4. How Do Mutations in DNA Affect the Function of Genes?  Mutations result from Nucleotide Substitutions, Insertions, or Deletions  Mutations may have a variety of effects on protein structure and function  Mutations provide the raw material for evolution Chapter 10.

5. MUTATION  Process of mutation may result in a gene coding for a new protein  Mutations are not good or bad, they do provide the raw material of change.  There are many different types of mutations. Chapter 10.

6. Nucleotide substitution can result in either a change or no change in amino acid of the codon.

7. MUTATION  Point Mutation - change in one nucleotide.  Insertion - new nucleotide pairs are inserted into DNA molecule  Deletion - nucleotide pairs are removed from DNA molecule Chapter 10.

8. Different Types of Mutations

9. MUTATION  Effect of Mutation on protein structure and function  No change for protein gene codes for.  No change in function of protein even though change results in use of different amino acid (amino acids are functionally equivalent to each other).  Codes for a new amino acid that is functionally different from original.  Mutation codes for a stop codon, results in non-functional gene and protein. Chapter 10.

11. - Change in a single nucleotide of a genetic sequence - Possible results: - Base Substitution - Same Sense Mutation - Missense Mutation - Nonsense Mutation - Frame Shift Mutation - Insertion - Deletion

12. Base Substitution

13. Same Sense Mutation

14. Mutations that result in replacement of one amino acid for another within a polypeptide chain. Mutant polypeptides still retain some residual biologic activity of the wildtype. Missense Mutation

15. Nonsense Mutation Nonsense mutations: Mutations that result in replacement of an amino acid codon with a termination codon production of an amino-terminal fragment of the normal polypeptide often result in complete loss of activity of the gene product.

16. Fitness effects of mutations Some mutations are selectively neutral (i.e., they have no fitness effects), including: Mutations in non-coding DNA (i.e., regions not coding for protein product, transcription factor binding site, etc) Silent substitutions (i.e., mutations in coding regions that do not result in amino acid substitutions, because of redundancy in the genetic code)

17. Fitness effects of mutations, continued Mutations that change amino acid sequences tend to be selectively non-neutral, including: Point mutations: Missense mutations: cause amino acid substitution Nonsense mutations: amino acid changed to stop codon (UAA, UAG, UGA), signalling termination of translation

19. Fitness effects of mutations, continued Other mutations that change amino acid sequences include: Insertions and deletions (indels) Indels not in multiples of 3 nucleotides cause frameshift mutations Indels of 3 nucleotides, cause insertion/deletion of amino acid or nonsense mutation

22. Summary

23. Exposure to UV causes adjacent thymines to become dimer and disrupting their normal base pairing. An enzyme cuts out and removes the damaged DNA. DNA polymerase fits the gap by synthesizing new DNA, using the intact strand as a template. DNA ligase seals the remaining gap by joining the old and bew DNA. Non-ionizing Radiation Mutation

27. Mutation and Selection  Mutations are heritable changes in the genome.  Spontaneous mutations in individual bacteria are rare.  Some mutations cause changes in phenotypic characteristics.  In microbial genetics, specific reference organisms are referred to as wild-type strains, and descendants having mutations in their genomes are called mutants.  Variant forms of a specific genetic determinant are called alleles. Genotypic symbols - lower case, italicized abbreviations that specify individual genes, with a (+) superscript indicating the wild type allele (eg. genotypic symbol for the ability to produce  -galactosidase, required to ferment lactose, is lacZ +, and mutants are lacZ - ) Phenotypic symbols - capitalized and not italicized, to distinguish them from genotypic symbols. (Eg. The lactose-fermenting phenotype is designated LAC +, and inability to ferment lactose is LAC - )

29. Spontaneous and Induced Mutations The mutation rate in bacteria is determined by - accuracy of DNA replication - occurrence of damage to DNA - effectiveness of mechanisms for repair of damaged DNA. For a particular bacterial strain under defined growth conditions, the mutation rate for any specific gene is constant and is expressed as the probability of mutation per cell division. the proportion of mutants usually increases progressively as the size of the bacterial population increases.

30. Reversion and Suppression Forward mutations - mutations that convert the phenotype from wild-type to mutant Reverse mutations - mutations that change the phenotype from mutant back to wild-type, such bacteria are called revertants. Reverse mutations that restore the exact nucleotide sequence of the wild-type DNA are true revertants – are identical to wild- type strains both genotypically and phenotypically.

