Microbes and biotechnology Option F.1

Assessment statements F.1.1 Outline the classification of living organisms into three domains. F.1.2 Explain the reasons for the reclassification of living organisms into three domains. F.1.3 Distinguish between the characteristics of the three domains. F.1.4 Outline the wide diversity of habitat in the Archae, as exemplified by methanogens, thermophiles and halophiles. F.1.5 Outline the diversity of Eubacteria, including shape and cell wall structure. F.1.6 State, with one example, that some bacteria form aggregates that show characteristics not seen in individual bacteria. F.1.7 Compare the structure of the cell walls of Gram-positive and Gram-negative Eubacteria. F.1.8 Outline the diversity of structure in viruses including: naked capsid versus enveloped capsid; DNA versus RNA; and single stranded versus double stranded DNA or RNA. F.1.9 Outline the diversity of microscopic eukaryotes, as illustrated by Saccharomyces, Amoeba, Plasmodium, Paramecium, Euglena and Chlorella.

The five kingdoms Established in 1959 by Robert Whittaker Bacteria: single-celled organisms with no organized nucleus and no membrane-bound organelles Protista: single-celled organisms with an organized nucleus and organelles each surround by a membrane Fungi: multicellular organisms which obtain their food using extracellular digestion and have cell walls of chitin Plants: multicellular organisms which obtain their food by photosynthesis and have cell walls of cellulose Animals: multicellular organisms which obtain their food by feeding on other organisms and have no cell wall

Five kingdoms grouped into two categories Prokaryotes: bacteria which have no organized nucleus and no membrane-bound organelles Eukaryotes: all of the other kingdoms which have an organized nucleus

The three domains Attempt by Carl Woese to improve the accuracy of the classification system based on studies of rRNA Eubacteria: ‘true’ bacteria, prokaryotes with no organized nucleus and no membrane-bound organelles. Archaea: archaebacteria or ‘ancient’ bacteria are also prokaryotes; most live in extreme environments Eukarya: single-celled and multicellular organisms which all have their DNA contained in a nucleus; plants, animals, protists, and fungi

Reasons for reclassification into three domains rRNA is a molecule common to all organisms Performs same function in all organisms Coded for by DNA By looking at variation in the sequence of rRNA, evolutionary relationships became apparent Eubacteria and Archaea have different molecules making up their cell walls E and A have different molecular structure of their cell membranes E and A have different sequences of nucleotides in their rRNA

Characteristics of the three domains Histones Introns Size of Ribosomes Cell membrane lipids Peptidoglycan in cell walls Organelles Eukarya Present Large subunits (80S) Unbranched hydrocarbons Absent Archaea Histone-like proteins Present in some Small subunits (70S) Some branched hydrocarbons Eubacteria

Homework What is the RNA world hypothesis? Give supporting and refuting details for the hypothesis. NO OPINIONS!!!

Diversity of habitat of Archaebacteria Methanogens – use carbon dioxide to make methane; strict anaerobes; live in guts of termites and cattle, Siberian tundra, swamps rice fields, and in large intestines of dogs, pigs, and humans Thermophiles – live in sulfur hot springs where the pH is very acidic and temperatures are up to 90°C; some live in hydrothermal vents up to 105°C Halophiles – live in the Dead Sea, the Great Salt lake, and evaporated salt water ponds

Diversity of Eubacteria Varieties of shape Chain (strepto) Cluster (staphylo) Pair (diplo) Aggregation Vibrio fischeri emit light when in large groups, but not alone Bioluminescence caused by turning on of gene by increased amount of signal molecule Exist in light organs of squid Bacteria senses high density based upon amt. of signal present called quorum sensing Variety of cell wall Gram + Stain purple Gram – Stain pink Three main shapes Spheres (cocci) Rods (bacilli) Helices (spirilla)

Comparison of gram + and gram - bacteria Cell wall structure Gram + bacteria (stain purple) Gram – bacteria (stain pink) Complexity Simple Complex Amount of peptidoglycan (protective sugar network) Large amount Small amount Peptidoglycan placement In outer layer of bacteria Covered by outer membrane (protects from antibiotics) Outer membrane Absent Present with lipopolysaccharides (toxic to host) attached

Diversity of structure of viruses Are viruses alive? Not cellular Cannot reproduce without a host Basic structure Nucleic acid DNA (double- or single-stranded) RNA (double- or single-stranded) Several enzymes Protein coat (capsid) Naked Enveloped by membrane

