1. N.De Lappe 1, D.Morris 2, G.Doran 1, J.O Connor 1, G.Corbett-Feeney 1,2, M.Cormican 1,2. National Salmonella Reference Laboratory, Medical Microbiology department, University College Hospital Galway, Galway 1 Bacteriology department, National University of Ireland Galway, Galway, Ireland 2 Genomic Structure of Salmonella Enteritidis typing phage 3 AMENDED ABSTRACT S.Enteritidis is the most common Salmonella serotype causing human infection worldwide. The Salmonella Enteritidis phage typing scheme of Ward et al uses 16 typing phage. Recently we reported that the typing phage comprise 6 siphoviridae, 6 podoviridae and 4 myoviridae. To date no complete S.Enteritidis phage sequence has been published. Typing phage 3 (a siphovirus) is recorded as having been originally isolated from a lysogenic strain. We have characterised phage 3 by determination of the complete genome sequence and by other methods. This double-stranded DNA phage contains 42,752 bp with a GC content of 49.85%. Phage 3 shows high homology to the recently sequenced virulent S.Typhimurium phage KS7 over large sections of the genome. Sequence analysis revealed 53 putative open reading frames. The genome is arranged in a modular format including seperate head, tail and DNA replication regions. A gene with homology to the lipopolysaccharide binding tailspike protein of phage P22 was detected in the tail region. Phage 3 encodes the necessary machinery to replicate its own DNA including DNA polymerase and DNA helicase. It also has a transcriptional regulator gene similar to phage immunity proteins and a DNA-binding protein similar to the phage anti-repressor ant. The presence of a terminase gene suggests the use of a headful packaging mechanism for DNA packaging. A novel lysozyme gene was also identified. We were unable to create a lysogen of PB406 (PT1b) with phage 3 although lysogens were obtained with the podo- and myoviridae. Analysis of this widely used phage may help in understanding phage-host interactions in this important pathogen. It may also have potential in a therapeutic role, e.g. use of the novel lysozyme. INTRODUCTION  S.Enteritidis is the most common Salmonella serovar causing human infection worldwide.  Recently we reported that the typing phage comprise 6 siphoviridae, 6 podoviridae and 4 myoviridae.  To date, no complete S.Enteritidis phage sequence has been published.  Typing phage 3 is recorded as having been originally isolated from a lysogenic strain.  We have characterised  3 by determination of it’s complete genome sequence and by other methods. METHODS AND MATERIALS   3 was propagated on PB406 (PT1b) using the host overlay method.  DNA was prepared using a modification of the Qiagen lambda midi kit and reconstituted in 10mM Tris-cl. Fimer-directed sequencing of phage DNA was performed by Fidelity systems, Gaithersburg, MD (Slesarev, Mezhevaya et al. 2002).  The quality of the phage sequence was confirmed by digesting  3 DNA with HindIII and electrophoresing the fragments. The digest was compared with a theoretical digest with HindIII using the REBASE website.  Putative open reading frames (orfs) were detected and annotated using the gene prediction program Glimmer. All potential orfs were compared to all other non-redundant sequences in the NCBI database using the BlastX program.  PCR was performed on  3 DNA, using primers designed to amplify  3 orfs, and the amplicons sequenced. These were compared to the directly sequenced  3 genome.  An attempt was made to lysogenise PB406 (PT1b) with  3. RESULTS   3 is a member of the family Siphoviridae, species Jersey. Heads measure 62.5nm and are icosahedral. Tails are rigid, non-contractile, measure 120 x 7nm, have striations with a periodicity of 4nm, and carry a 20nm-wide baseplate with spikes. There is evidence that the baseplate has 6 spikes of 13nm in length.  The digestion pattern of  3 with HindIII correlated with the theoretical digest performed with REBASE.  All PCR amplicons had identical sequences to the directly sequenced  3 genome.   3 contains 42,752 bp with a GC content of 49.85% and shows high homology to the recently sequenced virulent S.Typhimurium phage KS7 over large sections of the genome.  Sequence analysis revealed 53 putative open reading frames. The genome is arranged in a modular format including separate head, tail and DNA replication regions.  A gene with homology to the lipopolysaccharide binding tailspike protein of phage P22 was detected in the tail region. Previous work in this laboratory has shown that  3 adsorbs to the lipopolysaccharide of Salmonella serovars with antigenic structures 4,12 and 9,12.   3 encodes the necessary machinery to replicate its own DNA including DNA polymerase and DNA helicase.  The presence of a terminase gene suggests the use of a headful packaging mechanism for DNA packaging.  A novel lysozyme gene was also identified.  We were unable to create a lysogen of PB406 (PT1b) with  3 although lysogens were obtained with the podo- and myoviridae. This suggests that  3 is a virulent phage.  3, like other siphoviridae in the Enteritidis phage typing scheme, tends to produce clear rather than opaque lysis with test isolates, a characteristic of virulent phage. Opaque lysis is caused by the re-growth of lysogens and is indicative of temperate phage. DISCUSSION Most temperate phages depend almost totally on the host replication machinery for their lytic as well as their prophage replication cycles. In contrast many of the lytic phages encode their own DNA polymerases. It would seem unlikely that this phage was originally isolated from a lysogenic strain. Analysis of this widely used phage may help in understanding phage-host interactions in this important pathogen. ACKNOWLEDGEMENTS Hans Ackermann (Univ. Laval, Quebec) performed the Electron Microscopy. Gayle Philip (Nat. Univ. of Ireland, Maynooth) provided help with the Glimmer Gene Prediction program. REFERENCES 1. Delcher, A. L., D. Harmon, et al. (1999). "Improved microbial gene identification with GLIMMER." Nucleic Acids Res 27(23): 4636-41. 2. Slesarev, A. I., K. V. Mezhevaya, et al. (2002). "The complete genome of hyperthermophile Methanopyrus kandleri AV19 and monophyly of archaeal methanogens." Proc Natl Acad Sci U S A 99(7): 4644-9. Electron micrograph of phage 3 Tail DNA Replication and Repair LysisHead FIG. x. Schematic illustration of the phage 3 genome. Boxes depict ORFs determined by Glimmer. ORFs directed in rightward orientation are drawn above the black line; ORFs directed in the leftward direction are drawn below the line.