Bacterial Genetics Lecture Notes PDF

1 BACTERIAL GENETICS Assist. Prof. Dr. Güner EKİZ DİNÇMAN Faculty of Pharmacy Department of Pharmaceutical Microbiology 25.09.2023  2 Depending on intracellular or extracellular factors, micro...

1 BACTERIAL GENETICS Assist. Prof. Dr. Güner EKİZ DİNÇMAN Faculty of Pharmacy Department of Pharmaceutical Microbiology 25.09.2023  2 Depending on intracellular or extracellular factors, microorganisms may change their main charachteristics. These changes may be: I. Phenotypic II. Genotypic  3 I. PHENOTYPIC CHANGES Phenotypic changes refer to observable changes in an organism's characteristics or traits. These changes can be influenced by both genetic factors and environmental conditions. Phenotypic traits include physical attributes (e.g., color, shape), biochemical properties, and behaviors. § Do not cause genetic changes § Is reversible  4 Phenotypic changes in bacteria: Changes in these structures are also categorized under the phenotypic changes. § Flagella § Capsule § Fimbria § Spores § Cell periphery (protoplast-spherioplast) § Colony properties (mucoid, smooth, rough) § Staining properties § Enzymes § Others (pigments, granules)  5 II. GENOTYPIC CHANGES The term "genotypic" refers to changes that occur at the genetic level within an organism. Ø These changes are alterations in the DNA sequence, which can be caused by mutations, insertions, deletions, or rearrangements. the action or process of changing the position, time, or order of something § Changes in the genetic structure § Transfer to next generation § Irreversible (or long lived) § Result of mutation or recombination  6 As a result of genotypic changes, organism can obtain some specific properties: - virulence the severity or harmfulness of a disease or poison - toxin production - resistance to chemicals and chemotherapeutics  7 MUTATION § Changes in the nucleotide sequence of DNA § Alters the structure and function of protein to be synthesized § The organism that has gone through mutation – mutant § Mutation does not always result in a phenotypic change 8  9 Mutation mechanisms Point mutation § A single nucleotide change Generally, purine-purine (A ↔ G) Transition pyrimidine-pyrimidine (T ↔ C) purine-pyrimidine (A ↔ T) Transversion § Only a single amino acid difference in the synthesized protein, does not cause a major alteration in protein function § Most point mutations do not actually cause any phenotypic change  10 Genetic region mRNA codons amino acids Genetic region mRNA codons amino acids  11 Mutation mechanisms Base-pair substitutions Base-pair substitutions are a type of point mutation where one nucleotide base pair is replaced by another. These mutations can have various effects on the resulting protein and the organism's phenotype. Here are the main types of base-pair substitutions:  12 1. Silent Mutations Definition: A substitution that does not change the amino acid in the protein sequence. Example: AGC to AGT, both code for Serine. Impact: Usually no effect on the protein function or phenotype. 2. Missense Mutations Definition: A substitution that changes one amino acid in the protein sequence. Example: GAG to GUG; Glutamic acid to Valine. Impact: Can be neutral, beneficial, or harmful depending on the role of the amino acid. 3. Nonsense Mutations Definition: A substitution that changes an amino acid codon to a stop codon. Example: UGG to UGA; Tryptophan to Stop. Impact: Usually results in a nonfunctional protein.  13 Possible effects of base-pair substitution in a gene encoding a protein. Three different protein products are possible from changes in the DNA for a single codon.  