Homework: Fill in chart Organism Nutrition Locomotion Cell wall Chloroplasts Cilia or flagella Saccharomyces Amoeba Plasmodium Paramecium Euglena Chlorella

Saccharomyces (yeast) Ferments carbohydrates in flour or malt and gain energy from this digestion Carbon dioxide gas (in bread) and ethanol (in beer) are by-produces Fungi Secrete enzymes outside their cells and absorb the produces of digestion back into the cell Have chitin in cell wall

Amoeba Fluid state of cytoplasm enables it to change its shape easily Pseudopodia wrap around a prey in order to trap it in a food vacuole for intracellular digestion

Plasmodium Parasitic heterotroph Mosquitoes carry and infect humans causing malaria

Paramecium Ciliated heterotroph Intracellular digestion Food taken into oral groove and passes to gullet

Euglena Both autotrophic and heterotrophic Contains chlorophyll Has eyespot which facilitates movement towards light Can absorb food from outside the cell Has flagellum

Chlorella Single-celled green algae Non-motile Cell wall of cellulose

Microbes and biotechnology Option F.2

Assessment Statements F.2.1 List the roles of microbes in ecosystems, including producers, nitrogen fixers and decomposers. F.2.2 Draw and label a diagram of the nitrogen cycle. F.2.3 State the roles of Rhizobium, Azotobacter, Nitrosomonas, Nitrobacter and Pseudomonas denitrificans in the nitrogen cycle. F.2.4 Outline the conditions that favour denitrification and nitrification. F.2.5 Explain the consequences of releasing raw sewage and nitrate fertilizer into rivers. F.2.6 Outline the role of saprotrophic bacteria in the treatment of sewage using trickling filter beds and reed bed systems. F.2.7 State that biomass can be used as raw material for the production of fuels such as methane and ethanol. F.2.8 Explain the principles involved in the generation of methane from biomass, including the conditions needed, organisms involved and the basic chemical reactions that occur.

Role of microbes in ecosystems Producers Change inorganic molecules into organic molecules Algae and some bacteria use chlorophyll to trap sunlight Chemosynthetic bacteria use chemical energy Nitrogen fixers Bacteria remove nitrogen gas from the atmosphere and fix it into nitrates which are useable by producers Decomposers Breakdown detritus and release inorganic nutrients back into the ecosystems

The nitrogen cycle Mutualistic nitrogen fixation: bacteria forms symbiotic relationship with a host plant and fix nitrogen for it (Ex.Rhizobium) Free-living nitrogen fixation: live in soil (Ex. Azotobacter) Industrial nitrogen fixation: burning of fossil fuels to produce fertilizer Nitrification: ammonia turned into nitrites by bacteria (Ex. Nitrosomonas) and to turn nitrites into nitrates (Ex. Nitrobacter) Active transport of nitrates: nitrates taken in by roots Plants and animals: plants use nitrates to make their own proteins; animals feed on plants, digest and rearrange proteins to make their own proteins Death and excretion: products of digestion and dead bodies contain molecules which contain nitrogen Putrefaction: decomposers break down complex proteins and release nitrogen gas into the atmosphere Denitrification: bacteria remove nitrates and nitrites and put nitrogen gas back into the atmosphere (Ex. Pseudomonas denitrificans)

Conditions which favor nitrification and denitrification Carried out by Nitrosomonas (ammonia into nitrate) Carried out by Nitrabacter (nitrite into nitrate) Available oxygen Neutral pH Warm temperature Denitrification Carried out by Pseudomonas denitrificans (nitrates into nitrogen gas) No available oxygen High nitrogen input

Consequences of releasing raw sewage and nitrate fertilizer into rivers High nitrates and phosphates fertilize the algae present in water Increased growth of algae Algae are decomposed by aerobic bacteria which use up the oxygen in the water (biochemical oxygen demand – BOD) Water becomes low in oxygen and fish and other organisms which need oxygen die

Sewage treatment by saprotrophic bacteria Stages of sewage treatment: Inorganic materials are removed and organic matter is left 90% of the organic matter is removed by saprotrophic bacteria

Trickling filter system Bed of stones 3-6 feet wide Saprotrophic bacteria adhere to the stones and act on the sewage trickled over them until it is broken down Cleaner water trickles out of the bottom of the bed This flows to another tank where the bacteria are removed The water is further treated with chlorine to finish the disinfectant process

Reed bed Waste water provides water and the nutrients to the growing reeds Reeds are then harvested for compost Breakdown of organic waste is again accomplished by saprotrophic bacteria Nitrate and phosphates released as a result of bacterial action are used as fertilizer by the reeds Can only handle small sewage flow

Water Alert!

