Bacterial Genetics Lecture Notes PDF

Bacterial Genetics 02/12/2024 1  Learning Objectives 1) Understand microbial gene and its functions. 2) Describe the microbial extrachromosomal genetic material. 3) Differentiate between genotypic and phenotypic variations....

Bacterial Genetics 02/12/2024 1  Learning Objectives 1) Understand microbial gene and its functions. 2) Describe the microbial extrachromosomal genetic material. 3) Differentiate between genotypic and phenotypic variations. 4) Define different types of mutations. 5) Describe and discuss the methods of gene transfer in bacteria. 6) Describe the structure, life cycle and the uses of bacteriophages. 7) Understand the process of genetic engineering and its applications. 02/12/2024 2  Bacterial Genome It is the sum of genes of an organism. The bacterial genes are carried on: 1. Chromosome 2. Extra-chromosomal material such as: Plasmids, Transposons and Prophages 02/12/2024 3  Bacterial Chromosome Bacterial cells are haploid having a single chromosome and a single copy of each gene. Eukaryotic cells (such as human cells) are diploid, they have a pair of each chromosome and therefore have two copies of each gene. In diploid cells, one copy of a gene (allele) may be expressed as a protein (i.e., be dominant), whereas another allele may not be expressed (i.e., be recessive). In haploid cells, any gene that has acquired a mutation will result in a cell synthesizing either a mutant protein or no protein at all depending on the type of mutation. 02/12/2024 4  Extra-chromosomal material 1- Plasmids Plasmids are extrachromosomal, double-stranded, circular DNA molecules that are capable of replicating independently of the bacterial chromosome (not all). Sometimes Plasmids can be integrated into the bacterial chromosome and is called “episome”. Plasmids occur in both gram-positive and gram-negative bacteria 02/12/2024 5  Several different types of plasmids can exist in one cell: 1. Transmissible plasmids Can be transferred from cell to cell by conjugation. They are large since they contain about a dozen genes responsible for synthesis of the sex pilus and for the enzymes required for transfer. They are usually present in a few (1–3) copies per cell. 2. Non-transmissible plasmids They are small since they do not contain the transfer genes; they are frequently present in many (10–60) copies per cell. 02/12/2024 6  Plasmids carry the genes for the following functions and structures of medical importance: 1. Exotoxins, such as the enterotoxins, anthrax toxin, exfoliative toxin, tetanus toxin. 2. Pili (fimbriae), which mediate the adherence of bacteria to epithelial cells. 3. Antibiotic resistance, which is mediated by a variety of enzymes, such as the β-lactamase. 4. Resistance to heavy metals, ultraviolet light, which is mediated by DNA repair enzymes. 5. Bacteriocins production, which are toxic proteins produced by certain bacteria that are lethal for other bacteria. Bacteria that produce bacteriocins have a selective advantage in the competition for food sources over those that do not. 02/12/2024 7  2-Transposons“Jumping genes” Transposons: are pieces of DNA that move readily from one site to another either within or between the DNAs of bacteria, plasmids, and bacteriophages. Transposons can code for drug-resistant enzymes, toxins, or a variety of metabolic enzymes and can either cause mutations in the gene into which they insert or alter the expression of nearby genes. 02/12/2024 8 Transposition: movement of transposons 1- Replicative transposition: Transposons move by replicating their DNA and inserting the new copy into another site. Meaning In the replicative pathway, the original transposon remains in place while new copies are mobile 2- Direct transposition: Transposons are excised from the site without replicating and then inserted into the new site. (Cut and Paste). 02/12/2024 9  3- Bacteriophage They are viruses that infect bacterial cells. They are highly specific to their host cell. Structure of Bacteriophage: 1. Head: is formed of protein capsid which encloses phage DNA. 2. Tail: a hollow core surrounded by contractile sheath which ends in Base Plate. 3- Base plate: is attached to tail fibers that are used for attachment of the phages to cell wall receptors. 02/12/2024 10 02/12/2024 11  Importance of Bacteriophages It is involved in several procedures: 1. Gene transfer: Transfer of genetic material from one bacterial strain to another by transduction. 2. Lysogenic conversion: In which phage's own DNA confers new properties to the cell, such as production of certain bacterial exotoxins. 