Bacterial Genetics Chapter 8

Diversity in Bacteria Bacteria use three different mechanism to adapt to changing environments Mutation Gene transfer Regulation of gene expression

Spontaneous Mutation Spontaneous mutations occur in natural environment Occur infrequently and randomly Rate of mutation probability that a mutation will be observed in a given gene each time the cell divides Rate is generally between 1 in 10,000 and 1 in a trillion Low rate is due to cellular repair mechanisms

Spontaneous Mutation Mutations are stable heritable changes in the base sequence of DNA Can occur from Base substitutions Removal or addition of nucleotides Transposable elements

Spontaneous Mutation Base substitutions Most common type of mutation Results from mistakes during DNA synthesis Incorrect base is incorporated into DNA Point mutations Occur when one base pair is changes Missense mutation Mutation resulting from amino acid substitution is called Nonsense mutation Mutation that changes a amino acid codon to a stop codon

Spontaneous Mutation Removal and addition of nucleotides Shifts the translational reading frame Shifts the codons Mutation called frameshift mutation Affects all amino acids downstream from addition or deletion Mutations frequently result in premature stop codons

Spontaneous Mutation Transposable elements Special segments of DNA that move spontaneously from gene to gene Elements called transposons Transposons disrupt proper function of gene Gene or gene product generally non functional

Induced Mutations Mutations are essential for understanding genetics Mutations can be intentionally produced to demonstrate function of particular gene or set of genes These mutations are termed induced Mutations can be induced via Chemical mutagens Transposition Radiation

Induced Mutations Chemical mutagens Nitrous acid Alkylating agents Converts amino group to a keto group Changes cytosine to uracil Uracil binds to adenine while cytosine binds to guanine Alkylating agents Largest group of chemical mutagens Alters hydrogen bonding of bases Nitrosoguanine is common alkylating agent

Induced Mutations Chemical mutagens Base analogs Chemicals that are structurally similar to the nitrogenous bases but have slightly altered base pairing properties Base analogs include: 2-aminopurine which incorporates in the place of adenine but binds with cytosine 5-bromouracil which incorporates in the place of thymine but binds with guanine

Induced Mutations Chemical mutagens Intercalating agents Molecules that insert themselves between adjacent bases Creates space between bases Extra base is often added to fill space Ethidium bromide is common intercalating agent Potential carcinogen

Induced Mutations Transposition Common procedure used to induce mutation in laboratory Gene that receives transposon will undergo a knockout mutation Termed insertion mutation

Induced Mutations Radiation Two types Ultraviolet light X rays Causes covalent bonding between adjacent thymine bases Forms thymine dimers which distorts DNA X rays Cause breaks and alterations in DNA Breaks that occur on both strand are often lethal

Repair of Damaged DNA Repair of base substitution Cells develop two methods of repair Proofreading DNA polymerase has proofreading function Able to excise incorrect base and replace with correct one Mismatch repair Endonuclease enzyme removes short stretch of nucleotide DNA polymerase fills gap DNA ligase joins ends Mismatch repair

Repair of Damaged DNA Repair of thymine dimers Two mechanisms Light repair A.k.a photoreactivation Enzyme uses visible light to break covalent bonds between bases Dark repair A.k.a excision repair Endonuclease excises damages section New section replicated and joined to original strand Thymine dimer repair

Repair of Damaged DNA Repair of modified bases SOS repair Enzyme cuts DNA backbone and removes base DNA polymerase incorporates new base SOS repair Last ditch effort to bypass damage Damage induces SOS system Produces new DNA polymerase Highly error prone Mutations can arise from synthesis with new enzyme

Mutations and Their Consequences Mutation provide organism way to respond to changing environments Environment selects for cells suited to survive Environment does not cause mutation

Mutation Selection Major problem in induced mutation is identifying bacteria with desired mutation Techniques used include Direct selection Involves inoculating population of bacteria on medium on which only mutants will grow Used to select antimicrobial resistant organisms Indirect selection Required to isolate organisms that require growth factor that parent strain does not Replica plating Replica Plating

Mutation Selection Testing for cancer causing chemicals Many mutagens are also carcinogens Cancer causing agents Microbes used to test potential carcinogenic activity Tests are based on effect chemical has on microbial DNA Ames test common chemical carcinogen test Test rate of reversion of Salmonella auxotroph Also test potential lethality

Mechanisms of Gene Transfer Genes are naturally transferred between bacteria using three mechanisms DNA-mediated transformation Transduction Conjugation

Mechanisms of Gene Transfer Gene exchange in bacteria Two event occur in gene exchange 1. Donor DNA is transferred and accepted by the recipient cell Donor DNA is transferred one of three ways Transformation Transduction Conjugation 2. Donor DNA is integrated onto the recipient cell’s chromosome

Mechanisms of Gene Transfer DNA-mediated transformation Definition: the transfer of naked DNA from one bacterium to another Discovered by Fredrick Griffith in 1928 while working with Streptococcus pneumoniae

Mechanisms of Gene Transfer Griffith realized S. pneumonic existed in two forms Encapsulated, virulent form Smooth in appearance Nonencapsulated, avirulent form Rough in appearance Griffith wondered if injections with the smooth strain could be used as a vaccine against pneumonia He injected mice with the different strains and recorded his results

Griffith Experiment

Mechanisms of Gene Transfer DNA-mediated transformation Involves the transfer of naked DNA from the environment to the recipient cell Cells rupture during the stationary and death phase The chromosome breaks into small pieces and explode through the ruptured cell wall Recipient cell picks up piece of the naked DNA The naked DNA is integrated onto the recipient chromosome Replaces the homologous gene on the chromosome of the recipient cell

Mechanisms of Gene Transfer DNA-mediated transformation Natural transformation occurs when bacterial cells are “competent” Competence is a condition in which bacterial cells are capable of taking up and integrating larger fragments of DNA Competence occurs during the late log, early stationary phase

Natural transformation occurs in four stages Entry of the DNA Only single strands enter, double strands are degraded Integration of the donor DNA Donor DNA is integrated by hydrogen bonding Enzymes cleave recipient DNA Donor DNA is put in place Mismatch repair Repair mechanism remove either donor or recipient DNA that doesn’t match Repairs with correct nucleotides Cell multiplication Transformed cells multiply under selective conditions in which non-transformed cell will not grow Transformation

Mechanisms of Gene Transfer Transduction Bacterial DNA that is transferred from donor to recipient via a bacterial virus (bacteriophage) Two types of transduction Generalized Any gene of donor can be transferred Specialized Only specific genes can be transferred

Transduction is a mis-packaging of DNA during viral replication The mis-packaged phage infects a new bacterial cell and insert the donor DNA into the recipient cell The donor DNA is integrated and mismatched pairs are repaired Transduction

Mechanisms of Gene Transfer Conjugation – only form of gene exchange in which donor survives Conjugation is mediated by a plasmid Plasmid is self replicating extrachromosomal piece of DNA Can code for traits that give bacteria advantage Conjugation requires direct contact between cells Cells must be of opposite mating types Donor cells carry a plasmid that codes for fertility factor or “F factor” This cell is designated F+ Recipient cell does not carry a plasmid This cell is designated F-

During conjugation, the plasmid is replicated in the donor cell and is transferred to the recipient After plasmid is transferred, F- cell becomes F+ Conjugation

In some F+ cells, the F factor integrates onto the host chromosome Converts F+ to Hfr Hfr= High frequency of recombination Conjugation between Hfr and F- cell results in only a portion of the F factor being replicated and transferred F- remain F- F- has new information but may not have the F factor gene Conjugation - Hfr