Game Plan

1
Game Plan
Lab Review temp and UV results Chemical
growth control Streak plates Prelab Open Lab
Lecture Biofilms Review of basic
genetics Bacterial gene structure Gene
regulation Mutations
2
Biofilms

• Biofilm - organized microbial system of layers of
  microbial cells embedded in a polysaccharide
  matrix of microbial origin associated with
  surfaces
• Medicine - on catheters, teeth, middle ear
  infections, GI, GU tract, lungs (cystic
  fibrosis), biomedical products
• Aquatic environments - on algae, rocks, ships
• Industry - pipes, air conditioning vents, plastics

3
Biofilms
Industrial biofilm
Dental biofilm
Intestinal biofilm
4
Biofilm formation
5
Why form a biofilm?
1. Protection from antibiotics, toxins, and
immune cells
Effect of tobramycin (A) and ciprofloxacin (B) on
survivial of planktonic cells
(circle) resuspended biofilm cells
(triangle) biofilm colony cells (squares) Open
symbols are untreated
6
Why form a biofilm?
1. Protection from antibiotics, toxins, and
immune cells 2. Source of nutrients/ limits on
nutrients
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Why form a biofilm?

• 1. Protection from antibiotics, toxins,
• and immune cells
• 2. Source of nutrients/ limits
• on nutrients
• 3. Favorable microenvironment
• Highly hydrated
• Low oxygen

8
Why form a biofilm?

• 1. Protection from antibiotics, toxins,
• and immune cells
• 2. Source of nutrients/ limits
• on nutrients
• 3. Favorable microenvironment
• Highly hydrated
• Low oxygen
• 4. Stability - can detach and leave

Figure 8.1a
9
Why form a biofilm?

• 1. Protection from antibiotics, toxins,
• and immune cells
• 2. Source of nutrients/limits
• on nutrients
• 3. Favorable microenvironment
• Highly hydrated
• Low oxygen
• 4. Stability - can detach and leave
• 5. Community
• Gene transfer, signal transduction, quorum sensing

10
Review of DNA processing
Figure 8.2
11
Genetics terminology
Genetics- the study of genes Genes- a segment of
DNA that codes a functional production
(protein) Genome- all of the genetic material
in a cell Genomics- molecular study of
genomes Genotype- genes of an organism
Phenotype- physical expression of the genes
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DNA structure
Figure 8.3b
13
DNA replication

• Enzymes DNA polymerase, DNA ligase
• primase to make RNA primers,
• accessory enzymes (topoisomerase,
• gyrase, helicase)
• Antiparallel 5 to 3 synthesis results in
• leading and lagging strands
• Semi-conservative replication

Figure 8.5
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Bacterial DNA replication
Figure 8.6 - Overview
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Pit stop
The E. coli genome replicates every 45 minutes,
but divides every 26 minutes in ideal
conditions. How do you reconcile these two
facts?
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RNA transcription

• Enzymes RNA polymerase
• Promoters and terminators start and end
• process
• 5 to 3 synthesis
• Types tRNA, mRNA, rRNA

Figure 8.7
17
RNA processing in eukaryotes
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Translation and the Genetic Code

• Players ribosomes ( rRNA), tRNA,
• and mRNA
• Codons
• Anticodons

19
Translation and the Genetic Code

• Players ribosomes ( rRNA), tRNA,
• and mRNA
• Codons
• Anticodons

20
Translation
21
Translation
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Universal Genetic Code
Gene 5 ATG GTC CGA GCC CGC TAA GGC 3
3 TAG CAG GCT CGG GCG ATT CCG 5
Figure 8.8
23
Universal Genetic Code
Gene 5 ATG GTC CGA GCC CGC TAA GGC 3
3 TAG CAG GCT CGG GCG ATT CCG 5 RNA 5 AUG
Figure 8.8
24
Universal Genetic Code
Gene 5 ATG GTC CGA GCC CGC TAA GGC 3
3 TAG CAG GCT CGG GCG ATT CCG 5 RNA 5 AUG GUC
Figure 8.8
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Universal Genetic Code
Gene 5 ATG GTC CGA GCC CGC TAA GGC 3
3 TAG CAG GCT CGG GCG ATT CCG 5 RNA 5 AUG
GUC CGA
Figure 8.8
26
Universal Genetic Code
Gene 5 ATG GTC CGA GCC CGC TAA GGC 3
3 TAG CAG GCT CGG GCG ATT CCG 5 RNA 5 AUG
GUC CGA GCC
Figure 8.8
27
Universal Genetic Code
Gene 5 ATG GTC CGA GCC CGC TAA GGC 3
3 TAG CAG GCT CGG GCG ATT CCG 5 RNA 5 AUG
GUC CGA GCC CGC
Figure 8.8
28
Universal Genetic Code
Gene 5 ATG GTC CGA GCC CGC TAA GGC 3
3 TAG CAG GCT CGG GCG ATT CCG 5 RNA 5 AUG
GUC CGA GCC CGC UAA
Figure 8.8
29
Universal Genetic Code
Gene 5 ATG GTC CGA GCC CGC TAA GGC 3
3 TAG CAG GCT CGG GCG ATT CCG 5 RNA 5 AUG
GUC CGA GCC CGC UAA GGC 3 Protein

