Microbial Genetics

Genes for the Germs

10

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 m cell by looping

and supercoiling– E. coli

• Over 4,000 genes

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

© H

. P

otte

r-D

. D

ress

ler/

Vis

ual

s U

nlim

ited

.

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

Figure 10.3: A TEM of bacterial plasmids.

Cou

rtes

y of

the

CD

C

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– 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 ()

• 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

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

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