31. Selection -After gene transfer -For strain carries gene of interest. - On specific media – replica plating technique, colonies patching Detection of Mutant Phenotypes  Selective and differential media are helpful for isolating bacterial mutants.  Selective media - permit mutants to grow, but not wild-type strains.  Differential media - permit both wild-type & mutant bacteria to grow and form colonies that differ in appearance.

32. Identification of Mutant Cells Positive (direct selection detects mutant cells because they grow or appear different. Negative (indirect) selection detects mutant cells because they do not grow.

33. Detection of Mutant Phenotypes Consider a wild-type strain of E coli that is susceptible to the antibiotic streptomycin (genotype Str s ) and can utilize lactose as the sole source of carbon (phenotype LAC + ). a)Selective media containing streptomycin: b)Selective media containing chemical analogs of lactose which can be converted to toxic metabolites byproducts: c)Minimal media with lactose as the sole source of carbon: c) Differential media such as MacConkey-lactose agar or eosin-methylene blue- lactose agar:

34. Detection of Mutant Phenotypes Consider a wild-type strain of E coli that is susceptible to the antibiotic streptomycin (genotype Str s ) and can utilize lactose as the sole source of carbon (phenotype LAC + ). a)Selective media containing streptomycin: Str r mutants grow, wild-type Str s bacteria are killed. a)Selective media for LAC- mutants of E coli made by incorporating chemical analogs of lactose that are converted into toxic metabolites by LAC+ bacteria but not by LAC- mutants. Thus Lac- mutants grow, LAC+ wild-type bacteria are killed. b)Minimal media with lactose as the sole source of carbon: LAC+ wild-type strains will grow, but LAC- mutants cannot grow. c) Differential media such as MacConkey-lactose agar or eosin-methylene blue (EMB)-lactose agar: LAC+ wild-type and LAC- mutant strains of E coli can be distinguished by their color.

35. Conditional and suppressive mutations Mutations that inactivate essential genes in haploid organisms are usually lethal. Such potentially lethal mutations can often be studied if their expression is controlled by manipulation of experimental conditions. Temperature-sensitive and cold-sensitive mutations are examples of conditional mutations, as are suppressible mutations. A conditional lethal phenotype indicates that the mutant gene is essential for viability. Eg. a mutation that increases the thermo-lability of an essential gene product may prevent bacterial growth at 42°C, although the mutant bacterium can still grow at 25°C. Conversely, cold-sensitive mutants express the mutant phenotype at low temperature, but not at high temperature.

36. Mutation creates new alleles. Natural selection affects the rate of fixation or loss of alleles in populations.

37. Mutation.

39. Purifying selection removes deleterious mutations.

40. Rare mutations with positive fitness consequences increase in frequency through natural selection--may eventually become fixed. Mutation continually introduces new alleles All allele copies in a population share a common ancestor (the “coalescent”).

41. Extra-chromosomal Elements

42. Bacteriophages  bacterial viruses or phages  Extrachromosomal Elements  Can survive outside host cell  Infect bacteria: Replcate  lysis of cell – lytic Intergrate without cell death = lysogenic

43. Infection of E. coli by Phage  Virulent phage replicate and kill their host by lysing or breaking it open  phage can infect cells but don’t necessarily kill  Two paths of reproduction Lytic mode: infection progresses as in a virulent phage Lysogenic mode: phage DNA is integrated into the host genome

44. Bacteriophages  Infectious agents, replicate as obligate intracellular parasites in bacteria  Morphologically different - polyhedral, filamentous, and complex (polyhedral heads with tails attached)  consist of a protective shell (capsid) surrounding the tightly packaged nucleic acid genome  genomes vary in size (~ 2 to 200 kb); either dsDNA, ssDNA, or RNA  genes encodes proteins - for replication & phage assembly

45. Lysis plaques of  phage on E. coli bacteria lawn

46. Plasmid  First plasmid described was discovered in Japan in Shigella species during an outbreak of dysentery in the early 1940‘s 3 main components: Origin of replication Selectable marker Restriction enzyme site(s) Enzymes that cut at specific sequence on DNA Plasmid – small, circular, extrachromosomal DNA which replicates independently of host chromosomal DNA

47. Plasmids Content  Replication factors  Genes

48. Ori Region  Ori, actual site of replication  Proteins that assist in replication (varies)  Recognition sequences for control factors  The ori determines the Range

49. The Large Virulence Plasmid of Shigella flexneri

50. Plasmids  Discrete, extrachromosomal genetic elements in bacteria  Usually much smaller than bacterial chromosome  Size varies from 100 kbp  Mostly supercoiled, circular, ds DNA molecules  Replicate independently of the chromosome  Exist in multiple copies in bacterial (the average number of plasmid per bacterial is called copy number).  Usually encode traits that are non-essential for bacterial viability.