Homework Fill in the blank: The three wastewater treatment plants in Mobile have a total design flow of _____ million gallons per day. Read the following article: http://www.usaid.gov/our_work/environment/water/us_japan_init.html What has been done in the countries aided by this project?

Sewage treatment video

Biomass can be used as raw material for the production of fuels such as methane and ethanol To make biogas, manure and cellulose are put into a digester without oxygen Anaerobic decomposition is performed by bacteria which occur naturally in the manure Manure and cellulose are broken down into organic acids and alcohol Organic acids and alcohol are broken down into carbon dioxide, hydrogen, and acetate Finally two type of bacteria work on these to produce methane which can be used to run electrical machinery Ammonia and phosphate are byproducts and can be used as fertilizer

Conditions to be kept constant in digester No free oxygen Constant temperature of 95 degrees F pH (not too acidic) Bacteria required for methanogenesis Acidogenic bacteria convert organic matter to organic acids and alcohol Acetogenic bacteria make acetate Methanogenic bacteria create the methane

Topic F.3

Assessment Statements F.3.1 State that reverse transcriptase catalyses the production of DNA from RNA. F.3.2 Explain how reverse transcriptase is used in molecular biology. F.3.3 Distinguish between somatic and germ line therapy. F.3.4 Outline the use of viral vectors in gene therapy. F.3.5 Discuss the risks of gene therapy.

Background information Genetic material of virus can be RNA or DNA An RNA virus is called a retrovirus Flow of genetic information in a retrovirus is from RNA back to DNA The enzyme which enables backwards transcription is reverse transcriptase

HIV life cycle HIV attaches to a host cell RNA of the virus and the enzyme reverse transcriptase enter the host cell Reverse transcriptase copies viral RNA into cDNA (complementary DNA) cDNA makes a second strand which is a complement to the first strand of DNA; viral RNA is destroyed New double-stranded viral DNA enters the nucleus of the host cell If HIV is active, it will use this DNA to make more HIV viruses; they will then burst out of the cell and infect other cells

How reverse transcriptase is used in molecular biology To make therapeutic proteins such as insulin and somatostatin Human DNA molecule is taken from a pancreas cell mRNA copies the DNA without the introns Reverse transcriptase produces a new single strand of DNA called cDNA The single strand replicates to make double-stranded DNA using DNA polymerase Inserted into plasmid Bacteria cell is stimulated to take up plasmid Bacteria multiply and produce insulin Insulin is harvested and used by diabetics

somatic and germ line therapy Gene therapy aims to replace defective genes with effective ones which give the message to make the correct protein Genes are delivered by vectors which are viruses that have been genetically engineered to infect certain cells in the patient Somatic therapy – affects only the patient involved; may be possible to cure single-gene defects such as cystic fibrosis and hemophilia Germ line therapy – changes gamete DNA, which would be passed to offspring

use of viral vectors in gene therapy Treatment of a disease called severe combined immune deficiency disease (SCID) Children do not produce enzyme ADA and thus have no immune system In 1998 stem cells were withdrawn from kids with SCID Cells mixed with a virus carrying normal form of gene that produces ADA Virus transferred normal copy of gene into stem cells Stem cells then infused back into bone marrow Immune systems were restored

risks of gene therapy Virus vector may enter another cell by mistake Virus vector might put gene in wrong place and cause mutation Genes could be over-expressed and too much protein produced Virus vector might stimulate immune reaction Virus vector might be transferred from person to person Children might be more sensitive

Benefits of gene therapy Possibility of curing a disease caused by a single-gene or multiple genes

Microbes and food production

Assessment Statements F.4.1 Explain the use of Saccharomyces in the production of beer, wine and bread. F.4.2 Outline the production of soy sauce using Aspergillus oryzae. F.4.3 Explain the use of acids and high salt or sugar concentrations in food preservation. F.4.4 Outline the symptoms, method of transmission and treatment of one named example of food poisoning.

