Marine phage as vectors of gene transfer between marine bacteria and bacterial pathogens

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Marine phage as vectors of gene transfer between
marine bacteria and bacterial pathogens Peter
R. Weigele Jonathan King, PI Department of
Biology, MIT
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The big questions
To what extent do marine bacterial and
bacteriophage communities act as a reservoir for
virulence genes? Is phage infection an important
pathway of gene flow between marine and
terrestrial bacteria? (via phage encoded genes
and lateral gene transfer)
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Phages encode genes which make their host
pathogenic
Examples Function in lysogenized pathogen bor
more resistant to mammalian serum gipA
survives better in Peyers patches lom
improved binding to mammalian cells sod
superoxide dismutase virulent in mice sopE
stimulates mammalian cell ruffling and cell
invasion stx shiga-like toxin kills mammalian
cells
see Hendrix et al. (2000) Trends in Microbiology
8504
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Bacteriophages have a role in human disease
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Hypothetical flow of virulence genes between
populations
virulence
non-pathogens
pathogens
virulence
virulence
all genes
phage
virulence genes enhance fitness of host and/or
phage
benign bacteria
converted to pathogens
...phages are ubiquitous and may be an ideal
vector for virulence
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collect intact phage communities concentration of
phage by anion exchange purification by CsCl step
gradient
screen for virulence genes multiplex PCR for
known PEVG plaque lift and Southern
hybridization dilution and random amplification
characterize community visualize by TEM detect
subtypes by PCR
sequence phage genomes single phage
genome metagenomic approaches culture free
sequencing
elucidate physiology of phage infection e.g. host
range
...seek to characterize individual phages
isolated from community rather than use
metagenomic approach
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The complete genome and structural proteins of
the Syn9, a bacteriophage infecting Synechococcus
WH8109 learn from actual infectious phage
propagating on a marine cyanobacterial host
rather than environmental sequence dataset
also have genome and analyses for Syn5
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Syn9 host Synechococcus WH8109 contractile
tail 177,300 bp 225 orfs
200 nm
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Protein composition of Syn9 virions
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Map of Syn9 Genome
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Whole genome comparisons of T4 and T4-like
cyanophage
DNA metabolism
Virion structural proteins
T4 genes (43) cyanophage genes photosynthesis
genes
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Syn9 genomic sequence findings
Syn9 contains structural and DNA
replication/metabolism genes conserved with
enterophage T4 Possible bioactive natural
products- tryptophan halogenases Insights into
bacterial redox and respiratory pathways phage
allows host to deal with oxidative light
stress, starvation ...the phage has a number of
host-like genes that may be specific
adaptations to growth on a marine cyanobacterium
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Phage encoded antimicrobial or toxin?
pyrrolonitrin biosynthetic pathway in
Pseudomonas fluorescens
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Syn9 encodes three enzymes of the oxidative
pentose phosphate pathway, (survivability of
cells?)
Gluc. 6-phos. glucose-6-phosphate dehydrogenase
Gnd P-gluconate dehydrogenase
TalC P-gluconate dehydrogenase
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Syn9 has genes involved in photosynthesis,
electron transport,and oxidative stress
PsbA- photosystem II, D1 protein PsbD-
photosystem II, D2 protein
PetE- plastocyanin- copper binding, electron
transfer protein Plastoquinol terminal oxidase-
energy-dissipating oxidase
Thioredoxin- oxidative stress,
regulation Glutaredoxin- oxidative stress,
regulation
Syn9 carries genes to deal with the
movement/maintenance of excitation energy
prevent formaton of free radicals
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30Å reconstruction of bacteriophage Syn9 capsid
Image courtesy of James Conway, U. of Pittsburgh
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What's REALLY in the Water?

• Characterizing phage and the occurrence of
  phage-encoded toxin genes in aquatic environments

Myrriah S. Chávez Massachusetts Institute of
Technology Summer 2006
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Why do some bacterial strains acquire the toxin
genes while others do not?
Do these uninfected strains carry a latent form
of the toxin?
Where are these toxin encoding phages? What
types of niches harbor these phages?
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Phage Diversity in the Charles River

• Introduction
• Summer project goals
• Phage diversity
• Methods
• Purifying dsDNA tailed bacteriophages from water
• Concentrating phage (column chromatography)
• Isolating phage and bacterial host
• Purifying plaques
• Characterizing phage particles (Transmission
  Electron Microscopy, polyacrylamide gel
  electrophoresis, restriction enzyme digest)
• Identify virulence genes (PCR)
• Results

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Introduction Summer Goals

• Identify phage encoded virulence genes
• To study bacteriophage diversity in various
  freshwater environments
• Characterize phage
• Shape of capsid
• Type of phage (siphoviradae, podoviridae,
  myoviridae)
• Size of genome
• Similarity to other phage
• Identify bacterial host

Do these bacteriophages carry virulence genes?
Are phages agents of toxin gene transfer?
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Concentrating whole phage communities from
environmental waters

• Collected water from the Charles River Estuary
  and Willow Pond (Mt. Auburn cemetary)
• Concentrated whole phage communities with
  DEAE-cellulose, a positively charged resin that
  binds phage particles
• Eluted phage using a high salt buffer (20mM Tris,
  ph 7.5, 5MMm MgCl2, .5mM NaCl).

