1. 10
Microbial Genetics
Genes for the Germs

2. Deadly Diarrhea 1968 Guatemala
Bloody dysentery hit 100,000 people, with 12,000 deaths
Caused by Shigella
Standard antibiotics had no effect
Arose through genetic selection of antibiotic-resistant strains

3. Bacterial DNA The bacterial chromosome DNA Double helix
Closed, circular loop
Free in cytoplasm
If extended into line, 1.5 mm long
Shrinks to fit inside 1 mm cell by looping and supercoiling
E. coli
Over 4,000 genes
© H. Potter-D. Dressler/Visuals Unlimited.
Figure 10.1: A) An electron micrograph of an E. coli cell immediately after disruption. The tangled mass is the organism’s DNA. B) The loops in the structure chromosome, viewed head-on

4. Bacterial DNA The bacterial chromosome Replication
Unwinding by helicase and/or gyrase
Single strands held apart by single-stranded DNA binding protein
New synthesis initiated by primase
All new DNA synthesized by DNA polymerase
Synthesis is semi-conservative

5. Bacterial DNA: Replication
Figure 10.2: Replication of the E. coli chromosome

6. Bacterial DNA Plasmids Extrachromosomal independent units
Closed, circular, double-stranded DNA
May confer selective advantage to microbe
“R” factors
Courtesy of the CDC
Figure 10.3: A TEM of bacterial plasmids.

7. Gene Mutations Mutation is permanent change in an organism’s DNA
Change is passed from originator to all its progeny
Method by which some drug resistance occurs

8. Gene Mutations Causes of mutation Spontaneous errors by DNA polymerase
Estimated at 1 observable change in every billion replications
Over 1 billion bacteria in an observable colony
Hence, at least 1 mutant, perhaps drug resistant, in the population
That resistant survivor can now successfully replicate to occupy the niche where all the other susceptible bacteria have died
Mutagens
Increase spontaneous error rate of DNA polymerase
Therefore, increase presence of mutants
Examples
Ultraviolet radiation
Chemicals

9. Gene Mutations: Mutagens
Figure 10.4: How nitrous acid causes bacterial mutations

10. Gene Mutations Causes of mutation Transposons
Changes in the way proteins are encoded may alter the way in which antibiotics bind
Antibiotics no longer effective against the mutated target
Transposons
Small segments of DNA that can move from one position to another in the chromosome
Jumping genes in corn; Barbara McClintock’s Nobel prize
Insertions of DNA into new spots in chromosome may also alter protein function

11. Gene Recombinations Transfer of genetic information between bacteria
Conjugation
Two live bacteria
F+ donor cell
F(ertility) factor
F plasmid
Sex pili
F- recipient cell
Conjugation bridge
Replication and passage of DNA through bridge
Frequently F plasmids also encode genes for drug resistance
Hfr bacteria
Many genera: Escherichia, Salmonella, Shigella

12. Gene Recombinations: Conjugation
Figure 10.5: Bacterial conjugation

13. Gene Recombinations: Conjugation
Figure 10.5: Bacterial conjugation, cont’d.

14. Gene Recombinations Transduction
Gene transfer with the assistance of bacterial viruses
Bacteriophages
Also known as phages (F)
Insert DNA into cytoplasm of bacteria
Turn bacteria into phage factories, executing phage DNA program
Phage production is usually lytic
Sometimes random segment of host DNA packaged in progeny
That segment of host DNA is delivered to new host
Result is transfer of DNA from one bacterium to another
DNA may encode drug resistance

15. Gene Recombinations: Generalized Transduction
Figure 10.7: Generalized transduction

16. Gene Recombinations: Generalized Transduction
Figure 10.7: Generalized transduction, cont’d.

17. Gene Recombinations Transformation
Acquisition of genes from surrounding environment
Requires competent cells
Requires only naked DNA
Some of this DNA may encode drug resistance genes
Fig. 10.9: Bacterial transformation

18. Gene Recombinations In today’s world
Gene transfers have resulted in proliferation of drug resistant bacteria
Staphylococcus aureus
Part of normal flora
Harmful if they penetrate skin
Open wounds
Piercings
Damaged hair follicles
Cuts, scratches
Many S. aureus are acquiring multiple drug resistance genes: MRSA
Diseases
Boils
Abscesses
Pneumonia
Septicemia
Endocarditis
Toxic shock
Now appearing in clinics: VRSA (vancomycin-resistant S. aureus)

19. Genetic Engineering Manipulation of DNA sequences in vitro
Cutting and splicing DNA segments together in new combinations that never existed before in nature
Not possible until the 1970s

20. Genetic Engineering The beginning of genetic engineering Endonucleases
Restriction enzymes
Expressed by bacteria to restrict infection by phages
Cut DNA at specific sequences
Create sticky ends
Sticky ends can be put back together in new combinations

21. Genetic Engineering: Restriction Enzymes
Fig : A) A restriction enzyme cuts through two strands of a DNA molecule to produce two fragments. B) The recognition sites of several restriction enzymes

22. The first recombinant DNA molecule
Genetic Engineering
The first recombinant DNA molecule
Cut gene from SV40 with restriction enzyme
Cut E. coli plasmid with same enzyme
Pasted to sticky DNAs together with ligase
Created new plasmid
Figure 10.11: Construction of a recombinant DNA molecule

23. The first recombinant DNA molecule
Genetic Engineering
The first recombinant DNA molecule
Figure 10.11: Construction of a recombinant DNA molecule, cont’d.

24. Genetic Engineering The implications
US government guidelines on recombinant DNA technology
New field of biotechnology
Food production
New medicines
Pollution control
New vaccines
Threat of new bioterrorism agents
Recombinant human insulin produced from bacteria
Recombinant Factor VIII , produced in bacteria, for hemophiliacs
Recombinant human growth factor
Advances in agriculture, medical diagnostics, forensic science