The successful making of bread, beer, and wine Yeast was discovered as the fungus responsible for products Saccharomyces cerevisae Yeast uses sugars for energy and reproduces quickly by ‘budding’ Bud breaks off and forms a new yeast cell Turns glucose into two molecules of ethanol and two molecules of carbon dioxide gas as waste product

Beer Source of glucose: grain Sweet liquid wort is made from malt Hops are added and liquid is boiled and cooled Fermentation by yeast produces beer containing ethanol and carbon dioxide

Wine Crushed grapes and yeast are put into a tank Ethanol stays in the tank, while carbon dioxide escapes

Bread Fermentation of sugars in the dough by yeast Carbon dioxide makes the dough rise Baking in the oven kills the yeast, stops fermentation and evaporates the ethanol

Production of soy sauce Soak soy beans, boil and drain Mix a mash of soy beans with toasted wheat Add a culture of Aspergillus oryzae Incubate for 3 days at 85°F Add salt and water and ferment for 3-6 months Filter and pasteurize

Food preservation with sugar Increasing sugar content of food preserves it because it is a dehydrating environment for bacteria, yeast, and mold Boil fruit with sugar to kill microorganisms and dissolve sugar Add some pectin so it will gel Seal in hot sterile jars No need to refrigerate

Food preservation with acid Leave vegetables in a salt solution, then strain and rinse Place raw vegetables in sterile jars Pour hot vinegar and spices over the vegetables creating an acid environment Put on lids and process in a hot water-bath to give a tight seal by creating a vacuum so that no mold can grow

Salmonella poisoning Symptoms occur 12-72 hrs after infection and last 4-7 days Diarrhea Fever Abdominal cramps Small # develop Reiter’s syndrome Transmission Improper hand washing after going #2 Improper cooking of food Improper hand washing after touching feces of pets Improper hand washing after handling reptiles Raw meat cut on cutting board transferred to other food Irrigation with contaminated water Raw eggs Unpasteurized milk or dairy products

Treatment Treat dehydration by drinking lots of water, preferably with sugar and salt Intravenous fluid Antibiotics if passed to blood What food product was there a recent recall of due to food poisoning? What should be done to keep another such outbreak from causing sickness and death? http://www.washingtonpost.com/wp-dyn/content/article/2009/05/03/AR2009050301828.html

Metabolism of microbes

Assessment Statements F.5.1 Define the terms photoautotroph, photoheterotroph, chemoautotroph and chemoheterotroph. F.5.2 State one example of a photoautotroph, photoheterotroph, chemoautotroph and chemoheterotroph. F.5.3 Compare photoautotrophs with photoheterotrophs in terms of energy sources and carbon sources. F.5.4 Compare chemoautotrophs with chemoheterotrophs in terms of energy sources and carbon sources. F.5.5 Draw and label a diagram of a filamentous cyanobacterium. F.5.6 Explain the use of bacteria in the bioremediation of soil and water.

Definitions Photoautotroph – an organism that uses light energy to generate ATP and produces organic compounds from inorganic substances (Anabaena) Photoheterotroph – an organism which uses light energy to generate ATP and obtains organic compounds from other organisms (Rhodobacter sphaeroides) Chemoautotroph – an organism that uses energy from chemical reactions to generate ATP and produces organic compunds from inorganic substances (Nitrosomonas) Chemoheterotroph – an organism that uses energy from chemical reactions to generate ATP and obtains organic compounds from other organisms (Saccharomyces)

Photoautotroph Photoheterotroph Energy source Light Light and organic compounds Carbon source Carbon dioxide Organic compounds

Chemoautotroph Chemoheterotroph Energy source Inorganic compounds Organic compounds Carbon source Carbon dioxide

Anabaena Filamentous cyanobacterium Lives on grass and in freshwater ponds Two distinct and interdependent cell types Heterocysts Photosynthetic cells Heterocysts fix nitrogen from dinitrogen in the air into nitrogen compounds such as ammonia which is used to make proteins Photosynthetic cells produce carbohydrates Akinete - spore-like cell capable of withstanding certain environmental extremes and of germination

Bioremediation Use of bacteria and fungi to treat environments such as soil or water contaminated with polluting agents such as pesticides, oil and industrial solvents The microorganisms derive energy for their own growth and reproduction

F.6 Microbes and disease

Assessment Statements F.6.1 List six methods by which pathogens are transmitted and gain entry to the body. F.6.2 Distinguish between intracellular and extracellular bacterial infection using Chlamydia and Streptococcus as examples. F.6.3 Distinguish between endotoxins and exotoxins. F.6.4 Evaluate methods of controlling microbial growth by irradiation, pasteurization, antiseptics and disinfectants. F.6.5 Outline the mechanism of the action of antibiotics, including inhibition of synthesis of cell walls, proteins and nucleic acids. F.6.6 Outline the lytic life cycle of the influenza virus. F.6.7 Define epidemiology. F.6.8 Discuss the origin and epidemiology of one example of a pandemic. F.6.9 Describe the cause, transmission and effects of malaria, as an example of disease caused by a protozoan. F.6.10 Discuss the prion hypothesis for the cause of spongiform encephalopathies.

