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Type IV pili (T4P) are thin, flexible filaments exposed on the cell surface of gram-negative bacteria and are involved in pathogenesis-related processes, including cell adsorption, biofilm formation, and twitching motility. Bacteriophages often use these filaments as receptors to infect host cells. Here, we describe the identification of a protein that inhibits T4P assembly in Pseudomonas aeruginosa, discovered during a screen for host factors influencing phage infection. We show that expression of PA2560 (renamed PlzR) in P. aeruginosa inhibits adsorption of T4P-dependent phages. PlzR does this by directly binding the T4P chaperone PilZ, which in turn regulates the ATPase PilB and results in disturbed T4P assembly. As the plzR promoter is induced by cyclic di-GMP, PlzR might play a role in coupling T4P function to levels of this second messenger., (© 2024. The Author(s).)

Spread of multidrug-resistant Pseudomonas aeruginosa strains threatens to render currently available antibiotics obsolete, with limited prospects for the development of new antibiotics. Lytic bacteriophages, the viruses of bacteria, represent a path to combat this threat. In vitro-directed evolution is traditionally applied to expand the bacteriophage host range or increase bacterial suppression in planktonic cultures. However, while up to 80% of human microbial infections are biofilm-associated, research towards targeted improvement of bacteriophages' ability to combat biofilms remains scarce. This study aims at an in vitro biofilm evolution assay to improve multiple bacteriophage parameters in parallel and the optimisation of bacteriophage cocktail design by exploiting a bacterial bacteriophage resistance trade-off. The evolved bacteriophages show an expanded host spectrum, improved antimicrobial efficacy and enhanced antibiofilm performance, as assessed by isothermal microcalorimetry and quantitative polymerase chain reaction, respectively. Our two-phage cocktail reveals further improved antimicrobial efficacy without incurring dual-bacteriophage-resistance in treated bacteria. We anticipate this assay will allow a better understanding of phenotypic-genomic relationships in bacteriophages and enable the training of bacteriophages against other desired pathogens. This, in turn, will strengthen bacteriophage therapy as a treatment adjunct to improve clinical outcomes of multidrug-resistant bacterial infections., (© 2024. The Author(s).)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

RNA sequencing of phage-infected bacterial cultures offers a snapshot of transcriptional events occurring during the infection process, providing insights into the phage transcriptional organization as well as the bacterial response. To better mimic real environmental contexts, we performed RNA-seq of Pseudomonas aeruginosa PAO1 cultures infected with phage LUZ19 in a mammalian cell culture medium to better simulate a phage therapy event and the data were compared to lysogeny broth medium. Regardless of the media, phage LUZ19 induces significant transcriptional changes in the bacterial host over time, particularly during early infection ( t = 5 min) and gradually shuts down bacterial transcription. In a common response in both media, 56 P. aeruginosa PAO1 genes are differentially transcribed and clustered into several functional categories such as metabolism, translation and transcription. Our data allowed us to tease apart a medium-specific response during infection from the identified infection-associated responses. This reinforces the concept that phages overtake bacterial transcriptome in a strict manner to gain control of the bacterial machinery and reallocate resources for infection, in this case overcoming the nutritional limitations of the mammalian cell culture medium. From a phage therapy perspective, this study contributes towards a better understanding of phage-host interaction in human physiological conditions and demonstrates the versatility of phage LUZ19 to adapt to different environments.

The bacterial DNA gyrase complex (GyrA/GyrB) plays a crucial role during DNA replication and serves as a target for multiple antibiotics, including the fluoroquinolones. Despite it being a valuable antibiotics target, resistance emergence by pathogens including Pseudomonas aeruginosa are proving problematic. Here, we describe Igy, a peptide inhibitor of gyrase, encoded by Pseudomonas bacteriophage LUZ24 and other members of the Bruynoghevirus genus. Igy (5.6 kDa) inhibits in vitro gyrase activity and interacts with the P. aeruginosa GyrB subunit, possibly by DNA mimicry, as indicated by a de novo model of the peptide and mutagenesis. In vivo, overproduction of Igy blocks DNA replication and leads to cell death also in fluoroquinolone-resistant bacterial isolates. These data highlight the potential of discovering phage-inspired leads for antibiotics development, supported by co-evolution, as Igy may serve as a scaffold for small molecule mimicry to target the DNA gyrase complex, without cross-resistance to existing molecules., Competing Interests: Declaration of interests The authors declare no conflict of interest., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

As part of the ongoing renaissance of phage biology, more phage genomes are becoming available through DNA sequencing. However, our understanding of the transcriptome architecture that allows these genomes to be expressed during host infection is generally poor. Transcription start sites (TSSs) and operons have been mapped for very few phages, and an annotated global RNA map of a phage - alone or together with its infected host - is not available at all. Here, we applied differential RNA-seq (dRNA-seq) to study the early, host takeover phase of infection by assessing the transcriptome structure of Pseudomonas aeruginosa jumbo phage ɸKZ, a model phage for viral genetics and structural research. This map substantially expands the number of early expressed viral genes, defining TSSs that are active ten minutes after ɸKZ infection. Simultaneously, we record gene expression changes in the host transcriptome during this critical metabolism conversion. In addition to previously reported upregulation of genes associated with amino acid metabolism, we observe strong activation of genes with functions in biofilm formation ( cdrAB ) and iron storage ( bfrB ), as well as an activation of the antitoxin ParD. Conversely, ɸKZ infection rapidly down-regulates complexes IV and V of oxidative phosphorylation ( atpCDGHF and cyoABCDE ). Taken together, our data provide new insights into the transcriptional organization and infection process of the giant bacteriophage ɸKZ and adds a framework for the genome-wide transcriptomic analysis of phage-host interactions.

