TEAM 1TEAM 1Investigation of bacteriophages of the Investigation of bacteriophages of the

bird pathogen,bird pathogen, Bordetella avium Bordetella avium

IntroductionIntroduction

What is What is Bordetella aviumBordetella avium??• Bacteria that causes upper respiratory disease bordetellosis in avian species (chicken, turkey, etc.)

• Gram negative, non-fermentable, aerobic and motile

• Infects commercially grown turkeys throughout the world

Hosts for Hosts for B. aviumB. avium

SO …what?SO …what?

B. aviumB. avium infections infections result in severe result in severe economic losses in all economic losses in all poultry-producing poultry-producing regions of the world.regions of the world.

What is a bacteriophage?What is a bacteriophage?

A bacteriophage is a virus that solely A bacteriophage is a virus that solely infects bacteria.infects bacteria.

What is a virus?What is a virus? Obligate parasite, cannot replicate itselfObligate parasite, cannot replicate itself Needs a host cell for survivalNeeds a host cell for survival

Structure of BacteriophageStructure of Bacteriophage•Phage head: composed of coat protein and genome in the core

•Genome: DNA codes for enzymes and proteins necessary to replicate more viruses

•Tail Sheath: DNA travels from head to bacteria through sheath

•Tail fiber: helps anchor the phage on the cell membrane

Life cycles of a Temperate PhageLife cycles of a Temperate Phage

Two life cycles: Lytic cycle – viruses lyse the cell after replicating in the host cellLysogenic cycle – viral DNA integrates into host cell DNA to replicate, but no new viruses are synthesized

http://www.msu.edu/course/lbs/145/s02/graphics/campbell_18.5.gif

Ba1-1 and Ba1-2Ba1-1 and Ba1-2

• First identified as Ba1• After sequencing, two phage chromosomes were found Ba1-1, Ba1-2• Under electron microscope, look exactly the same

SEM of PhageSEM of Phage

About 2/3 of the chromosomes are the same

Ends- enzymes

Center- structure

GoalGoalTo determine which To determine which

phage(s) were in each phage(s) were in each B. avium B. avium strainstrain

Methods and MaterialsMethods and Materials

Behavior in Live BacteriaBehavior in Live Bacteria

How did we check for How did we check for active phage?active phage?Spontaneous lysisSpontaneous lysis197N infection197N infection

Spontaneous LysisSpontaneous Lysis

Grow single colonies of bacteria in Grow single colonies of bacteria in Brain Heart Infusion (BHI) brothBrain Heart Infusion (BHI) broth

Add culture to melted BHI top agar Add culture to melted BHI top agar and spread over a BHI plateand spread over a BHI plate

Incubate at 30Incubate at 30°C for 18-24 hours, °C for 18-24 hours, examine for plaquesexamine for plaques

Cartoon of a plate with plaquesCartoon of a plate with plaques

plaquebacteria

Testing bacterial strains for Testing bacterial strains for phage that infect 197Nphage that infect 197N

Add two drops chloroform to cultures Add two drops chloroform to cultures to kill bacteria, leaving only phageto kill bacteria, leaving only phage

Combine this culture with 197N Combine this culture with 197N culture, plate and incubateculture, plate and incubate

Examine for plaquesExamine for plaques

Polymerase Chain ReactionPolymerase Chain Reaction

Goal: amplification of a small Goal: amplification of a small amount of DNAamount of DNA

Ingredients: template DNA, Ingredients: template DNA, primers, nucleotides, and primers, nucleotides, and thermostable Taq Polymerasethermostable Taq Polymerase

Polymerase Chain ReactionPolymerase Chain Reaction

Ingredients run through a series of Ingredients run through a series of heating and cooling cyclesheating and cooling cycles Denaturation Denaturation –– DNA separated into DNA separated into

two strandstwo strands Annealing Annealing – Primers attach– Primers attach Polymerization Polymerization – free bases attach– free bases attach

Our PrimersOur Primers

Beginning primers unique to Ba1-Beginning primers unique to Ba1-1 and Ba1-2 (left)1 and Ba1-2 (left)

Recombinase primers from Ba1-1 Recombinase primers from Ba1-1 and Ba1-2 (right)and Ba1-2 (right)

Tail Fiber primer identical in both Tail Fiber primer identical in both strains (middle)strains (middle)

Gel ElectrophoresisGel Electrophoresis

Method used to analyze PCRMethod used to analyze PCR PCR product injected into gel wellsPCR product injected into gel wells Apply electrical fieldApply electrical field DNA travels from the negative electrode DNA travels from the negative electrode

to the positiveto the positive Travels through gel based on sizeTravels through gel based on size

Gel ElectrophoresisGel Electrophoresis

Results read as dark lines in the gelResults read as dark lines in the gel Fragment size read against a 1 kb (1000 Fragment size read against a 1 kb (1000

base pair) ladderbase pair) ladder Ethidium Bromide makes discrete bands Ethidium Bromide makes discrete bands

visible under UVvisible under UV

Agarose Gel & Gel Agarose Gel & Gel ElectrophoresisElectrophoresis

ResultsResults

Results for spontaneous Results for spontaneous lysis and infection of 197 Nlysis and infection of 197 N