3. Genetic engineering 02/12/2024 12  Interaction between Bacteriophage & Bacterial Cell Bacteriophage cycle is of one of the following forms: 1- Bacteriolysis (Lytic cycle): Bacteriophage cycle ends in lysis of the bacterial host cell and release of new formed phages. This occurs by virulent or lytic phage. 2-Lysogeny: Bacteriophage cycle doesn’t end in lysis of the bacterial host cell but the phage DNA is integrated into the bacterial chromosome. Occurs by "the temperate phage", which does not replicate and lyse the bacteria. The integrated non lytic form which persists in the cell is called "prophage". The prophage replicates within the bacterial chromosome and transferred to the progeny of the cell. 02/12/2024 13 02/12/2024 14  Bacterial Variation Changes of the normal character of the bacterium Two types: 1. Phenotypic Variation 2. Genotypic Variation 02/12/2024 15  Phenotypic Variation Definition: Changes of certain bacterial characters in response to environmental factors without change in its genetic makeup. It is Not heritable and reversible when environmental cause is removed. Examples: 1. Change in colonial morphology when bacteria grow on unsuitable media, e.g. (change from smooth to rough colony). 2. Loss of pigment production by Staphylococci when grown in anaerobic conditions 3.Bacterial sporulation and vegetation. 02/12/2024 16  Genotypic Variation Changes of bacterial characters due to change in its genetic makeup irreversible and heritable. Types of genotypic variation: 1- Mutations A mutation is a change in the base sequence of DNA that can result in the insertion of a different amino acid or stop codon into a protein and the appearance of an altered phenotype. Mutation may be: A. Spontaneous: occurs as a result of error of replication. B. Induced: by physical agents, e.g. light, ultraviolet rays or gamma rays, or by chemical agents as alkylating agents, nitroso compounds. Medical Importance: Induced mutation is used for manipulation of the microbial genome to get mutant of low virulence that can be used as vaccine, or mutant producing large amounts of antibiotics. 02/12/2024 17 02/12/2024 18  Mutations result from three types of molecular changes 1- Base Substitution 2- Frameshift 3- Transposons and transposition 02/12/2024 19  1-Base substitution This occurs when one base is inserted in place of another. It takes place at the time of DNA replication, either because the DNA polymerase makes an error or due to exposure to a mutagen. Base Substitution can result in: Missense mutation: When the base substitution results in a codon that simply causes a different amino acid to be inserted. Nonsense mutation: When the base substitution generates a termination codon that stops protein synthesis prematurely. Nonsense mutations almost always destroy protein function. 02/12/2024 20 02/12/2024 21  2-Frameshift mutation This occurs when one or more base pairs are added or deleted which shifts the reading frame on the ribosome and results in incorporation of the wrong amino acids “downstream” from the mutation and in the production of an inactive protein. 3-Transposons or insertion sequences When they are inserted into the DNA they cause marked changes in the genes into which they insert and in adjacent genes. 02/12/2024 22  Gene transfer 1- Transfer of DNA within bacterial cells 2- Transfer between 2 different cells Gene Transfer within bacterial cell Transposons transfer DNA from one site on the bacterial chromosome to another site or to a plasmid. 02/12/2024 23  The transfer of genetic information from one cell to another can occur by three methods: conjugation, transduction, and transformation. Medical Importance of Gene transfer, the two most important consequences of DNA transfer are: (1) The transfer of a transposon to a plasmid and the subsequent transfer of the plasmid to another bacterium by conjugation contribute significantly to the spread of antibiotic resistance. (2) Several important exotoxins are encoded by bacteriophage genes and are transferred by transduction. 02/12/2024 24  Gene transfer methods 1. Conjugation 2. Transduction 3. Transformation 02/12/2024 25  1. Conjugation Conjugation is the process by which one bacterium transfers genetic material to another through direct contact during which DNA is transferred from the donor to the recipient cell. A direct connection between the cytoplasm of the donor and recipient cells occur via the conjugation pilus or tube. The newly acquired DNA can recombine into the recipient’s DNA and become a stable component of its genetic material. R plasmids that encodes the β-lactamases of Staphylococcus aureus, Escherichia coli, & Klebsiella pneumoniae can be transferred by conjugation. 