The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and posttranscriptional regulation through the RNA-binding proteins Hfq, RsmA, and RsmN have been studied in detail, global information about stable RNA-protein complexes in this human pathogen is currently lacking. Here, we implement gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73% of all transcripts and ∼40% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa , including 6S RNA with RNA polymerase and associated product RNAs (pRNAs). We observe specific clusters of noncoding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain-containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased cosedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded noncoding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell. IMPORTANCE Stable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbor some of the largest genomes known in bacteria, encoding ∼5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong colocalization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser., (Copyright © 2021 Gerovac et al.)

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.

The emergence of phage-resistant mutants is a key aspect of lytic phages-bacteria interaction and the main driver for the co-evolution between both organisms. Here, we analyze the impact of PA5oct jumbo phage treatment on planktonic/cell line associated and sessile P. aeruginosa population. Besides its broad-spectrum activity and efficient bacteria reduction in both airway surface liquid (ASL) model, and biofilm matrix degradation, PA5oct appears to persist in most of phage-resistant clones. Indeed, a high percentage of resistance (20/30 clones) to PA5oct is accompanied by the presence of phage DNA within bacterial culture. Moreover, the maintenance of this phage in the bacterial population correlates with reduced P. aeruginosa virulence, coupled with a sensitization to innate immune mechanisms, and a significantly reduced growth rate. We observed rather unusual consequences of PA5oct infection causing an increased inflammatory response of monocytes to P. aeruginosa . This phenomenon, combined with the loss or modification of the phage receptor, makes most of the phage-resistant clones significantly less pathogenic in in vivo model. These findings provide new insights into the general knowledge of giant phages biology and the impact of their application in phage therapy.

Although the evolution of tailed bacteriophages has increasingly been better understood through comparisons of their DNA sequences, the functional consequences of this evolution on phage infectious strategies have remained unresolved. In this study, we comprehensively compared the transcriptional strategies of two related myoviruses, PAK_P3 and PAK_P4, infecting the same Pseudomonas aeruginosa host strain. Outside of the conservation of their structural clusters, their highly syntenic genomes display only limited DNA similarity. Despite this apparent divergence, we found that both viruses follow a similar infection scheme, relying on a temporal regulation of their gene expression, likely involving the use of antisense transcripts, as well as a rapid degradation of 90% of the host non-ribosomal mRNA, as previously reported for PAK_P3. However, the kinetics of the mRNA degradation is remarkably faster during PAK_P4 infection. Moreover, we found that each virus has evolved specific adaptations, as exemplified by the distinct patterns of their core genes expression as well as the specific manipulation of the expression of iron-related host genes by PAK_P4. This study enhances our understanding of the evolutionary process of virulent phages, which relies on adjusting globally conserved ancestral infection mechanisms.

Bacterial endotoxins have high immunogenicity. Phage biology studies as well as therapeutic phage applications necessitate highly purified phage particles. In this study, we compared combinations of seven different endotoxin removal strategies and validated their endotoxin removal efficacy for five different phages (i.e. four Pseudomonas aeruginosa phages and one Staphylococcus aureus phage). These purification strategies included Endotrap HD column purification and/or CsCl density centrifugation in combination with Endotrap purification, followed by organic solvent (1-octanol), detergent (Triton X-100), enzymatic inactivation of the endotoxin using alkaline phosphatase and CIM monolytic anion exchange chromatography. We show that CsCl density purification of the P. aeruginosa phages, at an initial concentration of 10 12 -10 13 pfu/ml, led to the strongest reduction of endotoxins, with an endotoxin removal efficacy of up to 99%, whereas additional purification methods did not result in a complete removal of endotoxins from the phage preparations and only yielded an additional endotoxin removal efficacy of 23 to 99%, sometimes accompanied with strong losses in phage titer., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Phage-mediated metabolic changes in bacteria are hypothesized to markedly alter global nutrient and biogeochemical cycles. Despite their theoretic importance, experimental data on the net metabolic impact of phage infection on the bacterial metabolism remains scarce. In this study, we tracked the dynamics of intracellular metabolites using untargeted high coverage metabolomics in Pseudomonas aeruginosa cells infected with lytic bacteriophages from six distinct phage genera. Analysis of the metabolomics data indicates an active interference in the host metabolism. In general, phages elicit an increase in pyrimidine and nucleotide sugar metabolism. Furthermore, clear phage-specific and infection stage-specific responses are observed, ranging from extreme metabolite depletion (for example, phage YuA) to complete reorganization of the metabolism (for example, phage phiKZ). As expected, pathways targeted by the phage-encoded auxiliary metabolic genes (AMGs) were enriched among the metabolites changing during infection. The effect on pyrimidine metabolism of phages encoding AMGs capable of host genome degradation (for example, YuA and LUZ19) was distinct from those lacking nuclease-encoding genes (for example, phiKZ), which demonstrates the link between the encoded set of AMGs of a phage and its impact on host physiology. However, a large fraction of the profound effect on host metabolism could not be attributed to the phage-encoded AMGs. We suggest a potentially crucial role for small, 'non-enzymatic' peptides in metabolism take-over and hypothesize on potential biotechnical applications for such peptides. The highly phage-specific nature of the metabolic impact emphasizes the potential importance of the 'phage diversity' parameter when studying metabolic interactions in complex communities.

In all domains of life, the catalysed degradation of RNA facilitates rapid adaptation to changing environmental conditions, while destruction of foreign RNA is an important mechanism to prevent host infection. We have identified a virus-encoded protein termed gp37/Dip, which directly binds and inhibits the RNA degradation machinery of its bacterial host. Encoded by giant phage фKZ, this protein associates with two RNA binding sites of the RNase E component of the Pseudomonas aeruginosa RNA degradosome, occluding them from substrates and resulting in effective inhibition of RNA degradation and processing. The 2.2 Å crystal structure reveals that this novel homo-dimeric protein has no identifiable structural homologues. Our biochemical data indicate that acidic patches on the convex outer surface bind RNase E. Through the activity of Dip, фKZ has evolved a unique mechanism to down regulate a key metabolic process of its host to allow accumulation of viral RNA in infected cells.