Figure 8.8
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Universal Genetic Code
Gene 5 ATG GTC CGA GCC CGC TAA GGC 3
3 TAG CAG GCT CGG GCG ATT CCG 5 RNA 5 AUG
GUC CGA GCC CGC UAA GGC 3 Protein
Met
Figure 8.8
31
Universal Genetic Code
Gene 5 ATG GTC CGA GCC CGC TAA GGC 3
3 TAG CAG GCT CGG GCG ATT CCG 5 RNA 5 AUG
GUC CGA GCC CGC UAA GGC 3 Protein
Met Val
Figure 8.8
32
Universal Genetic Code
Gene 5 ATG GTC CGA GCC CGC TAA GGC 3
3 TAG CAG GCT CGG GCG ATT CCG 5 RNA 5 AUG
GUC CGA GCC CGC UAA GGC 3 Protein
Met Val Arg
Figure 8.8
33
Universal Genetic Code
Gene 5 ATG GTC CGA GCC CGC TAA GGC 3
3 TAG CAG GCT CGG GCG ATT CCG 5 RNA 5 AUG
GUC CGA GCC CGC UAA GGC 3 Protein
Met Val Arg Ala
Figure 8.8
34
Universal Genetic Code
Gene 5 ATG GTC CGA GCC CGC TAA GGC 3
3 TAG CAG GCT CGG GCG ATT CCG 5 RNA 5 AUG
GUC CGA GCC CGC UAA GGC 3 Protein
Met Val Arg Ala Arg
Figure 8.8
35
Universal Genetic Code
Gene 5 ATG GTC CGA GCC CGA TAA GGC 3
3 TAG CAG GCT CGG GCT ATT CCG 5 RNA 5 AUG
GUC CGA GCC CGA UAA GGC 3 Protein
Met Val Arg Ala Arg Stop (formylmethionin
e in bacteria)
Figure 8.8
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DNA processing in bacteria- simultaneous
transcription/translation
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Game Plan
Lecture Gene regulation and the
operon Mutations Bring books next class
for APO-2
Lab Open Lab LAB EXAM NEXT CLASS
38
Review of DNA processing
Figure 8.2
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What changes a bacterium from planktonic to
sessile (i.e. what causes biofilm formation)?
Genes being turned on and off! Example Different
ial expression of 2.9-17 P. aeruginosa genes
between planktonic and biofilm cells
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When are genes on and off?

• Constitutive genes genes are constantly on
  (60-80)
• Regulated genes can be repressed and induced

The Jacob and Monad Operon Model
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Regulation of bacterial genes

• Response to nutrients in the environment
• Example Inducible genes
• Lactose operon

Figure 8.12a
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Regulation of bacterial genes

• Response to nutrients in the environment
• Example Inducible genes
• Lactose operon
• Lac operon induction needs lactose AND
• low glucose

43
Regulation of bacterial genes

• Response to nutrients in the environment
• Example Inducible genes
• Lactose operon
• Lac operon induction needs lactose AND
• low glucose

Figure 8.13 - Overview
44
Regulation of bacterial genes

• Response to nutrients in the environment
• Example Repressible genes
• Tryptophan operon

Figure 8.12b
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Regulation of bacterial genes

• 2. Response to accumulation of metabolic
  products
• Example Feedback inhibition
• Regulation of enzymes to synthesize
• threonine

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Regulation of bacterial genes

• 3. Quorum sensing of the environment
• QS ability of bacteria to communicate and
  coordinate
• behavior (through gene expression) by release of
  signaling
• molecules (phermones)

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Regulation of bacterial genes

• 3. Quorum sensing of the environment
• Example Biofilm genes

• Gram negative
• homoserine lactones (HSL), a signaling
  molecule, results in loss of flagella,
  induction of virulence genes
• Gram positives peptides

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Regulation of bacterial genes

• 3. Quorum sensing of the environment
• Example Sporulation genes- low nutrients and QS
  induce
• sporuation genes and shut down competence
  genes

Bacillus subtilis sporulation
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Regulation of bacterial genes

• 3. Quorum sensing of the environment
• Example Virulence genes- P. aeruginosa uses QS
  to sense
• high cell density and activate virulence genes
  to cause
• disease

50
If you inoculate a flask of TSB with a single E.
coli, after 24 hours will all the cells be
identical? Why or why not?
51
Mutations base pair substitutions

• 1. Missense and nonsense
• mutations
• Effect on protein
• No change, altered function, loss of function
• Examples
• Sickle cell disease (mis)
• Cystic fibrosis (non)
• 2. Silent mutations
• Effect on protein
• No change in protein

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Mutations frameshifts

• 3. Frameshift mutations
• Effect on protein
• Many possible outcomes
• Rarely no change
• Examples (trinucleotide repeat diseases)
• Fragile X Syndrome
• Huntingtons Disease

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Mutagens
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Base analogs
Figure 8.18 - Overview
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Mutations radiation
Figure 7.5
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Mutations UV pyrimidine dimers
Figure 7.5
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Mutations UV pyrimidine dimers

• Solutions
• Photoreactivation
• - Photolyases

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Mutations UV pyrimidine dimers

• Solutions
• Photoreactivation
• - Photolyases remove
• dimers
• Nucleotide excision repair
• - Can repair other damage

Figure 7.5
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Mutations UV pyrimidine dimers

• Solutions
• Photoreactivation
• - Photolyases remove
• dimers
• Nucleotide excision repair
• - Can repair other damage
• When all else fails
• SOS! repair system
• - Slows cell division,
• but results in mutations

60
Mutations ionizing radiation
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Independent Study

• 1. Review material!
• 2. Preview the mechanisms of genetic transfer in
  microbes
• transformation, conjugation and transduction.
  (see Figures 8.24, 8.26,
• and 8.27)
• Print out APO-2 Have you thanked a microbe
  today?
• Print out information for student presentations
• - In Student Presentation folder
• 1. List of individual assignments
• 2. Presentation guidelines