Microbial Genetics Unit 7 PDF

Microbial genetics Unit 7 Ve más allá 1 Bacterial genome Bacterial genome: genetic information contained in a cell. ✓ Chromosome: DNA containing the genes ✓ extra chromosomal genetic elements: plasmids, bacteriophages Genes: sequen...

Microbial genetics Unit 7 Ve más allá 1 Bacterial genome Bacterial genome: genetic information contained in a cell. ✓ Chromosome: DNA containing the genes ✓ extra chromosomal genetic elements: plasmids, bacteriophages Genes: sequences of nucleotides that have a biological function and encodes proteins Bacteria are usually haploid (one copy of their chromosomes)  Genotype and phenotype Phenotype ✓ Manifestation of the bacterial genotype ✓ Morphological or physiological visual characteristics of the bacteria ✓ Determined by the interaction between the expression of its genome and the external influence of the environment in which they grow ✓ Not every character is encoded genetically Genotype ✓ Specific set of genes of an organism: specific genes or complete DNA ✓ Inherited characteristics ✓ Not all genes are expressed phenotypically Phenotypic variations Definition: Produced by environmental pressure with no impact on the genome…….. ✓ High frequency, affecting the entire population exposed to the environmental pressure. ✓ Reversible, returning to the original state when the generating cause dissapears. ✓ Not inherited, there is no change in the DNA. Phenotypic variations Types/Examples: ✓ Morphological: mucus layer in media with sucrose. ✓ Chromogenic: red pigment at 22 °C but not at 37 °C (Serratia marcescens)  Genotypic variations. Mutations Definitions: ✓ A mutation is a persistent change of DNA bases without addition of exogenous DNA. They arise: 1. Spontaneously 2. By mutagens 3. By transposable elements ✓ The microorganisms that have undergone a specific mutation are called mutants. ✓ The microorganisms before becoming mutants are called wild-type. Genotypic variations. Mutations Bacterial mutations High-speed duplication. Most species in about half an hour can double their population. Within 24 hours a single bacterium can generate a population of >105 bacteria. The DNA repair system produces higher frequency of mutations in bacteria than in other organisms (lack of time for repairing). Genotypic variations. Mutations Many mutants are incompatible with life, which is not a great biological loss due to the high-speed duplication. Some specific mutations can provide the mutant a biological advantage over the wild population, especially in certain environments such as the presence of antibiotics. Genotypic variations. Mutations Characteristics: Low frequency: can be increased by a mutagenic agent. Irreversible. Inherited by the progeny because the new character is printed on the DNA. Specific: affect one or a few specific characters. Spontaneous mutation occurs before the mutated population become predominant due to the selector agent. Genotypic variations. Mutations Types of mutations Substitution A substitution is a mutation that replaces one base with another Deletion Deletions are mutations in which a section of DNA is lost or deleted. Insertion Insertions are mutations in which extra base pairs are inserted into a new place in the DNA.  Genotypic variations. Mutations Mutations can affect multiple bases or a single base (point mutations). The protein translation through mRNA can produce an altered protein (amino acid change: missense mutation) or no protein (the mutation generates a premature termination codon: nonsense mutation) Single-base mutations can generate a codon that encodes the same amino acid and the protein does not vary. It would be a silent mutation. http://www.sbs.utexas.edu/psaxena/MicrobiologyAnimations/Animations/Mutations/PLAY_mutations.html 2 Genotypic variations. Mutations Effect of the mutation in protein translation WILD GENE MUTATED GENE mRNA Ribosome NO PROTEIN TERMINAL CODON mRNA SAME SAME AMINOACID PROTEIN DIFFERENT DIFFERENT PROTEIN AMINOACID Genotypic variations. Mutations Spontaneous mutations: random change in the DNA arising from errors in replication Induced mutations: results from exposure to known mutagens ▪ Physical agents: Ionizing and ultraviolet radiation. ▪ Chemical agents. ✓ Substitutions: Nitrous acid (transforms adenine in hypoxanthine that binds to cytosine), 5-bromouracil (replaces thymine and binds to guanine) ✓ Insertions: Acridine Orange. Transposable elements. Movement of pieces of DNA around the genome. They produce both deletions and insertions. ✓ Insertion sequences. ✓ Transposons Genotypic variations. Example of mutagenic agents Adenine Nitrous acid Thymine Hypoxanthine X Cytosine Adenine-thymine becomes hypoxanthine-cytosine Acridine orange: N Double-stranded DNA ATGCCTAAGCTAC ATGCCTNAAGNCTAC [ TACGGATTCGATG TACGGA−TTC−GATG Intercalant agent  Transposable Elements DNA fragments that must always be integrated with other DNA. High mobility within the DNA where they are integrated, even DNA can jump from one DNA to another. They can provide new information or not, but always generate a DNA reorganization. They consist of: ▪ IR (inverted repeats): small nucleotide sequences located at the ends and are repeated and inverted. ▪ Genes for transposases: Enzymes to transpose (cutting and rejoining) Transposases cut DNA by recognition of an area called DR (direct repeats), consisting of a few nucleotides repeated but not inverted. They cut and bind the IR. Transposable Elements Types of Transposable Elements Insertion sequences (IS) Short sequence of DNA containing only the genes for those enzymes required for transposition and bounded at both ends by identical or very similar sequences of nucleotides in reversed orientation known as inverted repeats. Complex transposon They have two IS at both ends, usually found in an inverted position. Between them there are other genes that may determine the acquisition of biological functions. Non-IS-dependent non-complex transposons Similar to IS but larger in size, with genes that encode biological functions. Transposable Elements INSERTION SEQUENCE recipient DNA IS recipient DNA DR IR IR DR (GTCGACAGCT) (ATCGCATAGT) (TGATACGCTA) (GTCGACAGCT) tnpA COMPLEX TRANSPOSON IS Other genes IS Genotypic variations. Mutations- Repair systems In general, a mutation has negative consequences for a colony of bacteria. Mechanisms to avoid mutations or repair them: Bacteria have mutation repair systems that are less developed than in higher organisms Reversion: A reverse mutation returns to the wild type genotype. Very rare. DNA Repair system: ✓ Specific enzymes: Photolyase corrects the damage done by UV radiation. ✓ Excision-repair system: The damaged chain gets cut (endonuclease) and re-synthesized (polymerase and ligase). ✓ Recombination system: damaged chain is replaced by the homologous fragment and synthesized. ✓ SOS repair system: regulatory system of proteins/enzymes involved in repair processes. Genotypic variations. Mutations Mutant (resistant) Wild type ] In 24h wild type population of > 105 Selector (antibiotic): Select only the mutated strain (resistant) ] In 24h mutated population of > 105 Mutagen agent: generates mutations. Selector agent: selects a spontaneous mutation that confers the mutant an advantage in the presence of this selector agent. Genotypic variations. Mutations Examples of mutations According to their biological consequences: Alterations of surface components such as capsule or flagella may produce changes in virulence. Loss of ability to use certain carbon sources such as lactose. Loss of the ability to synthesize certain growth factors. Auxotrophic mutants (unable to grow in media without supplement). For instance, auxotrophic for arginine only grows in media with arginine. Resistance to antibiotics. For example a mutation in the gene of a porin can alter the cell permeability to an antibiotic (antibiotic resistance). GREAT GLOBAL HEALTH PROBLEM Horizontal gene transfer between bacteria Genetic horizontal transfer Genetic transfer: Entry of foreign DNA within the bacteria from the surroundings. ✓ Environment: Transformation ✓ Another bacteria: Conjugation ✓ Bacteriophage: Transduction Genetic Transfer Genetic transfer: Entry of foreign DNA within the bacteria. ✓ Environment: Transformation ✓ Another bacteria: Conjugation ✓ Bacteriophage: Transduction  Transformation The uptake by a cell of a naked DNA molecule or fragment from the medium and the incorporation of this molecule into the recipient chromosome in a heritable form. Random process that takes place after the lysis of bacteria. Any portion of a genome may be transferred between bacteria. In natural conditions, this transformation usually occurs between bacteria that share the same ecological niche. When a bacterium is able to take up DNA from the environment and be transformed is called a competent bacteria (artificially: electrical shock or calcium chloride). Transformation Transformation of Streptococcus mitis by Streptococcus pneumoniae DNA These species are related, both Streptococci. Their DNAs have many areas with high homology. Shared ecological niche: Pharynx. Occurrence of pseudoespecies with mixed characteristics. Cell lysis S. mitis incorporates Recombination of S. pneumoniae (free DNA) exogenous DNA DNA Genetic Transfer Genetic transfer: Entry of foreign DNA within the bacteria. ✓ Environment: Transformation ✓ Another bacteria: Conjugation ✓ Bacteriophage: Transduction Conjugation Conjugation: transfer of genetic material between two bacteria by direct contact between them. The