Test 3 Flashcards
-Nutrition
- The Metabolism of Microbes
- > Metabolism
- > Two types of reactions: Catabolism and Anabolism
-Building Blocks and Energy
-Metabolism – ‘change’ pertaining to all chemical and physical
workings of a cell
Two types of chemical reactions:
–>Catabolism – degradative; breaks the bonds of larger molecules
forming smaller molecules; releases energy
–>Anabolism – biosynthesis; process that forms larger
macromolecules from smaller molecules; requires energy input
- Enzymes as builders: anabolic rxns – Synthesis/Condensation Reactions
- > How much water is released per each bond formed
-Enzymes as recyclers: catabolic rxns
- Sensitivity of Enzymes to Their Environment
- -> Labile vs denaturation
• Synthesis or condensation reactions – anabolic reactions to form covalent bonds between smaller substrate molecules, require ATP, release one molecule of water for each bond formed
• Hydrolysis reactions – catabolic reactions that break down substrates into small molecules; requires the input of water to break bonds
– Energy released can be harvested and used elsewhere
• Activity of an enzyme is influenced by the cell’s environment
–> Enzymes operate under temperature, pH, and osmotic pressure of organism’s habitat
• When enzymes are subjected to changes in organism’s habitat they become unstable
– Labile: chemically unstable enzymes
– Denaturation: weak bonds that maintain the shape of the apoenzyme are broken
- Enzymes
- > What are they? - > How do they promote a reaction?
- > Is it permanently altered?
- Enzyme Structure
- > Simple enzyme
- > Holoenzyme
- > Apoenzyme
- > Cofactors (Metallic and coenzymes)
- Apoenzymes: Specificity and the Active Site
- > Induced Fit
- Enzymes are biological catalysts that increase the rate of a chemical reaction by lowering the energy of activation (the resistance to a reaction)
- The enzyme is not permanently altered in the reaction
- Enzyme promotes a reaction by serving as a physical site for specific substrate molecules to position
• Simple enzymes – consist of protein alone
• Conjugated enzymes or holoenzymes – contain protein and nonprotein molecules
– Apoenzyme – protein portion
– Cofactors – nonprotein portion
• Metallic cofactors: iron, copper, magnesium
• Coenzymes, organic molecules: vitamins
-Apoenzyme: Exhibits primary, secondary, tertiary, and some, quaternary structure
• Induced Fit: Site for substrate binding is active site, or catalytic site
A temporary enzyme-substrate union occurs when substrate
moves into active site
-Anaerobic Respiration
- Fermentation
- -> Alcholic and Acidic Fermentation
- Photosynthesis: The Earth’s Lifeline
- -> Light-dependent and Light-independent
• Anaerobic Respiration: Functions like aerobic respiration except it utilizes oxygen containing ions, rather than free oxygen, as the final electron acceptor
– Nitrate (NO3-) and nitrite (NO2-)
• Most obligate anaerobes use the H+ generated during glycolysis and the Kreb’s cycle to reduce some compound other than O2
• Fermentation: Incomplete oxidation of glucose or other carbohydrates in the
absence of oxygen
• Uses organic compounds as terminal electron acceptors
• Yields a small amount of ATP
• Production of ethyl alcohol by yeasts acting on glucose
• Formation of acid, gas, and other products by the action of various bacteria on pyruvic acid
-Alcoholic Fermentation: yeast –> produce ethyl alcohol
-Acidic Fermentation: humans and homolactic bacteria –> produce lactic acid
-Pyruvate Fermentation: mixed acids - escherichia and shigella
-Photosynthesis: The ultimate source of all the chemical energy in cells comes from the sun (occurs in 2 stages)
** 6CO2 + 6H2O –> C6H12O6 + 6O2 (by way of light) **
• Light-dependent – photons are absorbed by chlorophyll, carotenoid, and phycobilin pigments
– Water split by photolysis, releasing O2 gas and provides electrons to drive photophosphorylation
– Released light energy used to synthesize ATP and NADPH
• Light-independent reaction – dark reactions – Calvin cycle – uses ATP to fix CO2 to ribulose- 1,5-bisphosphate and convert it to glucose
- Metabolic Statregies: Aerobic, Anaerobic, and Fermentation
- Aerobic Respiration: Glycolysis, TCA, ETC and Oxidative Phosphorylation
- The Formation of ATP and Chemiosmosis
- The Terminal Step
- Theoretic ATP Yield for Aerobic Respiration
- Strategies: Nutrient processing is varied, yet in many cases is based on three catabolic pathways that convert glucose to CO2 and gives off energy
- Aerobic respiration – glycolysis, the Kreb’s cycle, respiratory chain
- Anaerobic respiration – glycolysis, the Kreb’s cycle, respiratory chain; molecular oxygen is not the final electron acceptor
