Bacterial Genetics BIOL3400 PDF

Chapter 08 Bacterial Genetics See separate PowerPoint slides for all figures and tables pre- inserted into PowerPoint without notes.  Chapter 8 topics Bacterial Genetics Horizontal Transfer Spontaneous mutations Induced mutations Transduction Mobile gene pool ...

Chapter 08 Bacterial Genetics See separate PowerPoint slides for all figures and tables pre- inserted into PowerPoint without notes.  Chapter 8 topics Bacterial Genetics Horizontal Transfer Spontaneous mutations Induced mutations Transduction Mobile gene pool  Introduction Through natural selection, organisms adapt to ever-changing environments Bacteria have two general mechanisms to adjust to new circumstances Regulation of gene expression Genetic change E. coli often used as model system of genetic change Easy to grow, inexpensive, rapid accumulation of large numbers ©Dr. Gopal Murti/Science Source  Genetic Change in Bacteria (1) Two mechanisms of genetic change in bacteria Mutation: changes in existing nucleotide sequence Horizontal gene transfer: movement of DNA from one organism to another Changes are passed to progeny by vertical gene transfer Genetic Change in Bacteria a) Mutation b) Horizontal gene transfer  Genetic Change in Bacteria Deletion of gene for tryptophan biosynthesis yields a mutant that only grows if tryptophan is supplied Growth factor required; mutant termed auxotroph Auxo = "increase“ ; troph = "nourishment" Prototroph does not require growth factors Proto = "first" Geneticists compare mutants to wild type Typical phenotype of strains isolated from nature For example, wild-type E. coli strain is prototroph Strains designated by three-letter abbreviations Trp− cannot make tryptophan Streptomycin resistance designated Str R  Genetic Change in Bacteria Change in organism’s DNA alters genotype Sequence of nucleotides in DNA Bacteria are haploid, so only one copy, no backup Change in genotype often changes observable characteristics, or phenotype Also influenced by environmental conditions  Spontaneous Mutations Spontaneous mutations are genetic changes that result from normal processes Occur randomly at infrequent characteristic rates Mutation rate: probability of mutation per cell division Typically between 10−4 and 10−12 for a given gene Mutations passed to progeny Occasionally change back to original state: reversion Also occurs spontaneously at low frequencies Spontaneous Mutations Base substitution most common Incorrect nucleotide incorporated during DNA synthesis Point mutation is change of a single base pair Environment selects Spontaneous Mutations Base substitution: three possible outcomes Silent (synonymous) mutation: wild-type amino acid Missense mutation: different amino acid Resulting protein often does not function normally Nonsense mutation: Specifies stop codon Yields shorter, often non-functional protein Jump to Spontaneous Mutations - Figure 8.3 Long Description  Spontaneous Mutations Deletion or addition of nucleotides Impact depends on number of nucleotides involved Three pairs changes one codon Wild type One amino acid more or less Impact depends on location within protein One or two pairs yields frameshift mutation Mutant Different set of codons translated Often results in premature stop codon Shortened, nonfunctional protein  Spontaneous Mutations Transposons (jumping genes): pieces of DNA that can move from one location to another in a cell’s genome; process of transposition Insertional inactivation: gene into which transposon jumps is inactivated; function disrupted Most transposons have transcriptional terminators Block expression of downstream genes in operon  Common Mutagens Induced mutations result from outside influence Agent that induces change is mutagen Geneticists may use mutagens to increase mutation rate Two general types: chemical agent, radiation Agent Action Result Chemical Mutagens Chemicals that modify nucleobases Chemical modifications change base-pairing Nucleotide substitution properties of nucleobases Base analogs Base-pairing properties differ from those of Nucleotide substitution nucleobases normally found in DNA Intercalating agents Insert between base pairs, pushing them apart Addition or subtraction of nucleotides Transposons Randomly insert into DNA Insertional inactivation Radiation Ultraviolet (UV) light Causes thymine dimers to form Errors during repair process X rays Cause single- and double-strand breaks in DNA Deletions  Induced Mutations Chemicals that modify nucleobases Change