51. Plasmid Genes

52. F plasmids  codes for sex factor of bacteria  also called conjugative plasmids  function: - genes promote transfer of plasmid - donor to recipient - genes code for proteins required for their replication  usually large plasmids (>40 Kbp), small copy number (1 to several per chromosome)  partition themselves among daughter cells during cell division similar to bacterial chromosome

53. R plasmids:  medically important, eg, resistant to Penicillin (carries genes of the Bla operon)  In early 1940's, Penicillin was introduced for general use 1946 - 14% of Staphylococcus aureus were penicillin resistant 1947 - 38% PenR 1969 - 59% PenR 1970's - almost 100% PenR  resistance to one or several antibiotics (R factor)

54. Col plasmids:  produce colicins, a type of bacteriocin that affect sensitive cells (Col - ) & inhibit growth  may or may not be self-transmissible  ColE1 is mobilizable but non-conjugative  size : <7.5 Kbp  high copy numbers (typically 10-20 per chromosome)  rely on their bacterial host to provide some functions required for replication  are distributed randomly between daughter cells at division.

55.  Many plasmids control medically important properties of pathogenic bacteria, contain genes that code for : a) resistance to one or several antibiotics b) production of toxins eg. heat-labile & heat-stable enterotoxins of E. coli, Shiga toxins of Shigella exfoliative toxin of S. aureus tetanus toxin of C. tetani c) synthesis of cell surface structures required for adherence or colonization  Some plasmids are cryptic = no recognizable effects on the bacterial host  Comparing plasmid profiles = for assessing possible relatedness of individual clinical isolates of a particular bacterial species for epidemiological studies Function of plasmids

56. Plasmid DNA replication  Plasmid replication by - Theta model (either uni- or bidirectional) or - Rolling circle  Replicon - DNA molecules that can replicate autonomously (plasmids, chromosomes, phage)  Replicon must have on origin of replication, called ori  Functions of the ori region: Host range - narrow or broad host ranges  Broad-host-range plasmids = encode all of their own proteins required for replication initiation  Regulation of copy number Stringent - low copy number (F factor) Relaxed - high copy number (pBR322 =16 copies; pUC =30 to 50) Requires host proteins for replication

57. Theta Model Replication fork Ori Rep Replication fork Ori Rep Replication bubble

58. Rolling circle replication  Enable rapid synthesis of multiple copies of circular DNA or RNA (plasmid or phage genomes).  A striking feature: = one strand is replicated first (which protrudes after being displaced) and the second strand is replicated after completion of the first one.

59. Mechanism of Rolling circle DNA replication  An initiator protein encoded by the plasmid DNA nicks one strand of the ds plasmid at the ori site.  The initiator protein binds to the 5' PO 4 end of the nicked strand  The free 3' OH end serve as a primer for DNA synthesis by DNA polymerase III, using the un-nicked strand as a template.  The 5' PO 4 ssDNA strand is displaced by helicase PcrA in the presence of the initiation protein.  Continued DNA synthesis can produce multiple ss linear copies of the original DNA in a continuous head-to-tail series called a concatemer.  These linear copies are converted to ds circular plasmid by: the initiator protein makes another nick to terminate synthesis of the first (leading) strand. DNA polymerase III replicate the ss ori to make complementary strand, RNA primer removed, DNA ligase joins the ends to make ds circular plasmid.