- Fermentation – glycolysis, organic compounds are the final electron acceptors
- Aerobic: Series or enzyme-catalyzed reactions in which electrons are transferred from fuel molecules (glucose) to oxygen as a final electron acceptor
- Glycolysis – glucose (6C) is oxidized and split into 2 molecules of pyruvic acid (3C), NADH is generated
- TCA – processes pyruvic acid and generates 3 CO2 molecules , NADH and FADH2 are generated
- Electron transport chain – accepts electrons from NADH and FADH; generates energy through sequential redox reactions called oxidative phosphorylation
- Glycolysis: Takes place in the cytoplasm of both prokaryotes and eukaryotes
- -> Net result: 2 ATP, 2 NADH, 2 pyruvic acid
-Electron Transport and Oxidative Phosphorylation: Final processing of electrons and hydrogen and the major generator of ATP
• Location: mitochondria of eukaryotes or cell membrane of prokaryotes
• Chain of redox carriers that receive electrons from reduced carriers (NADH and FADH2)
• ETC shuttles electrons down the chain, energy is released and subsequently captured and used by ATP synthase complexes to produce ATP – Oxidative phosphorylation
• Chemiosmosis – as the electron transport carriers shuttle electrons, they
actively pump hydrogen ions (protons) across the membrane setting up a gradient of hydrogen ions – proton motive force
• Hydrogen ions diffuse back through the ATP synthase complex causing it to rotate, causing a 3-D change resulting in the production of ATP
-The Terminal Step: • Oxygen accepts 2 electrons from the ETC and then picks up 2 hydrogen ions from the solution to form a molecule of water. Oxygen is the final electron acceptor
– Cytochrome oxidase – useful in bacterial identification
** 2H+ + 2e- + 1⁄2O2 → H2O **
• Theoretical Yield: Each NADH can produce 3 ATP, Each FADH2 can produce 2 ATP
- Biological Oxidation and Reduction
- -> what does the process salvage?
- Electron and Proton Carriers
- Adenosine Triphosphate: ATP
- Formation of ATP (3 ways)
- -> Substrate-level, oxidative, photophosphorylation
- Redox reactions – always occur in pairs
- There is an electron donor and electron acceptor which constitute a redox pair
- Process salvages electrons and their energy
- Released energy can be captured to phosphorylate ADP or another compound
• Electron and Proton Carriers: Repeatedly accept and release electrons and
hydrogen to facilitate the transfer of redox energy
• Most carriers are coenzymes: NAD, FAD, NADP, coenzyme A, and compounds of the respiratory chain
•ATP: Metabolic “currency”
• Three part molecule consisting of:
– Adenine – a nitrogenous base
– Ribose – a 5-carbon sugar
– 3 phosphate groups
• Removal of the terminal phosphate releases energy
• ATP utilization and replenishment is a constant cycle in active cells
- ATP can be formed by three different mechanisms:
1. Substrate-level phosphorylation – transfer of phosphate group from a phosphorylated compound (substrate) directly to ADP
2. Oxidative phosphorylation – series of redox reactions occurring during respiratory pathway
3. Photophosphorylation – ATP is formed utilizing the energy of sunlight
- Overview of Enzyme Characteristics
- Metabolic Pathways
- What is energy?
- The Pursuit and Utilization of Energy
- Cell Energetics
- -> Endergonic vs exergonic
-Composed mostly of protein (may require nonprotein cofactors), act as organic catalysts to speed up rate of reactions, lowers the activation energy required for a chemical reaction to proceed, enable metabolic reactions to proceed at a speed compatible with life, have unique characteristics such as shape, specificity, and function, provide an active site for substrates, are much larger size than substrates, do not become integrated into reaction products, are not used up/permanently changed, can be recycled and function in extremely low concentrations, are greatly affected by temp and pH, can be regulated by feedback and genetic mechanisms
- Many tasks within the cell require multiple steps.
- Each step is catalyzed by a different enzyme
- Metabolic pathway - The sum of the steps from starting point through finished products
- Energy: the capacity to do work or to cause change
- Forms of energy include – Thermal, radiant, electrical, mechanical, atomic, and chemical
-Energetics: Cells manage energy in the form of chemical reactions that make or break bonds and transfer electrons
-Endergonic reactions – consume energy (Anabolic)
Energy + A + B –> C (by way of enzyme)
• Exergonic reactions – release energy (catabolic)
X + Y –> Z + Energy (by way of enzyme)
• Energy released is temporarily stored in high energy phosphate molecules. The energy of these molecules is used in endergonic cell reactions.