process is different for Gram-positive and Gram- negative bacteria: ▪ Gram-Negative: Due to the presence of plasmids or factors capable of encoding F pili. ▪ Gram-Positive: Due to the generation of cell aggregates between donor and recipient cells. Conjugation GRAM-NEGATIVE CONJUGATION Bacteria with F factor (F +) are donor cells able to produce F pili that contact surface receptors of bacteria without the F factor (F-) (recipient cell). Through this bridge, DNA passes from the donor cell to the recipient cell. This DNA can be plasmidic DNA (transfer of an independent self- replicating F plasmid), or chromosomic DNA (when the F plasmid is integrated into the chromosome and replicates along with it). The transferred DNA can have the F factor gene (the recipient cell becomes F +) or be absent (would remain F-).  Conjugation GRAM-POSITIVE CONJUGATION Less frequent than in Gram-negative. A sex pilus may not be required. Cell aggregates are produced between donor and recipient cell (not pili). The recipient bacteria secrete some small peptides or pheromones that stimulate the expression of adhesins (proteins) in the donor cells. These adhesins interact with host cell receptors forming cell aggregates (cell adhesion). Intercellular bridges are established, and DNA is transferred through them. Conjugation GRAM-NEGATIVE CONJUGATION Donor Recipient cell cell GRAM-POSITIVE CONJUGATION Donor Recipient cell cell Adhesins Pheromones Genetic Transfer Genetic transfer: Entry of foreign DNA within the bacteria. ✓ Environment: Transformation ✓ Another bacteria: Conjugation ✓ Bacteriophage: Transduction Transduction Transference of DNA induced by bacteriophages. Bacteriophages are viruses that infect bacteria. They fix to the bacterial surface and drill "injecting" the viral DNA: ✓ Virulent phages: use the enzymatic machinery to synthesize bacterial phage structures. They create viral progeny breaking the bacteria to get out (lytic cycle). ✓ Attenuated phages: The DNA of the phage is integrated into the cell genome cells and does not produce lysis (lysogenic cycle) Fuente:Phage lambda life cycle es.svg. Zlir’a 2014. Disponible en: commons.wikimedia.org/wiki/File:Phage_lambda_life_cycle_es.svg?uselang=es  Transduction Lytic cycle: Some viral capsids can uptake fragments of bacterial DNA. Some phages will only have bacterial DNA (transducing particles). When they infect bacteria, a generalized transduction will occur as they carry a large number of bacterial genes. Lysogenic cycle: At some point, the prophage separates from the bacterial DNA to initiate a lytic cycle. Sometimes they drag some bacterial genes. The new progeny of prophage genes transmit both viral and bacterial genes (specialized transduction). The success of both types depends on the ability of the DNA of the donor to be integrated with the DNA of the new cell (recipient bacteria) or self-replicating. If none of this happens it will get lost in successive divisions (abortive transduction). Generalized transduction Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Phage DNA Donor (host) chromosome (1) Involves a lytic phage 1 Cell A (donor) Infection as usual Parts of phage Separated piece of host DNA Mistaken packaging of a host (2) 2 gene Defective phage (it cannot Newly assembled phage incorporating piece of host DNA initiate a lytic cycle) Lysis (3) 3 Transducing DNA from donor particle Acts as a carrier of genetic (4) 4 information from the original Cell B (recipient) Incorporated into chromosome bacterium to another cell. (5) 5 Cell survives and utilizes transduced DNA Specialized Transduction Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Prophage within (1) 1 the bacterial chromosome of Lysogenic phage lysogenized cell Inserts as prophage Incorrect excision: (2) 2 excised phage DNA Aberrant excision contains some bacterial DNA. Pick up adjacent gene New viral particles Defective phage (3) 3 are synthesized. (4) 4 Acts as a carrier of genetic Infection of recipient information from the original cell transfers bacterial DNA to a new cell. bacterium to another cell. Bacterial Bacterial chromosome chromosome containing both virus (5) 5 containing only and donor DNA donor DNA Recombination results in two possible outcomes. Genetic Transfer Exogenous DNA bacteria can react in three ways: ✓ Degradation: The new DNA is destroyed. Not recognized as its own DNA and restriction endonucleases destroy it. ✓ Circulation: The new DNA can be methylated avoiding the action of endonucleases. Then it can: ▪ Replicate independently of bacterial DNA (transmitted to daughter cells). ▪ Do not replicate and will be lost in successive divisions (abortive process) ✓ Recombination: DNA genetic exchange between bacterial DNA and the exogenous DNA (mixed genetic information).