Complete Sequence and Evolutionary Genomic Analysis of the Pseudomonas aeruginosa Transposable Bacteriophage D3112

Abstract

Bacteriophage D3112 represents one of two distinct groups of transposable phage found in the clinically relevant, opportunistic pathogen Pseudomonas aeruginosa. To further our understanding of transposable phage in P. aeruginosa, we have sequenced the complete genome of D3112. The genome is 37,611 bp, with an overall G؉C content of 65%. We have identified 53 potential open reading frames, including three genes (the c repressor gene and early genes A and B) that have been previously characterized and sequenced. The organization of the putative coding regions corresponds to published genetic and transcriptional maps and is very similar to that of enterobacteriophage Mu. In contrast, the International Committee on Taxonomy of Viruses has classified D3112 as a -like phage on the basis of its morphology. Similarity-based analyses identified 27 open reading frames with significant matches to proteins in the NCBI databases. Forty-eight percent of these were similar to Mu-like phage and prophage sequences, including proteins responsible for transposition, transcriptional regulation, virion morphogenesis, and capsid formation. The tail proteins were highly similar to prophage sequences in Escherichia coli and phage Phi12 from Staphylococcus aureus, while proteins at the right end were highly similar to proteins in Xylella fastidiosa. We performed phylogenetic analyses to understand the evolutionary relationships of D3112 with respect to Mu-like versus -like bacteriophages. Different results were obtained from similarity-based versus phylogenetic analyses in some instances. Overall, our findings reveal a highly mosaic structure and suggest that extensive horizontal exchange of genetic material played an important role in the evolution of D3112.

Genome sequence comparison and superinfection between two related Pseudomonas aeruginosa phages, D3112 and MP22

“…To amplify the D3112 ORF17 and the junction regions between ORF46 and ORF47, appropriate oligonucleotide primer pairs were designed based on the annotation information for the D3112 genome (Wang et al, 2004). Each 0.5 kb DNA fragment was amplified and directly sequenced using the same primers.…”

“…This direct cloning and sequencing has enabled us to define both junction sequences between the PA14 and MP22 genomes, and thus determine the left and right ends of the MP22 prophage, which are 59-TGT-39. Because sequence variations as well as imprecise excision could occur during prophage excision (Darzins & Casadaban, 1989b;Wang et al, 2004), our approach may be a valid method to determine the functional MP22 genome, based on the observation that a functional phage lysate could be obtained from the established lysogen PA14M (see Fig. 7).…”

“…This suggests that MP22 is capable of integrating into different locations on the P. aeruginosa genome as in other P. aeruginosa phages such as D3112 and B3 (Braid et al, 2004;Wang et al, 2004). Therefore, instead of trimming the heterogeneous sequences, we directly cloned both left-and right-end regions from the genome of one established MP22 lysogen (PA14M).…”

“…In particular, the type IV pili (TFP) have been shown to function as receptors for various P. aeruginosa phages, including filamentous phage Pf (Bradley, 1973b), single-stranded RNA phage PP7 (Bradley, 1967), Bradley B-type phage PO4 (Bradley, 1973a), and temperate transducing phages such as F116 (Byrne & Kropinski, 2005), DMS3 (Budzik et al, 2004), B3 (Roncero et al, 1990) and D3112 (Wang et al, 2004). To determine whether TFP serve as receptors for MP22, we created a deletion mutant of the TFP gene, pilA.…”

“…Another phage, DMS3, which is capable of generalized transduction within and between P. aeruginosa strains, is useful for exchange or transfer of genetic markers (Budzik et al, 2004). Moreover, the growing information on the genome sequences of these phages permits comparative genomic analyses (Braid et al, 2004;Wang et al, 2004) and comprehensive taxonomy (Rohwer & Edwards, 2002). These new perspectives on the phylogenetic aspects of the phages may be exploited to control P. aeruginosa infections.…”