Bacterial Gene Expression

Regulation of Bacterial Gene Expression

• Constitutive genes are expressed at a fixed rate.
• Other genes are expressed only as needed.
• Inducible genes are expressed only as needed.
• Repressible genes are expressed only as needed.
• Catabolite repression is when another gene is expressed only as needed.

Pre-transcriptional Control

• Repression inhibits gene expression and also decreases enzyme synthesis.
• Repressors mediate gene expression and are proteins that block transcription.
• The default position of a repressible gene is on.
• Induction turns on gene expression.
• An inducer initiates gene induction.
• The default position of an inducible gene is off.

Operon Model of Gene Expression

• Promoter: segment of DNA where RNA polymerase initiates transcription of structural genes, such as the TATA box.
• Operator: segment of DNA that controls transcription of structural genes (stop/go).
• Operon: set of operator promoter sites and structural genes.
• Two types of operon: inducible and repressible.

Operon Model of Gene Expression – Inducible Operon

• Structural genes are not transcribed in an inducible operon unless an inducer is present.
• lac operon
• In the absence of lactose, the repressor binds to the operator, which prevents transcription.
• In the presence of lactose, lactose (inducer) binds to the repressor; the repressor cannot bind to the operator, and transcription occurs.

Operon Model of Gene Expression – Repressible Operon

• Structural genes are transcribed in repressible operons until they are turned off.
• Excess tryptophan is a corepressor that binds and activates the repressor to bind to the operator, stopping tryptophan synthesis.

Changes in Genetic Material

• Mutation: a permanent change in the base sequence of DNA.
• Mutations may be neutral (silent), beneficial, or harmful.
• Mutagens: agents that cause mutations.
• Spontaneous mutations occur in the absence of a mutagen.
• Spontaneous mutations occur at a rate of about 1 mutation per million replicated genes

Types of Mutations

• The most common mutations are base substitutions (point mutations).
• Results in a change in one base in DNA.
• Silent mutations
• Missense mutations
• Nonsense mutations
• Frameshift mutations
• Base addition or deletion mutations

Missense Mutation

• Base substitution that results in a change in an amino acid.

Nonsense Mutation

• Base substitution results in a nonsense (stop) codon.

Frameshift Mutation

• Insertion or deletion of one or more nucleotide pairs in a gene.
• Shifts the translational "reading frame".

Chemical Mutagens

• Nitrous acid causes adenine to bind with cytosine instead of thymine.
• Mutagens can increase mutation rate up to 1000x.
• Nucleoside analog incorporates into DNA and can cause mistakes in base pairing.
• Nucleoside analogs are chemotherapeutic agents
• Intercalating agents (aflatoxin, ethidium bromide) cause frameshift mutations.

Radiation Mutagen

• Ionizing radiation (X rays and gamma rays) causes the formation of ions that can oxidize nucleotides and break the deoxyribose-phosphate backbone.
• This causes deletion mutations.
• UV radiation (non-ionizing radiation) causes thymine dimers (intra-strand bonding).

Radiation Repair

• Photolyases separate thymine dimers. Uses enzymes that use energy from visible light to fix UV light damage.
• Nucleotide excision repair removes incorrect bases with enzymes to fill them in correctly. Repairs all mutations, and cuts the nonmethylated strand (defective new strand).
• Methylase adds a methyl group to the adenine of the sequence 5'-GATC-3' in newly synthesized DNA.

Frequency of Mutation

• Spontaneous mutation rate is 1 in 10^6 (10^-6).
• Mutagens increase the rate by a factor of 10 to 1000, resulting in a rate of 10^-3 to 10^-5.

Identifying Mutants

• Positive (direct) selection detects mutant cells. The cells grow or appear different than unmutated cells.
• Negative (indirect) selection detects mutant cells that cannot grow or perform a certain function.
• Auxotroph are mutants, the mutant has a nutritional requirement absent in the parent.
• Replica plating can be used to identify auxotrophs.

Identifying Chemical Carcinogens

• Many mutagens are also carcinogens.
• The Ames test exposes mutant Salmonella bacteria to mutagenic substances to measure the rate of reversal of the mutation.
• This indicates the degree to which a substance is mutagenic.
• In the Ames tests animal liver cell extracts are combined with Salmonella auxotroph. The mixture is exposed to a test substance, and examined for signs of mutation in Salmonella, i.e. colonies that have reverted from his- to his+.

Genetic Transfer and Recombination

• Genetic recombination creates genetic diversity.
• Crossing over: Two chromosomes break and rejoin; insertion of foreign DNA into the chromosome.

Vertical vs Horizontal Gene Transfer

• Vertical gene transfer happens when genes are transferred from an organism to its offspring: a generation of cells.
• Horizontal gene transfer happens when genes are transferred between cells of the same generation.
• In transformation, genes are transferred from one bacterium to another as "naked" DNA.
• Conjugation: plasmids are transferred from one bacterium to another, requiring cell-to-cell contact via sex pili.
• Transduction: DNA is transferred from a donor cell to a recipient via a bacteriophage (transducing phage).

Conjugation in Bacteria

• Donor cells carry the plasmid (F factor) and are called F+ cells.
• Recipient cell (F-) becomes F+.
• F factor integrates into the chromosome, these are Hfr cells.
• R plasmids (R factors) are also transferred via conjugation.

Transduction in Bacteria

• Transfers DNA from a donor cell to a recipient via a bacteriophage (transducing phage).
• Generalized transduction: Random bacterial DNA is packaged inside a phage and is transferred to a recipient cell.
• Specialized transduction: Specific bacterial genes are packaged inside a phage and are transferred to a recipient cell.

Plasmids

• Plasmids are self-replicating circular pieces of DNA.
• They have 1 to 5% the size of a bacterial chromosome
• Often code for proteins that enhance the pathogenicity of a bacterium.
• Type of plasmid: Resistance factors (R factors). Plasmids which encode antibiotic resistance.

Genes and Evolution

• Mutations and recombination create cell diversity.
• Diversity is the raw material for evolution.
• Natural selection acts on populations of organisms to ensure the survival of organisms fit for a particular environment.
• Some mutations can be beneficial and over time, genetic mutations create genetic diversity, which keeps populations healthy. Many mutations have no effect at all.