Microbial Genetics

Chapter 9(p. 251-265)

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Learning Objectives

• Explain the regulation of gene expression in bacteria by induction and repression

• Classify mutations by type, define mutagen.• Discuss two ways mutations can be repaired• Outline the methods of direct and indirect selection of

mutants• Identify the purpose and outline the procedure for

Ames test• Compare the mechanisms of genetic recombination in

bacteria: transformation, conjugation, and transduction

Regulation of Bacterial Gene Expression

• Constitutive enzymes are expressed at a fixed rate (i.e, they are on all the time)

• Other enzymes are expressed only as needed. They are usually under tight control– Repressible enzymes– Inducible enzymes

• Groups of coordinately expressed and regulated genes are called operons

• Regulatory proteins bind to operators• Transcription can be turned on or off

Regulation of Transcription

Inducible Operons:Lac operon

a. In absence of the substrate (lactose) the operon is off.

b. When substrate is present the operon is on.

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Repressible Operons:Arg operon

a. Nutrient product (arginine) is being used by the cell. The operon is on.

b. Nutrient product builds up, the operon is off.

Mutations

• Change in the genetic code• Wild type: strain expressing natural, non-mutated

characteristic• Mutant: strain expressing mutated gene• Mistakes during replication or damage to DNA• Wrong bases incorporated

– Transition AG or CT

– Transversion A(C or T) or G(C or T)

• Insertion or deletion of bases• Most are lethal, some are beneficial (e.g. drug resistance)

Replica Plating

Point MutationsA single base is altered in the sequence

• Silent mutation: TAT to TAC >> Tyr to Tyr• Missense mutation: TAT to TTT >> Tyr to Phe• Nonsense mutation: TAT to TAA >> Tyr to stop

Potential Reading Frames• Since triplet codons are read, there are three

reading frames in the forward direction.

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Frame Shift Mutations

• Addition or deletion of 1 or 2 bases knocks the sequence out of frame

• The whole amino acid sequence changes, usually results in a truncated (shortened) protein

• If the gene is essential, the mutation is lethal.• Insertions or deletions in multiples of three may

be tolerated• These are back in frame again• Can still be a big problem (Cystic Fibrosis, ∆508)

Causes of Mutations• Spontaneous mutations during replication (1 in 105-

1010)• Induced mutations• Physical: Electromagnetic radiation– X-rays, gamma rays nick DNA– UV light causes T-T dimers to form

• Chemicals– Analogs of bases– Base-modifying chemicals• Nitrosoguanidine, nitrous acid

– Intercalators insert between bases• Cause frameshift mutations

• Quick test for mutagen strength.

• His- Salmonella typhimurium

• Mutagen reverts cells to His+

• The degree of mutagenicity is calculated

Ames Test

Repair of Mutations

• Proofreading by DNA polymerase III

• Photoactivation or light repair by DNA photolyase

• Excision repair by DNA polymerase I and ligase.

DNA Recombination

• Horizontal gene transfer:Transformation (chromosomal DNA fragments)Conjugation (plasmids)Transduction (bacterial viruses)

Transformation

• Transfer of “naked” DNA.• Griffith worked with Streptococcus pneumoniae

Encapsulated: smooth colony appearance (S), virulent

Lacking a capsule: rough colony appearance (R), non-virulent

• Dead virulent bacteria were able to “transform” live non-virulent bacteria

The Transforming Principle

Dies

Survives

EffectCell Type

No capsule

LiveRstrain

(b)Rough (R)

Strain ofColony

Effect

Dies(a)

Smooth (S)

Strain ofColony

Cell Type

Capsule

LiveSstrain

(c)

Heat-killedS strain

Survives

(d)

Heat-killed S strain

Live R strain

Live S and R strainsisolated from deadmouse

Competence

• Non-specific ability to take up exogenous soluble fragments of DNA

• Some bacteria are always naturally competent• Others regulate competence• Still others need to be coaxed– Calcium chloride, chemicals, heat-shock, or

electroporation

Sex pilus contracts,bringing cells together.

F factor Bacterialchromosome

PhysicalConjugation

Sex pilus makes contact with F* recipient cell.

F+

F–

F+

F–

Conjugation

• Plasmid-directed transfer of plasmid DNA

• Requires cell contact via pilus

F Factor

• Donor (F+) cell makes a copy of the F factor

• F factor is transferred into the recipient (F+) cell via pilus

• Medical importance: R factors transfer resistance to antibiotics

Hfr Strains

• High frequency of recombination

• Integrated F factor (episome)• Conjugal transfer• Incorporation of new genes

into the chromosome• Used earlier to map the

genome.

Transduction

• Bacterial virus (bacteriophage) serves as the carrier of DNA from the donor cell to a recipient cell

• Generalized transduction• Specialized transduction

Generalized Transduction

•Phage injects its DNA into a cell•Phage DNA serves as a

template for new phage DNA and protein synthesis•Packaging of a random

fragment of bacterial DNA, and transfer to newly infected bacterial cells

Specialized Transduction

• Only certain genes transferred.

• Medical importance: toxins of Corynebacteria diphtheriae, Clostridium spp., and Streptococcus pyogenes are transferred by specialized transduction

Telling Them Apart

• Transformation – involves competency, transfers naked DNA (chromosomal or plasmid)

• Conjugation - involves cell contact through a pilus, transfers plasmid DNA

• Transduction - involves bacteriophage, transfers chromosomal DNA (specific or non-specific)

• DNase sensitivity (transformation)• 0.2 µm membranes (conjugation)

Transposons• Jumping genes• Mobile genetic

elements• Move from place to

place in the genome, plasmids, and viral genomes

• Disrupt genes when they land

• May mobilize other genes (like antibiotic resistance)

(1)

(2) (4)(3)

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Pathogenicity Islands

• Found in pathogens, improve pathogenicity:Yersinia pestis: ability to scavenge ironStaphylococcus aureus: ability to produce exotoxin

• Discovered because of the different G+C concentration and presence of bacteriophage or transpozon sequences.