base-pairing properties; increase chance of incorrect nucleotide incorporation Alkylating agents add alkyl groups onto nucleobases Nitrosoguanidine adds methyl group to guanine May base-pair with thymine  Induced Mutations Radiation can be used as mutagen Ultraviolet light causes thymine dimers (covalent bonds between adjacent thymines) Distorts molecule; replication and transcription stall Mutations result from cell’s SOS repair mechanism X rays cause single- and double-strand breaks in DNA Double-strand breaks often lethal Can alter nucleobases  Induced Mutations Transposition Transposons can be introduced intentionally to generate mutations Transposon inserts into cell’s genome Generally inactivates gene into which it inserts Repair of Damaged DNA Enormous amount of spontaneous and mutagen-induced damage to DNA If not repaired, can lead to cell death; cancer in animals For example, in humans, two genes associated with breast cancer code for DNA repair enzymes; mutations in either result in high probability of breast cancer Mutations are rare because alterations in DNA generally repaired before being passed to progeny Repair of Errors in Nucleotide Incorporation During replication, DNA polymerase sometimes incorporates wrong nucleotide Mutation prevented by repairing before DNA replication Two mechanisms of repair: proofreading and mismatch repair Proofreading by DNA polymerases; checks accuracy Can back up, remove incorrect nucleotide Inserts correct nucleotide Very efficient but not perfect Repair of Errors in Nucleotide Incorporation Mismatch Repair fixes errors 1. missed by DNA polymerase The wrong nucleotide is incorporated during DNA synthesis. Enzyme cuts sugar-phosphate backbone of new DNA strand 2. Near the site of the mismatched Another enzyme degrades short base, an enzyme cuts the sugar- phosphate backbone of the unmethylated strand. region of DNA strand with error Methylation of DNA indicates 3. An enzyme degrades a template strand short stretch of the strand that had the error. Newly synthesized strand is 4. unmethylated DNA polymerase synthesizes a new stretch, incorporating the correct nucleotide. DNA polymerase, DNA ligase fill in and seal the gap 5. DNA ligase joins the 3' end of the newly synthesized segment to the original strand. Screening for Possible Carcinogens Carcinogens cause many cancers; most are mutagens Animal tests expensive, time-consuming; quicker and cheaper to test mutagenic effect of chemicals in microbiological systems Mutagens increase low frequency of spontaneous reversions Ames test measures effect of chemical on reversion rate of histidine- requiring Salmonella auxotroph Uses direct selection on glucose-salts plate Only prototrophs that have undergone reversion can grow If chemical is mutagenic, reversion rate increases relative to control (more colonies grow) Rat liver extract may be added since non-carcinogenic chemicals may be converted to carcinogens by animal enzymes Additional tests on mutagenic chemicals to determine if they are also carcinogenic Horizontal Gene Transfer as a Mechanism of Genetic Change (1) Recombinants acquire genes from other cells by horizontal gene transfer Can demonstrate with auxotrophs Combine two strains that cannot grow on glucose-salts medium His − , Trp− and Leu− , Thr − Spontaneous mutants unlikely; simultaneous mutations required Colonies that can grow on glucose- salts medium most likely acquired genes from other strain Screening for Possible Carcinogens Horizontal Gene Transfer as a Mechanism of Genetic Change Genes naturally transferred by three mechanisms DNA-mediated transformation: “naked” DNA taken up from the environment Transduction: bacterial DNA transfer by a virus Conjugation: DNA transfer during cell-to-cell contact Horizontal Gene Transfer as a Mechanism of Genetic Change DNA-mediated transformation Recipient cell takes up naked DNA; the DNA Donor cell lyses; DNA released is then integrated into the chromosome Transduction Bacteriophage-infected donor cell lyses, releasing Recipient cell acquires donor DNA from the phage particles; error during infection process bacterial DNA-containing phage coat; the generates phage coat that contains bacterial DNA DNA then integrates into the chromosome Conjugation Donor cell physically contacts Recipient cell acquires donor DNA during cell-to-cell contact; if recipient cell then directly transferred DNA is a plasmid, then integration into the transfers DNA chromosome is not required for it to be passed to daughter