Pseudo-pac site sequences used by phage P22 in generalized transduction of Salmonella

Abstract

Salmonella enterica Serovar Typhimurium (Salmonella) and its bacteriophage P22 are a model system for the study of horizontal gene transfer by generalized transduction. Typically, the P22 DNA packaging machinery initiates packaging when a short sequence of DNA, known as the pac site, is recognized on the P22 genome. However, sequences similar to the pac site in the host genome, called pseudo-pac sites, lead to erroneous packaging and subsequent generalized transduction of Salmonella DNA. While the general genomic locations of the Salmonella pseudo-pac sites are known, the sequences themselves have not been determined. We used visualization of P22 sequencing reads mapped to host Salmonella genomes to define regions of generalized transduction initiation and the likely locations of pseudo-pac sites. We searched each genome region for the sequence with the highest similarity to the P22 pac site and aligned the resulting sequences. We built a regular expression (sequence match pattern) from the alignment and used it to search the genomes of two P22-susceptible Salmonella strains- LT2 and 14028S- for sequence matches. The final regular expression successfully identified pseudo-pac sites in both LT2 and 14028S that correspond with generalized transduction initiation sites in mapped read coverages. The pseudo-pac site sequences identified in this study can be used to predict locations of generalized transduction in other P22-susceptible hosts or to initiate generalized transduction at specific locations in P22-susceptible hosts with genetic engineering. Furthermore, the bioinformatics approach used to identify the Salmonella pseudo-pac sites in this study could be applied to other phage-host systems.

Figure 1:. Salmonella pseudo-pac site candidate sequences identified using P22 reads mapped to the Salmonella genome

A) Coverage plot of the Salmonella enterica Serovar Typhimurium LT2 (LT2) genome with sequencing reads from purified P22. Black vertical lines indicate the eight initiation sites for generalized transduction and the locations of the associated pseudo-pac sites along the LT2 genome. The exact locations of the pseudo-pac site sequences identified in this study are highlighted in pink on subsets of the LT2 read coverage plot associated with each site. The sequence of the pseudo-pac site candidate associated with each site is displayed above its respective read coverage plot. B) A multiple sequence alignment (MSA) generated with ClustalW of the eight pseudo-pac site candidate sequences, the P22 pac site and the respective neighboring genome sequences. The location of the 12 bp pac site consensus region, identified in Casjens et al. (1987) and further characterized by Wu et al. (2002), is defined above the MSA. The regular expression pattern built from the pseudo-pac site candidate sequences is displayed below the MSA.

Figure 2:. Salmonella pseudo-pac site sequences identified using regular expression searches

A-C) Coverage plots of the Salmonella enterica Serovar Typhimurium LT2 (LT2) and Salmonella enterica Serovar Typhimurium 14028S (14028S) genomes with sequencing reads from purified P22. The additional generalized transduction site present in 14028S but not LT2 is shaded in grey. The regular expression (Regex) patterns used to search the Salmonella genomes for additional pseudo-pac sites are displayed above their associated plots. Black vertical lines indicate the locations of the pseudo-pac site sequences that were previously identified in this study. Orange and pink dashed lines indicate the locations of regex matches on the forward and reverse Salmonella genome strands, respectively. The additional pseudo-pac sites identified with the regex searches, sites 9–11, are indicated in dashed boxes below their respective pattern match locations. D) A summary table with information about each pseudo-pac site identified in this study numbered in order of detection. Sites 1–8 were initially visually identified using P22 read coverages mapped to the Salmonella genome. Sites 9–11 were identified using the regex searches and were confirmed visually with read coverages.