Chapter 5 Microbial Biotechnology
Interesting Facts • Microbes have existed on the earth for over 3.5 billion years • 50% of the living matter is comprised of micoorganisms • Less than 1% of all bacteria have been identified, cultivated and studied in the laboratory • Yet we are literally surrounded by microbes all the time
What bacteria look like spheres or cocci growing in chains cork-screw rods
Microorganisms as tools for biotechnology • Ways of getting foreign genes into a bacterial cell • Transformation • Naked DNA taken up by cell from the environment • Electroporation • Electric shock opens up the cell wall and allows DNA to enter the cell from the environment
E. coli bacterium transformed with a gene from a jellyfish The jellyfish gene encodes a “green fluorescent protein” (GFP) which allows you to easily see a bacterial cell that has been transformed and expresses the protein In this case, the gene is referred to as a reporter gene because it is reporting on the location of the bacterium.
Cloning and Expression Techniques • Bacterial fusion or hybrid proteins for synthesis and isolation of recombinant proteins • use recombinant DNA method to insert the gene for a protein of interest into a plasmid containing a gene for a well-known protein that serves as a “tag” for the protein of interest • the tag protein then allows for the isolation and purification of the recombinant protein as a fusion protein
Expression vectors • Plasmid vectors for making fusion proteins are called expression vectors because they enable bacterial cells to produce or express large amounts of protein • vectors have gene encoding • Maltose-binding protein
Fusion Proteins-how they are made and recovered Recover from culture medium and purify DNA peptide protein of interest mRNA Maltose-binding protein
Microbial Proteins as Reporters Luciferase (lux gene) Light organ filled with bacterium Vibrio fisheri
Ethanol fermentation • Anaerobic reactions • Conversion of sugar from grains and fruits to EtOH • grains beer • grapes wine and vinegar • By manipulating rate of biochemical reaction in the culture, brewers can control the EtOH content. • Ethanol fermentation microorganism • Saccharomyces cervisiae (yeast fungi)
Fermentation reactions Many enzyme proteins required for the conversion of glucose to ethanol
Same yeast used to make EtOH is used to make bread • Yeast uses sugar in dough to make EtOH and CO2 • CO2 gets trapped in dough and causes it to “rise”. • Baking the dough causes to gas to escape, leaving “holes” behind in the bread. • Cooking the bread completely causes all the EtOH to evaporate • sourdough bread has some of the EtOH retained in the bread because it is undercooked.
stopped Fermentation-anaerobic metabolism • Lactic acid fermentation • Lactococcus lactis (bacterium) • Used to make • sauerkraut from cabbage • yogart • vinegar • citric acid in fruit • methanol and acetone
Therapeutic proteins • Insulin is part of a class of proteins called hormones • It is produced by cells in our pancreas and secreted into the bloodstream to stimulate uptake of blood glucose into body cells such as muscle tissue • Allowing blood glucose levels to remain high causes health problems • high blood pressure • poor blood circulation • cataracts
Human insulin consist of 2 polypeptides • A subunit (21 amino acids) • B subunit (30 amino acids) • The 2 peptides bind to each other by reversible chemical bonds • Pancreas cells produce the 2 subunits as a single polypeptide chain and then enzymatically cut the polypeptide to produce the 2 subunits, which then fold properly into an active insulin protein. Enzymatic cleavage in cells of body Functionally-active hormone
b-gal Recombinant insulin antibody Lactose in culture medium insulin subunit Combine genes to make fusion protein bead b-galactosidase protein
Protein DNase Interferons and Interleukins Superoxide dismutase Function digests DNA stimulates cell growth binds and destroys harmful free radicals Therapeutic Proteins from Recombinant Bacteria Application cystic fibrosis treat different cancers; leukemia minimize tissue damage after heart attack Protein
Target sites of antimicrobials (antibiotics) in bacterial cells erythromycin tetracycline pennicilin ciprofloxacin fostriencin rifampicin
Vaccines • First vaccine developed by Edward Jenner in 1796 • used a live cowpox virus to vaccinate humans (himself) against smallpox • based on claims that milkmaids exposed to cowpox virus in udder infections on cow never got smallpox disease. • Exposure to cowpox fluid stimulated immune system of human volunteers to develop protection against smallpox
Vaccines • Today, DPT vaccine is given to infants to protect them from • diptheria toxin • pertussis toxin • tetanus toxin • Another common vaccine is MMR • measles • mumps • rubella • Polio vaccine
Benchmarks in disease control stopped
Polio vaccine • Many people don’t have their children immunized these days because they think that polio doesn’t exist or pose a real threat now. • Virus is still around and children can still succumb to the disease. • Question: Is the risk of developing a side-effect or getting the disease from the vaccine worth the risk of avoiding the disease when exposed to a natural source of the virus?