As interest in the therapeutic and biotechnological potentials of bacteriophages has grown, so has value in understanding their basic biology. However, detailed knowledge of infection cycles has been limited to a small number of model bacteriophages, mostly infecting Escherichia coli. We present here the first analysis coupling data obtained from global next-generation approaches, RNA-Sequencing and metabolomics, to characterize interactions between the virulent bacteriophage PAK_P3 and its host Pseudomonas aeruginosa. We detected a dramatic global depletion of bacterial transcripts coupled with their replacement by viral RNAs over the course of infection, eventually leading to drastic changes in pyrimidine metabolism. This process relies on host machinery hijacking as suggested by the strong up-regulation of one bacterial operon involved in RNA processing. Moreover, we found that RNA-based regulation plays a central role in PAK_P3 lifecycle as antisense transcripts are produced mainly during the early stage of infection and viral small non coding RNAs are massively expressed at the end of infection. This work highlights the prominent role of RNA metabolism in the infection strategy of a bacteriophage belonging to a new characterized sub-family of viruses with promising therapeutic potential.

Pseudomonas aeruginosa causes chronic lung infections in people with cystic fibrosis (CF) and acute opportunistic infections in people without CF. Forty-two P. aeruginosa strains from a range of clinical and environmental sources were collated into a single reference strain panel to harmonise research on this diverse opportunistic pathogen. To facilitate further harmonized and comparable research on P. aeruginosa, we characterized the panel strains for growth rates, motility, virulence in the Galleria mellonella infection model, pyocyanin and alginate production, mucoid phenotype, LPS pattern, biofilm formation, urease activity, and antimicrobial and phage susceptibilities. Phenotypic diversity across the P. aeruginosa panel was apparent for all phenotypes examined, agreeing with the marked variability seen in this species. However, except for growth rate, the phenotypic diversity among strains from CF versus non-CF sources was comparable. CF strains were less virulent in the G. mellonella model than non-CF strains (P = 0.037). Transmissible CF strains generally lacked O-antigen, produced less pyocyanin and had low virulence in G. mellonella. Furthermore, in the three sets of sequential CF strains, virulence, O-antigen expression and pyocyanin production were higher in the earlier isolate compared to the isolate obtained later in infection. Overall, this full phenotypic characterization of the defined panel of P. aeruginosa strains increases our understanding of the virulence and pathogenesis of P. aeruginosa and may provide a valuable resource for the testing of novel therapies against this problematic pathogen.

Pf1-like bacteriophages (family Inoviridae) of Pseudomonas aeruginosa can contribute to bacterial short term evolution and virulence. Here we examine Pf1-like (pro)phage diversity and prevalence among different P. aeruginosa isolates. Pf1-like prophages in sequenced genomes of P. aeruginosa were analyzed and grouped into four clades: Pf4, Pf5, Pf7 and Pf-LES. P. aeruginosa strains (n=241) were screened for the presence of universal (primers PfUa and PfUb) and specific Pf1-like genetic elements (Pf1, Pf4 and Pf5). More than half of the strains contained at least one Pf1-like genetic element (60%); universal elements were detected in 56% of the strains, Pf4 in 22%, Pf1 in 18% and Pf5 in 7%. Infectivity experiments confirmed that strains yielding PCR products with either universal or Pf4 specific primers can release infective virions. Based on the high prevalence of Pf1-like (pro)phages, it is necessary to further examine their involvement in P. aeruginosa virulence., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.

Immediately after infection, virulent bacteriophages hijack the molecular machinery of their bacterial host to create an optimal climate for phage propagation. For the vast majority of known phages, it is completely unknown which bacterial functions are inhibited or coopted. Early expressed phage genome regions are rarely identified, and often filled with small genes with no homology in databases (so-called ORFans). In this work, we first analysed the temporal transcription pattern of the N4-like Pseudomonas-infecting phages and selected 26 unknown, early phage ORFans. By expressing their encoded proteins individually in the host bacterium Pseudomonas aeruginosa, we identified and further characterized six antibacterial early phage proteins using time-lapse microscopy, radioactive labelling and pull-down experiments. Yeast two-hybrid analysis gaveclues to their possible role in phage infection. Specifically, we show that the inhibitory proteins may interact with transcriptional regulator PA0120, the replicative DNA helicase DnaB, the riboflavin metabolism key enzyme RibB, the ATPase PA0657and the spermidine acetyltransferase PA4114. The dependency of phage infection on spermidine was shown in a final experiment. In the future, knowledge of how phages shut down their hosts as well ass novel phage-host interaction partners could be very valuable in the identification of novel antibacterial targets., (© 2014 John Wiley & Sons Ltd.)

Addressing the functionality of predicted genes remains an enormous challenge in the postgenomic era. A prime example of genes lacking functional assignments are the poorly conserved, early expressed genes of lytic bacteriophages, whose products are involved in the subversion of the host metabolism. In this study, we focused on the composition of important macromolecular complexes of Pseudomonas aeruginosa involved in transcription, DNA replication, fatty acid biosynthesis, RNA regulation, energy metabolism, and cell division during infection with members of seven distinct clades of lytic phages. Using affinity purifications of these host protein complexes coupled to mass spectrometric analyses, 37 host complex-associated phage proteins could be identified. Importantly, eight of these show an inhibitory effect on bacterial growth upon episomal expression, suggesting that these phage proteins are potentially involved in hijacking the host complexes. Using complementary protein-protein interaction assays, we further mapped the inhibitory interaction of gp12 of phage 14-1 to the α subunit of the RNA polymerase. Together, our data demonstrate the powerful use of interactomics to unravel the biological role of hypothetical phage proteins, which constitute an enormous untapped source of novel antibacterial proteins. (Data are available via ProteomeXchange with identifier PXD001199.).

