Microbial Genetics Lesson 5 PDF

9/3/2024 Le...

9/3/2024 Learning objectives: ▪ Describe the importance of DNA Microbial ▪ Describe microbial genetics and the central dogma of molecular biology ▪ Differentiate vertical from horizontal gene transfer ▪ Describe the mechanisms of horizontal gene transfer Genetics ▪ Explain how mechanisms of horizontal gene transfer contributes to the spread of antibiotic resistance genes in the clinical and environmental setting Irish A. Rabano Instructor IV Centro Escolar University – Manila Introducing Genes and Genomes Microbial genetics ▪ Genetics ▪ is the study of the mechanisms of heritable information in microorganisms ▪ study of heredity in general and of genes in particular ▪ study of inherited traits, rooted in DNA, and their variations and transmission ▪ Heredity ▪ Transmission of inherited traits from generation to generation ▪ Genes ▪ A section of a DNA molecule whose sequence of building blocks instructs a cell to produce a particular protein ▪ Genomes ▪ The complete set of genetic instructions in the cells of a type of organism. ▪ DNA is the information molecule for all living organisms. All of the DNA of an organism is called its genome. ▪ Genomics – The field that analyzes and compares genomes  9/3/2024 Genetic Information of Microbes Nucleoid-associated proteins (NAPs) ▪ Bacterial genome ▪ The bacterial genome is usually contained in a circular DNA molecule which is supercoiled and localized within the nucleoid of the cell. ▪ Size of bacterial genome: usually less than 500,000 base pairs (500 kb) to 5 million base pairs (5 Mb) ▪ (The major pattern in bacterial genome size is that, on average, free-living species have larger genomes than parasitic species which in turn have larger genomes than obligate pathogens) ▪ Exceptions: ▪ Some bacteria have two or more chromosomes ▪ Vibrio, Burkholderia, Leptospira and Brucella species ▪ Some chromosomes may be linear ▪ Borrelia burgdorferi  Bacteria have a cell cycle with a duration on the order of tens of minutes. ▪ Some chromosomes may be large  As a consequence, genome folding and transcription are intimately coupled with genome ▪ Bacillus megaterium may be as large as 30 MB replication. (Brocken et al., 2018) Operon - (Bacterial Gene Organization) a set of functionally related structural genes adjacent to a ▪ A typical bacterial genome has common transcriptional control site (promoter and operator) approximately 4,400 genes (humans 25,000) ▪ The genome of bacteria encodes: ▪ Biochemical functions necessary for survival ▪ Virulence factors for pathogenic bacteria ▪ Non-coding regions  9/3/2024 - any DNA molecule regardless of its specific nature (diCenzo and Finan, 2017) Plasmid Genetic Information of Microbes Extrachromosomal DNA – found in bacteria, archaea, some fungi ▪ Bacterial DNA (Deoxyribonucleic acid) – usually small, closed circular DNA molecules ▪ Circular chromosome + plasmid Exist and replicate independently of chromosome Contain few genes that are non-essential – confer selective advantage to host (e.g., drug resistance, E. coli: plasmid encodes toxins produced and bacterial attachment to intestinal cells) Plasmids are used in genetic engineering as gene transfer vectors  9/3/2024 DNA is a nucleic acid and nucleotides are the building blocks of all nucleic acids. 1 2 3 Three essential components: 1. Nitrogenous base 2. Phosphate group 3. Pentose sugar (a 5-carbon sugar) Polymers - a substance or material consisting of very large molecules called macromolecules, composed of many repeating subunits or monomers Lipids – fatty acids, Carbohydrates – monosaccharides, Proteins – amino acids Nucleic acids - nucleotide What is the structure of a DNA? Orientation? Phosphate of one nucleotide attaches to the 3rd C-OH group of the sugar of the 2nd nucleotide, thereby forming 5’ → 3’ linkage Nitrogenous base Purines – Guanine and adenine The phosphate residue is attached to the 5′ carbon of the sugar Pyrimidines – Cytosine, The hydroxyl group is attached to the thymine, and uracil 3′ carbon of the sugar The nitrogenous bases is attached in the 1’ carbon of the sugar  9/3/2024 Central Dogma of Molecular Biology The central dogma of molecular biology is a theory stating that genetic information flows DNA only in one direction, from DNA, to RNA, to protein, or RNA directly to protein. RNA 1. Sugar = 1. Sugar = ribose ▪ Central Dogma deoxyribose 2. Bases = A,C, ▪ Replication – Transcription - Translation 2. Bases = A,C, G, U G, T 3. Single 3. Double stranded stranded 4. Leaves the 4. Stays in nucleus nucleus DNA replication is semi-conservative DNA REPLICATION DNA must be replicated in order to ensure that each new cell receives the correct number of chromosomes.  