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Chapter 9 Microbial Genetics Genetics: The science of heredity • Research in Genetics takes place on several levels – – – –

Organismal Cell Chromosome Molecular

Genetic material is found in: DNA & RNA It can be found in: Prokaryotic organisms

– Nucleoid – Plasmids

Eukaryotic organisms

– Chromosome – Mitochondria – Chloroplasts – Plasmids

Viruses

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DNA RNA

Chromosome – Cellular structure -packaged DNA molecule Gene – A segment of DNA that codes for functional product (protein) Genotype – The sum of all genes- Genetic make-up Phenotype - Manifestation of genotype

The size and packaging of genomes E.coli 4,288 genes —- 1 um cell size. Human cell: 20,000 – 25,000 genes Size of the stretched out DNA – 1mm Structure of DNA Composed of nucleotides (bases) • Purine (adenine, guanine) hold by 3 H bonds • Pyrimidine (cytosine, thymine) hold by 2 H bonds Deoxyribose sugar Phosphate group Orientation: 3’ to 5’

-Helicase opens the Double helix – unzipping hydrogen bonds -Bases of single stranded DNA exposed -Replication fork is formed -Synthesis of the new strand by attachment of complementary nucleotides -Semiconservative replication (DNA contains one old and one new strand) -The strands are synthesized in the 5’ to 3’ direction -DNA polymerase I removes RNA primers -Lygase joins the fragments

DNA Replication – overall process

Leading strand is synthesized continuously Lagging strand is synthesized in pieces (Okazaki fragments)

Enzymes involved in DNA replication Helicase: Untwists the DNA helix by braking the H bonds Primase: Synthesis of RNA primer DNA polymerase III: adding bases to the new DNA Ligase: joining of the of DNA fragments Topoisomerase I, II: Supercoiling

The Flow of Genetic Information: • • •

DNA to RNA to PROTEIN

DNA Replication: reproduction of cells Transcription: DNA is copied to RNA Translation: polypeptides synthesized from RNA nucleotide sequences to amino acid sequence of protein

Sugar component:

DNA RNA deoxyribose ribose G -----------C C------------G T-------------A A------------ U (uracyl)

Process of transcription 1) 2) 3) 4) 5) 6) 7)

RNA polymerase binds to the promoter Unwinds the double helix of DNA One DNA strand acts as a template for synthesis of RNA RNA polymerase puts free nucleotides together forming RNA chain As new RNA grows, polymerase moves along the DNA When the RNA polymerase reaches the sites called terminator, transcription ends RNA polymerase and new RNA strand are released

Translation • Codons are groups of three nucleotides (mRNA) - The sequence in the codon determines which amino acid will be incorporated into a protein

- Translation is taking place in ribosomes

The beginning of protein synthesis 1) 2) 3) 4)

Ribosome moves along mRNA and reads the codons Transfer RNA (tRNA) has a sequence of three bases – anticodons complementary to codons For each amino acid there is a specific tRNA The first tRNA recognizes the start codon and brings methionine

Elongation and termination • Peptide bonds are formed between amino acids -polypeptide is formed • Blank tRNA discharged • When nonsense codon is reached, translation is terminated • Ribosome is separated into its subunits

Simultaneous transcription and translation More than one ribosome reads one mRNA molecule Most genes (75%) are constitutive – their products are produced constantly Other genes are regulated so that they are transcribed and translated only when needed Transcription and translation in Eukaryotes The introns – non-functional segments of DNA are transcribed but not translated The genes need to be processed The introns are cut out

Mechanisms of Genetic Control Repression: Inhibition of gene expression. Mediated by a protein – repressor Inducer: a substance that induces transcription

Operon model of gene expression - Inducible operon

- Lactose operon: regulates lactose metabolism, must be activated by inducers Functional gene consists of: Regulatory gene:

Structural genes

Operon Promoter - RNA polymerase initiates transcription Operator – go or stop signal - B-galactosidase - Lac permease - Transacetylase

Changes in the Genetic Material Mutation: change of the genetic code It can be:

Spontaneous Induced beneficial: mutant enzyme - enhanced activity lethal: lost activity of the enzyme

Categories of mutation Point mutation: substitution, insertions, and deletions of a single base in the DNA Missense mutation: substitution of one amino acid Nonsense mutation: creating a stop codon in the middle of protein Frameshift mutation: deletion or insertion of a DNA fragment; causes changes in many amino acids

MUTAGENS Chemicals: converts adenine into form that pairs with C instead of T. Nucleoside analogs: molecules that are structurally similar to normal bases. Radiation: X-rays, gamma rays, errors in replication; physical damage of DNA. Horizontal gene transfer –