Unlabelled: Pseudomonas aeruginosa bacteriophage ϕKZ is the type representative of the giant phage genus, which is characterized by unusually large virions and genomes. By unraveling the transcriptional map of the ∼ 280-kb ϕKZ genome to single-nucleotide resolution, we combine 369 ϕKZ genes into 134 operons. Early transcription is initiated from highly conserved AT-rich promoters distributed across the ϕKZ genome and located on the same strand of the genome. Early transcription does not require phage or host protein synthesis. Transcription of middle and late genes is dependent on protein synthesis and mediated by poorly conserved middle and late promoters. Unique to ϕKZ is its ability to complete its infection in the absence of bacterial RNA polymerase (RNAP) enzyme activity. We propose that transcription of the ϕKZ genome is performed by the consecutive action of two ϕKZ-encoded, noncanonical multisubunit RNAPs, one of which is packed within the virion, another being the product of early genes. This unique, rifampin-resistant transcriptional machinery is conserved within the diverse giant phage genus., Importance: The data presented in this paper offer, for the first time, insight into the complex transcriptional scheme of giant bacteriophages. We show that Pseudomonas aeruginosa giant phage ϕKZ is able to infect and lyse its host cell and produce phage progeny in the absence of functional bacterial transcriptional machinery. This unique property can be attributed to two phage-encoded putative RNAP enzymes, which contain very distant homologues of bacterial β and β'-like RNAP subunits., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Artilysins constitute a novel class of efficient enzyme-based antibacterials. Specifically, they covalently combine a bacteriophage-encoded endolysin, which degrades the peptidoglycan, with a targeting peptide that transports the endolysin through the outer membrane of Gram-negative bacteria. Art-085, as well as Art-175, its optimized homolog with increased thermostability, are each composed of the sheep myeloid 29-amino acid (SMAP-29) peptide fused to the KZ144 endolysin. In contrast to KZ144, Art-085 and Art-175 pass the outer membrane and kill Pseudomonas aeruginosa, including multidrug-resistant strains, in a rapid and efficient (∼ 5 log units) manner. Time-lapse microscopy confirms that Art-175 punctures the peptidoglycan layer within 1 min, inducing a bulging membrane and complete lysis. Art-175 is highly refractory to resistance development by naturally occurring mutations. In addition, the resistance mechanisms against 21 therapeutically used antibiotics do not show cross-resistance to Art-175. Since Art-175 does not require an active metabolism for its activity, it has a superior bactericidal effect against P. aeruginosa persisters (up to >4 log units compared to that of the untreated controls). In summary, Art-175 is a novel antibacterial that is well suited for a broad range of applications in hygiene and veterinary and human medicine, with a unique potential to target persister-driven chronic infections., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The potential of bacteriophage therapy to treat infections caused by antibiotic-resistant bacteria has now been well established using various animal models. While numerous newly isolated bacteriophages have been claimed to be potential therapeutic candidates on the basis of in vitro observations, the parameters used to guide their choice among billions of available bacteriophages are still not clearly defined. We made use of a mouse lung infection model and a bioluminescent strain of Pseudomonas aeruginosa to compare the activities in vitro and in vivo of a set of nine different bacteriophages (PAK_P1, PAK_P2, PAK_P3, PAK_P4, PAK_P5, CHA_P1, LBL3, LUZ19, and PhiKZ). For seven bacteriophages, a good correlation was found between in vitro and in vivo activity. While the remaining two bacteriophages were active in vitro, they were not sufficiently active in vivo under similar conditions to rescue infected animals. Based on the bioluminescence recorded at 2 and 8 h postinfection, we also define for the first time a reliable index to predict treatment efficacy. Our results showed that the bacteriophages isolated directly on the targeted host were the most efficient in vivo, supporting a personalized approach favoring an optimal treatment.

Unlabelled: In contrast to the rapidly increasing knowledge on genome content and diversity of bacterial viruses, insights in intracellular phage development and its impact on bacterial physiology are very limited. We present a multifaceted study combining quantitative PCR (qPCR), microarray, RNA-seq, and two-dimensional gel electrophoresis (2D-GE), to obtain a global overview of alterations in DNA, RNA, and protein content in Pseudomonas aeruginosa PAO1 cells upon infection with the strictly lytic phage LUZ19. Viral genome replication occurs in the second half of the phage infection cycle and coincides with degradation of the bacterial genome. At the RNA level, there is a sharp increase in viral mRNAs from 23 to 60% of all transcripts after 5 and 15 min of infection, respectively. Although microarray analysis revealed a complex pattern of bacterial up- and downregulated genes, the accumulation of viral mRNA clearly coincides with a general breakdown of abundant bacterial transcripts. Two-dimensional gel electrophoretic analyses shows no bacterial protein degradation during phage infection, and seven stress-related bacterial proteins appear. Moreover, the two most abundantly expressed early and late-early phage proteins, LUZ19 gene product 13 (Gp13) and Gp21, completely inhibit P. aeruginosa growth when expressed from a single-copy plasmid. Since Gp13 encodes a predicted GNAT acetyltransferase, this observation points at a crucial but yet unexplored level of posttranslational viral control during infection., Importance: Massive genome sequencing has led to important insights into the enormous genetic diversity of bacterial viruses (bacteriophages). However, for nearly all known phages, information on the impact of the phage infection on host physiology and intracellular phage development is scarce. This aspect of phage research should be revitalized, as phages evolved genes which can shut down or redirect bacterial processes in a very efficient way, which can be exploited towards antibacterial design. In this context, we initiated a study of the human opportunistic pathogen Pseudomonas aeruginosa under attack by one its most common predators, the Phikmvlikevirus. By analyzing various stages of infection at different levels, this study uncovers new features of phage infection, representing a cornerstone for future studies on members of this phage genus.