- Enzymes as builders
- -> Anabolic Rxns
- -> Catabolic Rxns
- Senzitivity of Enzymes to Their Environment
- -> Labile vs Denaturation
- Regularity of Enzyme Action
- -> Constitutive Enzymes vs Regulated Enzymes
- Regulation of Enzyme Activity
- -> Genetic Control : Enzyme repression vs enzyme induction
- -> Direct Control : Competitive inhibition vs noncompetitive inhibition
- Anabolic: Synthesis or condensation reactions
– anabolic reactions to form covalent bonds between smaller substrate molecules, require ATP, release one molecule of water for each bond formed
-Catabolic: Hydrolysis reactions – catabolic reactions that break down substrates into small molecules; requires the input of water to
break bonds – Energy released can be harvested and used elsewhere
-Sensitivity: Activity of an enzyme is influenced by the cell’s environment
– Enzymes operate under temperature, pH, and osmotic pressure of organism’s habitat
• When enzymes are subjected to changes in organism’s habitat they become unstable
– Labile: chemically unstable enzymes
– Denaturation: weak bonds that maintain the shape of the apoenzyme are broken
- Constitutive enzymes – always present, always produced in equal amounts or at equal rates, regardless of the amount of substrate
- Regulated enzymes – not constantly present; production is turned on (induced) or turned off (repressed) in response to changes in the substrate concentration
Genetic control of Enzyme activity: Enzyme repression – inhibits at the genetic level by controlling synthesis of key enzymes
• Enzyme induction – enzymes are made only when suitable substrates are present
Direct control of Enzyme activity: Competitive inhibition – substance that resembles the normal substrate competes with the substrate for the active site
• Noncompetitive inhibition – enzymes are regulated by the binding of molecules other than the substrate away from the active site
– Allosteric inhibition
-Microbial Metabolism: Concept Summary
• Cells need to build macromolecules and break these down….dynamic process
– Enzymes are catalytic proteins that make these reactions go faster
• Enzymes required for life. Sensitive to environment. If you do screw up essential enzymes, the cell will die.
• Cell functions require energy. Potential energy stored in organic molecules (i.e. glucose)
– The breakdown of glucose (or other molecules) releases energy that is harvested and stored as ATP
– Reactions that carry this out: Glycolysis, Kreb’s cycle, electron transport
– Energy harvesting processes, involve the transfer of electrons
• Aerobic respiration
– O2 serves as final acceptor of electron. Splits to form water.
• Sometimes this results in formation of oxygen radical species.
– CO2 is released during process. Carbons are the remnants of glucose that started at the beginning.
• Fermentation: In the absence of oxygen, some aerobic organisms can function entirely on glycolysis
– Requires the recycling of NADH– doing so results in the production and accumulation of byproducts: Acid or alcohol and sometimes CO2
• Photosynthesis:
– Energy from light used to make ATP (light reactions).
– ATP used to construct sugars from CO2 (dark reactions/Calvin cycle)
-Microbial Genetics
- Genetics and Genes
- -> Three categories of genes
- Levels of Structure and Function of the Genome
- -> Genome of cells vs viruses
- Chromosomes and Genes
- Genotypes and Phenotypes
• All living things + viruses have genomes
• The genome defines what an organism is
– How/where a microbe can grow
– Whether it is pathogenic, antibiotic resistant
• Gene expression, genome maintenance, and genetic transfer is fundamental to understanding microbes
– Vaccines, antibiotic resistance, environmental habitats, microbial control strategies
Genetics – the study of heredity
The science of genetics explores:
1. Transmission of biological traits from parent to offspring
2. Expression and variation of those traits
3. Structure and function of genetic material
4. How this material changes
• Genome – sum total of genetic material of a cell (chromosomes + mitochondria/chloroplasts and/or plasmids)
– Genome of cells – DNA
– Genome of viruses – DNA or RNA
• DNA complexed with protein constitutes the genetic material as chromosomes
• Gene - the fundamental unit of heredity responsible for a given trait
– Three basic categories of genes:
1. Genes that code for proteins – structural genes
2. Genes that code for RNA
3. Genes that control gene expression – regulatory genes
- All types of genes constitute the genetic makeup – genotype
- The expression of the genotype creates observable traits – phenotype
- Genomes Vary in Size
- -> Smallest virus
- -> E. coli
- -> Human cell
-DNA
–> Basic unit
–> Parts of DNA
–>Shape?
How many bonds for A-T? G-C?
-The Overall Replication Process
- DNA Replication
- -> DNA Pol I
- -> DNA Pol III
- -> Primase
- -> Helicase?
- -> Leading vs Lagging strand?
