Biochemical characterization of malate synthase G of P. aeruginosa.

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.

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

Giant bacteriophages phiKZ 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 phiKZ mutant). These data form the basis for future structural studies and provide insights into the relatedness of these large phages.

The adsorption of Pseudomonas aeruginosa bacteriophage phiKMV 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.

A procedure for systematic identification of bacteriophage-host interactions of P. aeruginosa phages.

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.

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.

General M13 phage display: M13 phage display in identification and characterization of protein-protein interactions.

In M13 phage display, proteins and peptides are exposed on one of the surface proteins of filamentous phage particles and become accessible to affinity enrichment against a bait of interest. We describe the construction of fragmented whole genome and gene fragment phage display libraries and interaction selection by panning. This strategy allows the identification and characterization of interacting proteins on a genomic scale by screening the fragmented "proteome" against protein baits.

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.

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.

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.

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

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.

A standardized approach for accurate quantification of murein hydrolase activity in high-throughput assays.

Current spectrophotometers measure murein hydrolase activity simultaneously under many conditions and in small intervals. A correct interpretation of these large data sets requires clear and standardized criteria. Furthermore, there is a need for a uniform unit definition to express enzymatic activity, because application of variable definitions seriously hampered comparison between different studies.

Genomic analysis of Pseudomonas aeruginosa phages LKD16 and LKA1: establishment of the phiKMV 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)).

Whole genome phage display selects for proline-rich Boi polypeptides against Bem1p.

Interaction selection by biopanning from a fragmented yeast proteome displayed on filamentous phage particles was successful in identifying proline-rich fragments of Boi1p and Boi2p. These proteins bind to the second "src homology region 3'' (SH3) domain of Bem1p, a protein of Saccharomyces cerevisiae involved in bud formation. Target Bem1p was a doubly-tagged recombinant, Bem1([Asn142-Ile551]), which strongly interacts in ELISA with a C-terminal 75 amino acids polypeptide from Cdc24p exposed on phage.

The structural proteome of Pseudomonas aeruginosa bacteriophage phiKMV.

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.

Genome comparison of Pseudomonas aeruginosa large phages.

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.

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.

Revisiting the yeast chromosome VI DNA sequence reveals a correction merging YFL007w and YFL006w to a single ORF.

The overall contiguity of the Saccharomyces cerevisiae chromosome VI sequence assembly was assessed by systematic long-range PCR, PCR product size determination and sequencing. Using S. cerevisiae strain FY1679 total genomic DNA as template, 41 overlapping PCR products were generated, covering the complete 270 kb chromosome VI sequence.

In vivo selectively infective phage as a tool to detect protein interactions: evaluation of a novel vector system with yeast Ste7p-Fus3p interacting proteins.

The selectively infective phage (SIP) approach allows rapid identification of interacting proteins by linking protein-protein interaction to phage infectivity. Infection of E. coli by filamentous phage depends on viral g3p.

Selection and identification of dense granule antigen GRA3 by Toxoplasma gondii whole genome phage display.

Toxoplasma gondii is a ubiquitous, unicellular, eukaryotic parasite with a complex intracellular life cycle capable of invading and chronically infecting a wide variety of vertebrate host species, including man. Although normally opportunistic in healthy adults, it is a lethal pathogen in immunocompromised humans, particularly in AIDS patients. We present the application of a genomic phage display as a tool for the direct identification of antigens with potential value in diagnosis and/or as subunit vaccine components.