Bacterial Genetics - II PDF

How do bacterial genomes change?  Mechanisms contributing to DNA variation in bacteria 1. Mutation (a change in one or many base pairs occurs in the nucleotide sequence of a gene) 2. Recombination (new genes are introduced into the genome  Mutation...

How do bacterial genomes change?  Mechanisms contributing to DNA variation in bacteria 1. Mutation (a change in one or many base pairs occurs in the nucleotide sequence of a gene) 2. Recombination (new genes are introduced into the genome  Mutation Bacterial cells multiply by copying their genomes and dividing into two new cells Every time this happens there will be a small number of errors in the sequence. These errors are known as mutations Spontaneous mutations - very rare (one in every 106 divisions) Induced mutations – exposure to mutagenic agents Eg. X-rays, UV, chemicals A stable inheritable alteration in any genome is termed mutation  Effects of mutations Differences in individual DNA bases in a gene can change the amino acids in the corresponding protein May cause a phenotypic change Useful (enhanced or new activity) OR Harmful (inactivation or lower activity)  Types of mutations Point mutation/single base-substitution Insertions and deletions (Frame shift mutation) Inversions Point mutation/single base-substitution Point mutations are either silent, missense or nonsense code for a stop codon code for a code for the same amino different acid amino acid  Frameshift mutations Deletion or insertion of one or more bases (that are not a multiple of three)- change every amino acid after the point of the mutation  Inversions Inverted orientation of a segment of DNA within the chromosome  Exchange of Genetic Information in bacteria (Genetic recombination) Introduction of new genes into the genome from a different cell Induces an unexpected inheritable change New genetic material may be introduced by; 1. Conjugation 2. Transduction HORIZONTAL GENE TRANSFER 3. Transformation Image curtesy: Wellcome Genome Campus Advanced Courses and Scientific Conferences  Conjugation New genetic material are introduced in the form of plasmids Most frequently found in Gram negative bacteria (Escherichia and Pseudomonas) A recipient cell that has received DNA as a result of conjugation is called a transconjugant  Self-transmissible vs mobilizable plasmids Self-transmissible plasmids Can transfer themselves encode all the functions they need to move among cells - “Transfer (tra) Genes” Many naturally occurring plasmids are self-transmissible Mobilizable plasmids Not self-transmissible (encode only some of the proteins required for transfer) need the help of self- transmissible plasmids in the same cell to move  Any bacterium harboring a self-transmissible plasmid is a potential donor Conjugation Requires direct contact between cells rolling-circle replication  Conjugation F+ (male) bacteria possess fertility (F) plasmid During conjugation, F+ bacteria synthesize a pilus (‘F’ or sex pilus) F pilus can attach to F- bacteria (female) F+ bacteria DONOR CELL F- bacteria RECIPIENT CELL One strand of F plasmid DNA is passed to the recipient cell through the F pilus Complementary strand is synthesized in the recipient cell Recipient is converted in to an F + bacterium  Types of Plasmids Fertility plasmid (F-plasmids) Ability to conjugate and transfer genetic information –self transmissible Degradative plasmids Ability to digest several types of unusual substances Resistance plasmids Resistance to antibiotics Bacteriocin plasmids Produce bacteriocins - proteins inhibitory to other bacteria Virulence plasmids e.g toxins, capsule – Bacillus anthracis adherence factors, enterotoxins – E.coli  Transduction Bacterial DNA transferred from donor to recipient via bacteriophage When bacterial cells are infected by phages, DNA from the bacterial chromosome or plasmid can be incorporated into phage nucleic acid This acquired DNA is transferred by progeny of the phage to susceptible recipient cell  There are two types of transduction in bacteria: 1. Generalized transduction Essentially any region of the bacterial DNA can be transferred from one bacterium to another 2. Specialized transduction Only certain genes, those close to the attachment site of the prophage, can be transferred  Transformation Transfer of free or ‘naked’ DNA (chromosomal or plasmid DNA) from a lysed donor bacterium to a recipient [bacterial cells take up free DNA directly from their environment] Cell that has taken up the incoming DNA is referred to as a transformant  Naturally transformable Bacteria that are naturally capable of taking up free DNA Only a few bacterial genera are naturally transformable eg. Bacillus subtilis, Streptococcus pneumoniae, Helicobacter pylori Competent Bacteria at the stage in which they can take up DNA, and integrate it into chromosome Naturally competent Bacteria that are naturally capable of reaching competent state Artificially Induced Competence laboratory treatments: electroporation (DNA can be forced into them by a strong electric field), heat shock, Ca2+ treatment of cells, removal of the cell wall to generate protoplasts  Discovery of Transformation - Griffith’s Experiment pathogenic strains -smooth colonies (polysaccharide capsule) Non pathogenic strains – rough colony forming Fredrick Griffith (1928) worked with Streptococcus pneumoniae  Effects of genetic changes Mutation and gene transfer accelerate the rate of bacterial evolution Genetic variation May cause a more severe disease (outbreaks) May cause a less severe disease May make a bacterium resistant to antibiotics Steps involved in the characterization of a bacterial isolate prior to molecular investigation  Molecular Biology Manipulations with DNA Molecular hybridization techniques (Southern blotting, Northern blotting, DNA probes) DNA sequencing (Sanger sequencing/dideoxynucleotide sequencing, Whole bacterial genome sequencing, High-throughput DNA sequencing) Polymerase chain reaction (PCR) – Conventional PCR, Real-time PCR, Quantitative real- time PCR (qPCR), Reverse Transcription-PCR DNA fingerprinting and Molecular subtyping (Pulsed-field gel electrophoresis (PFGE), Rapid amplification of polymorphic DNA (RAPD), PCR–RFLP analysis of conserved genes, PCR ribotyping, REP–PCR, ERIC–PCR, BOX–PCR, Amplification fragment length polymorphism (AFLP), Multilocus variable-number tandem repeat analysis (MLVA), Multilocus sequence typing (MLST) Plasmid profiling Restriction endonuclease analysis (REA) Ribotyping DNA microarray technology