• Smallest virus – 4-5 genes
• E. coli – single chromosome containing 4,288 genes; 1 mm; 1,000X
longer than cell
• Human cell – 46 chromosomes containing 31,000 genes; 6 feet; 180,000X longer than cell
• Basic unit of DNA structure is a nucleotide
• Each nucleotide consists of 3 parts:
– A 5 carbon sugar – deoxyribose
– A phosphate group
– A nitrogenous base – adenine, guanine, thymine, cytosine
• Nucleotides covalently bond to form a sugar-phosphate backbone
– Each sugar attaches to two phosphates – 5′ carbon and 3′ carbon
• Two strands twisted into a double helix
• Nitrogenous bases of one strand span the center of the molecule to
pair with an appropriate complementary base on the other
strand – Adenine binds to thymine with 2 hydrogen bonds
– Guanine binds to cytosine with 3 hydrogen bonds
• Antiparallel strands 3′ to 5′ and 5′ to 3′
• Order of bases constitutes the DNA code
- Each strand provides a template for the exact copying of a new strand
- Replication occurs on both strands simultaneously
- Creates complementary strands
- Semiconservative replication process
• Begins at an origin of replication
• Helicase unwinds and unzips the DNA double helix
• An RNA primer is synthesized at the origin of replication by Primase
• DNA polymerase III adds nucleotides in a 5′ to 3′ direction
– Leading strand – synthesized continuously in 5′ to 3′ direction
– Lagging strand – synthesized 5′ to 3′ in short segments; overall direction is 3′ to 5′
• DNA polymerase I removes the RNA primers and replaces them with DNA
-Overall Bacterial Replication
- Helicase
- Primase
- DNA Pol III
- DNA Pol I
- Ligase
- Gyrase
-Application of the DNA code
1) The chromosome to be replicated is unwound by a helices, forming a replication fork with two template strands
2) This template for the leading strand is 3’ to 5’, which allows DNA pol III to add nucleotides in the 5’ to 3’ direction toward the replication fork –> synthesized as the continuous strand.
3) The template for the lagging strand runs 5’ to 3’ (opposite tot he leading strand) so to make the new stand in the 5’ to 3’ orientation, synthesis must proceed backward away from the replication fork
4) Before synthesis of the lagging stand can start, a primate adds an RNA primer to direct the DNA pol III, synthesis produces unlinked segments of RNA primer and new DNA called Okazaki fragments
5) DNA pol I removes the RNA primers and fills in the correct complementary DNA nucleotides at the open sites
6) Unjoined ends of the nucleotides (a nick) must be connected by a ligase
- Helicase: Unzipping the DNA helix
- Primase: Synthesizing an RNA primer
- DNA Pol III: Adding bases to the new DNA chain; proofreading the chain for mistakes
- DNA Pol I: Removing RNA primers, replacing gaps between Okazaki fragments with correct nucleotides, repairing mismatched bases
- Ligase: Final binding of nicks in DNA during synthesis and repair
- Gyrase: Supercoiling
- Application of the DNA code: Information stored on the DNA molecule is conveyed to RNA molecules through the process of transcription
- The information contained in the RNA molecule is then used to produce proteins in the process of translation
- RNAs
- Structure?
- ->mRNA
- ->tRNA
- What structure is important?
- What does the anticodon complement?
- ->rRNA
• Single-stranded molecule made of nucleotides
– 5 carbon sugar is ribose
– 4 nitrogen bases – adenine, uracil, guanine, cytosine
– Phosphate
- mRNA: carries DNA message through complementary copy; message is in triplets called codons
- tRNA: made from DNA; secondary structure creates loops; bottom loop exposes a triplet of nucleotides called anticodon which designates specificity and complements mRNA; carries specific amino acids to ribosomes
- rRNA: component of ribosomes where protein synthesis occurs
- Transcription: The First Stage of Gene Expression
- -> What steps (4)
- -> Approximately how long is the transcript
-Translation: The Second Stage of Gene Expression
- Gene-Protein Connection
- > Primary sequence
- > redundancy
- > How many amino acids and how many combinations?
- RNA polymerase binds to promoter region upstream of the gene
- RNA polymerase adds nucleotides complementary to the template strand of a segment of DNA in the 5′ to 3′ direction
- Uracil is placed as adenine’s complement
- At termination, RNA polymerase recognizes signals and releases the transcript
▪ 100-1,200 bases long
1) Initiation: Promoter Binding
2) RNA pol adds nucleotides 5’ –> 3’
3) Termination: release of transcript
LOOK AT THE CHART
• All the elements needed to synthesize protein are brought together on the ribosomes • The process occurs in five stages: – initiation – elongation – termination – protein folding – post-translational processing
- DNA and RNA composed of 4 letters: ATCG or AUGC
- Each 3 nucleotide sequence (triplet) codes for 1 amino acid
- 20 amino acids, 64 triplet combinations
- degeneracy
- Primary sequence defines the protein identity, and resulting secondary and tertiary structure
- The Master Genetic Code
- Interpreting the DNA Code
- Translation
- -> What are the 3 stop codons?
