N.De Lappe 1, D.Morris 2, G.Doran 1, J.O Connor 1, G.Corbett-Feeney 1,2, M.Cormican 1,2.

1
Genomic Structure of Salmonella Enteritidis
typing phage 3
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

• RESULTS
• Y3 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 Y3 with HindIII
  correlated with the theoretical digest performed
  with REBASE.
• All PCR amplicons had identical sequences to the
  directly sequenced Y3 genome.
• Y3 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 Y3 adsorbs to the
  lipopolysaccharide of Salmonella serovars with
  antigenic structures 4,12 and 9,12.
• Y3 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 Y3 although lysogens were obtained
  with the podo- and myoviridae. This suggests that
  Y3 is a virulent phage. Y3, 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.

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 Y3 by determination of its
  complete genome sequence and by other methods.

Electron micrograph of phage 3

• METHODS AND MATERIALS
• Y3 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 Y3 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 Y3 DNA, using primers
  designed to amplify Y3 orfs, and the amplicons
  sequenced. These were compared to the directly
  sequenced Y3 genome.
• An attempt was made to lysogenise PB406 (PT1b)
  with Y3.

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.
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.