Escherichia coli bacteriophage T7 is a founding member of a large clade of podoviruses encoding a single-subunit RNA polymerase (RNAP). Phages of the family rely on host RNAP for transcription of early viral genes; viral RNAP transcribes non-early viral genes. T7 and its close relatives encode an inhibitor of host RNAP, the gp2 protein. Gp2 is essential for phage development and ensures that host RNAP does not interfere with viral RNAP transcription at late stages of infection. Here, we identify host RNAP inhibitors encoded by a subset of T7 clade phages related to ϕKMV phage of Pseudomonas aeruginosa. We demonstrate that these proteins are functionally identical to T7 gp2 in vivo and in vitro. The ability of some Pseudomonas phage gp2-like proteins to inhibit RNAP is modulated by N-terminal domains, which are absent from the T7 phage homolog. This finding indicates that Pseudomonas phages may use external or internal cues to initiate inhibition of host RNAP transcription and that gp2-like proteins from these phages may be receptors of these cues., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Although horizontal gene transfer plays a pivotal role in bacteriophage evolution, many lytic phage genomes are clearly shaped by vertical evolution. We investigated the influence of minor genomic deletions and insertions on various phage-related phenotypic and serological properties., Findings: We collected ten different isolates of Pseudomonas aeruginosa bacteriophage ϕKMV. All sequenced genomes (42-43 kb, long direct terminal repeats) are nearly identical, which intuitively implied strongly similar infections cycles. However, their latent periods vary between 21 and 28 minutes and they are able to lyse between 5 and 58% of a collection of 107 clinical P. aeruginosa strains. We also noted that phages with identical tail structures displayed profound differences in host spectra. Moreover, point mutations in tail and spike proteins were sufficient to evade neutralization by two phage-specific antisera, isolated from rabbits., Conclusion: Although all analyzed phages are 83-97% identical at the genome level, they display a surprisingly large variation in various phenotypic properties. The small overlap in host spectrum and their ability to readily escape immune defences against a nearly identical phage are promising elements for the application of these phages in phage therapy.

Aims: To select and evaluate an appropriate outer membrane (OM) permeabilizer to use in combination with the highly muralytic bacteriophage endolysin EL188 to inactivate (multi-resistant) Pseudomonas aeruginosa., Methods and Results: We tested the combination of endolysin EL188 and several OM permeabilizing compounds on three selected Ps. aeruginosa strains with varying antibiotic resistance. We analysed OM permeabilization using the hydrophobic probe N-phenylnaphtylamine and a recombinant fusion protein of a peptidoglycan binding domain and green fluorescent protein on the one hand and cell lysis assays on the other hand. Antibacterial assays showed that incubation of 10(6) Ps. aeruginosa cells ml(-1) in presence of 10 mmol l(-1) ethylene diamine tetraacetic acid disodium salt dihydrate (EDTA) and 50 μg ml(-1) endolysin EL188 led to a strain-dependent inactivation between 3·01 ± 0·17 and 4·27 ± 0·11 log units in 30 min. Increasing the EL188 concentration to 250 μg ml(-1) further increased the inactivation of the most antibiotic resistant strain Br667 (4·07 ± 0·09 log units)., Conclusions: Ethylene diamine tetraacetic acid disodium salt dihydrate was selected as the most suitable component to combine with EL188 in order to reduce Ps. aeruginosa with up to 4 log units in a time interval of 30 min., Significance and Impact of the Study: This in vitro study demonstrates that the application range of bacteriophage encoded endolysins as 'enzybiotics' must not be limited to gram-positive pathogens., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

The article continues a study of pseudolysogeny in Pseudominas aeruginosa infected with phiKZ-like phages of the EL species. Analysis was performed for several newly isolated virulent mutants of EL phages (EL and RU) that were virulent (capable of causing lysis of bacteria infected with the wild-type phage) and a lower extent of opalescence of negative colonies (NCs). Wile-type recombinants were detected in crosses of virulent mutants of phages EL and RU to confirm the polygenic control of virulence. Since a deletion mutation was found in one of the virulent EL mutants and high genetic instability was characteristic of another mutant, a mobile genetic element was assumed to play a role in mutagenesis. Pseudolysogeny of bacteria provides for horizontal gene transfer between different bacterial strains. Hence, sequencing of the phage genome and demonstration of the lack of toxic gene products are insufficient for the phage to be included into a therapeutic mixture. To use live phages, it is essential to study in detail the possible consequences of their interaction with host bacteria.

We examined the genetic diversity of lytic Pseudomonas aeruginosa bacteriophage PB1 and four closely related phages (LBL3, LMA2, 14-1 and SN) isolated throughout Europe. They all encapsulate linear, non-permuted genomes between 64 427 and 66 530 bp within a solid, acid-resistant isometric capsid (diameter: 74 nm) and carry non-flexible, contractile tails of approximately 140 nm. The genomes are organized into at least seven transcriptional blocks, alternating on both strands, and encode between 88 (LBL3) and 95 (LMA2) proteins. Their virion particles are composed of at least 22 different proteins, which were identified using mass spectrometry. Post-translational modifications were suggested for two proteins, and a frameshift hotspot was identified within ORF42, encoding a structural protein. Despite large temporal and spatial separations between phage isolations, very high sequence similarity and limited horizontal gene transfer were found between the individual viruses. These PB1-like viruses constitute a new genus of environmentally very widespread phages within the Myoviridae.

The "phiKMV-like viruses" comprise an important genus of T7 related phages infecting Pseudomonas aeruginosa. The genomes of these bacteriophages have localized single-strand interruptions (nicks), a distinguishing genomic trait previously thought to be unique for T5 related coliphages. Analysis of this feature in the newly sequenced phage phikF77 shows all four nicks to be localized on the non-coding DNA strand. They are present with high frequencies within the phage population and are introduced into the phage DNA at late stages of the lytic cycle. The general consensus sequence in the nicks (5'-CGACxxxxxCCTAoh pCTCCGG-3') was shown to be common among all phiKMV-related phages.

