Chapter 8 Microbial Genetics part B. Genetics Study of what genes are. How DNA replicate. DNA DNA How information is expressed. Regulation of gene expression.

Chapter 8

Microbial Genetics

part B

Genetics• Study of what genes are.

• How DNA replicate. DNA DNA

• How information is expressed.

• Regulation of gene expression

• How they carry information.

• Changes in the genetic material

• Genes and evolution

RNA polymerase Translation

Gene (DNA) mRNA Protein

5’ ….ATGCCCTGAAAAGAGTCT…..3’

DNA polymerase

• The cell conserves energy by making only those proteins needed at a particular time.

– Constitutive enzymes are produced at a fixed rate• Enzymes of glycolysis are example

• Genes encoded these enzymes – constitutive genes– 60 – 80% of cell genes

– Other enzymes are produced only as needed

• Inducible enzymes– Induction – The process that turns on the transcription of a gene or

genes

• Repressive enzymes – Repression – the regulatory mechanism that inhibits (turns off) gene

expression and decrease the synthesis of enzymes

Regulation of Bacterial Gene Expression

Regulation of Bacterial Gene Expression

• Regulatory genes (I) code for regulatory proteins– Often repressor proteins

• Repressor proteins then attach to a DNA segment known as the operator (O). – By binding to the operator, the repressor protein prevents the

RNA polymerase from creating messenger RNA.– Blocking of gene expression is called repression

Figure 8.13

Repressor protein

Regulation of Bacterial Gene Expression

• Repressor protein activity depends on the presence or absence of an effector substance.

• Induction – When the cells are exposed to the compound (substrate) which has to be processed

• Repression - When the cells are exposed to a particular end-product.

Figure 8.13

EnzymesSubstrate Product

Regulation of Gene Expression - Induction

Figure 8.14.2

When Lactose is absent:

- Repressor is active- No transcription

When Lactose is present into the cell: - It is converted to allolactose (inducer)- Repressor is inactive - Active transcription – mRNA is synthesize

Lac operon is called inducible operon The lac operon is an operon required for the transport and metabolism of lactose in Escherichia coli and

some other enteric bacteriaEffector substance – Lactose

I – Regulatory gene – encode repressor protein - active; effector substance – substrate

Induced by presence of Lactose (substrate)

Regulation of Gene Expression - Repression

Figure 8.14.3

Tryptophan operon is repressible operonTrp operon encodes the genes for the synthesis of tryptophan

Effector substance – Tryptophan

Absence of tryptophan

- Repressor is inactive- Transcription is on

Presence of tryptophan- Tryptophan (corepressor) binds with the repressor protein – active repressor- No transcription

I – Regulatory gene – encode repressor protein – inactive; Effector substance – product

Repressed by presence of Tryptophan ( product)

Regulation of Gene Expression - Catabolite repression

Figure 8.15

Glucose effect or catabolite repression – inhibition of the metabolism of alternative carbon sources by glucose

Regulation of Lac operon also depend on the level of glucose in the medium

Glucose concentration in the medium controls the intracellular level of the small molecule cyclic AMP (cAMP), when glucose is no longer available, cAMP accumulates in the cell.

Lactose (disaccharide) hydrolysis glucose + galactose

Regulation of Gene Expression: Catabolite repression

CAP – catabolite activator molecule

Lactose presentGlucose is absent cAMP level is high

Transcription is stimulated

Lactose presentGlucose presentcAMP level is low

Transcription is not stimulated

Regulation of Gene Expression - Epigenetic Control

• Epigenetic control of gene expression - the switching on and switching off of genes. – Methylating certain nucleotides – turned gene expression off

– Methylated (off) genes are passed to offspring cells

– Not permanent

– Signals from the outside can work through the epigenome to change a cell's gene expression

• Contributes to cellular differentiation and development

• Aberrant epigenetic control contributes to disease (particularly to cancer)

• Biofilm behavior

Transcription ofmiRNA occurs.

miRNA binds to targetmRNA that has at least sixcomplementary bases.

mRNA is degraded.

DNA

miRNA

mRNA

• MicroRNAs control a wide range of activities in cells.

Regulation of Gene Expression – Post-transcriptional control

Changes in the genetic material

1. Mutation • A randomly derived change to the nucleotide sequence

of the genetic material of an organism.

2. Genetic Transfer and Recombination

• Genetic recombination refers to the exchange between two DNA molecules.

– It results in new combinations of genes on the chromosome.

• Mutation occur spontaneously – spontaneous mutation

• Mistakes of DNA polymerase

• Genetic recombination

– Mutation rate is the probability that a gene will mutate when a cell divides; the rate is expressed as 10 to a negative power.

• Spontaneous mutation rate = 1 in 109 replicated base pairs (frequency – 10-9 ) or 1 in 106 replicated genes (10-6 )

• Mutations usually occur randomly along a chromosome.

– A low rate of spontaneous mutations is beneficial in providing the genetic diversity needed for evolution.