StrainStrain Spontaneous Spontaneous LysisLysis

Infection of Infection of 197 N197 N

WamplerWampler 00 ++197 N197 N 00 00JBBAJBBA ++ 00

Gel of Repressor PrimerGel of Repressor Primer

Results for PCRResults for PCR

StrainStrain Unk-1Unk-1 Rec 1Rec 1 Tail fiberTail fiber Rep-2Rep-2 Rec 2Rec 2

WamplerWampler ++ ++ ++ ++ 00

197 N197 N 00 00 ++ ++ 00

G24G24 00 00 00 00 00

DiscussionDiscussion

DiscussionDiscussion

Interpretation of results Interpretation of results

Effectiveness of methodsEffectiveness of methods

Future applications and extensions of our Future applications and extensions of our workwork

Reasons for TestsReasons for Tests

Behavior of phage in strain of Behavior of phage in strain of B.aviumB.avium Spontaneous lysis testSpontaneous lysis test 197N infection test197N infection test

Which part of phage DNA in bacterial DNA?Which part of phage DNA in bacterial DNA? PCR & gel electrophoresis using primers that PCR & gel electrophoresis using primers that

amplified pieces of DNA from different phagesamplified pieces of DNA from different phages

Spontaneous LysisSpontaneous Lysis

Which strains Which strains made plaques?made plaques?

What does that What does that mean?mean?

What if there were What if there were no plaques?no plaques?

T4T4WamplerWampler239239

Phage present Phage present and lyticand lytic

Phage absentPhage absentPhage highly Phage highly lysogeniclysogenicNot enough Not enough phagephage

Infection of 197NInfection of 197N

Which strains Which strains made plaques?made plaques?

What does that What does that mean?mean?

What if there were What if there were no plaques?no plaques?

T4T4WamplerWampler239239Ba011Ba011Ba177Ba177DBL260DBL260ATCCATCC

Phage present Phage present and able to infect and able to infect 197N197N

Phage absentPhage absentPhage unable to Phage unable to infect 197Ninfect 197NNot enough Not enough phagephage

The plates don’t tell…The plates don’t tell…

If plaques…If plaques…which phage active in that strain?which phage active in that strain?

If no plaques…If no plaques…does the bacterial strain still contain does the bacterial strain still contain some phage DNA? Which pieces?some phage DNA? Which pieces?

T4 and WamplerT4 and Wampler

•Positive in four primers

•Spontaneous Lysis, Infection results also positive

•Probably contain both phages

D4, D10, and D27D4, D10, and D27

All PCR primers yielded positiveAll PCR primers yielded positive Probably contains phagesProbably contains phages

Spontaneous Lysis, Infection results Spontaneous Lysis, Infection results negativenegative Could be phage debrisCould be phage debris May be in lysogenic cycleMay be in lysogenic cycle Conditions may not be right for lysisConditions may not be right for lysis

G24G24

All PCR Primers yield negativeAll PCR Primers yield negative Spontaneous Lysis, Infection Spontaneous Lysis, Infection

negativenegative Probably does not contain Probably does not contain

phagesphages

197N and ATCC197N and ATCC

Some negative, some positiveSome negative, some positive ATCC—Unk-1, Rec 1, TF positiveATCC—Unk-1, Rec 1, TF positive

Infection positiveInfection positive Probably Ba 1-1, not Ba 1-2Probably Ba 1-1, not Ba 1-2

197N—Rep-2, TF positive197N—Rep-2, TF positive Infection positiveInfection positive Probably Ba 1-2,not Ba 1-1Probably Ba 1-2,not Ba 1-1

What’s next?What’s next?

Experiments only the beginningExperiments only the beginning More PCRMore PCR

Results not perfect, more PCR means more Results not perfect, more PCR means more accuracyaccuracy

Different primersDifferent primers More Spontaneous Lysis and InfectionMore Spontaneous Lysis and Infection

Don’t fully understand conditions for lysisDon’t fully understand conditions for lysis

More Future ProjectsMore Future Projects

Compare DNA/Genes of Ba 1-1 and Compare DNA/Genes of Ba 1-1 and 1-21-2 Similar genes code for proteins common Similar genes code for proteins common

in both phages—head, tail fiber…in both phages—head, tail fiber… Different genes will code for differencesDifferent genes will code for differences

—enzymes—enzymes Researchers can discover what makes Researchers can discover what makes

the strains differentthe strains different

AcknowledgementsAcknowledgements

Thank you to…Thank you to… Dr. TempleDr. Temple Holly KuzmiakHolly Kuzmiak Kelly PrescottKelly Prescott Octawia WojcikOctawia Wojcik Drew Biology DepartmentDrew Biology Department Dr. MiyamotoDr. Miyamoto