- Polyribosomal Complex
- Splicing of Eukaryotic pre-mRNA
- The Master Genetic Code: Represented by the mRNA codons and the amino acids they specify
- Code is universal among organisms
- Code is redundant
- Transcription produces an mRNA complementary to the DNA gene
- During translation, tRNAs use their anticodon to interpret the mRNA codons and bring in the amino acids
-Translation: Ribosomes assemble on the 5′ end of an mRNA transcript
• Ribosome scans the mRNA until it reaches the start codon, usually AUG
• A tRNA molecule with the complementary anticodon and methionine amino acid enters the P site of the ribosome and binds to the mRNA
– Process repeats along transcript
– Stop codon releases peptide
• A second tRNA with the complementary anticodon fills the A site
• A peptide bond is formed is formed between the amino acids on the neighboring tRNAs
• Termination codons – UAA, UAG, and UGA – are codons for which there is no corresponding tRNA
• When this codon is reached, the ribosome falls off and the last tRNA is removed from the polypeptide
• Polyribosomal complex allows for the synthesis of many protein molecules simultaneously from the same mRNA molecule.
-Splicing: • Removal of introns and connection of exons
• Does not occur in Prokaryotes
- Regulation of Protein Synthesis and Metabolism
- > What are operons?
- ->Inducible vs repressible operons?
- > catabolic vs anabolic operon?
-3 parts of operon: regulator, control locus, and structural locus?
- Lactose Operon: Inducible Operon
- -> b-galactosidase, permease, transacetylase
- Lactose Operon: Inducible Operon
- Arginine Operon: Repressible
• Genes are regulated to be active only when their products are required
• In prokaryotes this regulation is coordinated by operons, a set of genes, all of which are regulated as a single unit
– Inducible – operon is turned ON by substrate: catabolic operons - enzymes needed to metabolize a nutrient are produced when needed
– Repressible – genes in a series are turned OFF by the product synthesized; anabolic operon –enzymes used to synthesize an amino acid stop being produced when they are not needed
Made of 3 segments:
1. Regulator – gene that codes for repressor
2. Control locus – composed of promoter and operator
3. Structural locus – made of 3 genes each coding for an enzyme needed to catabolize lactose –
b-galactosidase – hydrolyzes lactose
permease – brings lactose across cell membrane
b-galactosidase transacetylase – uncertain function
-Operon: Lactose must be enzymatically broken down prior to oxidative phosphorylation
•Lac Operon: Normally off
– In the absence of lactose, the repressor binds with the operator locus and blocks transcription of downstream structural genes
• Lactose turns the operon on by acting as the inducer
– Binding of lactose to the repressor protein changes its shape and causes it to fall off the operator. RNA polymerase can bind to the promoter. Structural genes are transcribed.
-Arginine: Normally on and will be turned off when the product of the pathway is no longer required
• When excess arginine is present, it binds to the repressor and changes it. Then the repressor binds to the operator and blocks arginine synthesis. Arginine is the corepressor.
- Mutations: Changes in the Genetic Code
- Causes of Mutations: Spontaneous vs induced
- Categories of Mutations
- ->Point
- ->Missense
- ->Nonsense
- ->Silent
- ->Back
- ->Frameshift
- The Master Genetic Code
- -> Represent by what?
- ->Is it universal?
- ->Is it redundant?
-Mutations: A change in phenotype due to a change in genotype (nitrogen base sequence of DNA) is called a mutation
• A natural, nonmutated characteristic is known as a wild type (wild strain)
• An organism that has a mutation is a mutant strain, showing variance in morphology, nutritional characteristics, genetic control mechanisms, resistance to chemicals, etc.
• Mutations can be spontaneous or induced by exposure to mutagenic chemicals (mutagens)
-Causes: Spontaneous mutations – random change in the DNA due to errors in replication that occur without known cause
• Induced mutations – result from exposure to known mutagens, physical (primarily radiation) or chemical agents that interact with DNA in a disruptive manner
- Point mutation – addition, deletion, or substitution of a single base
- Missense mutation – causes change in a single amino acid
- Nonsense mutation – changes a normal codon into a stop codon
- Silent mutation – alters a base but does not change the amino acid
- Back-mutation – when a mutated gene reverses to its original base composition
- Frameshift mutation – when the reading frame of the mRNA is altered
- Represented by the mRNA codons and the amino acids they specify
- Code is universal among organisms
- Code is redundant
- Consequences of mutations
- ->Protein Truncations
- -> Inactive Proteins
- ->Super Active Proteins
- Repair of Mutations
- -> DNA Pol
- -> Mismatch Repair
- -> Light Repair
- -> Excision Repair
-The Ames Test
- Positive and Negative Effects of Mutations
- -> What mutations are the basis of change in a population?
