1. Kelly Doran, Ph.D.
Assistant Professor of Pediatric Infectious Diseases
UCSD, School of Medicine

2. Microbial Genetics Lectures
Mutation ( )
Types of mutations
Detection of mutations
Recombination and Plasmids ( )
Recombination
Plasmids
Conjugation
Transposable Elements
Transformation

3. Genetics = branch of biology that deals with heredity, especially the mechanisms of hereditary transmission and the variation of inherited characteristics among similar or related organisms.
At the most basic level is the study of genes

4. Genes are the fundamental unit of heredity
DNA sequence in the chromosome
Transcribed into mRNA
Translated into proteins which make cells work

5. Genes are copied (DNA replication) almost exactly from parent cell to daughter cell (and from parent to offspring)
The copying of genes from one generation to the next is crucially important
Too many mistakes (MUTATIONS) and gene integrity is lost and the system falls apart

6. Maybe mistakes are not such a bad thing?
Anyone who has never made a mistake has never tried anything new. - Albert Einstein
A mistake may turn out to be the one thing necessary to a worthwhile achievement
- Henry Ford

7. Bacterial Genetics
In bacterial populations mutations are constantly arising due to errors made during replication.
If there is any selective advantage for a particular mutation (e.g. antibiotic resistance), the mutant will quickly become the major component of the population due to rapid growth rate of bacteria.
Bacteria have mechanisms by which genes can be transferred to other bacteria. Thus, a mutation arising in once cell can be passed on to other cells.

9. Mutation = A stable, heritable change in the genomic nucleotide sequence

10. How do mutations occur?
Spontaneous mutations - Arise occasionally in all cells; are often the result of errors in DNA replication (random changes)
Errors in replication which cause point mutations; other errors can lead to frameshifts
Point mutation - mismatch substitution of one nucleotide base pair for another
Frameshift mutation - arise from accidental insertion or deletion within coding region of gene, results in the synthesis of nonfunctional protein

11. Types of Mutations
Point mutation = affects only 1 bp at a single location
Silent mutation = a point mutation that has no visible effect because of code degeneracy
Missense mutation = a single base substitution in the DNA that changes a codon from one amino acid to another
Nonsense mutation = converts a sense codon to a nonsense or stop codon, results in shortened polypeptide

12. Base-pair substitution – missense mutation

13. (Silent mutation)
Missense mutation
Nonsense mutation

14. Types of Mutations
Frameshift mutation = arise from accidental insertion or deletion within coding region of gene, results in the synthesis of nonfunctional protein

15. Frame-shift mutation - Insertion

16. Frameshift mutation - Deletion

17. Other Types of Mutations
Insertion/deletion mutation = Larger stretch of DNA added or deleted from a gene that alters gene expression
Forward mutation = a mutation that alters phenotype from wild type
Reverse mutation = a second mutation which may make the mutant appear wt (in same gene)

18. How do Mutations occur? Induced mutations-caused by mutagens
Mutagens – Molecules or chemicals that damage DNA or alter its chemistry and pairing characteristics
Base analogs are incorporated into DNA during replication, cause mispairing
Modification of base structure (e.g., alkylating agents)
Intercalating agents insert into and distort the DNA, induce insertions/deletions that can lead to frameshifts
DNA damage so that it cannot act as a replication template (e.g., UV radiation, ionizing radiation, some carcinogens)

19. Mutations affect bacterial cell phenotype
Morphological mutations-result in changes in colony or cell morphology
Lethal mutations-result in death of the organism
Conditional mutations-are expressed only under certain environmental conditions
Biochemical mutations-result in changes in the metabolic capabilities of a cell
1) Auxotrophs-cannot grow on minimal media because they have lost a biosynthetic capability; require supplements
2) Prototrophs-wild type growth characteristics
Resistance mutations-result in acquired resistance to some pathogen, chemical, or antibiotic

20. Mutant Detection
In order to study microbial mutants, one must be able to detect them and isolate them from the wild-type organisms
Visual observation of changes in colony characteristics
Mutant selection-achieved by finding the environmental condition in which the mutant will grow but the wild type will not (useful for isolating rare mutations)
Screen for auxotrophic mutants: A lysine auxotroph will only grow on media that is supplemented with lysine

21. Mutant Detection
Mutants are generated by treating a culture of E. coli with a mutagen such as nitrosoguanidine
The culture will contain a mixture of wild-type and auxotrophic bacteria
Out of this population we want to select for a Lysine auxotrophic mutant

22. Isolation of a Lysine Auxotroph
minus lysine
complete
Lysine auxotrophs
do not grow
All strains grow

23. Microbial Recombination and Plasmids (p285-306)
Conjugation
Transposable Elements
Transformation

24. Bacterial Recombination A process by which one or more nucleic acid molecules are rearranged or combined to produce a new nucleotide sequence
Types of recombination
General recombination involves exchange between homologous DNA sequences
Site-specific recombination is the nonhomologous insertion of DNA into a chromosome; often occurs during viral genome or transposon integration into the host, a process catalyzed by enzymes specific for the host sequence
Replicative recombination accompanies replication and is used by some genetic elements that move about the genome

25. DNA Recombination:
Horizontal gene transfer-transfer of genes from one independent organism to another
Vertical gene transfer-transmission of genes from parents to offspring)