Transformation • Fragments of the DNA are transferred from one microorganism to another. Incorporated into genom • Experimentally shown by Frederick Griffith in England 1928

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Conjugation • “Sexual contact” between two mating cells • Donor cell has sex pilli and F+ (fertility factor) a gene located on the plasmid • DNA is transferred from one cell to another through sex pillus

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Transduction- Virus mediated (DNA Transfer)

Plasmids: Small circular self-replicating DNA elements – – – –

Conjugative plasmids Enable survival under challenging conditions Production of toxins (kill other bacteria) Resistance factors (antibiotics)

Chapter 10 Genetic Engineering Biotechnology: Application of biological systems (microorganisms) to obtain a product. Recombinant DNA technology: procedures by which a fragment of DNA of one organism is incorporated into the genome of a different organism Goals of Genetic Engineering • Create organisms that synthesize products humans need (insulin) • Eliminate undesirable phenotypic traits Tools and Techniques of Genetic Engineering • • • • •

Restriction enzymes – major tool Analysis of DNA – gel electrophoresis Nucleic A hybridization: two complementary parts of DNA together DNA sequencing Polymerase Chain Reaction - PCR

Restriction Enzymes DNA cutting enzymes • Recognize and cut specific fragments of DNA • Leave single stranded sticky ends of DNA • DNA from different sources cut w/ same restriction enzyme will produce the same type of sticky end • DNA is cut on a specific Palindromic (identical sequences when read in opposite directions) DNA Gel electrophoresis • Separation of DNA fragments based on their size & weight • In agarose gel, DNA fragments are subjected to an electrical current • DNA molecule has a negative charge, moves toward the positive pole • Smaller fragments move faster

DNA sequencing: A process in which exact sequence of nucleotides in a DNA segment is determined

Polymerase chain reaction, PCR: Technique by which small amount of specific DNA fragment can be amplified in vitro

One PCR cycle has 3 basic steps: • • •

Denaturation: 940C – separation of DNA strands Priming: 50–65 0C, primer attached to complementary strand of DNA Extension: 720C

What is needed?

– PCR machine - thermal cycler – Target DNA that serves as a template – Supply of 4 nucleotides – DNA polymerase – Primers (short fragments of DNA)

**DNA polymerase extends the molecule by adding nucleotides •Typically we use 20-40 cycles – millions of copies of DNA Recombinant DNA Technology: • A selected gene is removed from the genetic donor • This gene is incorporated into a vector (plasmid or virus) • The vector is inserted into the cloning host (bacteria, yeast) Vectors: DNA molecules into which a segment of foreign DNA can be incorporated (plasmids, transposons and viruses) – Self-replicating – Circular shape – Proper size – to be able to accept foreign DNA – Must have a promoter & a gene for antibacterial resistance Applications of Recombinant DNA Technology Pharmaceutical and Therapeutic Applications – Protein synthesis – Vaccines, DNA vaccines – Genetic screening – DNA fingerprinting – Gene therapy

Hormone insulin, needed by diabetics. Gene was cloned into E. coli Before it was obtained from the pancreases of slaughtered animals Somatostatin, Human hormone used for treatment of giantism Before - 500,000 sheep brains were needed to produce 5 mg of somatostatin. Today - 8L culture of genetically engineered bacteria to obtain the equivalent amount

Agricultural applications Transgenic organisms: recombinant plants and animals altered by addition of genes from other organisms Improving Crops – Herbicide resistance: Gene for resistance to glyphosate was incorporate, that don’t kill plants. – Salt tolerance – Freeze resistance – Pest resistance: Tomato Plant with Bacillus thuringiensis toxin – Improvements in nutritional value and yield

Transgenic Animals

Many human genes have better expression in animals than in bacteria • The product (protein) can be collected in milk or semen

• Foreign genes are inserted into an embryo by using a virus or an injection

Transgenic Plants

Many human genes have better expression in animals than in bacteria • The product (protein) can be collected in milk or semen • Foreign genes are inserted into an embryo by using a virus or an injection

Antisense DNA and RNA • Antisense strand of DNA recognizes and binds to the complementary mRNA fragment • This results in blocking the expression (translation) of the harmful gene • Antisense drugs are being researched to treat cancers and other diseases

Inserting foreign DNA into cells TRANSFORMATI ON

o Plasmid from the surrounding environment is taken up by a cell o Cells have to be made competent - by soaking them in calcium chloride

SCREENI NG OFBACTERI ATHATCONTAI NSFOREI GNDNA

o The vector (plasmid) contains the gene for ampicillin resistance o Just transformed cells can grow on medium containing ampicillin

SYNTHETI CDNA

o DNA synthesis machine o Short fragments of DNA can be synthesized o Sequence of the DNA fragment that will be synthesize must be known.