cells Horizontal Gene Transfer as a Mechanism of Genetic Change - Figure 8.19 Transferred DNA replicated only if a replicon with origin of replication Chromosomes, plasmids DNA fragments can be added to recipient chromosome by a) Non-integrated DNA fragment homologous recombination Donor DNA replaces complementary region of recipient cell’s DNA b) Integrated DNA fragment Jump to Horizontal Gene Transfer as a Mechanism of Genetic Change - Figure 8.19 Long Description Transduction Transduction: transfer of bacterial genes by bacteriophages (phages) Phages infect bacterial cells Attaches to cell and injects its nucleic acid Phage enzymes cut bacterial DNA into small pieces Bacterial cell enzymes produce phage nucleic acid and a phage coat – components of new phage particles Phage particles are released from bacterial cell Generalized transduction results when a fragment of bacterial DNA enters the phage protein coat Produces a transducing particle Transduction Transducing particle may attach to another bacterial cell and inject the DNA it contains New DNA may be integrated into chromosome 1. A bacteriophage attaches to a 1. A transducing particle specific receptor on a host cell. attaches to a specific receptor on a host cell. 2. The phage DNA enters the cell. 2. The bacterial DNA is The empty phage coat remains on the outside or the bacterium. injected into a cell. 3. Enzymes encoded by the phage 3. The injected bacterial DNA genome cut the bacterial DNA integrates into the into small pieces. chromosome by homologous recombination. 4. Phage nucleic acid is replicated and coat proteins synthesized. 4. Bacteria multiply with new genetic material. 5. During construction of viral Replaced host DNA is degraded. particles, bacterial DNA can mistakenly enter a protein coat. This creates a transducing particle that carries bacterial b) The process of transduction DNA instead of phage DNA a) Formation of a transducing particle The Mobile Gene Pool - Figure 8.26 Much variation in gene pool of a single species Less than 50% of E. coli genes found in all strains Termed core genome of species Remaining make up mobile gene pool Can move from one DNA molecule to another Plasmids, transposons, genomic islands, phage DNA  The Mobile Gene Pool Plasmids common in microbial world Usually dsDNA with origin of replication Generally encode nonessential information, but may allow survival in particular environment Few to many genes Low-copy-number (one or a few per cell) to high-copy-number (many, up to 500) Most have narrow host range; some replicate in many different species Plasmids in same compatibility group cannot be maintained in the same cell Mobilizable plasmid requires conjugative plasmid for transfer The Mobile Gene Pool Transposons provide mechanism for moving DNA Can move into other replicons in same cell Simplest is insertion sequence (IS) Encodes only transposase enzyme, inverted repeats Composite transposons include one or more genes Integrate via non-homologous recombination Jump to The Mobile Gene Pool - Figure 8.28 Long Description  The Mobile Gene Pool Genomic islands: large DNA segments in genome that originated in other species Nucleotide composition very different from genome G-C base pair ratio characteristic for each species Characteristics encoded by genomic islands include Use of specific energy sources Acid tolerance Ability to cause disease Pathogenicity islands Bacterial Defenses Against Invading DNA Restriction-modification systems degrade foreign DNA Restriction enzyme cuts DNA at specific sequence Modification enzyme protects cell’s own DNA by adding methyl groups Bacterial Defenses Against Invading DNA CRISPR systems include small segments of phage DNA that recognize the specific DNA if it invades the cell again First invasion: complex of Cas proteins cuts DNA into short fragments Inserted into chromosome at CRISPR array; integrated DNA fragment called a spacer Subsequent invasion: transcription of CRISPR array generates crRNAs that direct DNA-cutting enzymes to invading DNA Bacterial Defenses Against Invading DNA 1. Infected bacterial cell Fragments of invading DNA are captured and then integrated into CRISPR array in cell’s genome. 2. Surviving bacterial cell and its descendants CRISPR array is transcribed and the RNA processed to generate crRNAs that bind to a Cas nuclease to form a complex. 3. Surviving bacterial cell or its descendants re-encounter the same phage crRNA-guided Cas nuclease degrades invading DNA. END OF CHAPTER 8