• Consequences: Protein misfolds – protein does not adopt proper structure and is inactive for intended function
• Protein truncations
– incomplete protein
• Inactive protein
– enzyme no longer capable of catalyzing reaction
– structural protein not correct shape, less stable, etc
• Super active protein
– Enzyme regulation disrupted. Enzyme always on
• Since mutations can be potentially fatal, the cell has several enzymatic repair mechanisms in place to find and repair damaged DNA
– DNA polymerase – proofreads nucleotides during DNA replication
– Mismatch repair – locates and repairs mismatched nitrogen bases that were not repaired by DNA polymerase
– Light repair – for UV light damage
– Excision repair – locates and repairs incorrect sequence by removing a segment of the DNA and then adding the correct nucleotides
- Ames Test: Any chemical capable of mutating bacterial DNA can similarly mutate mammalian DNA
- Agricultural, industrial, and medicinal compounds are screened using the Ames test
- Indicator organism is a mutant strain of Salmonella typhimurium that has lost the ability to synthesize histidine
- This mutation is highly susceptible to back-mutation
- Mutations leading to nonfunctional proteins are harmful, possibly fatal
- Organisms with mutations that are beneficial in their environment can readily adapt, survive, and reproduce – these mutations are the basis of change in populations
- Any change that confers an advantage during selection pressure will be retained by the population
-DNA Recombination Events
- Conjugation
- -> What is the recipient cell?
- Transformation vs High frequency recombination
- Vibrio cholera
- Genetic recombination – occurs when an organism acquires and expresses genes that originated in another organism
- > 3 means for genetic recombination in bacteria:
1. Conjugation
2. Transformation
3. Transduction
• Conjugation – transfer of a plasmid or chromosomal fragment from a donor cell to a recipient cell via a direct connection
– Gram-negative cell donor has a fertility plasmid (F plasmid, F′ factor) that allows the synthesis of a conjugative pilus
– Recipient cell is a related species or genus without a fertility plasmid
– Donor transfers fertility plasmid through pilus
• High-frequency recombination – donor’s fertility plasmid has been integrated into the bacterial chromosome – When conjugation occurs, a portion of the chromosome and a portion of the fertility plasmid are transferred to the recipient
• Transformation – chromosome fragments from a lysed cell are
accepted by a recipient cell; the genetic code of the DNA fragment is acquired by the recipient
• Donor and recipient cells can be unrelated
• Useful tool in recombinant DNA technology
-Vibrio cholera in search of DNA: Type IV pilus involved in natural DNA transformation
- Transduction
- -> Generalized vs Specialized vs Lateral
-Transposons
•Transduction – bacteriophage serves as a carrier of DNA from a donor cell to a recipient cell
• Two types:
– Generalized transduction – random fragments of disintegrating host DNA are picked up by the phage during assembly; any gene can be transmitted this way
– Specialized transduction – a highly specific part of the host genome is regularly incorporated into the virus
–> Generalized: Phage inserts viral DNA into bacterium –> enzymes break down host DNA and copy viral DNA –> new phages occasionally encapsulate some ghost DNA –> phage inserts bacterial DNA into a new host
–> Viral DNA splices into host DNA –> then activated, viral DNA is cut out and covid, sometimes with a snip host of DNA –> new phages encapsulate the excised DNA –> viral and bacterial genes enter a new host
–> Lateral: Viral DNA splices into host DNA –> Viral DNA replicates with adjacent host DNA –> Replicated DNA fills all the new phages –> Phage inserts bacterial DNA into a new host
- Special DNA segments that have the capability of moving from one location in the genome to another – “jumping genes”
- Cause rearrangement of the genetic material
- Can move from one chromosome site to another, from a chromosome to a plasmid, or from a plasmid to a chromosome
- May be beneficial or harmful
-Practical Concerns in Microbial Control
- Antimicrobial Agents’ Modes of Action (aka cellular targets)
- -> Cell wall
- -> Cell membrane’
- -> Protein and Nucleic Acid Synthesis
- -> Proteins
- Methods of Control (Physical)
- Thermal Death Measurements (Time and Point)
- Moist Heat Methods
- Nonpressurized Steam
- Boiling Water
- Pasteurization
- Dry Heat
- Cold
- Desiccation
- Radiation (ionizing vs nonionizing)
- Application of Ionizing Radiation
- Application of Nonionizing Radiation
–> What leads to coagulation of proteins and what leads to denaturation?
Selection of method of control depends on circumstances:
– Does the application require sterilization?
– Is the item to be reused?
– Can the item withstand heat, pressure, radiation, or chemicals?
– Is the method suitable?
– Will the agent penetrate to the necessary extent?
– Is the method cost- and labor-efficient and is it safe?
