on the virion of CB7, allowing the identification of 26 structural proteins-20 of which were found to be shared with the type phages of the genera of Vequintavirus and Seunavirus. The results of this study provide greater insights into the phages of the Certrevirus genus as well as the subfamily Vequintavirinae.

A Tailspike with Exopolysaccharide Depolymerase Activity from a New Providencia stuartii Phage Makes Multidrug-Resistant Bacteria Susceptible to Serum-Mediated Killing.

Providencia stuartii is emerging as a significant drug-resistant nosocomial pathogen, which encourages the search for alternative therapies. Here, we have isolated Providencia stuartii phage Stuart, a novel podovirus infecting multidrug-resistant hospital isolates of this bacterium. Phage Stuart is a proposed member of a new Autographivirinae subfamily genus, with a 41,218-bp genome, direct 345-bp repeats at virion DNA ends, and limited sequence similarity of proteins to proteins in databases. Twelve out of the 52 predicted Stuart proteins are virion components. We found one to be a tailspike with depolymerase activity. The tailspike could form a highly thermostable oligomeric ß-structure migrating close to the expected trimer in a nondenaturing gel. It appeared to be essential for the infection of three out of four P. stuartii hosts infected by phage Stuart. Moreover, it degraded the exopolysaccharide of relevant phage Stuart hosts, making the bacteria susceptible to serum killing. Prolonged exposure of a sensitive host to the tailspike did not cause the emergence of bacteria resistant to the phage or to serum killing, opposite to the prolonged exposure to the phage. This indicates that phage tail-associated depolymerases are attractive antivirulence agents that could complement the immune system in the fight with P. stuartiiIMPORTANCE The pace at which multidrug-resistant strains emerge has been alarming. P. stuartii is an infrequent but relevant drug-resistant nosocomial pathogen causing local to systemic life-threatening infections. We propose an alternative approach to fight this bacterium based on the properties of phage tailspikes with depolymerase activity that degrade the surface bacterial polymers, making the bacteria susceptible to the immune system. Unlike antibiotics, phage tailspikes have narrow and specific substrate spectra, and by acting as antivirulent but not bactericidal agents they do not cause the selection of resistant bacteria.

Analyzing Phage-Host Protein-Protein Interactions Using Strep-tag® II Purifications.

After injecting their genome into the bacterial host cell, bacteriophages need to convert the host metabolism toward efficient phage production. For this, specific proteins have evolved which interact with key host proteins to inhibit, activate or redirect the function of these proteins. Since 70% of the currently annotated phage genes are hypothetical proteins of unknown function, the identification and characterization of these phage proteins involved in host-phage protein-protein interactions remains challenging. Here, we describe a method to identify phage proteins involved in host-phage protein-protein interactions using a combination of affinity purifications and mass spectrometry analyses. A bacterial strain is engineered in which a bacterial target protein is fused to a Strep-tag® II at the C-terminal end. This strain is infected with a specific bacteriophage, followed by an affinity purification of the tagged protein which allows the copurification of all bacterial and phage specific interacting proteins. After SDS-PAGE analysis and an in-gel trypsin digestion, the purified interacting proteins are identified by mass spectrometry analysis. The identification of phage proteins involved in interactions provides first hints toward the elucidation of the biological function of these proteins.

Screening for Growth-Inhibitory ORFans in Pseudomonas aeruginosa-Infecting Bacteriophages.

Like all viruses, bacteriophages heavily depend on their host's physiology for reproduction. Therefore, phages have evolved numerous proteins that influence the host metabolism to facilitate the infection process. Some of these proteins strongly perturb the host cell, ultimately leading to cell death. These growth-inhibitory phage proteins presumably target key metabolic processes, which may provide a basis for innovative phage-derived antibacterials. Unfortunately, most of these proteins are the so-called ORFans, since they have no known function or sequence homology to any other gene. We here describe a screening method for the identification of growth-inhibitory ORFans of bacteriophages infecting gram-negative bacteria (e.g., Pseudomonas aeruginosa), using the pUC18-mini-Tn7T-Lac vector system, which allows for stable single-copy integration of the phage ORFans in the Pseudomonas genome under the control of an IPTG-inducible promoter. Furthermore, we describe a method to examine the effect of the phage proteins in different hosts, using different vector copy numbers. Finally, we explain how to investigate the effect of ORFan expression on the host morphology using time-lapse microscopy.

Novel N4-Like Bacteriophages of Pectobacterium atrosepticum.

Pectobacterium atrosepticum is an economically important phytopathogen that is responsible for potato blackleg and soft rot, and for which current control strategies are limited. In this study, stem samples of potato crops exhibiting blackleg were taken from three farms in Co. Cork, Ireland, and they were found to be infected with P. atrosepticum. Three closely related bacteriophages (phages) that are specific to this phytopathogen were isolated and characterized, namely vB_PatP_CB1, vB_PatP_CB3, and vB_PatP_CB4 (abbreviated as CB1, CB3, and CB4). Both CB1 and CB3 were determined to infect 12 strains and CB4 10 strains of the 19 strains of P. atrosepticum tested. Morphology, latent periods, burst sizes, and their stability at various temperatures and pHs were also examined. Genome sequencing of the three phages revealed that they shared a minimum nucleotide identity of 93% with each other. Their genomes exhibited an Enquartavirinae genome organization, possessing several conserved proteins that were associated with phages of this group, like the type species Escherichia virus N4. Tandem electrospray ionization-mass spectrometry (ESI-MS/MS) allowed for the identification of ten structural proteins that form the virion of CB1, six that are conserved in phage N4. Biocontrol experiments demonstrated that the phages suppress soft rot formation upon co-inoculation with P. atrosepticum on whole tubers. The results of this study indicate that CB1 related phages could be good candidates for phage-based control.

