Principles of Bacterial Genetics

1. Chapter 11 Principles of Bacterial Genetics Professor Bharat Patel

2. Chapter 10 Thirteenth Edition Madigan / Martinko Stahl / Clark Principles of Bacterial Genetics Professor Bharat Patel

3. NOTE 1. The following is a summary and are not full notes for the Lecture on “Principles of Genetics”. This summary is a study guide only and it is therefore recommended that students attend and take notes during the lectures. 2. There are differences in the content of the chapters of the two different editions of the recommended text book 3. The lecture & summary may not follow the same content as is in the book chapter 4. There is extra content that has been sourced from other resources

4. CONTENT The lecture content is divided into 3 parts: I. Bacterial Chromosomes & Plasmids • Physical location of the genes II. Mutation • Alterations in the genetic material • Chemical, Physical III. Genetic Transfer • Gene transfer & exchange mechanisms • Conjugation • Transduction • Transformation • Gene exchange mechanisms

5. Note: • Most of the techniques described here were used between 1950-80, but advances in the past three decades in cloning and sequencing has revolutionised studies on genomes & gene organisation: • Developments in molecular biology: • Manual sequencing & Automated 1st generation sequencers • 1970 – 2008: $1-2 million per microbial genome • 2nd generation sequencers (current) • Since 2009: $5,000 per microbial genome • 3rd generation sequencers • early next year, • semi-conductor real-time technology • $1,000 per human genome • Genomes OnLine Database (GOLD)- http://genomesonline.org – lists all genome sequencing projects.

6. I. Genetics of Bacteria and Archaea Lecture Content • 11.1 Genetic Map of the Escherichia coli Chromosome • 11.2 Plasmids: General Principles • 11.3 Types of Plasmids and Their Biological Significance

7. 11.1 Genetic Map of the Escherichia coli Chromosome Escherichia coli a model organism for the study of biochemistry, genetics, and bacterial physiology The E. coli chromosome (strain MG1655, derivative of K-12) was been mapped using • Conjugation (initial mapping) • Transduction (phage P1) • Molecular cloning & sequencing • Next Generation Sequencing (NGS) (most recent) E. coli is (gram -ve) is inefficient at transformation unlike Bacillus (gram +ve)

8. Circular Linkage Map of the Chromosome of E. coli K-12 Original map used distance (centisomes) 0 – 100 mins, 0 = arbitrary & set at thrABC (based on transfer by conjugation) Also shows kilobase pairs (kb) from sequencing studies Replication starts at oriC (84min) Figure 11.1

9. 11.1 Genetic Map of the Escherichia coli Chromosome • Some Features of the E. coli Chromosome • Many genes encoding enzymes of a single biochemical pathway are clustered into operons • Operons are equally distributed on both strands • Transcription can occur clockwise or anticlockwise • ~ 5 Mbp in size • ~ 40% of predicted proteins are of unknown function • Average protein size is ~ 300 amino acids • Insertion sequences (IS elements) are present

10. Genomes of pathogenic E. coli contain PAIs. Genome size is indicated in the centre. The outer ring shows gene by gene comparison with all 3 strains: common genes (green),genes in pathogens only (red),genes only in 536 (blue) Fig13.13

11. Pan Genome Versus Core Genome Figure 13.14 Core genome is in black & is present in all strains of the same species. The pan genome includes elements (genes) that are present in one or more strains but not in all strains. one coloured wedge = single insertion two coloured wedges = alternative insertions possible at the site but only can be present

12. Plasmids

13. 11.2 Plasmids: General Principles Plasmid Plasmid Plasmids: Genetic elements that replicate independently of the host chromosome • Small circular or linear DNA molecules • Range in size from 1 kbp to > 1 Mbp; typically less than 5% of the size of the chromosome • Carry a variety of nonessential, but often very helpful, genes • Abundance (copy number) is variable

14. 11.2 Plasmids: General Principles A cell can contain more than one plasmid, but it cannot be closely related genetically due to plasmid incompatibility • Many Incompatibility (Inc) groups recognized • Plasmids belonging to same Inc group exclude each other from replicating in the same cell but can coexist with plasmids from other groups • Borrellia burgdorferi (causes Lyme disease) - 17 different circular & liner plasmids