14 Mutation mechanisms Frameshifts and Other Insertions - deletions § the genetic code is read from one end of the nucleic acid in consecutive blocks of three bases (codons), any deletion or insertion of a single base pair results in a shift in the reading frame ü Frameshift mutations often have serious consequences. § a new nucleotide in the DNA nucleotide sequence (insertion) § removal of a nucleotide (deletion) § The codons starting from that nucleotide changes causing an alteration in the synthesized protein structure and function  15 Mutation mechanisms Frameshift Mutation Definition: A frameshift mutation occurs when one or more nucleotides are inserted or deleted from a DNA sequence, causing a shift in the reading frame of the genetic code. Impact: Frameshift mutations usually have a drastic effect, often resulting in a completely nonfunctional protein. Types: Insertion Frameshift: Addition of one or more nucleotides. Deletion Frameshift: Removal of one or more nucleotides. Example: Original: AUG-AAA-GGG-CCC-TAA (Start-Lys-Gly-Pro- Stop) Insertion: AUG-AAA-AGG-GCC-CTA-A (Frameshift) Deletion: AUG-AA-GGG-CCC-TAA (Frameshift)  16 Shifts in the reading frame of mRNA caused by insertions or deletions  17 Mutation mechanisms Other Insertions - deletions Definition: Insertions or deletions that do not cause a frameshift but add or remove a specific number of amino acids in the protein. Impact: The impact varies depending on the location and the number of nucleotides involved. Types: In-Frame Insertion: Addition of nucleotides in multiples of three. In-Frame Deletion: Removal of nucleotides in multiples of three. Example: Original: AUG-AAA-GGG-CCC-TAA (Start-Lys-Gly-Pro-Stop) In-Frame Insertion: AUG-AAAAAA-GGG-CCC-TAA (Extra Lys) In-Frame Deletion: AUG-GGG-CCC-TAA (Lys removed)  18 Mutations 1. Spontaneous mutations Occurs rarely and naturally (eg. during DNA replication) 2. Induced mutations Occurs by the effect of physical and chemical factors (mutagens) an agent, such as radiation or a chemical substance, which causes genetic mutation Ø The process of inducing mutations through treatment with a mutagen is known as mutagenesis. Ø The different mutagenic agents may be classified into two broad groups: 1. Physical mutagens 2. Chemical mutagens  19 Mutagens Physical mutagens: UV light X rays Chemical Mutagens: Base analogs (5 Bromo Uracil, 2 Amino Purines) Nitroazide Nitroguanidine Ethyl-methane sulphonate Acridines  20 Chemical and physical mutagens & their modes of action a) 5-Bromouracil can base-pair with guanine, causing AT to GC substitutions. (b) 2-Aminopurine can base-pair with cytosine, causing AT to GC substitutions.  21 DNA Repair and the SOS System § Cells have a variety of different DNA repair processes to correct mistakes or repair damage. § If damaged DNA can be corrected before the cell division, no mutation will occur. § Some types of DNA damage (e.g., large-scale damage from highly mutagenic chemicals/large doses of radiation), may cause lesions that interfere with replication. § If such lesions are not removed before replication occurs, DNA replication will stall and lethal breaks in the chromosome will result. stop or cause to stop making progress § Stalled replication as well as certain types of major DNA damage activate the SOS repair system.  22 DNA Repair and the SOS System SOS System The SOS system is a bacterial DNA repair system that is activated in response to significant DNA damage, such as that caused by UV radiation or chemical agents. Components: The SOS system is regulated by two proteins, LexA and RecA RecA Protein: Binds to single-stranded DNA and stimulates the cleavage of LexA repressor. LexA Repressor: Inhibits the transcription of SOS genes under normal conditions.  23 Mechanism of the SOS response. - DNA damage activates RecA protein, which in turn activates the protease activity of LexA. - The LexA protein then cleaves itself. - LexA protein normally represses the activities of the recA gene and the DNA repair genes uvrA and umuCD. - When LexA inactivated, these genes become highly active.  24 GENE TRANSFER IN BACTERIA Recombination § Incorporation of extrachromosomal (foreign) DNA into the chromosome occurs by recombination. § There are two types of recombination: § homologous § nonhomologous § Homologous (legitimate) recombination occurs between closely related DNA sequences and generally substitutes one sequence for another. § The process requires a set of enzymes produced (in E. coli) by the rec genes. § Nonhomologous (illegitimate) recombination occurs between dissimilar DNA sequences and generally produces insertions or deletions or both. § This process usually requires specialized recombination enzymes, such as those produced by many transposons and lysogenic bacteriophages.  