Conjugation between different bacterial genera

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Conjugation between different bacterial genera
Examining conjugation among gram negative and
gram positive bacteria
Research by Kenneth Ladd Seldeen
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Purpose

• Examine likelihood of conjugation between two
  bacteria of different genera

• Examine likelihood of conjugation between a
  bacteria possessing a gram negative cell wall
  with a bacteria possessing a gram positive cell
  wall.

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Overview

• What is conjugation?

• The differences between gram negative and gram
  positive
• The experiment

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Conjugation
How its done
Defined The transfer of genetic material from
one cell to another involving cell to cell
contact
Sex pilus An appendage on a bacteria cell used
for transfer of genetic information
Plasmid Self replicating independent strand of
DNA
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More on the Plasmid

• Self replicating works independently from host
  cell

• Holds specific information

• Provides information for sex pilus and transfer
  (I.e. the F plasmid, most plasmids have this
  ability or associate with another plasmid with
  this ability)

• Can provide bacteria with antibiotic resistance

• Can code any DNA sequence This allows for the
  production of gene protein products such as
  insulin

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Overview

• What is conjugation?

• The differences between gram negative and gram
  positive

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Bacterial Cell walls
Gram positive cell walls
Gram negative cell walls

• Composed primarily of Peptidoglycan

Car

Peptidoglycan (def.) The structural molecule of
bacterial cell walls consisting of peptides and
carbohydrates

• Plasma membrane

Outer Membrane Peptidoglycan Plasma
membrane Cytoplasm
T
Carbohydrate backbone
Peptide bridge
Cytoplasm
Teichoic acids
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Overview

• What is conjugation?

• The differences between gram negative and gram
  positive
• The experiment

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Purpose
restated

• Examine likelihood of conjugation between two
  bacteria of different genera

• Examine likelihood of conjugation between a
  bacteria possessing a gram negative cell wall
  with a bacteria possessing a gram positive cell
  wall.

Hypothesis Conjugation will occur between
bacteria of different genera (gram negative to
gram negative transfers)
Hypothesis Conjugation will not occur
Reasoning
Reasoning
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Materials

• Bacteria
• Media
• Equipment

Bacteria
Bacteria
Trypticase soy agar Ampicillin agar Manitol salt
agar EMB agar

• Escherichia coli
• Serratia marcesens
• Staphylococcus aureus
• Staphylococcus epidermidis

Media
Media

• Wire loops
• Bunsen burner
• Sterilized water
• Graduated cylinder
• Scale
• Incubator set to 26oC

Equipment
Ampicillin Agar
E. coli
Trypticase soy agar
S. marcesens
S. aureus
Creating Amp agar .1 mg/ml concentration
Manitol Salt Agar
EMB agar
S. epidermidis

• Kills off bacteria not resistant to ampicillin
• ampicillin dissolved into agar at a .1ml/mg
  concentration

• Gram negative rod
• typical diseases include food poisoning
  cause of fecal pollution
• widely used for experimental purposes because of
  fast generation time
• found in host flora

• Gram negative rod
• Typically only causes nosocomial infections
• Known for producing a red pigment

• Gram positive, arrange as a cluster of spheres
• Known to cause food poisoning, Impetigo, and
  other diseases

• Concentration of 7 salt dissolved in agar
• Differentiates for Staphylococci

• Causes E. coli to produce a green metallic sheen
• Allows for the identification of viable E. coli

• Gram positive, arranged in a cluster of spheres
• Typically survives harmlessly on skin

• General use media
• Useful for stock cultures and controls
• Easy to make

• Mix 100 mg of dry ampicillin in 100 ml of
  sterilized water
• Place 10 ml of solution into 90 ml of luke warm
  Trypticase soy agar

Will require Both Ampicillin resistant and
susceptible strain for experiment
Will require both an ampicillin resistant and
susceptible strain useful because red pigment
easy to identify
Will require One ampicillin susceptible strain.
Will require one ampicillin susceptible strain
YES
More?
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Methodology
Part I Gram negative to gram negative conjugation
Part II Gram negative to gram positive conjugation
Experiment I E. coliR to S. marcesensS Experiment
II S. marcesensR to E. coliS
Experiment II S. marcesensR to E. coliS
Experiment III S. marcesensR to S.
aureusS Experiment IV S. marcesensR to S.
epidermidisS Experiment V E. coliR to S.
aureusS Experiment VI E. coliR to S. epidermidisS
Experiment III -- VI
Controls
Test agar
Test Agar
Stock
EMB to identify E.coli growth
Manitol Salt Agar to check for growth of staph.
Mix Plate
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Results
Experiment II S. marcesensR to E. coliS
Experiment I E. coliR to S. marcesensS
Experiment III-VI Gram (-) to Gram ()
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Results
Qualitative Examination
Experiment Exp. I Gram (-) to Gram (-) Exp.
II Gram (-) to Gram (-) Exp. III Gram (-) to
Gram () Exp. III Gram (-) to Gram () Exp. III
Gram (-) to Gram () Exp. III Gram (-) to Gram
()
YesConjugationNo
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Discussion
What was accomplished?

• Proved conjugation between different bacterial
  genera.

• Proved, however, that conjugation does not occur
  through gram () cell wall.

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Applications
A little more then just something nice to know

• Information sheds light on the limits of
  conjugation

• Industrial uses I.e. Proteins found in
  gram (-) bacteria can be freely transferred
  between one another

• Provides some evidence of how freely antibiotic
  resistance can be transferred between bacteria

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Limitations

• Experiment is lacking in repetition

• The mysterious disappearance of fourth
  generation Staphylococci

• Contamination

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Further research
The Case of the mysterious Ampicillin-philic
Bacteria
Problem
Observations
Further questions
For future senior researchers
Update!
Unknown bacteria growing uniformly on ampicillin
test plates
Hypothesis
Bacteria linked to amp. solution
Results
Conclusion
Possible sites of contamination
Materials and Methods

• Why were these bacteria ampicillin resistant

• Uniform growth of bacteria
• growth on amp agars only
• Asceptic technique used and agars autoclaved

Sites of contamination Observations
Bacteria linked to distilled water

• Ampicillin solution
• Nozzle of bottle holding ampicillin solution
• sterilized water used to create ampicillin
  solution
• Graduated cylinder used to transfer amp solution
  to agar

Using swabs, sterile pipettes and trypticase soy
agars, general procedure includes swabbing
possible sites of contamination or extracting
samples from the site of contamination, placing
it on the agar and observing growth
No growth detected

• Why did the amp solution work the first time,
  however, later, produce the bacteria?

Graduated cylinder Nozzle of amp bottle Amp
solution Distilled water used to make amp solution
For purposes of anonymity, names will not be
used, however, water used in this experiment was
collected from room 214 - a.k.a the
virology/genetics room
1 colony detected
No growth detected

• Just what kind of bacteria are these?

Growth detected large colonies colonies similar
to ones on amp agars
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I would like to express my thanks to
Dr. Barbara Collins Assistance with
everything Dr. Dennis Revie Donation of E. coli
bacteria Erik Kiszczak Donation of S. marcesens
bacteria Matt Romeo Donation of
Staphylococcus Amber Larson Donation of EMB agar
solution Sheenah Hellmers Donation of Manitol
salt agars Allison Adams Laboratory
Assistance Roman Achberger Laboratory
Assistance C.L.U. Amgen Various Laboratory
Supplies
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