60. Plasmid amplification and curing  Plasmid amplification by chloramphenicol treatment - inhibits protein synthesis - inhibit chromosomal but not plasmid replication. - Chromosomal replication requires new protein synthesis but plasmid replication uses only stable bacterial replication proteins. - Plasmids replicated to high copy number because no repressor protein to control copy number  Plasmid curing - with acridine orange - inhibits plasmid but not chromosomal replication - unknown how this occurs

62. Plasmid is an ideal structure for genetic engineering because  Simple in structure  Easy to extract & isolate in the lab  Easy for genetic manipulation & transformed back into bacteria  Contains genetic information which can be used by the bacteria  Most plasmid present in high copy number  Plasmid codes for antibiotic resistant gene eg. Ampicillin, Ap r or Tetracyclin Tc r - selection of bacteria with transformed plasmid.  Non-essential for bacteria’s growth, thus possible to manipulate plasmid DNA without affecting bacteria growth.

63. Exchange of Genetic Information in bacteria  Medically important - rapid emergence and dissemination of antibiotic resistance plasmids - flagellar phase variation (eg. Salmonella) - antigenic variation of surface antigens (eg. Neisseria & Borrelia)  Sexual processes in bacteria involve transfer of genetic information from a donor to a recipient, results in: - substitution of donor alleles for recipient alleles - addition of donor genetic elements to the recipient genome.  3 major types of genetic transfer found in bacteria: a) Transformation b) Transduction c) Conjugation In all three cases, recombination between donor and recipient DNA result in formation of stable recombinant genomes

64. Types of transfers:  Non-transmissible = cannot initiate contact with recipient or transfer DNA Conjugative = can initiate contact with recipient bacterium Mobilizable = can prepare its DNA for transfer Self-transmissible = is both conjugative & mobilizable  4 stages of plasmid transfer: a) Effective contact b) Mobilization - preparation for DNA transfer c) DNA transfer d) Formation of F in recipient  Donation - a conjugative plasmid (F) can provide conjugative function to a mobilizable plasmid (eg. ColE1) such that both plasmids can be transferred.  Plasmid conduction - a self-transmissible plasmid (F) can recombine with a non-mobilizable plasmid and transfer the co-integrate.

65. Fin genes & plasmid transfer  fin genes - fertility inhibition - codes for repressor that prevents transcription of genes required for transfer.  F plasmid has 1 fin gene so transfer system is always ‘ON’  R plasmid has 2 fin genes so cannot always transfer. - in new recipients (repressor is absent) so transfer can occur soon after receiving the R plasmid but after time (when repressor is made) transfer can't occur

67.   introducing DNA from donor to recipient  result in uptake and integration of fragments of donor DNA into recipient genome.  produce stable hybrid progeny.  is most likely to occur when the donor and recipient bacteria the same or closely related species. (a) Bacterial Transformation

68. "Transformation" is simply the process where bacteria manage to "uptake" a piece of external DNA. Usually, this process is used in the laboratory to introduce a small piece of PLASMID DNA into a bacterial cell. Bacteria transformation in the lab

69.  Bacteriophage infect donor bacterium  form rare abnormal bacteriophage particles contain DNA from donor bacteria.  abnormal bacteriophage infect recipient bacteria & inject DNA into recipient  donor DNA integrated / recombined into recipient DNA resulting in transduced bacterium. Bacteria Transduction

70. Bacterial Conjugation  Transfer of DNA between 2 bacteria in contact with each other  Contact between donor and recipient (initiated by sex pili)  DNA transfer through a conjugation bridge  Mediated by a plasmid  Called an F-factor (fertility factor) or conjugative plasmid

73. Several important properties of F  F is a self-replicating plasmid and is maintained in a dividing cellular population.  Cells carrying F produce pili, minute proteinaceous tubules that allow the F+ cells to attach to other cells maintaining contact.  F+ cells can transfer its F plasmid to a F- cell, turning the recipient cell into an F+ cell. F+ cells are usually inhibited from making contact with each other.  Occasionally, F can integrate into the host bacterial chromosome and transfer the host chromosomal markers to the recipient cell.

74.  consist of methylases = methylate the adenine or cytosine residues at specific sequences in their own DNA  corresponding restriction endonucleases cleave foreign DNA which are not methylated at the same target sequences.  RM systems = protect bacteria against invasion by phages or plasmids.  Barrier to genetic exchanges between different bacterial strains or species.  Recent evidence suggests that plasmid-borne RM systems = strategy to ensure plasmid maintenance in a host strain since cells that lose these plasmid (and the corresponding protective methylase gene) are killed by restriction enzyme, which attacks the newly replicated & unmodified chromosomal DNA. Restriction-modification (RM) systems