25 Recombination § A part of or all of the genetic material in bacteria can be transferred from one cell to another § Uptake of DNA molecule from of one bacterium (donor cell) by another bacterium (recipient cell) § The exogenous material from the donor cell is called the exogenote, and its response in the recipient cell is called endogenote § The newly formed bacterium is recombinant  26 Recombination Recombinant bacteria: - Express the properties of both the donor and the recipient cell  27 Mechanisms of genetic transfer between cells The exchange of genetic material between bacterial cells may occurs in 3 main ways: 1. Transformation 2. Transduction 3. Conjugation  28 Transformation § The process by which bacteria take up fragments of naked DNA and incorporate them into their genomes. § competent cell: a cell that is able to take up DNA and be transformed (naturally capable of taking up exogenous DNA, including Haemophilus influenzae, Streptococcus pneumoniae, Bacillus spp., and Neisseria spp.) § E. coli and most other bacteria lack the natural ability for DNA uptake, and competence must be induced by chemical methods or electroporation (use of high-voltage pulses) to facilitate uptake of plasmid and other DNA.  29 Mechanism of transformation in a gram-positive bacterium. (a) Binding of double-stranded DNA by a membrane-bound DNA-binding protein (b) Passage of one of the two strands into the cell while nuclease activity degrades the other strand (c) The single strand in the cell is bound by specific proteins, and recombination with homologous regions of the bacterial chromosome is mediated by RecA protein (d) Transformed cell  30 Transduction § The process by which foreign DNA is introduced into a cell by a virus or viral vector (bacteriophage) § Bacteria infected with bacteriophages; - uptakes the DNA from the donor - incorporates the DNA into its chromosome by recombination  31 Bacteriophages § Infect and replicate within bacteria and archeae § Specific to certain bacterial types § Multiple bacteriophages may use a single bacterium as host § Bacteria have specific receptors for bacteriophages  32 Replication of bacteriophages § Virulent phages lyse bacteria (lytic phage) § Temperate phages infect bacteria (no lysis, lysogenic phage) § Replicate within the bacterial genome and transferred to the offspring  33 Lytic Cycle Characteristics: Immediate Replication: Upon infection, the phage immediately uses the bacterial machinery to replicate. Destruction of Host: The bacterial cell is lysed to release new phage particles. Steps: 1.Attachment: The phage attaches to the bacterial cell. 2.Injection: The phage injects its DNA into the bacterial cell. 3.Replication: The phage DNA directs the synthesis of new viral components. 4.Assembly: New phage particles are assembled. 5.Lysis: The bacterial cell is lysed, and new phages are released.  34 Lysogenic Cycle Characteristics: Integration: Phage DNA integrates into the bacterial chromosome and becomes a prophage. Dormancy: The phage remains dormant and replicates along with the bacterial chromosome. Steps: 1.Attachment: The phage attaches to the bacterial cell. 2.Injection: The phage injects its DNA into the bacterial cell. 3.Integration: Phage DNA integrates into the bacterial chromosome. 4.Propagation: The prophage is replicated along with the bacterial DNA during cell division. 5.Induction: Under certain conditions, the prophage may excise itself and enter the lytic cycle.  35 Lytic Cycle vs Lysogenic Cycle Key Differences: 1.Outcome for Host: 1. Lytic: Destruction of the bacterial cell. 2. Lysogenic: Temporary coexistence with the bacterial cell. 2.Phage Replication: 1. Lytic: Immediate replication and assembly. 2. Lysogenic: Delayed replication; can switch to the lytic cycle later. 3.Genetic Exchange: 1. Lytic: No integration into the host genome. 2. Lysogenic: Integration allows for gene transfer. 4.Applications: 1. Lytic: Used in phage therapy. 2. Lysogenic: Studied for their role in bacterial evolution and gene transfer.  