Background: Malate synthase catalyzes the second step of the glyoxylate bypass, the condensation of acetyl coenzyme A and glyoxylate to form malate and coenzyme A (CoA). In several microorganisms, the glyoxylate bypass is of general importance to microbial pathogenesis. The predicted malate synthase G of Pseudomonas aeruginosa has also been implicated in virulence of this opportunistic pathogen., Results: Here, we report the verification of the malate synthase activity of this predicted protein and its recombinant production in E. coli, purification and biochemical characterization. The malate synthase G of P. aeruginosa PAO1 has a temperature and pH optimum of 37.5 degrees C and 8.5, respectively. Although displaying normal thermal stability, the enzyme was stable up to incubation at pH 11. The following kinetic parameters of P. aeruginosa PAO1 malate synthase G were obtained: Km glyoxylate (70 microM), Km acetyl CoA (12 microM) and Vmax (16.5 micromol/minutes/mg enzyme). In addition, deletion of the corresponding gene showed that it is a prerequisite for growth on acetate as sole carbon source., Conclusion: The implication of the glyoxylate bypass in the pathology of various microorganisms makes malate synthase G an attractive new target for antibacterial therapy. The purification procedure and biochemical characterization assist in the development of antibacterial components directed against this target in P. aeruginosa.

Pseudomonas aeruginosa bacteriophage phiKMV requires type IV pili for infection, as observed from the phenotypic characterization and phage adsorption assays on a phage infection-resistant host strain mutant. A cosmid clone library of the host (P. aeruginosa PAO1) genomic DNA was generated and used to select for a clone that was able to restore phiKMV infection in the resistant mutant. This complementing cosmid also re-established type IV pili-dependent twitching motility. The correlation between bacteriophage phiKMV infectivity and type IV pili, along with its associated twitching motility, was confirmed by the resistance of a P. aeruginosa PAO1DeltapilA mutant to the phage. Subcloning of the complementing cosmid and further phage infection analysis and motility assays suggests that a common regulatory mechanism and/or interaction between the ponA and pilMNOPQ gene products are essential for bacteriophage phiKMV infectivity.

The binding affinity of the N-terminal peptidoglycan binding domain of endolysin KZ144 (PBD(KZ)), originating from Pseudomonas aeruginosa bacteriophage varphiKZ, has been examined using a fusion protein of PBD(KZ) and green fluorescent protein (PBD(KZ)-GFP). A fluorescence recovery after photobleaching analysis of bound PBD(KZ)-GFP molecules showed less than 10% fluorescence recovery in the bleached area within 15 min. Surface plasmon resonance analysis confirmed this apparent high binding affinity revealing an equilibrium affinity constant of 2.95 x 10(7)M(-1) for the PBD(KZ)-peptidoglycan interaction. This unique domain, which binds to the peptidoglycan of all tested Gram-negative species, was harnessed to improve the specific activity of the peptidoglycan hydrolase domain KMV36C. The chimeric peptidoglycan hydrolase (PBD(KZ)-KMV36C) exhibits a threefold higher specific activity than the native catalytic domain (KMV36C). These results demonstrate that the modular assembly of functional domains is a rational approach to improve the specific activity of endolysins from phages infecting Gram-negatives.

A collection of 15 newly isolated (bacterio)phages infecting the opportunistic pathogen Pseudomonas aeruginosa was established to investigate their global diversity and potential in phage therapy. These phages were sampled in 14 different countries traversing four continents, from both natural environments and hospital sewage. They all display unique DNA and protein profiles and cluster morphologically into six groups within the three major families of the Caudovirales. Extensive host range studies on a library of 122 AFLP-genotyped clinical P. aeruginosa strains (of which 49 were newly isolated at the University Hospital of Leuven, Belgium) showed that the phages lysed 87% of the strains. Infection analysis of outer membrane mutants identified 10 phages as type IV pili-dependent. More detailed information about the evolutionary relatedness of the phages was gathered by de novo peptide sequencing of major virion proteins using tandem Matrix-Assisted Laser Desorption/Ionization Time of Flight technology. Applying this technique for the first time to viruses, seven groups of closely related phages were identified without the need of prior knowledge of genome content and/or electron microscopic imaging. This study demonstrates both the epidemic population structure of P. aeruginosa and the global spread of P. aeruginosa phage species, and points at the resistance of two clinically predominant, widespread P. aeruginosa strains against phage attack.

Immediately after bacteriophage infection, phage early proteins establish optimal conditions for phage infection, often through a direct interaction with host-cell proteins. We implemented a yeast two-hybrid approach for Pseudomonas aeruginosa phages as a first step in the analysis of these - often uncharacterized - proteins. A 24-fold redundant prey library of P. aeruginosa PAO1 (7.32x10(6) independent clones), was screened against early proteins (gp1 to 9) of phiKMV, a P. aeruginosa-infecting member of the Podoviridae; interactions were verified using an independent in vitro assay. None resembles previously known bacteriophage-host interactions, as the three identified target malate synthase G, a regulator of a secretion system and a regulator of nitrogen assimilation. Although at least two-bacteriophage infections are non-essential to phiKMV infection, their disruption has an influence on infection efficiency. This methodology allows systematic analysis of phage proteins and is applicable as an interaction analysis tool for P. aeruginosa.