9/3/2024 DNA REPLICATION Purpose: create a duplicate copy of the double stranded DNA Site: DNA replication occurs in the nucleoid Template: Both strands (leading and lagging strands) Control: Origin of replication STAGES OF DNA REPLICATION: 1. Initiation 2. Elongation 3. Termination TRANSCRIPTION TRANSLATION DNA to mRNA to protein DNA to mRNA to protein Synthesis of proteins from the information Double stranded DNA must be TRANSCRIBED into a single stranded mRNA contained in a molecule of messenger RNA (mRNA).  9/3/2024 In an mRNA, the instructions for building a polypeptide are RNA nucleotides (AUGC) read in groups of three = Codons TRANSLATION Translation is the decoding of an mRNA message into a polypeptide chain (protein) Purpose: convert a sequence of mRNA into a sequence of amino acids to make proteins Site: ribosomes in the cytoplasm Template: mRNA Key components: tRNA, ribosomes, mRNA What are the ways that genes can be transferred? 1. REPRODUCTION - Binary fission Exchange of ▪ Vertical gene transfer - Transfer of genetic material from parental organism to progeny Gene Transfer refers to the Genetic transfer of DNA containing functional genes between any two How do they get genetic variability? 2. Horizontal Gene Transfer - Transfer of genetic material organisms Information between unrelated individuals ▪ Transformation: Bacteria take up DNA from their environment ▪ Conjugation: Bacteria directly transfer genes to another cell ▪ Transduction: Bacteriophages (bacterial viruses) move genes from one cell to another  9/3/2024 Transposable Elements Conjugation ▪ A transposable element (TE, transposon, or jumping gene) ▪ Transposons can insert into plasmids which can be transferred to recipient cells by conjugation ▪ a donor bacterium transfers a copy of a ▪ Designed to move from one location to another within a DNA molecule by a process known as transposition. plasmid to a recipient bacterium, through a pilus via direct cell to cell contact  Transposable elements are flanked by inverted repeats and often contain multiple antibiotic resistance ▪ Donor cell – has (F+) conjugative genes plasmid ▪ All transposable elements code for the enzyme transposase, the enzyme responsible allowing ▪ F (fertility) factor is a conjugative plasmid transposition to occur, and have short inverted repeats (IRs) at each end. transferred from cell to cell by conjugation ▪ F factor is an episome = genetic element that can insert into chromosome or replicate as circular plasmid ▪ Recipient cell – (F-) does not contain conjugative plasmid 30 Kinds of Transposition Transposable elements Transposition is the process by which transposons move within a genome from one location to another. ▪ A transposable element (TE, transposon, or jumping gene) There are two main kinds of transposition mechanisms, that determine how ▪ Designed to move from one location to transposable elements copy and move within the genome: another within a DNA molecule by a process known as transposition. 1. Replicative Transposition ▪ All transposable elements code for the (Copy-and-Paste Mechanism): enzyme transposase, the enzyme responsible allowing transposition to occur, and have short inverted repeats (IRs) at  The transposable element creates a copy of each end. itself at a new genomic location while leaving the original element intact at its original site. This mechanism increases the overall number of transposable elements within the genome.  