Cellular targets of physical and chemical agents:
- The cell wall – cell wall becomes fragile and cell lyses; some antimicrobial drugs, detergents, and alcohol
- The cell membrane – loses integrity; detergent, surfactants
- Protein and nucleic acid synthesis – prevention of replication, transcription, translation, peptide bond formation, protein synthesis; chloramphenicol, ultraviolet radiation, formaldehyde
- Proteins – disrupt or denature proteins; alcohols, phenols, acids, heat
Physical Methods: 1. Heat – moist and dry • Moist heat – lower temperatures and shorter exposure time; coagulation and denaturation of proteins • Dry heat – moderate to high temperatures; dehydration, alters protein structure; incineration 2. Cold temperatures 3. Desiccation 4. Radiation 5. Filtration
- Thermal death time (TDT) – shortest length of time required to kill all test microbes at a specified temperature
- Thermal death point (TDP) – lowest temperature required to kill all microbes in a sample in 10 minutes
-Moist Heat Methods:
• Steam under pressure – sterilization
• Autoclave 15 psi/121oC/10-40min
• Steam must reach surface of item being sterilized
• Item must not be heat or moisture sensitive
• Mode of action – denaturation of proteins, destruction of membranes and DNA
- Nonpressurized Steam: Tyndallization – intermittent sterilization for substances that cannot withstand autoclaving
- Items exposed to free-flowing steam for 30–60 minutes, incubated for 23 24 hours and then subjected to steam again
- Repeat cycle for 3 days
- Used for some canned foods and laboratory media
- Disinfectant
• Boiling at 100oC for 30 minutes to destroy non-spore-forming pathogens (Disinfection)
- Pasteurization – heat is applied to kill potential agents of infection and spoilage without destroying the food flavor or value
- 63°C–66°C for 30 minutes (batch method)
- 71.6°C for 15 seconds (flash method)
- Not sterilization – kills non-spore-forming pathogens and lowers overall microbe count; does not kill endospores or many nonpathogenic microbes
-Dry heat using higher temperatures than moist heat
• Incineration – flame or electric heating coil
– Ignites and reduces microbes and other substances
• Dry ovens – 150–180oC – coagulate proteins
- Cold: Microbiostatic – slows the growth of microbes
- Refrigeration 0–15oC and freezing <0oC
- Used to preserve food, media, and cultures
• Gradual removal of water from cells, leads to metabolic inhibition
– desiccation = drying
• Not effective microbial control – many cells retain ability to grow when water is reintroduced
• Lyophilization – freeze drying; preservation
• Ionizing radiation – deep penetrating power that has sufficient energy to cause electrons to leave their orbit, breaks DNA
– Gamma rays, X- rays, cathode rays
– Used to sterilize medical supplies and food products
• Nonionizing radiation – little penetrating power so it must be directly exposed
– UV light creates pyrimidine dimers, which interfere with replication
- Ionizining: Preserving food with ionizing radiation
- Nonionizing: Sterilizing air, water or surfaces
- Physical and Chemical Agents for Microbial Control
- -> Controlling Microorganisms
-Relative Resistance of Microbes (Highest, Moderate (4), Lowest (4))
- Terminology and Methods of Control
- -> Sterilization
- -> Disinfection
- -> Antiseptic
- -> Sanitization
- -> Degermation
- Microbial Death
- -> Microbicidal vs microbistatic?
-Factors That Affect Death Rate
• Physical, chemical, and mechanical methods to destroy or reduce undesirable microbes in a given area (decontamination)
• Primary targets are microorganisms capable of causing infection or spoilage:
– Vegetative bacterial cells and endospores
– Fungal hyphae and spores, yeast
– Protozoan trophozoites and cysts
– Worms
– Viruses
– Prions
• Highest resistance – Prions, bacterial endospores • Moderate resistance – Pseudomonas sp. – Mycobacterium tuberculosis – Staphylococcus aureus – Protozoan cysts • Least resistance – Most bacterial vegetative cells – Fungal spores and hyphae, yeast – Enveloped viruses – Protozoan trophozoites
- Sterilization – a process that destroys all viable microbes, including viruses and endospores
- Disinfection – a process to destroy vegetative pathogens, not endospores; inanimate objects
- Antiseptic – disinfectants applied directly to exposed body surfaces
- Sanitization – any cleansing technique that mechanically removes microbes from inanimate objects
- Degermation – reduces the number of microbes through mechanical means from body surfaces
• Hard to detect, microbes often reveal no conspicuous vital signs to begin with
• Microbial Death: Permanent loss of reproductive capability, even under optimum growth conditions
– Microbicidal – agent causes death of microbe
– Microbistatic – agent prevents microbe from growing, but does not kill it.
-Factors That Affect Death Rate: • Number of microbes • Nature of microbes in the population • Temperature and pH of environment • Concentration or dosage of agent • Mode of action of the agent • Presence of solvents, organic matter, or inhibitors
- Filtration
- Chemical Methods of Control
- Desirable Qualities of Chemicals
- -> High or low concentration?
- -> Solubility in what?
- -> Broad or narrow spectrum?