36 Lysogenic and lytic cycle phages  37 Transduction § Only occurs in similar bacterial types § Salmonella, Shigella, Escherichia, Pseudomonas, Vibrio, Proteus, Staphylococcus and Bacillus § Localized transduction: Bacteriophage attaches to a specific region of bacterial chromosome therefore transfer of a specific DNA region § Generalized transduction: Equal chances for the transfer of every single single in bacterial DNA  38 Conjugation § A form of horizontal gene transfer that requires cell-to-cell contact § A plasmid encoded mechanism that can mediate DNA transfer between unrelated cells, even between different genera § Conjugative plasmids use this mechanism to transfer copies of themselves and the genes they encode, such as those for antibiotic resistance, to new host cells. § In conjugation, donor cells contain F plasmid (F factor = Fertility factor) as well as its own DNA § These cells are F+ cells (male) § Cells without F factor, F- (female)  39 F Plasmid § F plasmid (F stands for “fertility”) is a circular DNA molecule § one region of the plasmid contains genes that regulate DNA replication § also contains a number of transposable elements that allow the plasmid to integrate into the host chromosome § F plasmid has a large region of DNA, the tra region, containing genes that encode transfer Genetic map of the F (fertility) functions plasmid of Escherichia coli § Only donor cells produce sex pilus (pili) Transfer of plasmid DNA by 40 conjugation § The transfer of the F plasmid converts an F− recipient cell into an F+ cell Pilus found on male cells attach to female cells and form a bridge for conjugation Single strand of F plasmid is transferred to the female cell via the bridge Both cells synthesize complimentary strands Both cells become F+  41 Conjugation E. coli Salmonella Shigella Pseudomonas Serratia Vibrio Streptomyces  42 Elements transferred from one bacterium to another Genetic material transferred from cell to cell; chromosome of donor bacterium or F factor Apart from the bacterial chromosome – plasmid and transposons  43 Plasmids § small DNA molecule within a bacterial cell that is physically separated from a chromosomal DNA and can replicate independently § most commonly found as small circular, double-stranded DNA molecules in bacteria  44 Plasmids § Can be transferred from one bacterium to another (via conjugation) § Causes functional alterations in recipient cells: - Resistance to antibiotics, heavy metals, UV - Enterotoxins - Hemolysins - Proteases - Pigment formation, coagulse and fibrinolysin production  45 Main plasmids 1. F factor (F plasmid = fertility factor): Carry genes for the formation of sex pili and for the initiation of conjugation (E.g., F- plasmid in E. coli) ü Function: Facilitates the transfer of genetic material from a donor to a recipient cell. 2. Col plasmid (colisinogenic factors): Carry genes that encode colicins, which are proteins that kill other bacteria (E.g., ColE1 plasmid in E. coli). ü Function: Provides a competitive advantage by killing off surrounding bacteria. 3. Degradative plasmids: Carry genes for the breakdown of unusual organic compounds (E.g., TOL plasmid in Pseudomonas putida) ü Function: Allows bacteria to utilize unusual substances as a source of carbon.  46 Main plasmids 4. R plasmid (Resistance Transfer Factor - RTF): Contain genes that provide resistance against antibiotics or other toxins (E.g., R100 plasmid conferring multiple antibiotic resistances). ü Function: Enables bacteria to survive in the presence of antibiotics. 5. Bacteriocinogenic Plasmids: Carry genes for bacteriocins, which are toxic proteins that inhibit the growth of similar or closely related bacterial strains (p9B plasmid in Lactococcus lactis). ü Function: Provides a competitive advantage in microbial communities. 6. Virulence plasmids: Contain genes that turn a bacterium into a pathogen (E.g., pXO1 and pXO2 in Bacillus anthracis). ü Function: Enables bacteria to infect and cause disease in a host organism.  47 Transposable (mobile) elements § Transposons: double stranded DNA sequence that can change its position within a genome, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size § are called jumping genes § smaller than plasmids § cannot replicate independently § can code drug resistance enzymes, toxins or metabolic enzymes § In bacteria, they play a significant role in the evolution and adaptability of the organism by facilitating gene transfer and rearrangement.