Mutation

• Based on the changes in DNA sequence mutations may be: • Base substitution

• Deletion

• Insertion

• Based on the phenotypic effect on the organism mutations may be:• Neutral

• Beneficial

• Harmful

• Organisms have mechanisms such as DNA repair to remove mutations

• Mutation is generally accepted by biologists as the mechanism by which natural selection acts– Generating advantageous new traits – offspring survive and multiply

– Generating disadvantageous traits - offspring tend to die out

Mutation

Base substitution (point mutation)

1. Missense mutation - Change in one base result in change in amino acid

2. Nonsense mutation - Change in one base results in a nonsense codon

Figure 8.17a, b

Mutation – deletion and insertion

• One or few nucleotide pairs are deleted or inserted in the DNA

• Frameshift mutation – shift the “translational reading frame”

Figure 8.17a, d

Identifying mutants - Replica Plating

Figure 8.21

Example: an auxotrophic mutant cannot synthesize Histidine100 colony

• Positive (direct) selection• Negative (indirect) selection

Mutagens• Mutagens are agents in the environment that cause permanent

changes in DNA. – Mutagens increase mutations to 10–5 or 10–3 per replicated gene

1. Chemical mutagens – Nitrous acid (alters Adenine), nucleoside analogs, benzopyrene

2. Ionizing radiation (X rays and gamma rays) causes the formation of ions that can react with nucleotides and the deoxyribose-phosphate backbone.

3. UV radiation causes thymine dimers

– Light-repair separates thymine dimers

» Photolyases – enzymes that can

repair UV-induced damage

The Ames Test for Chemical Carcinogens

Figure 8.22

• Many mutagens have been found to be carcinogens

• Ames test – – Mutated Salmonella his- (lost ability to synthesize histidine)

– Mutagenic substance may cause new mutation that reverse the original

mutation to his+ ( back mutation or reversions)

– Incubation with mutagen / Control – without mutagen

– Liver extract – supply all necessary activation enzymes

Genetic Transfer and Recombination

• Genetic recombination, – The rearrangement of genes from separate groups of genes– A molecule of nucleic acid is broken and then joined to a different one– It contributes to genetic diversity.

• Vertical gene transfer occurs during sexual reproduction– between generations of cells

• Horizontal gene transfer in bacteria – involves a portion of the cell’s DNA being transferred from

donor to recipient between cells of the same generation

Figure 8.23

Genetic Recombination • In eukaryotes, recombination occurs in meiosis - chromosomal

crossover - exchange of genes between two DNA molecules

• Crossover occurs when two chromosomes break and rejoin

Figure 8.25

Genetic Recombination

Donor Recipient

Recombinant cell

• When some of the donor’s DNA has been integrated into the recipient’s DNA, the resultant cell is called a recombinant

Genetic transfer1. Transformation

• During this process, genes are transferred from one bacterium to another as “naked” DNA in solution.

• This process was first demonstrated in Streptococcus pneumoniae

and occurs naturally among a few genera of bacteria.

Figure 8.24

Genetic transfer

2. Conjugation • Requires contact between living cells.

• Genetic donor cell is an F+;– F+ cells contain plasmids called F factors;

– F factors are transferred to the F– cells during conjugation.

• Recipient cells are F–

– Converted to F+

Figure 8.27a

Conjugation• When the plasmid becomes incorporated into the chromosome, the

cell is called an Hfr (high frequency of recombination) cell.

• During conjugation, an Hfr cell can transfer chromosomal DNA to an F– cell.

• Usually, the Hfr chromosome breaks before it is fully transferred

Figure 8.27b

Plasmids

• Plasmids are self-replicating circular molecules of DNA carrying genes that are not usually essential for the cell’s survival.

• Conjugative plasmids – Carries genes for sex pili and transfer of the plasmid

• Dissimilation plasmids – Encode enzymes for catabolism of unusual compounds

• Pathogenicity Plasmids – Carrying genes for toxins or bacteriocins

• R factors – Encode antibiotic resistance

• Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae)

2

3

4

5

6

Figure 8.28

Genetic transfer3. Transduction by a Bacteriophage (viruses that infect

bacteria).

• In this process, DNA is passed from one bacterium to another in a bacteriophage and is then incorporated into the recipient’s DNA. . In generalized transduction, any bacterial genes can be transferred.

Specialized transduction

Transposons

– Segments of DNA that can move from one region of DNA to another

– Contain insertion sequences for cutting and resealing DNA (transposase)

– Complex transposons carry other genes

Figure 8.30a, b

Phenotypic effect of mutation

• Neutral

• Beneficial – Appear new quality– Change metabolic requirement

• Loose the ability to metabolize some substrate

• Increase activity of some enzyme

• Harmful• Lethal effect

Evolution

• Evolution is the process of change in the inherited traits of a population of organisms from one generation to the next

. 1. Diversity is the precondition for evolution • Genetic mutation and recombination provide a diversity of

organisms

2. The process of natural selection (positive selection)

• Allows the growth of those best adapted to a given environment

Learning objectives

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

• Classify mutations by type, and describe how mutations are prevented and repaired.

• Define mutagen• Describe the effect of mutagens on the mutation rate.• Compare the mechanisms of genetic recombination in

bacteria.• Differentiate between horizontal and vertical gene transfer.• Describe the functions of plasmids and transposons.• Outline methods of direct and indirect selection of mutants.• Discuss how genetic mutation and recombination provide

material for natural selection to act on.