10 Microbial Genetics Genes for the Germs

Deadly Diarrhea 1968 Guatemala Bloody dysentery hit 100,000 people, with 12,000 deaths Caused by Shigella Standard antibiotics had no effect Arose through genetic selection of antibiotic-resistant strains

Bacterial DNA The bacterial chromosome DNA Double helix Closed, circular loop Free in cytoplasm If extended into line, 1.5 mm long Shrinks to fit inside 1 mm cell by looping and supercoiling E. coli Over 4,000 genes © H. Potter-D. Dressler/Visuals Unlimited. Figure 10.1: A) An electron micrograph of an E. coli cell immediately after disruption. The tangled mass is the organism’s DNA. B) The loops in the structure chromosome, viewed head-on

Bacterial DNA The bacterial chromosome Replication Unwinding by helicase and/or gyrase Single strands held apart by single-stranded DNA binding protein New synthesis initiated by primase All new DNA synthesized by DNA polymerase Synthesis is semi-conservative

Bacterial DNA: Replication Figure 10.2: Replication of the E. coli chromosome

Bacterial DNA Plasmids Extrachromosomal independent units Closed, circular, double-stranded DNA May confer selective advantage to microbe “R” factors Courtesy of the CDC Figure 10.3: A TEM of bacterial plasmids.

Gene Mutations Mutation is permanent change in an organism’s DNA Change is passed from originator to all its progeny Method by which some drug resistance occurs

Gene Mutations Causes of mutation Spontaneous errors by DNA polymerase Estimated at 1 observable change in every billion replications Over 1 billion bacteria in an observable colony Hence, at least 1 mutant, perhaps drug resistant, in the population That resistant survivor can now successfully replicate to occupy the niche where all the other susceptible bacteria have died Mutagens Increase spontaneous error rate of DNA polymerase Therefore, increase presence of mutants Examples Ultraviolet radiation Chemicals

Gene Mutations: Mutagens Figure 10.4: How nitrous acid causes bacterial mutations

Gene Mutations Causes of mutation Transposons Changes in the way proteins are encoded may alter the way in which antibiotics bind Antibiotics no longer effective against the mutated target Transposons Small segments of DNA that can move from one position to another in the chromosome Jumping genes in corn; Barbara McClintock’s Nobel prize Insertions of DNA into new spots in chromosome may also alter protein function

Gene Recombinations Transfer of genetic information between bacteria Conjugation Two live bacteria F+ donor cell F(ertility) factor F plasmid Sex pili F- recipient cell Conjugation bridge Replication and passage of DNA through bridge Frequently F plasmids also encode genes for drug resistance Hfr bacteria Many genera: Escherichia, Salmonella, Shigella

Gene Recombinations: Conjugation Figure 10.5: Bacterial conjugation

Gene Recombinations: Conjugation Figure 10.5: Bacterial conjugation, cont’d.

Gene Recombinations Transduction Gene transfer with the assistance of bacterial viruses Bacteriophages Also known as phages (F) Insert DNA into cytoplasm of bacteria Turn bacteria into phage factories, executing phage DNA program Phage production is usually lytic Sometimes random segment of host DNA packaged in progeny That segment of host DNA is delivered to new host Result is transfer of DNA from one bacterium to another DNA may encode drug resistance

Gene Recombinations: Generalized Transduction Figure 10.7: Generalized transduction

Gene Recombinations: Generalized Transduction Figure 10.7: Generalized transduction, cont’d.

Gene Recombinations Transformation Acquisition of genes from surrounding environment Requires competent cells Requires only naked DNA Some of this DNA may encode drug resistance genes Fig. 10.9: Bacterial transformation

Gene Recombinations In today’s world Gene transfers have resulted in proliferation of drug resistant bacteria Staphylococcus aureus Part of normal flora Harmful if they penetrate skin Open wounds Piercings Damaged hair follicles Cuts, scratches Many S. aureus are acquiring multiple drug resistance genes: MRSA Diseases Boils Abscesses Pneumonia Septicemia Endocarditis Toxic shock Now appearing in clinics: VRSA (vancomycin-resistant S. aureus)

Genetic Engineering Manipulation of DNA sequences in vitro Cutting and splicing DNA segments together in new combinations that never existed before in nature Not possible until the 1970s

Genetic Engineering The beginning of genetic engineering Endonucleases Restriction enzymes Expressed by bacteria to restrict infection by phages Cut DNA at specific sequences Create sticky ends Sticky ends can be put back together in new combinations

Genetic Engineering: Restriction Enzymes Fig. 10.10: A) A restriction enzyme cuts through two strands of a DNA molecule to produce two fragments. B) The recognition sites of several restriction enzymes

The first recombinant DNA molecule Genetic Engineering The first recombinant DNA molecule Cut gene from SV40 with restriction enzyme Cut E. coli plasmid with same enzyme Pasted to sticky DNAs together with ligase Created new plasmid Figure 10.11: Construction of a recombinant DNA molecule

The first recombinant DNA molecule Genetic Engineering The first recombinant DNA molecule Figure 10.11: Construction of a recombinant DNA molecule, cont’d.

Genetic Engineering The implications US government guidelines on recombinant DNA technology New field of biotechnology Food production New medicines Pollution control New vaccines Threat of new bioterrorism agents Recombinant human insulin produced from bacteria Recombinant Factor VIII , produced in bacteria, for hemophiliacs Recombinant human growth factor Advances in agriculture, medical diagnostics, forensic science