15. 11.2 Plasmids: General Principles • Some plasmids (episomes) can integrate into the cell chromosome; similar to prophage integration – replication is under the control of the host cell • Host cells can be cured of plasmids by agents that interfere with plasmid (but not cell) replication • Acridine orange or can be spontaneous • Conjugative plasmids can be transferred between suitable organisms via cell-to-cell contact • Conjugal transfer controlled by tra genes on plasmid • Plasmid replicate up to 10 times faster than host cell DNA due to their small size • unidirectional (one fork) or bi-directional (two forks)

16. 11.3 Types of Plasmids and Their Biological Significance • Genetic information encoded on plasmids is not essential for cell function under all conditions but may confer a selective growth advantage under certain conditions • Plasmids are transferred by conjugation (refer to Conjugation later) – provide cells with additional “coping and fighting” strategies

17. Examples of Phenotypes Conferred by Plasmids

18. Examples of Phenotypes Conferred by Plasmids

19. 11.3 Types of Plasmids and Their Biological Significance R plasmids • Resistance plasmids; confer resistance to antibiotics and other growth inhibitors • Widespread and well-studied group of plasmids • Many are conjugative Outer ring: resistance genes (str streptomycin, tet tetracylcine, sul sulfonamides, & other genes (tra transfer functions, IS insertion sequence, Tn10 transposon). Inner ring: Plasmid size = 94.3 kb

20. 11.3 Types of Plasmids and Their Biological Significance • In several pathogenic bacteria, virulence characteristics are encoded by plasmid genes

21. 11.3 Types of Plasmids and Their Biological Significance • Bacteriocins • Proteins produced by bacteria that inhibit or kill closely related species or even different strains of the same species • Genes encoding bacteriocins are often carried on plasmids

22. 11.3 Types of Plasmids and Their Biological Significance • Plasmids have been widely exploited in genetic engineering for biotechnology • Plasmids are transferred by conjugation (refer to Conjugation later) – provide cells with additional “coping and fighting” strategies

23. Mutation

24. II. Mutation • 11.4 Mutations and Mutants - definitions • 11.5 Molecular Basis of Mutation • 11.6 Mutation Rates • 11.7 Mutagenesis • 11.8 Mutagenesis and Carcinogenesis: The Ames Test

25. 11.4 Mutations and Mutants - definitions • Mutation • Heritable change in DNA sequence that can lead to a change in phenotype (observable properties of an organism) • Mutant • A strain of any cell or virus differing from parental strain in genotype (nucleotide sequence of genome) • Wild-type strain • Typically refers to strain isolated from nature Animation: The Molecular Basis of Mutations

26. 11.4 Mutations and Mutants – definitions (cont’d) • Selectable mutations • Those that give the mutant a growth advantage under certain environmental conditions • Useful in genetic research • Nonselectable mutations • Those that usually have neither an advantage nor a disadvantage over the parent • Detection of such mutations requires examining a large number of colonies and looking for differences (screening)

27. Selectable mutants: Antibiotic resistance colonies can be detected around a zone of clearance created by the inhibition of a sensitive bacterium Nonselectable mutants: Aspergilus nidulans produces different interchangeable spontaneously. Selectable and Nonselectable Mutations Figure 11.4

28. 11.4 Mutations and Mutants • Screening is always more tedious than selection • Methods available to facilitate screening • E.g., replica plating • Replica plating is useful for identification of cells with a nutritional requirement for growth (auxotroph) Animation: Replica Plating

29. Screening for Nutritional Auxotrophs Figure 11.5

30. 11.5 Molecular Basis (Types ) of Mutation • Induced mutations • Those made deliberately • Spontaneous mutations • Those that occur without human intervention • Can result from exposure to natural radiation or oxygen radicals • Point mutations • Mutations that change only one base pair • Can lead to single amino acid change in a protein or no change at all

31. Possible Effects of Base-Pair Substitution Figure 11.6

32. 11.5 Molecular Basis (consequences) of Mutation • Silent mutation • Does not affect amino acid sequence • Missense mutation • Amino acid changed; polypeptide altered • Nonsense mutation • Codon becomes stop codon; polypeptide is incomplete

33. 11.5 Molecular Basis of Mutation • Deletions and insertions cause more dramatic changes in DNA • Frameshift mutations • Deletions or insertions that result in a shift in the reading frame • Often result in complete loss of gene function