Gp181 (2237 amino acids) of Pseudomonas aeruginosa bacteriophage phiKZ (Myoviridae) is a structural virion protein, which bears a peptidoglycan hydrolase domain near its C-terminus. This protein is supposed to degrade the peptidoglycan locally during the infection process. Nine deletional mutants allowed delineation of the peptidoglycan hydrolase domain between amino acids 1880-2042 (gp181M8) and analysis of its biochemical properties. Gp181M8 tolerates a high ionic strength (>320mM) and is less sensitive to long thermal treatments compared to the similar phiKZ endolysin. Gp181M8 lysed all tested outer membrane-permeabilized Gram-negative species. The C-terminal distal end (amino acids 2043-2237) enhances the specific activity of gp181M8 threefold, resulting in a twelve times higher activity than commercial hen egg white lysozyme. These biochemical properties suggest that this novel peptidoglycan hydrolase domain may be suitable for enzybiotic applications.

The parameters influencing outer membrane permeability of Pseudomonas aeruginosa PAO1 under high hydrostatic pressure were quantified and optimized, using fusion between a specific A1gamma peptidoglycan-binding domain and green fluorescent protein (PBD-GFP). Based on the obtained data, optimal conditions were defined to assess the synergistic bactericidal action between high hydrostatic pressure and peptidoglycan hydrolysis by bacteriophage-encoded endolysins KZ144 and EL188. Under high hydrostatic pressure, both endolysins show similar inactivation of P. aeruginosa as the commonly used hen egg white lysozyme or slightly higher inactivation in the case of EL188 at 150 and 200 MPa. The partial contribution of pressure to the bacterial inactivation increases with higher pressure, while the partial contribution of the enzymes is maximal at the onset pressure of outer membrane permeabilization for the PBD-GFP protein (175 MPa). This study's results demonstrate the usefulness of this approach to determine optimal synergy for hurdle technology applications.

Pseudomonas aeruginosa bacteriophage endolysins KZ144 (phage phiKZ) and EL188 (phage EL) are highly lytic peptidoglycan hydrolases (210 000 and 390 000 units mg(-1)), active on a broad range of outer membrane-permeabilized Gram-negative species. Site-directed mutagenesis indicates E115 (KZ144) and E155 (EL188) as their respective essential catalytic residues. Remarkably, both endolysins have a modular structure consisting of an N-terminal substrate-binding domain and a predicted C-terminal catalytic module, a property previously only demonstrated in endolysins originating from phages infecting Gram-positives and only in an inverse arrangement. Both binding domains contain conserved repeat sequences, consistent with those of some peptidoglycan hydrolases of Gram-positive bacteria. Fusions of these domains with green fluorescent protein immediately label all outer membrane-permeabilized Gram-negative bacteria tested, isolated P. aeruginosa peptidoglycan and N-acetylated Bacillus subtilis peptidoglycan, demonstrating the broad range of peptidoglycan-binding capacity by these domains. Specifically, A1 chemotype peptidoglycan and fully N-acetylated glucosamine units are essential for binding. Both KZ144 and EL188 appear to be a natural chimeric enzyme, originating from a recombination of a cell wall-binding domain encoded by a Bacillus or Clostridium species and a catalytic domain of an unknown ancestor.

Lytic Pseudomonas aeruginosa phages LKD16 and LKA1 were locally isolated and morphologically classified as Podoviridae. While LKD16 adsorbs weakly to its host, LKA1 shows efficient adsorption (ka = 3.9 x 10(-9) ml min(-1)). LKA1, however, displays a narrow host range on clinical P. aeruginosa strains compared to LKD16. Genome analysis of LKD16 (43,200 bp) and LKA1 (41,593 bp) revealed that both phages have linear double-stranded DNA genomes with direct terminal repeats of 428 and 298 bp and encode 54 and 56 genes, respectively. The majority of the predicted structural proteins were experimentally confirmed as part of the phage particle using mass spectrometry. Phage LKD16 is closely related to bacteriophage phiKMV (83% overall DNA homology), allowing a more thoughtful gene annotation of both genomes. In contrast, LKA1 is more distantly related, lacking significant DNA homology and showing protein similarity to phiKMV in 48% of its gene products. The early region of the LKA1 genome has diverged strongly from phiKMV and LKD16, and intriguing differences in tail fiber genes of LKD16 and LKA1 likely reflect the observed discrepancy in infection-related properties. Nonetheless, general genome organization is clearly conserved among phiKMV, LKD16, and LKA1. The three phages carry a single-subunit RNA polymerase gene adjacent to the structural genome region, a feature which distinguishes them from other members of the T7 supergroup. Therefore, we propose that phiKMV represents an independent and widespread group of lytic P. aeruginosa phages within the T7 supergroup.

Study of two recently isolated giant bacteriophages Lu11 and OBP that are active on Pseudomonas putida var. Manila and Pseudomonas fluorescens, respectively, demonstrated their similarity in morphology, genome size, and size of phage particles, with giant bacteriophages of Pseudomonas aeruginosa assigned to the supergroup of phiKZ-like phages of the family Myoviridae designated in this manner according to the best studied phage phiKZ that belongs to the species of this group widely distributed in nature. Comparison of major polypeptide sizes of mature particles suggests the similarity of certain proteins in the phages examined. In OBP particles visualized with an electron microscope, an "inner body" was detected, which points to the specific DNA package intrinsic to phages of phiKZ group. In the meantime, phages Lul11 and OBP do not exhibit resemblance among themselves or with any of earlier described phiKZ-like phages in respect to other traits; particularly, they have no detectable DNA homology. Note that phage Lu11 of P. putida var. Manila exhibits very slight homology with phage Lin68 of the family of P. aeruginosa phiKZ-like phages detected only in blot hybridization. This suggests the possible involvement of these phages in interspecies recombination ("gene shuffling") between phages of various bacterial species. Results of partial sequencing of phage genomes confirmed the phylogenetic relatedness of phage OBP to phages of the phiKZ-supergroup, whereas phage Lu11 most probably belongs to a novel species that is not a member of supergroup phiKZ composition. The results of the study are discussed in terms of the evolution of these phages.

