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Bacterial genetics


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  • 2. Genetic Basis of Variation in Bacteria NB: Antibiotic resistance is one phenotype of genetic transfer between bacteria and that the same principles allow other genes like pathogenicity and virulence factors to spread. Bacteria change their DNA very easily and very readily. Aim: understanding how this occurs and the consequence it has on the changing variety of bacteria and bacterial pathogenicity.A.S. HOZA
  • 3. Genetic Basis of Variation in Bacteria 1. Vertical Inheritance of mutations Bacteria multiply exponentially. The generation time varies: 20 min. in perfect conditions hours in a real infection. Growing exponentially means one cell can turn into millions of bacteria. Daughters are identical to the parent – this is a clonal population, all are genetically identical.  A clone is represented on an agar plate by a single bacterial colony.A.S. HOZA
  • 4. Genetic Basis of Variation in Bacteria NB: But DNA changes. This affects the properties of the bacteria and creates a subclone within the population. Mutations occur at a low frequency, 1 in a million cells will have a mutation in any gene. Because bacteria grow so rapidly, this is actually a significant number.A.S. HOZA
  • 5. Genetic Basis of Variation in Bacteria Mutation Outcomes: 1) Deleterious: blocking or disrupting a gene causes a disadvantage (lethal, slow growth). This population dies out by being taken over by wild type (normal) bacteria. 2) Beneficial: mutation has added an advantageous function to the cell, like antibiotic resistance. Under the appropriate conditions, this advantageous mutation will be selected for and will overtake the other populations of bacteria. 3) Random/Spontaneous: no obvious effect on phenotype, silent mutations. These can accumulate and the sum can then lead to change in gene functionA.S. HOZA
  • 6. Genetic Basis of Variation in Bacteria Two kinds of physical mutations (occur at the same low spontaneous rate) 1) Point mutations: change of a single nucleotide 2) DNA rearrangements: shuffling of the genetic information insertions, deletions, inversions, or changes in structure (several thousand nucleotides)A.S. HOZA
  • 7. 2. Horizontal inheritance. DNA can be transferred from one bacteria to another and assuming stable inheritance this acquisition of genetic material will form a new subclone population. 1) Transformation – results from the release and uptake of naked DNA (e.g from lysed cells).  New DNA is incorporated into the chromosome.  This is the most inefficient form of transfer since the DNA is open to the damaging environment, and requires a high density of bacteria.A.S. HOZA
  • 8. Recombination refers to changes in genetic information Homologous recombination involves replacement of DNA sequence with a similar Sequence Bacteria may also acquire additional DNAA.S. HOZA
  • 9. Evidence for Bacterial TransformationA.S. HOZA
  • 10. Mechanism of Bacterial Transformation Natural transformation is limited to particular species Transformation requires specialized proteins in the recipient cells for competenceA.S. HOZA
  • 11. Generalized TransductionA.S. HOZA
  • 12. 2. Horizontal inheritance. 2) Transduction – bacterial genes are transferred in virus particles. Bacteriophages package DNA and inject DNA into other bacteria. More efficient because of protection of the DNA in a safe protein coat. However, the amount of DNA is limited by the capsid size. Furthermore, phage can only infect bacteria expressing the correct receptor, so there is a tropism to the transfer of DNA.A.S. HOZA
  • 13. Transduction Bacteriophage (phage) are viruses of bacteria - can be either lytic or temperate i. Lytic - always lyse (kill) host bacterial cell ii. Temperate - can stably infect and coexist within bacterial cell (lysogeny) until a lytic phase is inducedA.S. HOZA
  • 14. Life Cycle of a Bacteriophage (Bacteriophage Lambda)A.S. HOZA
  • 15. Lysogeny i. The phage genome during lysogeny is called the prophage, and the bacterial cell is called a lysogen ii. If the phage genome encodes an observable function, the lysogen will be altered in its phenotype – lysogenic conversion (e.g., diphtheria toxin in Corynebacterium diphtheriae)A.S. HOZA
  • 16. A.S. HOZA
  • 17. Specialized transduction i. Some prophages integrate into the bacterial genome at a specific location ii. When a prophage is induced to lytic phase, it may drag along a piece of the bacterial genome next to the integration site and move that bacterial sequence into the new recipient host cell, changing the recipients genome iii. Not very important medically since only selected genes can be transferredA.S. HOZA