- Levels of Chemical Decontamination (High, Intermediate, and Low)
- Factors that Affect Germicidal Activity of Chemicals
- Germicidal Categories
- Filtration: Physical removal of microbes by passing a gas or liquid through filter
- Used to sterilize heat sensitive liquids and air in hospital isolation units and industrial clean rooms
-Chemical Methods: – Disinfectants – antiseptics – sterilants – degermers – preservatives
-Desirable qualities of chemicals: • Rapid action in low concentration • Solubility in water or alcohol, stable • Broad spectrum, low toxicity • Penetrating • Noncorrosive and nonstaining • Affordable and readily available
• High-level germicides – kill endospores; may be sterilants
– Devices that are not heat sterilizable and intended to be used in sterile environments (body tissue)
• Intermediate-level – kill fungal spores (not endospores), tubercle bacillus, and viruses
– Used to disinfect devices that will come in contact with mucous membranes but are not invasive
• Low-level – eliminate only vegetative bacteria, vegetative fungal cells, and some viruses
– Clean surfaces that touch skin but not mucous membranes
-Factors: • Nature of the material being treated • Degree of contamination • Time of exposure • Strength and chemical action of the germicide
- Germicidal Categories:
1. Halogens
2. Phenolics
3. Chlorhexidine
4. Alcohols
5. Hydrogen peroxide
6. Aldehydes
7. Gases
8. Detergents & soaps
9. Heavy metals
10. Dyes
11. Acids and Alkalis
- Halogens
- -> Unstable in what?
- -> Inactivated in what?
- -> What does it do?
- -> What level?
- Phenolics
- -> What are they not?
- -> What does it do?
- -> What level?
- Chlorhexidine
- -> What outbreaks is it used to control?
- -> What level?
- -> What is it used as?
- -> Microbicidal or microbistatic?
- Alcohols
- -> What concentration is the best and why?
- -> What does it promote?
- -> What level is best?
- Hydrogen Peroxide
- -> Especially toxic to what?
- -> What does it do?
- -> What are strong solutions?
- Aldehydes
- -> What does it effect?
- -> What level?
- Gases and Aerosols
- -> What do they do?
- -> What level?
- Detergents and Soaps
- -> Cationic or anionic?
- -> What do they remove?
- -> What do soaps do?
–>What denatures proteins and what coagulates proteins?
• Halogens: Chlorine – Cl2 , hypochlorites (chlorine bleach), chloramines
– Denature proteins by disrupting disulfide bonds
– Intermediate level
– Unstable in sunlight, inactivated by organic matter
– Water, sewage, wastewater, inanimate objects
• Iodine - I2, iodophors (betadine)
– Denatures proteins by disrupting disulfide bonds
– Intermediate level
– Milder medical and dental degerming agents, disinfectants, ointments
• Phenolics: Disrupt cell walls and membranes and precipitate proteins
• Low to intermediate level
– bactericidal, fungicidal, virucidal, not sporicidal
– Lysol
– Triclosan – antibacterial additive to soaps
• Chlorhexidine: A surfactant and protein denaturant with broad microbicidal properties
• Low to intermediate level
• Hibiclens, Hibitane
• Used as an antiseptic
– in preoperative scrubs, skin cleaning, and burns
– in hospitals for handwashing
– Additive in cosmetic creams, toothpaste, eye drops, deordorants, mouthwash
• Antiseptic cleanser of choice to control Staphylococcus MRSA and Acinetobacter outbreaks in hospitals
- Alcohols: Ethyl, isopropyl in solutions of 50-95%
- Act as surfactants dissolving membrane lipids and coagulating proteins of vegetative bacterial cells and fungi
- Intermediate level
- Greatest microbicidal activity at 70% concentration because water is necessary to promote protein coagulation.
-Hydrogen Peroxide: Produce highly reactive hydroxyl-free radicals that damage protein and DNA while also decomposing to O2 gas – especially toxic to anaerobes
• Antiseptic at low concentrations; strong solutions are sporicidal – Bactericidal, viricidal, fungicidal, sporicidal
• Commonly used to sterilize medical equipment like endoscopes, and dental tools
- Aldehydes: Kill by alkylating protein and DNA – Glutaraldehyde in 2% solution (Cidex) used as sterilant for heat sensitive instruments
- High level – Formaldehyde – disinfectant, preservative, toxicity limits use
- Formalin – 37% aqueous solution
- Intermediate to high level
- Rapid and broad spectrum
- Gases and Aerosols: Ethylene oxide, propylene oxide
- Strong alkylating agents
- High level
- Sterilize and disinfect plastics and prepackaged devices, foods
- Quaternary ammonia compounds (quats) act as surfactants that alter membrane permeability of some bacteria and fungi – Cationic detergents
- Very low level
- Soaps – mechanically remove soil and grease containing microbes
- -> Denaturation: Halogens, Chlorhexidine
- -> Coagulation: Alcohols
- ->Phenolics causes protein precipitation