The structural proteome of phiKMV, a lytic bacteriophage infecting Pseudomonas aeruginosa, was analysed using two approaches. In one approach, structural proteins of the phage were fractionated by SDS-PAGE for identification by liquid chromatography-mass spectrometry (LC-MS). In a second approach, a whole-phage shotgun analysis (WSA) was applied. WSA uses trypsin digestion of whole phage particles, followed by reversed-phase HPLC and gas-phase fractionation of the complex peptide mixture prior to MS. The results yield a comprehensive view of structure-related proteins in phiKMV and suggest subtle structural differences from phage T7.

Pseudomonas aeruginosa phage EL is a dsDNA phage related to the giant phiKZ-like Myoviridae. The EL genome sequence comprises 211,215 bp and has 201 predicted open reading frames (ORFs). The EL genome does not share DNA sequence homology with other viruses and micro-organisms sequenced to date. However, one-third of the predicted EL gene products (gps) shares similarity (Blast alignments of 17-55% amino acid identity) with phiKZ proteins. Comparative EL and phiKZ genomics reveals that these giant phages are an example of substantially diverged genetic mosaics. Based on the position of similar EL and phiKZ predicted gene products, five genome regions can be delineated in EL, four of which are relatively conserved between EL and phiKZ. Region IV, a 17.7 kb genome region with 28 predicted ORFs, is unique to EL. Fourteen EL ORFs have been assigned a putative function based on protein similarity. Assigned proteins are involved in DNA replication and nucleotide metabolism (NAD+-dependent DNA ligase, ribonuclease HI, helicase, thymidylate kinase), host lysis and particle structure. EL-gp146 is the first chaperonin GroEL sequence identified in a viral genome. Besides a putative transposase, EL harbours predicted mobile endonucleases related to H-N-H and LAGLIDADG homing endonucleases associated with group I intron and intein intervening sequences.

The complete DNA sequence of a new lytic T7-like bacteriophage phiKMV is presented. It is the first genome sequence of a member of the Podoviridae that infects Pseudomonas aeruginosa. The linear G + C-rich (62.3%) double-stranded DNA genome of 42,519 bp has direct terminal repeats of 414 bp and contains 48 open reading frames that are all transcribed from the same strand. Despite absence of homology at the DNA level, 11 of the 48 phiKMV-encoded putative proteins show sequence similarity to known T7-type phage proteins. Eighteen open reading frame products have been assigned, including an RNA polymerase, proteins involved in DNA replication, as well as structural, phage maturation, and lysis proteins. Surprisingly, the major capsid protein completely lacks sequence homology to any known protein. Also, the strong virulence toward many clinical P. aeruginosa isolates and a short replication time make phiKMV attractive for phage therapy or a potential source for antimicrobial proteins.

The article continues a study of pseudolysogeny in Pseudominas aeruginosa infected with phiKZ-like phages of the EL species. Analysis was performed for several newly isolated vir mutants of EL phages (EL and RU) that were virulent (capable of causing lysis of bacteria infected with the wild-type phage) and a lower extent of opalescence of negative colonies (NCs). Wile-type recombinants were detected in crosses of virulent mutants of phages EL and RU to confirm the polygenic control of virulence. Since a deletion mutation was found in one of the virulent EL mutants and high genetic instability was characteristic of another mutant, a mobile genetic element was assumed to play a role in mutagenesis. Pseudolysogeny of bacteria provides for horizontal gene transfer between different bacterial strains. Hence, sequencing of the phage genome and demonstration of the lack of toxic gene products are insufficient for the phage to be included into a therapeutic mixture. To use live phages, it is essential to study in detail the possible consequences of their interaction with host bacteria. [ABSTRACT FROM AUTHOR]

Study of two recently isolated giant bacteriophages Lu11 and OBP that are active on Pseudomonas putida var. Manila and Pseudomonas fluorescens, respectively, demonstrated their similarity in morphotype, genome size, and size of phage particles, with giant bacteriophages of Pseudomonas aeruginosa assigned to the supergroup of ϕKZ-like phages of the family Myoviridae. This supergroup was designated in this manner according to the best studied phage ϕKZ that belongs to the species of this group widely distributed in nature. Comparison of major polypeptide sizes of mature particles suggests similarity of certain proteins in the phages examined. In OBP particles visualized with an electron microscope, an “inner body” was detected, which points to specific DNA package intrinsic to phages of ϕKZ group. In the meantime, phages Lu11 and OBP do not exhibit resemblance among themselves or with any of earlier described ϕKZ-like phages in respect to detectable DNA homology. Note that phage Lu11 of P. putida var. Manila exhibits very slight homology with phage Lin68 of the family of P. aeruginosa ϕKZ-like phages detected only in blot hybridization. This suggests the possible involvement of these phages in interspecies recombination (“gene shuffling”) between phages of various bacterial species. Results of partial sequencing of phage genomes confirmed the phylogenetic relatedness of phage OBP to phages of the ϕKZ supergroup, whereas phage Lu11 most probably belongs to a novel species that is not a member of supergroup ϕKZ composition. The results of the study are discussed in terms of the evolution of these phages. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosabacteriophage ?KMV is a T7-like lytic phage. Liquid chromatography-mass spectrometry of the structural proteins revealed gene product 36 (gp36) as part of the ?KMV phage particle. The presence of a lysozyme domain in the C terminal of this protein (gp36C) was verified by turbidimetric assays on chloroform-treatedP. aeruginosaPAO1 andEscherichia coliWK6 cells. The molecular mass (20,884 Da) and pI (6.4) of recombinant gp36C were determined, as were the optimal enzymatic conditions (pH 6.0 in 16.7 mM phosphate buffer) and activity (4800 U/mg). Recombinant gp36C is a highly thermostable lysozyme, retaining 26% of its activity after 2 h at 100°C and 21% after autoclaving. This thermostability could prove an interesting characteristic for food conservation technology. [ABSTRACT FROM AUTHOR]