Kirsten Hertveldt

Genomic Analysis of Pseudomonas aeruginosa Phages LKD16 and LKA1: Establishment of the φKMV Subgroup within the T7 Supergroup

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.

Muralytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages φKZ and EL

Pseudomonas aeruginosa bacteriophage endolysins KZ144 (phage φKZ) 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.

The Genome and Structural Proteome of YuA, a New Pseudomonas aeruginosa Phage Resembling M6

Pseudomonas aeruginosa phage YuA (Siphoviridae) was isolated from a pond near Moscow, Russia. It has an elongated head, encapsulating a circularly permuted genome of 58,663 bp, and a flexible, noncontractile tail, which is terminally and subterminally decorated with short fibers. The YuA genome is neither Mu- nor lambda-like and encodes 78 gene products that cluster in three major regions involved in (i) DNA metabolism and replication, (ii) host interaction, and (iii) phage particle formation and host lysis. At the protein level, YuA displays significant homology with phages M6, phiJL001, 73, B3, DMS3, and D3112. Eighteen YuA proteins were identified as part of the phage particle by mass spectrometry analysis. Five different bacterial promoters were experimentally identified using a promoter trap assay, three of which have a sigma54-specific binding site and regulate transcription in the genome region involved in phage particle formation and host lysis. The dependency of these promoters on the host sigma54 factor was confirmed by analysis of an rpoN mutant strain of P. aeruginosa PAO1. At the DNA level, YuA is 91% identical to the recently (July 2007) annotated phage M6 of the Lindberg typing set. Despite this level of DNA homology throughout the genome, both phages combined have 15 unique genes that do not occur in the other phage. The genome organization of both phages differs substantially from those of the other known Pseudomonas-infecting Siphoviridae, delineating them as a distinct genus within this family.

“φKZ-like viruses”, a proposed new genus of myovirus bacteriophages

SummaryThe proposed φKZ genus of myoviruses has 21 members. Phages are virulent, lyse Pseudomonas bacteria, and are characterized by very large heads and correspondingly high DNA contents. The genome of the type virus, φKZ, has 306 ORFs and over 280 kbp and is the second-largest phage genome known. The φKZ genus has very few relationships to other phages and includes three species and one possible species.

The intron-containing genome of the lytic Pseudomonas phage LUZ24 resembles the temperate phage PaP3.

The virulent Pseudomonas aeruginosa bacteriophage LUZ24 (45,625 bp) was isolated from hospital sewage. It belongs to the family of the Podoviridae, and carries a bidirectionally transcribed dsDNA genome delineated by two direct terminal repeats of 184 bp. In vitro transcriptional analysis identified seven sigma(70) promoters, revealing a bias towards stronger promoter strength in the late genomic region. Reverse transcription demonstrated in vivo splicing of a 668 bp Group I intron embedded inside the DNA polymerase gene. Using mass spectrometry, nine structural proteins were identified as part of the phage particle. The lytic characteristics of LUZ24 are evaluated against its genomic content, which displays an overall 71% sequence similarity to the temperate phage PaP3.

The adsorption of Pseudomonas aeruginosa bacteriophage φKMV is dependent on expression regulation of type IV pili genes.

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.

Analysis of outer membrane permeability of Pseudomonas aeruginosa and bactericidal activity of endolysins KZ144 and EL188 under high hydrostatic pressure

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 studys results demonstrate the usefulness of this approach to determine optimal synergy for hurdle technology applications.

Identification of Gal80p-interacting proteins by Saccharomyces cerevisiae whole genome phage display.

Networks of interacting proteins and protein interaction maps can help in functional annotation in genome analysis projects. We present the application of genomic phage display as a tool to identify interacting proteins in Saccharomyces cerevisiae. We have developed a large phagemid display library (approximately 7.7x10(7) independent clones) of sheared S. cerevisiae genomic DNA (12.1 Mbp genome size) fused to gene III (lacking the N1 domain) of the filamentous phage M13. Baits tagged with an N-terminal E-tag and a C-terminal His(6)-tag are prepared in a novel Escherichia coli expression system. Using E-Gal80-His(6) as bait, biopanning of the library resulted in the isolation of two different clones containing fragments of the known interacting partner Gal4p. In addition, three new ligands (Ubr1p, YCL045c and Prp8p) with potential physiological relevance were isolated. Interactions were confirmed by ELISA. These results demonstrate the accessibility of the S. cerevisiae genome to display technology for protein-protein interaction screening.

Identification and comparative analysis of the structural proteomes of phiKZ and EL, two giant Pseudomonas aeruginosa bacteriophages.

Giant bacteriophages ϕKZ and EL of Pseudomonas aeruginosa contain 62 and 64 structural proteins, respectively, identified by ESI‐MS/MS on total virion particle proteins. These identifications verify gene predictions and delineate the genomic regions dedicated to phage assembly and capsid formation (30 proteins were identified from a tailless ϕKZ mutant). These data form the basis for future structural studies and provide insights into the relatedness of these large phages. The ϕKZ structural proteome strongly correlates to that of Pseudomonas chlororaphis bacteriophage 201ϕ2‐1. Phage EL is more distantly related, shown by its 26 non‐conserved structural proteins and the presence of genomic inversions.

Representational Difference Analysis (RDA) of bacteriophage genomes

We implemented the Representational Difference Analysis (RDA) screening method to identify genome variations between related bacteriophages without the need for complete genome sequencing. The strategy, optimized on phiKMV and LKD16 and further evaluated on the newly isolated phage LUZ19, is based on three successive rounds of reciprocal RDA, with an increasing driver/tester molar ratio from 100/1 to 750/1. Using three relevant restriction endonucleases, only 4 to 6 sequences per restriction enzyme are necessary to provide sufficient discriminatory information to reveal the major genome variations between phages.