26. Mechanisms of horizontal gene transfer
Conjugation is direct transfer from donor bacterium to recipient while the two are temporarily in physical contact
Transformation is transfer of a naked DNA molecule
Transduction is transfer mediated by a bacteriophage (viruses that infect bacteria)

27. Plasmids
Plasmids are small ds DNA molecules, usually circular that can exist independently of the host chromosome. They have their own replication origin so can replicate automonously (episomes) and have relatively few genes (<30) that may or may not be essential to the host.
Bacterial cell
Plasmid DNA
Chromosomal DNA

28. Types of Plasmids
Conjugative plasmids have genes for pili and can transfer copies of themselves to other bacteria during conjugation
Fertility factor or F factor - These plasmids can also intergrate into the host chromosome or be maintained as an episome (independent of chromosome)
R factor - Also conjugative plasmids which have genes that code for antibiotic resistence for the bacteria harboring them. These do not integrate into the host chromosome.
Col Plasmids - harbor Bacteriocins which are proteins that destroy other bacteria (eg cloacins kill Enterobacter species)
Virulent plasmids - have genes which make bacteria more pathogenic because the bacteria is better able to resist host defenses or produce toxins/invasins

29. Bacterial Conjugation
The transfer of genetic information via direct cell-cell contact
This process is mediated by fertility factors (F factor) on F plasmids
Video Clip…

32. Evidence for Bacterial Conjugation
1946
Tatum
Lederberg
Demonstrated genetic recombination

33. The U-Tube Experiment
Met- Thr+ Leu+ Thi+
Met+ Thr- Leu- Thi-
Genetic recombination by conjugation requires direct physical contact between bacteria

34. Basic Bacterial Conjugation
F+ / F- mating
An F plasmid moves from the donor (F+) to a recipient (F-)
The F plasmid is copied and transferred via a sex pilus, the recipient becomes F+ and the donor remains F+
The F factor codes for pilus formation which joins the donor and recipient and for genes which direct the replication and transfer of a copy of the F factor to the recipient
The F factor can remain as a plasmid or it can integrate into the bacterial chromosome via IS sequences. This type of donor is called and Hfr strain (High frequency recombination)
F’- When the F factor in an Hfr strain leaves the chromosome, sometimes is makes an error in excision and picks up some bacterial genes

36. High Frequency Recombination
F+ Strain
HFR Strain
F Factor
Homologous
recombination
F plasmid
Bacterial
chromosome
Bacterial
chromosome

38. Transposable Elements
Transposons - DNA segments that carry genes that allow them to move about the chromosome (transposition)
Unlike plasmids or phages, they are unable to reproduce or exist apart from the host chromosome Tn
plasmid

39. Transposable Elements
Insertion sequences - IS elements- short sequence of DNA containing only genes required for transposition Flanked by inverted repeats (IR) - identical or similar sequences bp in reversed orientation
Transposase - enzyme that recognizes the IR and promotes transposition
Transposase (402 amino acids)
Inverted
repeat (IR) IR
IS10
1329 bp
Bacterial
chromosome

40. Transposable Elements
Composite transposon (Tn)- contains other genes in addition to transposase like antibiotic resistance genes or toxins
Bacterial
chromosome
IS10L
IS10R
Tn10
9,300 bp
Tetracycline
resistance
gene

41. Transposition MechanismIR
ACAGTTCAG
TGTCAAGTC
CTGAACTGT
GACTTGACA
Transposase
Insertion of Tn
into chromosomal
DNA target sequence
catalysed by
transposase
TCGAT
AGCTA
Chromosomal
DNA
Cut

42. Transposition MechanismIR
ACAGTTCAG
TGTCAAGTC
CTGAACTGT
GACTTGACA
Transposase
TCGAT
AGCTA
Gap filled by
DNA polymerase
and DNA ligase IR
ACAGTTCAG
TGTCAAGTC
CTGAACTGT
GACTTGACA
Transposase
TCGAT
AGCTA
TCGAT
AGCTA

44. Transposable Elements
Importance
Can insert within a gene to cause a mutation or stimulate DNA rearrangement leading to deletions of genetic material
Can have termination sequences to block translation or transcription
Can have promoters which activate genes near pt of insertion
Can move antibiotic resistance genes around
Can be on plasmids to aid in insertion of F plasmids into host chromosome
Some bear transfer genes (Tn916) and can move between bacteria through conjugation (conjugative transposon)

45. DNA Transformation
Transformation-a naked DNA molecule from the environment is taken up by the cell and incorporated in some heritable form. This process is random and any portion of the genome may be transferred
A competent cell is one that is capable of taking up DNA
Competent bacteria must be in a certain stage of growth (usually exponential) and secrete a small protein (competency factor) that stimulates production of new protein required for DNA uptake
Gene transfer by this process occurs in soils and marine environments so it is an important route of genetic exchange in nature
Artificial transformation - carried out in laboratory to transfer plasmid DNA, a common method for introducing recombinant DNA into bacterial cells. eg CaCl2 or electroporation

48. Oswald T. Avery

49. Plasmid Treatment R strain S strain S strain Competent cell

50. Similar to the F+ X F– cross
Hfr X F– Mating
Similar to the F+ X F– cross
conjugal
bridge
Sex pilus
Hfr
F - cell
F - cell

51. Ames Test for Mutagenicity
Auxotroph (tryp- mutant)
Selective media to look for mutants
Looking for reversion mutants