  • 18. A.S. HOZA
  • 19. Generalized transduction i. When a phage lyses the host bacterial cell, it normally packages phage genome into the capsid ii. Sometimes the capsid is accidently filled with random pieces of bacterial genome, possibly including plasmids iii. When the capsid injects the host genes into a new recipient, the new gene can recombine into the recipient genome and cause a change iv. Virulence and antibiotic resistance genes can be moved by generalized transductionA.S. HOZA
  • 20. A.S. HOZA
  • 21. Difference between lysogeny and generalized transduction ??????A.S. HOZA
  • 22. 2. Horizontal inheritance. 3) Conjugation –involves cell to cell contact. Two cells come into contact, a pore is formed and DNA is transferred from one to the other. Very efficient and rapid and is able to transfer large amounts of DNA.  This is the most prevalent form of DNA transfer. NB: CONJUGATION IS PROMISCUOUS.A.S. HOZA
  • 23. Conjugation - Plasmid transfer Plasmids are circular DNA molecules replicated independently of the bacterial chromosome Plasmids encode proteins that allow for their transfer to cells without the plasmid Plasmid transfer is accompanied by “rolling circle” replicationA.S. HOZA
  • 24. Conjugation - Formation of an Hfr cell Recombination between the plasmid and the chromosome leads to integration of the plasmid into the chromosome Or is that integration of the chromosome into the plasmid?A.S. HOZA
  • 25. Conjugation - Transfer of chromosomal genes The plasmid begins rolling circle replication and transfer into the recipient This time, the chromosomal DNA of the Hfr is dragged along The transferred chromosomal DNA may undergo homologous recombination into the recipient chromosomeA.S. HOZA
  • 26. 2. Horizontal inheritance. The DNA has to be stabilized in bacteria via two ways: 1) Genetic recombination – the incoming DNA is inserted into the chromosome and replicates within the bacteria’s own genome and is passed into the daughter’s cells. 2) Plasmid – the incoming DNA forms a plasmid, accessory genetic elements that replicate outside of the chromosome that have their own replication signals, independent of the chromosome. i.e.“minichromosome”.A.S. HOZA
  • 27. Gene transfer is extremely efficient. Example of how horizontal gene transfer has real world consequence: Vancomycin requires 5 genes to be altered for resistance– this took 30 years to generate. In the few years since resistance has developed, there has been 30 fold increase in resistance to vancomycin.A.S. HOZA
  • 28. 2. Horizontal inheritance. Why so efficient? Remember properties of bacterial cell 1) Single chromosome Can be double stranded linear or circular. 2) Bacteria are haploid. One copy of each gene. 3) Replication time is short, bacterial are small evolution is rapidA.S. HOZA
  • 29. DNA makes RNA makes PROTEIN and this can all be mutated This is a gene. There is a start codon and A stop codon. There is a promoter for the binding of RNA polymerase to transcribe the DNA RNA is taken to ribosome to make protein, which reads the RNA in codonsA.S. HOZA
  • 30. Point Mutations Mutations which affect codons: 1) Missense: One nucleotide change can alter the amino acid of that codon. 2) Nonsense: creates a truncated protein by inserting a stop codon early 3) Frameshift: insertion or deletion of one nucleotide, causes an out of frame shift reading by the ribosome usually result in a truncation.A.S. HOZA
  • 31. Gene expression can be altered as well. Mutations can occur outside the coding sequence E.g in ribosome binding sites, promoters, repressor binding site, transcription activator binding sites. Mutations can: Increase or decrease levels of protein expression or gene transcription depending on where the mutation occurs.A.S. HOZA
  • 32. How do nucleotide changes occur (physically)? 1) DNA polymerase is extremely accurate.  Only 1 in a billion misreadings occurs.  Genes are a thousand nucleotides, thus about 1 in a million genes will have a change in it.  But there are billions of bacteria mutations can then accumulate relatively rapidly. 2) Mutagens (chemicals) can change a base from one to another.A.S. HOZA
  • 33. 3) If DNA is damaged very heavily?  A system called SOS response corrects DNA damage. It also induces the expression of a number of compensatory genes One of is a proofreading protein which lowers the fidelity of DNA polymerase. Badly damage DNA causes intrinsic hypermutagenesis. This might be evolutionarily advantageous Since a bacteria which finds itself in toxic conditions can undergo massive DNA change and perhaps gain the ability to cope with that damage and survive.A.S. HOZA