A Lytic Providencia rettgeri Virus of Potential Therapeutic Value Is a Deep-Branching Member of the T5virus Genus.

Providencia rettgeri is emerging as a new opportunistic pathogen with high antibiotic resistance. The need to find alternative methods to control antibiotic-resistant bacteria and the recent advances in phage therapy motivate the search for new phages able to infect Providencia spp. This study describes the isolation and characterization of an obligatory lytic phage, vB_PreS_PR1 (PR1), with therapeutic potential against drug-resistant P. rettgeri PR1 is a siphovirus. Its virion DNA size (118,537 bp), transcriptional organization, terminal repeats (10,461 bp), and nicks in the 3'-to-5' strand are similar to those of phage T5. However, sequence similarities of PR1 to phages of the T5virus genus at the DNA and protein levels are limited, suggesting that it belongs to a new species within the Siphoviridae family. PR1 exhibits the ability to kill P. rettgeri antibiotic-resistant strains, is highly specific to the species, and did not present known genomic markers indicating a temperate lifestyle. The lack of homologies between its proteins and proteins of the only other sequenced Providencia prophage, Redjac, suggests that these two phages evolved separately and may target different host proteins.IMPORTANCE The alarming increase in the number of bacteria resistant to antibiotics has been observed worldwide. This is particularly true for Gram-negative bacteria. For certain of their strains, no effective antibiotics are available. Providencia sp. has been a neglected pathogen but is emerging as a multidrug-resistant bacterium. This has revived interest in bacteriophages as alternative therapeutic agents against this bacterium. We describe the morphological, physiological, and genomic characterization of a novel lytic virus, PR1, which is able to kill drug-resistant P. rettgeri clinical isolates. Genomic and phylogenetic analyses indicate that PR1 is a distant relative of T5virus genus representatives. The lack of known virulence- or temperate lifestyle-associated genes in the genome of PR1 makes this phage a potential candidate for therapeutic use. Analysis of its genome also improves our knowledge of the ecology and diversity of T5-like siphoviruses, providing a new link for evolutionary studies of this phage group.

Pseudomonas predators: understanding and exploiting phage-host interactions.

Species in the genus Pseudomonas thrive in a diverse set of ecological niches and include crucial pathogens, such as the human pathogen Pseudomonas aeruginosa and the plant pathogen Pseudomonas syringae. The bacteriophages that infect Pseudomonas spp. mirror the widespread and diverse nature of their hosts. Therefore, Pseudomonas spp. and their phages are an ideal system to study the molecular mechanisms that govern virus-host interactions. Furthermore, phages are principal catalysts of host evolution and diversity, which directly affects the ecological roles of environmental and pathogenic Pseudomonas spp. Understanding these interactions not only provides novel insights into phage biology but also advances the development of phage therapy, phage-derived antimicrobial strategies and innovative biotechnological tools that may be derived from phage-bacteria interactions.

Characterization and genomic analyses of two newly isolated Morganella phages define distant members among Tevenvirinae and Autographivirinae subfamilies.

Morganella morganii is a common but frequent neglected environmental opportunistic pathogen which can cause deadly nosocomial infections. The increased number of multidrug-resistant M. morganii isolates motivates the search for alternative and effective antibacterials. We have isolated two novel obligatorily lytic M. morganii bacteriophages (vB_MmoM_MP1, vB_MmoP_MP2) and characterized them with respect to specificity, morphology, genome organization and phylogenetic relationships. MP1's dsDNA genome consists of 163,095 bp and encodes 271 proteins, exhibiting low DNA (<40%) and protein (<70%) homology to other members of the Tevenvirinae. Its unique property is a >10 kb chromosomal inversion that encompass the baseplate assembly and head outer capsid synthesis genes when compared to other T-even bacteriophages. MP2 has a dsDNA molecule with 39,394 bp and encodes 55 proteins, presenting significant genomic (70%) and proteomic identity (86%) but only to Morganella bacteriophage MmP1. MP1 and MP2 are then novel members of Tevenvirinae and Autographivirinae, respectively, but differ significantly from other tailed bacteriophages of these subfamilies to warrant proposing new genera. Both bacteriophages together could propagate in 23 of 27 M. morganii clinical isolates of different origin and antibiotic resistance profiles, making them suitable for further studies on a development of bacteriophage cocktail for potential therapeutic applications.

Things Are Getting Hairy: Enterobacteria Bacteriophage vB_PcaM_CBB.

Enterobacteria phage vB_PcaM_CBB is a "jumbo" phage belonging to the family Myoviridae. It possesses highly atypical whisker-like structures along the length of its contractile tail. It has a broad host range with the capability of infecting species of the genera Erwinia, Pectobacterium, and Cronobacter. With a genome of 355,922 bp, excluding a predicted terminal repeat of 22,456 bp, phage CBB is the third largest phage sequenced to date. Its genome was predicted to encode 554 ORFs with 33 tRNAs. Based on prediction and proteome analysis of the virions, 29% of its predicted ORFs could be functionally assigned. Protein comparison shows that CBB shares between 33-38% of its proteins with Cronobacter phage GAP32, coliphages PBECO4 and 121Q as well as Klebsiella phage vB_KleM_Rak2. This work presents a detailed and comparative analysis of vB_PcaM_CBB of a highly atypical jumbo myoviridae phage, contributing to a better understanding of phage diversity and biology.