02/12/2024 26 02/12/2024 27  2. Transduction Transduction is the transfer of DNA by bacterial viruses (bacteriophage). During the growth of the virus within the cell, a piece of bacterial DNA is incorporated into the virus particle and is carried into the recipient cell at the time of infection. Within the recipient cell, the phage DNA can integrate into the cell DNA and the cell can acquire a new properties (a process called lysogenic conversion). This process can change a non-pathogenic organism into a pathogenic one. Diphtheria toxin, botulinum toxin and cholera toxin are all encoded by bacteriophages and can be transferred by transduction. 02/12/2024 28 02/12/2024 29  Types of transduction 1) The generalized transduction: Where the bacteriophages can pick up any portion of the bacterial chromosome. This occurs because the bacterial DNA is fragmented after phage infection and pieces of this DNA the same size as the viral DNA are incorporated into the virus particle. 2) The specialized transduction: The bacteriophages pick up only specific portions of the bacterial chromosome. 02/12/2024 30 02/12/2024 31  3. Transformation Transformation (Uptake of pure “naked “DNA ) Dying bacteria are constantly releasing free DNA which can be taken by other bacteria. Most bacteria are not naturally competent to be transformed by DNA, due to the presence of restriction endonuclease enzymes which digest any foreign DNA. Certain bacteria, such as Neisseria, Haemophilus, and Streptococci, synthesize receptors on the cell surface that play a role in the uptake of DNA from the environment. 02/12/2024 32  Competence can be induced artificially in the laboratory by treating cells with calcium chloride or heat shock which alter cell membrane permeability. There must be homology between donor &recipient bacteria. If the DNA is completely unrelated, the absence of homology prevents recombination and the DNA is degraded. DNA fragment recombined with bacterial DNA can propagate new genes coding for new character, e.g. virulence factors. 02/12/2024 33 02/12/2024 34  Recombination Once the DNA is transferred from the donor to the recipient cell by one of the three processes, it can integrate into the host cell chromosome by recombination. There are two types of recombination: 1) Homologous recombination, in which two pieces of DNA that have extensive homologous regions pair up and reunion. 2) Non-homologous recombination, in which little homology is necessary. 02/12/2024 35 02/12/2024 36  Genetic Engineering It is the modification of the genotypes of organisms by incorporation of new genes from entirely different species, thus allowing the manufacture of a genetic material in the laboratory. 02/12/2024 37  Recombinant DNA Recombinant DNA is the DNA molecule which contains a new gene. This molecule can be propagated in a host organism by gene cloning. Gene cloning means the insertion of a fragment of DNA, containing specific genes, into a vector (usually plasmid or phage); and the subsequent propagation of the recombinant DNA molecules in a host organism. The propagation process allows the formation of a population of identical cells containing identical recombinant DNA molecules. 02/12/2024 38  Technique of recombinant DNA technology 1. DNA extraction: separation of DNA that contains specific genes from human or animal. 2. Extraction of vectors DNA from bacteria and some other organisms (e.g. yeast). 3. Both specific DNA fragment and the vector are treated with restriction endonuclease enzyme which break them at specific recognition sites. Both ends of broken DNA are complementary (sticky or cohesive ends). 4. Mixing of the endonuclease treated vector and the specific DNA together in presence of DNA ligase enzyme results in the joining of the sticky ends of both the vector molecule and the DNA to be cloned. The resulting molecule is referred to as recombinant DNA. 5. The recombinant DNA molecule is transferred to a bacterial cell by transformation. This cell is allowed to multiply, creating many genetically identical bacteria (clones); each is able to produce the gene product. 6. From the clone culture, the gene product may then be harvested. Thus, microorganisms can be genetically engineered to produce substances that they would not normally manufacture. 02/12/2024 39 Technique to create recombinant DNA technology 02/12/2024 40  Technique to create recombinant DNA technology 02/12/2024 41  Applications of genetic engineering Production of many substances as hormones (insulin) and recombinant vaccines (hepatitis B vaccine). Diagnosis of some diseases through the use of nucleic acid probe which is a short sequence of single stranded DNA or RNA labeled with radioactive isotopes or enzyme. Gene therapy which involves insertion of a normal gene into human cells to replace a defective gene to correct a specific genetic disorder. 02/12/2024 42