The Pseudomonas aeruginosa generalized transducing phage φPA3 is a new member of the φKZ-like group of ‘jumbo’ phages, and infects model laboratory strains and clinical isolates from cystic fibrosis patients

Abstract

The Pseudomonas aeruginosa generalized transducing phage QPA3 is a new member of the QKZ-like group of 'jumbo' phages, and infects model laboratory strains and clinical isolates from cystic fibrosis patients Pseudomonas aeruginosa is an important pathogen in cystic fibrosis patients, and a model organism for the study of nosocomially acquired infections, biofilms and intrinsic multidrug resistance. In this study we characterize QPA3, a new generalized transducing bacteriophage for P. aeruginosa. QPA3 transduced chromosomal mutations between PAO1 strains, and infected multiple P. aeruginosa clinical isolates as well as the P. aeruginosa model laboratory strains PAK and PA14. Electron microscopy imaging was used to classify QPA3 in the order Caudovirales and the family Myoviridae. The genome of QPA3 was sequenced and found to contain 309 208 bp, the second-largest bacteriophage currently deposited in GenBank. The genome contains 378 ORFs and five tRNAs. Many ORF products in the QPA3 genome are similar to proteins encoded by P. aeruginosa phage QKZ and Pseudomonas chlororaphis phage 201Q2-1, and so QPA3 was classified genetically as a member of the QKZ-like group of phages. This is the first report of a member of this group of phages acting as a generalized transducer. Given its wide host range, high transduction efficiency and large genome size, the 'jumbo' phage QPA3 could be a powerful tool in functional genomic analysis of diverse P. aeruginosa strains of fundamental and clinical importance. INTRODUCTIONPseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of hospital-acquired infections (Trautmann et al., 2005). The organism primarily infects those with compromised immune systems. Individuals are most susceptible to infection when suffering from open wounds and chronic pulmonary conditions, such as cystic fibrosis (Harrison, 2007;Wagner & Iglewski, 2008). P. aeruginosa has a large genome, with over 5500 genes, and produces a wide variety of secondary metabolites, allowing it to survive in metabolically diverse environments, including soil, water, plants, insects and animals (Boman et al., 1972;Mahajan-Miklos et al., 2000;Stover et al., 2000). The organism can live as biofilms, colonies or as planktonic cells (Tolker-Nielsen & Molin, 2004;Wagner & Iglewski, 2008). Due to its metabolic diversity and its importance as a human pathogen, P. aeruginosa has become an organism of intense study, so genetic tools for this organism have become increasingly important.Generalized transducing bacteriophages are useful genetic tools for strain engineering and for functional genomic analysis in strains that are not naturally competent, such as P. aeruginosa (Wong et al., 2000). Several P. aeruginosa generalized transducing bacteriophages have been described, though the transduction efficiency of these bacteriophages, in particular DMS3, UT1 and F116, can be very low (Budzik et al., 2004;Kidambi et al., 1994;Ripp et al., 1994). Given such low transduction efficiency, occasional background spontaneous mu...

Phylogenomic Network and Comparative Genomics Reveal a Diverged Member of the ϕKZ-Related Group, Marine Vibrio Phage ϕJM-2012

“…Given that synteny blocks can be a strong indicator of conserved gene function (54,60,75), this suggests that distinct evolutionary forces acting on the genome may have altered its functional relatedness to the KZ-like phages. In addition, a potential reengineering of the regulatory system of JM-2012 can be evidenced by the loss of the conserved positional patterns of notable orthologs, such as RNAPs (54,76), that are highly associated with the evolution of transcriptional regulation among the KZ-like phages (23). With respect to the HPRs specific to JM-2012, our identification of regional features has two intriguing implications.…”

“…A mathematical model of reticulate networks based on ortholog clustering (15,20) and subsequent comparative analysis provides strong molecular support for the notion that JM-2012 diverged from a common ancestor of the KZ-related groups (23)(24)(25)(26)(27), most likely because of the imposition of distinct evolutionary forces upon its genome. Thus, given that KZ-like phages have limited genetic diversity and a narrow host range (23)(24)(25)(26)(27)(28)(29), these data provide novel insights into the diversity and pairwise similarity comparisons of each predicted protein of the JM-2012 genome (n ϭ 173) with BLAST in ACLAME; we used a Markov clustering (MCL) algorithm and the database of "viruses and prophages" (as of November 2012), within an E value threshold of 0.0001. Fifty-eight potential gene products of JM-2012 were found to be associated with protein families in ACLAME, while the remaining proteins were defined as "dummy" families, e.g., the unclassified protein families 1 to 115, and considered to assess the actual similarity between JM-2012 and those in the ACLAME database.…”

Bacteriophages of Ralstonia solanacearum: Their Diversity and Utilization as Biocontrol Agents in Agriculture

“…These include Pseudomonas aeruginosa phage KZ (280 kbp, Mesyanzhinov et al, 2002), EL (211 kbp, Herveldt et al, 2005), and PA3 (309 kbp, Monson et al, 2011), Vibrio parahaemolyticus phage KVP40 (245 kbp, Miller et al, 2003), Stenotrophomonas maltophila phage SMA5 (250 kbp, Chang et al, 2005), and Yersinia enterocolitica phage R1-37 (270 kbp, Kiljunen et al, 2005). Jumbo phages were also reported for Sinorhizobium meliloti (phage N3, 207 kbp, Martin and Long, 1984) and Bacillus megaterium (phage G, 670 kbp, Sun and Serwer, 1997).…”

A Novel Bacteriophage Targeting Cronobacter sakazakii Is a Potential Biocontrol Agent in Foods

“…The presence of multiple copies of RNA polymerase ␤/␤=-subunits in the genome and the lack of similarity of RNA polymerase ␤/␤=-subunits between the phage and host genomes suggest that the phage has different gene transcription mechanisms, and phage gene transcription may be dominant rather than gene transcription of the host. To further understand the functions of multiple copies of RNA polymerase ␤/␤=-subunits in the genome of phage CR5, three different Pseudomonas phage genomes, including those of 201phi2-1, phiKZ, and phiPA3 (40)(41)(42), were analyzed and compared. Pfam protein domain analysis of genes encoding RNA polymerase ␤/␤=-subunits in two phages, 201phi2-1 and phiPA3, revealed that a gene encoding the RNA polymerase ␤=-subunit in each phage genome (gp275 of 201phi2-1 and ORF54 of phiPA3) has a highly conserved protein sequence motif, Rbp1_domain_2 (NADFDGD), associated with Mg 2ϩ binding (40,42), while no Rbp1_domain_2 motif was detected in the phage phiKZ.…”

“…To further understand the functions of multiple copies of RNA polymerase ␤/␤=-subunits in the genome of phage CR5, three different Pseudomonas phage genomes, including those of 201phi2-1, phiKZ, and phiPA3 (40)(41)(42), were analyzed and compared. Pfam protein domain analysis of genes encoding RNA polymerase ␤/␤=-subunits in two phages, 201phi2-1 and phiPA3, revealed that a gene encoding the RNA polymerase ␤=-subunit in each phage genome (gp275 of 201phi2-1 and ORF54 of phiPA3) has a highly conserved protein sequence motif, Rbp1_domain_2 (NADFDGD), associated with Mg 2ϩ binding (40,42), while no Rbp1_domain_2 motif was detected in the phage phiKZ. Three aspartic acid (D) residues in the motif (in bold) have been suggested to be necessary for holding Mg 2ϩ in the active center of the RNA polymerase ␤=-subunit.…”