9/3/2024 Kinds of Transposition Transformation ▪ New genes are acquired directly from the environment often recombines with genes on 2. Non-replicative or Conservative bacterial chromosome Transposition (Cut-and-Paste ▪ Requirement: Mechanism) ▪ Donor cell – lysed and released naked DNA to environment  In non-replicative transposition, the ▪ Recipient cell – must be in a state of competence transposable element is physically ▪ Natural competence moved from one location to another ▪ genetically predisposed to be able to uptake without creating a copy. This mechanism foreign DNA can lead to the loss of the original element ▪ Artificial competence from its original site. ▪ May be induced in the laboratory ▪ Heat shock and electroporation  Once inside the cell, the DNA must be incorporated into the bacterial chromosome by RecA, for the genes to be expressed. How does bacterial transformation work in the laboratory? Why transform bacteria? Definition of terms: Restriction enzymes - cleaves 1) to make multiple copies of DNA called DNA cloning DNA sequences at sequence- specific sites, producing DNA 2) to make large amounts of specific human proteins, for example human fragments with a known insulin, which can be used to treat people with Type I diabetes. sequence at each end Ligase - a DNA-joining enzyme 3) to genetically modify a bacterium or other cell  9/3/2024 Bacteriophage Classified into two major groups: Virulent and Temperate Transduction  Transfer of genes from a donor to a recipient by a bacteriophage (replicating virus)  Does not require physical contact between the cell donating the DNA and the cell receiving the DNA (which occurs in conjugation)  There are two different types of transduction: generalized transduction and specialized transduction. Bacteriophage Bacteriophages are viruses that infect bacteria. Bacteriophages may have a lytic cycle or a lysogenic cycle, and a few viruses are capable of carrying out both. Generalized transduction In generalized transduction, the bacteriophages can pick up any portion of the host's genome ▪ Lytic (virulent and temperate phage) ▪ Lysogenic (temperate phage) ▪ Phage which multiply on bacteria and kill the ▪ Quiescent state in cell; does not result in  a bacterial host cell is infected with cell by lysis at the end of the life cycle immediate lysing of the host cell. either a virulent or a temperate ▪ Viral genome will integrate with host DNA bacteriophage engaging in the lytic and replicate along with it fairly harmlessly, cycle of replication or may even become established as a plasmid  During the assembly stage, random ▪ As the lysogenic cycle allows the host cell to pieces of bacterial DNA are continue to survive and reproduce, the virus mistakenly packaged into a phage is reproduced in all of the cell’s offspring. head, resulting in the production of ▪ The virus remains dormant until host a transducing particle conditions deteriorate  can bind to a new bacterial host ▪ the endogenous phages (known as cell and inject their DNA inside = prophages) become active gene expression ▪ initiate the reproductive cycle, resulting in lysis of the host cell  9/3/2024 Specialized transduction In contrast, with specialized transduction, the bacteriophages pick up only specific Application portions of the host's DNA. Transduction is used to insert the genes of choices in animals and plant cells to  Can only occur with temperate modify the genetic constituents and get the desired characteristics. bacteriophage; lysogenic cycle  Bacteriophage injects viral DNA inside It can be used for gene therapy. It has huge potential to cure genetic diseases.  DNA integrates host cell chromosome forming a It is an important tool in genetics and molecular biology research. prophage  When such lysogenic cell is exposed to certain stimulus, it causes induction of virus genome from host cell genome and begins lytic cycle  Induction – prophage is excised from bacterial chromosomes  Phage genome carries a part of bacterial DNA (that on its sides) Molecular Recombination Types of Recombination ▪ In each of the cases of Horizontal Gene Transfer, the process is only successful if the genes can be expressed by the altered cell. ▪ Thus, the donor DNA must be recombined with the recipients chromosome Homologous Recombination  Involves the exchange of For the genes to be expressed, genetic material between two similar or identical DNA the DNA must be recombined with the sequences, usually between recipient’s chromosome. homologous chromosomes or sister chromatids.  9/3/2024 Types of Recombination Site-specific recombination  DNA strand exchange takes place between DNA segments that possess at least a certain degree of sequence homology but no extensive homology.  The exchange of genetic material at END OF PRESENTATION. specific target sites on DNA molecules, mediated by specific enzymes recognizing unique DNA sequences.  Often used by viruses to insert their genome into the chromosomes of their host. This type of recombination is also used by transposable elements.