1st exposure 2nd exposure Antibody production days • Primary response is slow and may not be able to produce enough antibody-producing cells to destroy antigen if it is being produced by bacteria replicating in our bodies • Secondary response is much quicker, so there is a better chance of destroying antigen and bringing disease under control
1st exposure 2nd exposure Vaccine serves as 1st exposure to antigen Antibody production days How do vaccines work? • Vaccines induce the primary response without causing the disease so that when an individual is later exposed to the real disease-causing agent, a secondary response will occur and provide a more rapid and effective immunological response to neutralize the disease-causing agent.
Types of vaccines • Inactivated vaccines - killed virus) • polio • Attenuated vaccines - live virus but genetically engineered so as not to replicate in host • polio • Subunit vaccines - portion of infectious agent that elicits good antibody response (protein or lipid molecule) • hepatitis B virus protein
Current efforts to develop vaccines against emerging diseases • HIV • 33 million people affected • high mutation rate of HIV virus compromises efficacy of vaccines developed to date
HIV life cycle RNA virus Receptor protein on T-cell RNA→DNA T-cell is a type of white blood cell that participates in cell mediated immunity T-cell
Current efforts to develop vaccines against emerging diseases • Tuberculosis • 3000-year-old disease • 2-3 million deaths/year; 7th leading cause of death • current treatment regime is demanding: 4 drugs taken daily for 6-18 months: patients don’t complete regime • new bacterial strains are resistant to antibiotics that controlled previous strains • genome of Mycobacterium tuberculosis has been sequenced and new proteins have been discovered that are good candidates for developing vaccines against • TB has become the single largest cause of death among AIDS patients
How Human Genome Project has been used to fight human disease • Tools and methods developed to sequence human genome are now being used to sequence genomes of bacteria that cause disease • New proteins located on the bacterial cell surface were discovered by sequencing Streptococcus pneumoniae genome • These are now used as target antigens for subunit vaccines
Using microbial genome information to identify causative agents of outbreaks of disease • E. coli strain 0157:h7 produces a lethal toxin that causes 20,000 cases of food poisoning each year. • The gene sequence for the toxin is known • Strains recovered from patients producing the toxin can be detected and distinguished from harmless strains using polymerase chain reaction (PCR)
The USDA has set up labs (PulseNet) across the country to do rapid identification of bacteria using rapid molecular approaches • What methods did we use to identify causative agents of disease before we could easily test for specific genes? • Why are the new rapid methods preferred for this application?
Microbes as bioweapons • Bioterrorism is not new in this country. • In 1800s the U.S. military distributed blankets and other articles contaminated with smallpox virus to the Native Americans who were occupying land in the West that settlers wanted to take possession of. • Since the Native Americans had never been exposed to this virus, they had virtually no resistance to the disease. • The American military exploited this situation to debilitate as many Native Americans as possible.
So many Native Americans became infected with and died from exposure to smallpox that they were unable to hold on to their hunting grounds and tribal sites and the survivors were relegated to reservations. • Americans were terrorized after 9-11-01 when spores of anthrax bacteria were transmitted in the mail and 5 people died. • Anthrax bacteria were found in large-scale production in Tokyo in 2000 and being used to attempt to kill people in that city.
No evidence of any deaths from the release in Tokyo. Why? • Disease-causing strain carries 2 plasmids each containing a different toxin gene. • Both genes must be expressed to cause disease. Toxin gene 2 Bacillus anthracis Toxin gene 1
Strain produced and disseminated by terrorists in Toyko carried only one of the plasmids, so it was not pathogenic. • Bioterrorists are not particularly knowledgeable in the molecular biology of disease. • However, with the appropriate knowledge such as that which you have acquired in this course, a person could produce and disseminate a disease-causing microbe into the environment. • Safeguards now in place to restrict public access to critical research results
What would be an effective way to use pathogenic microbes to intentionally kill a large number of Americans or American allies? • Choice of agent? • Delivery system? • Dispersion mechanism? • Targets?
Potential Biological Weapons • Brucella • Bacillus anthrasis • Clostridium botulinum • Ebola or Marburg virus • Francisella tularensis • Influenza virus • Rickettsia • Variola virus (smallpox) • Yersinia pestis (plague)
Food pathogens as a target of bioterrorism • Foot-and-mouth disease (virus) • Africa swine fever virus • Stem rust fungus for cereal crops • Southern corn leaf blight (fungus) • Rice blast (fungus) • Potato blight virus
How could U.S. mount an effective response to bioweapons released in this country? • Use of biotechnology to detect bioweapons released into the environment • similar strategy as one currently used to detect and control infectious disease outbreaks that occur naturally? • Air, water and soil monitoring • rapid-response teams and programs • new strategies needed • sanitizing mail: uv or x-ray treatment before handling and distribution