SUBUNI TSVACCI NES

o A gene coding for the viral protein is cloned o Hepatitis B vaccine (Saccharomyces cerevisiae carries the virus gene on a plasmid) o Advantage: there is no chance of becoming infected during vaccination

DNAVACCI NES

o A single gene from pathogen is artificially copied and multiplied. o Gene is then injected into a muscle. Muscle cells take up this gene and use it as one of their own. o The immune system recognize that product as foreign, and start producing antibodies

GENETHERAPY

o Mostly preliminary work o Missing or defective genes replaced with normal copies o Possible treatment for: cystic fibrosis, sickle cell anemia, som types of hemophilia, some types of diabetes

GENETI CSCREENI NG

o Many genetic diseases can be detected by genetic engineering techniques o Technique: Southern blotting (Ed Southern) o Inherited forms of breast cancer can be detected.

Chapter 11 Physical and Chemical Control of Microbes The purpose of controlling microbial growth – To stop spreading the diseases or food spoilage Methods • Physical agents – Heat – Radiation • Chemical Agents – Gases – Liquids • Mechanical removal – Filtration • •

Air Liquids

Methods of Microbial Control Sterilization: Destruction of all forms of microbes including endospores by steam under pressure or ethylene oxide. Disinfection: Destruction of vegetative cells of pathogenic microorganisms by chemicals or physical methods. Pasteurization: Application of high temperature (720 C) for short period of time (15 sec). Reduce the number of microbes Antiseptic: Antimicrobial agent that is sufficiently non-toxic to be applied on living tissue. Sanitization: Lowering the number of microbes on eating and drinking utensils by heat or chemical disinfectant. Decontamination: Mechanical removal of microbes from organisms or non-living objects by washing. Terminology

Bactericidal: agent that destroys or kills bacteria Bacteriostatic: agent that inhibits bacterial growth Microbial Death

Permanent loss of reproductive capabilities The cell structures become dysfunctional

Factors that affect death rate

Time of exposure (lower temp. can be compensated with longer exposure) The number of microbes Microbial characteristics (endospore, vegetative cells) Agent used Environmental influences (suspending medium, pH)

Mode of Action of Antimicrobial Agents Plasma membrane: when damaged, cell content leaks into the surrounding medium Proteins enzyme, active sites inactivated: – Complete denaturation – Different shape – Blocking the active sites Nucleic Acid: radiation or some chemicals lethally damage the DNA or RNA . Physical methods of microbial control HEAT — Moist heat and dry heat. Mechanism: denaturing the enzymes — Most commonly used method of killing the microbes: Thermal death point: the lowest temp at which all the microbes are killed in 10 min Thermal death time: the minimal length of time needed to kill all bacteria at given temperature MOIST HEAT – non pressurized steam — Mechanism: coagulation of proteins — Boiling (1000C) for 10 min kills vegetative cells of bacteria, viruses, and fungi ****Hepatitis virus can survive up to 30 min of boiling; some bacterial spores can survive more than 20 h. Tyndalization: boiling the medium for 60 min repeatedly for 3 day AUTOCLAVES - steam under pressure — Provide high temp. and high pressure (Pressure: 1 atm, temp.: 1210 C) — All microbes are killed in 15 min — Steam should contact all surfaces — Time is different for larger volumes — Used for sterilization of: Culture media Equipment Biological waste PASTEURIZATION — Original pasteurization: 630 C for 30 min — Today’s pasteurization: high temperature short-time, 720 C for 15 sec. or — Ultra-high-temperature treatment: Exposure to 1340 C for 3 sec. then rapidly cooled