Methods of transmission of pathogens Food Water Aerial (water droplets in air) Anima vectors Puncture wounds Sexual contacts

Intracellular infections Example: Chlamydia Live inside a cell of the host (epithelial cells which line genital tract) When cells reproduce, bacterium splits out and moves into genital tract Does not produce toxins (some people are asymptomatic) Does not directly damage cells (may cause PID or infertility) Not targeted by immune system

Extracellular infections Example: Streptococcus Lives in the host but outside a host cell Produces toxins Damages cells (produces invasins which split open and dissolve host cells) Is targeted immediately by the immune system

Endotoxins and exotoxins Lipopolysaccharides in walls of Gram- bacterium that causes fever and aches Ex. Salmonella Exotoxins Specific proteins secreted by bacteria that causes symptoms such as muscle spasms and diarrhea Ex. Clostridium tetani and Vibrio cholerae

Methods to control bacterial growth Irradiation Gamma radiation kills all microbes Microwaves kill all microbes UV radiation is weakest; kills all bacteria but leaves endospores Disinfectants Kills bacteria but not endospores Damaging to mucous membranes and skin Antiseptics Less effective, but safe for skin Pasteurization Kills pathogens, but is bacteriostatic for nonpathogenic bacteria

Antibiotics Antimicrobial agents produces by microbes which inhibit or kill other microbes Cell wall synthesis inhibition – inhibit production of peptidoglycan (ex. penicillin) Protein synthsis inhibition – attack the bacterial ribosome (ex. streptomycin) Nucleic acid inhibition – affect synthesis of DNA and RNA or attach to DNA or RNA so message can’t be read (ex. rifampicin)

Epidemiology Study of the occurrence, distribution, and control of disease in a population Occurrence - # of people with disease Distribution – regions of the country/world where it is occurring Control – best strategies to prevent its spread

Pandemic Worldwide infections Spanish flu (1918) Asian flu (1957) Hong Kong flu (1968)

Spanish Flu Occurrence Killed 40-50 million people Distribution Across the globe Control gauze masks were issued and directed to be worn in public Public gatherings banned Public institutions closed Quarantine of patients with flu Public education about hygiene Use of disinfectants and sterilization methods Development of vaccines

Lytic life cycle of influenza virus Virus attaches to receptors on cell Virus is taken into an endosome by endocytosis into the cytoplasm Coat of virus is removed and viral RNA enters the cytoplasm Viral RNA enters nucleus and makes mRNA Some mRNA is transported to the cytoplasm where it makes viral proteins (translation) Viral proteins are transported back to the nucleus to form a capsid around viral RNA ER of the cell synthesizes viral envelope proteins Viral envelope proteins are packaged at the Golgi apparatus and transported to the cell membrane Viral nucleocapsid recognizes proteins on the membrane and buds off surround by the viral envelope proteins

Malaria Causes Plasmodium falciparum Plasmodium vivax Plasmodium ovale Plasmodium malariae Transmission By female Anopheles mosquito Plasmodia reproduce in gut of mosquito Egg sac ruptures and releases cells called sporozoites which travel to salivary glands When mosquito bites human, sporozoites enter bloodstream and travel to liver Develop further and burst out of the liver to invade red blood cells

Symptoms Anemia Bouts of fever chills Shivering Pain in the joints headache

Spongiform encephalopathies Holes form in brain tissue Similar to scrapie which is common in sheep It jumped species barrier and was transmitted to cattle in their feed Humans then contracted disease by eating contaminated meat Variant Creutzfeldt-Jakob disease

Prion hypothesis The casual agent for vCJD is caused by a virus with a nucleic acid core and a protein coat protecting and hiding the nucleic acid Protein causes disease Nucleic acids have not been found Have witnessed proteins causing normal proteins to change shape which argues against model that only nucleic acids can code for the shape of proteins