  • 34. Gross DNA Rearrangements: Majority of these changes are caused by transposable elements. These are segments of DNA that have the ability to move from one location in the chromosome to another. In the process of moving, they can generate changes in DNA structure. These changes are deletions, inversions, formation of circles, translocation or mobilization of other genes.A.S. HOZA
  • 35. Transposons - “Jumping genes” First described for eukaryotes by Barbara McClintock Simplest are insertion sequences Complex transposons have contributed to evolution of R plasmids with genes for multiple antibiotic resistancesA.S. HOZA
  • 37. Insertion sequences (IS) Insertion sequences (IS) are the generic transposable element. They are present in large quantities in all bacterial chromosomes (the number is variable). It is a defined sequence of DNA (700-3000 bp long) Has flanking inverted repeats and Has one or two genes that encodes a transposase – a protein involved in movement of this element from one location to another. •ORF encodes the transposase •Inverted repeats are identical and of variable length •Different IS exist (IS1, IS2, IS50….) •Function unknown?A.S. HOZA
  • 38. Insertion sequences (IS) The gene is expressed from an internal promoter. Transposase is a recombination enzyme that recognizes the IR and cuts the junction between the IR and normal DNA. It can cut one strand at each end and ligate the single stranded nicks to other locations in the chromosome. Or it can make a double strand cut and excise the element and move it into another location. They move at a low frequency, the same frequency as point mutations – so 1/million to 1/100 million will get a mutation in any geneA.S. HOZA
  • 39. 2 Types of transposition mechanisms I. Replicative transposition:  When the IS element copies itself and then the new copy inserts elsewhere in the chromosome. II. Conservative transposition (non replicative/cut-and paste):  When the IS element excises itself completely and jumps to another place in the chromosome.  It ligates the ends of the excision.  This process is either precise, or imprecise leaving or taking single nucleotides from the site.  This has the potential for frameshift mutations at the point of transposition.A.S. HOZA
  • 40. A.S. HOZA
  • 41. What are the consequences?? When a IS element jumps, i. it can totally destroy that gene’s function. ii. can have other consequences if that gene product effects other genes  (e.g. jumping into a repressor of an operon will cause the operon to be transcribed more because of disruption of the repressor).A.S. HOZA
  • 42. What are the consequences?? IS elements can directly alter gene expression. They have their own promoters that not only point inwards for their gene products, but outwards as well for nearby (quiescent) genes. Thus they can insert their strong promoters upstream near important genes (like b -lactamase gene). It’s a portable promoter. IS elements can insert in just about any sequence, for the most part random (occasionally some specificity).A.S. HOZA
  • 43. What are the consequences?? If the target of the IS is between genes c and d, there are two outcomes to this scenario 1) Inversion of the sequence, this could be significant if the arrangement of genes affects expression, (e.. the c gene promoter upregulates b gene expression once b gene is rearranged downstream of the c gene). 2) Deletion of the DNA is the other outcome, with the deleted piece forming an extrachromosomal circle. The consequence of this circle is that it carries a transposable element and can then target other locations in the chromosome, or it can interact with a plasmidA.S. HOZA
  • 44. What are the consequences??A.S. HOZA
  • 45. Composite transposons. These are when a chromosomal gene(s) is flanked by IS elements on both sides, allowing the transposition of that gene(s) to other parts of the genome. This arrangement is demonstrated in Figure 8 as the result of IS-mediated intramolecular inversion. Horizontal transfer of that gene will increase since the gene will more easily incorporate into plasmids or be packaged into phage. This could be dangerous if the gene encodes antibiotic resistance or virulence.]A.S. HOZA
  • 46. Genome Organization Replication The Genome of Escherichia coli Genomes of eurkaryotes are usually composed of multiple linear chromosomes Genomes of prokaryotes are often single circular chromosomes Prokaryotes are monoploidA.S. HOZA
  • 47. Flow of Genetic InformationA.S. HOZA