DRY HEAT STERILIZATION — Mechanism: oxidation — Flaming: inoculating loops — Hot-air sterilization: Oven @1700 C for 2h DESICCATION — Without water microbes cannot grow but can survive — Bacterial spores can survive for centuries — Survival depends on microbial type and organism’s environment i.e. Mycobacterium tuberculosis – long survival Neisseria gonorrhoeae – dies after a few hours of air drying LOW TEMPERATURES — Ordinary refrigeration (0-70 C) - bacteriostatic effect — Psychotrophs grow slowly — Pathogenic bacteria will not grow in the refrigerator — Rapid freezing: microbes become dormant, if you rapidly freeze the micro you can preserve the micro, that is what scientists do!! Lyophilization – frozen samples dried in vacuum — Slow freezing – more harmful RADIATION Ionizing radiation — Short wavelength, high energy — Emitted by radioactive elements (Co) — Mechanism of action: ionization of water which forms hydroxyl radicals which react with DNA — Used for sterilization of Medical supplies (plastic syringes, Petri plates etc.) Certain food (spices, meat, vegetables) Non-ionizing radiation — UV light, germicidal light used for disinfection — Mechanism of action: damage of DNA (forms thymine dimmers) & Toxic free radicals are formed — Sterilization of the air (hospital rooms, operating rooms, cafeteria) — Disadvantage: Poor penetration & Harmful for human eyes, skin FILTRATION — Removal of microbes from a solution — Membrane filters: pore size 0.2 or 0.45 um) OSMOTIC PRESSURE — High concentration of salt causes water to leave the cell — Used in preservation of food Chemical methods of microbial control Effectiveness of the disinfectant depends on:

Type of the chemical agent Type of microbes Concentration of a disinfectant

Time of contact pH of the medium Temperature Types of Disinfectants Halogens: Fluorine, bromine chlorine, and iodine o o o o

Iodine is the oldest antiseptic (skin disinfection) Chlorine: gas (Ca-hypochlorite; Na-hypochlorite- bleach) Mode of action: oxidizing agent - alters cellular components Disinfection of drinking water, swimming pools, household (bleach) Phenolics (derivatives of phenol)

o Used first time by Lister – carbolic acid o Mechanism of action: damages the plasma membrane, enzyme inactivation o Advantage: active even in the presence of organic compounds o Hexachlorophene (bisphenol) used in antimicrobial soaps Is antibacterial soap any better than regular soap? The antibacterial components of soaps need to be left on a surface for about two minutes in order to work. ALCOHOLS Ethanol or isopropanol 60% - 95% o Kills vegetative cells of bacteria and fungi (not spores and non-envelope viruses) o Mechanism of action: protein denaturation Is pure ethanol a better disinfectant than 70% ethanol? Why? 100% ethanol coagulates proteins in the cell wall 70% ethanol penetrates the cell wall and coagulates the proteins inside the cell HYDROGEN PEROXIDE 3% solution used as an antiseptic o Skin and wound cleansing o Mouthwash o Contact lens o Surgical implants o Endoscopes Chemicals with surface action: Detergents / Soaps Detergents are polar molecules - surfactants — Decrease the surface tension among molecules and water — Soaps and Detergents are not antiseptics: they break the oily film on the surface of skin — They have microbicidal power when mixed with quaternary ammonium compounds Heavy metals: Silver, mercury, copper, gold, arsenic o o o o o

Only mercury and silver have germicidal significance Mechanism of action: ions combine with sulfhydril groups - protein denaturation 1% Silver nitrate - antiseptic Copper sulfate - controls algal growth Can be toxic to humans

Evaluation of a disinfectant Filter paper method: -Paper disks are soaked in a solution of disinfectant and placed on a agar previously inoculated with a test organism -Observe the inhibition zone around the disk Aldehydes (formaldehyde, glutaraldehyde) Most effective antimicrobials Formalin: used for preservation of biological specimens – High level disinfectant – Toxic - carcinogenic Glutaraldehyde: Used for disinfection of hospital instruments – Mode of action: forms covalent cross-links with functional groups of proteins – Kills bacterial spores, fungal spores and viruses

Chapter #12 Antimicrobial Drugs Chemotherapy: Chemical substances used for treatment of infectious diseases Antibiotic: Substance produced by one microorganism that is inhibitory or toxic against other microorganism o o o o

Antimicrobial drugs have selective toxicity (harmful against microbes and not the host) Penicillin was the first antibiotic discovered by Alexander Fleming in 1928. Mold – Penicillium notatum. Its commercial use started in 1945 New antibiotics are being discovered by screening large number of microbes (400,000 screened; 3 useful drugs)

Spectrum of Antibacterial Activity An antibiotic can be effective against a – Narrow group of microbes (Penicillin G – against Gram positive bacteria) – Broad-spectrum antibiotic disadvantage- eliminates also the normal microflora Modes of antimicrobial action Inhibition of: – Cell wall synthesis – Protein synthesis – Cytoplasmic membrane synthesis

– Synthesis of essential metabolites – DNA synthesis Inhibitors of cell wall synthesis Penicillins and cephalosporins interfere with the formation of peptidoglycan layer in the cell wall of bacteria = Bactericidal. • Block the enzymes that cross-link N-acetyl glucosamin and N-acetyl muramic acid • Less effective against Gr negative bacteria, do not...