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 DNA polymerase RNA polymerase Translation Gene (DNA) mRNA Protein 5’ ….ATGCCCTGAAAAGAGTCT…..3’

Regulation of Bacterial Gene Expression 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 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 Repressor protein Figure 8.13

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. Enzymes Substrate Product Figure 8.13

Regulation of Gene Expression - Induction I – Regulatory gene – encode repressor protein - active; effector substance – substrate 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 bacteria Effector substance – Lactose 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 Induced by presence of Lactose (substrate) Figure 8.14.2

Regulation of Gene Expression - Repression I – Regulatory gene – encode repressor protein – inactive; Effector substance – product Tryptophan operon is repressible operon Trp 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 Repressed by presence of Tryptophan ( product) Figure 8.14.3

Regulation of Gene Expression - Catabolite repression Lactose (disaccharide)  hydrolysis  glucose + galactose 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. Glucose effect or catabolite repression – inhibition of the metabolism of alternative carbon sources by glucose Figure 8.15

Regulation of Gene Expression: Catabolite repression CAP – catabolite activator molecule Lactose present Glucose is absent cAMP level is high Transcription is stimulated Lactose present Glucose present cAMP 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 11

Regulation of Gene Expression – Post-transcriptional control MicroRNAs control a wide range of activities in cells. Transcription of miRNA occurs. miRNA binds to target mRNA that has at least six complementary bases. mRNA is degraded. DNA miRNA mRNA

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 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

Base substitution (point mutation) Missense mutation - Change in one base result in change in amino acid 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 Positive (direct) selection Negative (indirect) selection Example: an auxotrophic mutant cannot synthesize Histidine 100 colony Figure 8.21

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 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 Figure 8.22

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

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 8.23 22

Genetic Recombination When some of the donor’s DNA has been integrated into the recipient’s DNA, the resultant cell is called a recombinant Donor Recipient Recombinant cell Figure 8.25 23

Genetic transfer 1. 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)

Genetic transfer 3. 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. . 2 In generalized transduction, any bacterial genes can be transferred. Specialized transduction 3 4 5 6 Figure 8.28

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.