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• CsCl density gradient
• -Charles River Concentrate

20 Sucrorse p1.37 50mM tris 7.5 25mM
NaCl 10mM MgCl
Phages were further concentrated and purified
using a cesium chloride density gradient
Phage band

• Sequential dialysis phage buffer with 3M, 0.3M,
  and then 25mM NaCl

CsCl p1.65 50mM tris 7.5 25mM
NaCl 10mM MgCl
Densities Water 1.0 Lipid 0.98
Protein 1.34 DNA, RNA
1.7 Phage 1.4-1.65
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• Visualized phage particles in environmental
  concentrates using Transmission Electron
  Microscope (TEM).

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Isolating phage hosts
WPB3-WPB2

• Plated water samples on LB plates and allowed
  bacterial colonies to grow overnight at 30C
• Isolated 10 different strains of bacteria from
  freshwater environments

• Spread 10ul of C.R.W onto LB plates _at_ 30 degree
  Celsius

CRB1, 2, and 5 were subsequently identified as
Corynebacterium aquaticum
CRB1
CRB5
CRB2
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Plated phage concentrates on LB plates with
endogenous bacterial strains and allowed
lawns/plaques to develop overnight.
CRB5
E.Coli
WPB3
CRB2
CRB7
CRB4
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• Isolated plaques
• Purified plaques
• resuspend in 1ml sterile buffer (10mM Tris ph7.6,
  25mM Nacl, 5mM MgCl2)
• 2 drops chloroform
• Centrifuge
• Serially diluted phage stocks

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• Large scale purification of phage
• Choose 8 plates with big plaques
• Choose 3 plaques per plate
• Resuspend in 500mL buffer (add chlorofrom)
• Start 10mL o/n for 8 strains of bacteria
• Prepare 8 flasks w/ LB _at_ 30 deg. Celcius
• 150 dilution
• Add phage with OD .2-.3
• Add 5mL Chloroform (complete lysis occurred)
• Centriguge 10 min _at_ 8000rpm

• PEG Precipitation followed by CsCl gradients
• Four phage strains behaved well
• CRB5
• CRB7
• E.Coli Big
• E.Coli P1

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Characterization of phages

• SDS Page of virion proteins
• Restriction digested phage DNA resolved by
  agarose gels
• Visualize phage morphology by (TEM)

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ISOLATED PHAGE
CRB7 1.4E8 phage/ml

• CRB5 1.9E6 phage/ml

E.Co P1 2.03E9 phage/ml
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SDS page of virion proteins
200000
116250
97400
66200
45000
31000
21500
14400
6500
MM
E.Co big
E.Co P1
E.Co
CRB5
CRB5 Bact.
CRB7
CRB7 bact.
P22
T4
SYN9
E15
E34
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Screening for Virulence Genes via PCR

• NOT ALL strains of bacteria carry toxin genes
• Ctx- Cholera toxin
• Cnf-cytotoxic necrotizing factor
• Sxt- shiga toxin

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PCR Screening for Virulence Genes
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7.19.06 Gel3 2
1 100 bp ladder 2 B1-5-1 cdts1, cdts2, cdta1
cdta2 3 JJ055 cdts1, cdts2, cdta1 cdta2 4
Vibrio 569B ctxA3f, ctxA3r 5 1 JJ055 ctxA3f,
ctxA3r 6 2 B1-1-4 Ibe10f, Ibe10r 7 JJ055
Ibe10f, Ibe10r 8 JJ079 Hlyf, Hlyr 9 JJ055
Hlyf, Hlyr 10 JJ079 cnf1, cnf2 11 JJ055 cnf1,
cnf2 12 100 bp ladder
Controls worked, but no positive PCR reactions in
any of the four phage strains tested...
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Next steps
PCR screen total phage isolates Redesign
PCR primers to target broader range of virulence
genes homologs (conserved domains, degenerate
oligos, etc)
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Acknowledgements

• Jonathan King
• Peter Weigele
• Mandana Sassnafar
• Cammie Haase Pettingell
• Ryan Simkovsky
• King Lab
• Margaret Werner-Washburne

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Fourteen H.S. teachers from Central and Eastern
MA 5 day laboratory workshop and seminars on
environmental phages isolate phage from
Charles River Estuary using laboratory and
endogenous hosts purify phage characterize
morphology, DNA, and protein Pilot for broader
outreach project using phage as a vehicle for
students to do real science around the themes of
water borne illness in and the ecology of their
own local aquatic environments.
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Essential qualities of an outreach project
discovery ownership scientifically
relevant social impact
Desired outcomes
change self-perceptions about doing science learn
scientific method critical thinking use science
literacy and critical thinking in the real world
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Phage Fishing Teacher Workshop July 17-21,
2006 Department of Biology Massachusetts
Institute of Technology Peter Weigele Mandana
Sassanfar and Jonathan King
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Dottie and her team got plaques of varying
morphologies!
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Phages had characteristic protein banding
patterns on SDS-gels
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Acknowldegements
MIT Jonathan King Welkin Pope, WHOI/MIT-JP Myrriah
Chavez- U. of NM-MSRP Polz lab U. of
Pittsburgh Roger Hendrix Graham Hatfull James
Conway Outreach Mandana Sassanfar-MIT/HHMI Kathy
Vandiver- MIT/CEHS
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1.glucose-6-phosphate dehydrogenase 2.6-P-gluconat
e dehydrogenase 8. fructose-6-P transaldolase