34. Shifts in the Reading Frame of mRNA Figure 11.7

35. 11.5 Molecular Basis of Mutation • Genetic engineering allows for the introduction of specific mutations (site-directed mutagenesis)

36. 11.5 Molecular Basis of Mutation • Point mutations are typically reversible • Reversion • Alteration in DNA that reverses the effects of a prior mutation

37. 11.5 Molecular Basis of Mutation • Revertant • Strain in which original phenotype that was changed in the mutant is restored • Two types • Same-site revertant: mutation restoration activity is at the same site as original mutation • Second-site revertant: mutation is at a different site in the DNA • suppressor mutation that compensates for the effect of the original mutation

38. 11.6 Mutation Rates • For most microorganisms, errors in DNA replication occur at a frequency of 10-6to10-7 per kilobase • DNA viruses have error rates 100 – 1,000 X greater • The mutation rate in RNA genomes is 1,000-fold higher than in DNA genomes • Some RNA polymerases have proofreading capabilities • Comparable RNA repair mechanisms do not exist

39. 11.7 Mutagenesis • Mutagens: chemical, physical, or biological agents that increase mutation rates • Several classes of chemical mutagens exist • Nucleotide base analogs: resemble nucleotides • Chemical mutagens can induce chemical modifications • I.e., alkylating agents like nitrosoguanidine • Acridines: intercalating agents; typically cause frameshift mutations Animation: Mutagens

40. Nucleotide Base Analogs Figure 11.8

41. Chemical and Physical Mutagens and their Modes of Action

42. 11.7 Mutagenesis • Several forms of radiation are highly mutagenic • Two main categories of mutagenic electromagnetic radiation • Non-ionizing (i.e., UV radiation) • Purines and pyrimidines strongly absorb UV • Pyrimidine dimers is one effect of UV radiation • Ionizing (i.e., X-rays, cosmic rays, and gamma rays) • Ionize water and produce free radicals • Free radicals damage macromolecules in the cell

43. Wavelengths of Radiation Figure 11.9

44. 11.7 Mutagenesis • Perfect fidelity in organisms is counterproductive because it prevents evolution • The mutation rate of an organism is subject to change • Mutants can be isolated that are hyperaccurate or have increased mutation rates • Deinococcus radiodurans is 20–200 times more resistant to radiation than E. coli

45. 11.8 Mutagenesis and Carcinogenesis: The Ames Test • The Ames test makes practical use of bacterial mutations to detect for potentially hazardous chemicals • Looks for an increase in the rate of back mutation (reversion) of auxotrophic strains in the presence of suspected mutagen • A wide variety of chemicals have been screened for determining carcinogenicity

46. The Ames Test to Assess the Mutagenicity of a Chemical Disc, with added mutagen Disc, no added mutagen Auxotrophs with single point mutations will not grow in if the required nutrient (eg an amino acid) is not included in the medium. However, in the presence of an added mutagen, some of the cells will revert to wild type an will grow. Eg Histidine-requiring mutants of Salmonella entrica (above)- colonies grow on both plates due to spontaneous mutation but colonies appear on the RHS plate which contains a mutagen) Figure 11.11

47. DNA REPAIR

48. DNA Repair • Three Types of DNA Repair Systems • Direct reversal: mutated base is still recognizable and can be repaired without referring to other strand eg by photoreactivation fromUV damage in which T-T dimers are formed • Repair of single strand damage: damaged DNA is removed and repaired using opposite strand as template eg Excision repair • Repair of double strand damage: a break in the DNA Requires more error-prone repair mechanisms eg SOS repair

49. DNA Repair Pyrimidine dimers form due to exposure to UV radiation (260 nm) – an absorption maxima for DNA . There are 4 mechanisms by which pyrimidine dimers can be repaired – Refer to htp://trishul.ict.griffith.edu.au/courses/ss12bi/repair.html Note: Some of the these mechanisms are also used for repairing mutations caused by other mutagenic agents.

50. III. Genetic Exchange in Prokaryotes • 11.9 Genetic Recombination • 11.10 Transformation • 11.11 Transduction • 11.12 Conjugation: Essential Features • 11.13 The Formation of Hfr Strains and Chromosome Mobilization • 11.14 Complementation • 11.15 Gene Transfer in Archaea • 11.16 Mobile DNA: Transposable Elements