Microbiology - Esiobu Exam 2

Amphibolic Pathways

"amphi" greek for both sides Metabolic pathways that function both catabolically and anabolically. i.e. • Embden-Meyerhof pathway • Pentose phosphate pathway • Tricarboxylic acid (TCA) cycle

Lithotrophs

"litho" rock, "trophs" eaters An organism that uses reduced inorganic compounds as its electron source.

Repressible genes

-Are on until repressed - involved in biosynthesis - anabolism •Enzymes that function in biosynthetic pathways are products of repressible genes •Generally these enzymes are always present unless the end product in the biosynthetic pathway is available

Exceptions to Genetic code

-Some protists use a single stop codon; the other two code for amino acids instead -Some microbes incorporate two rare amino acids into polypeptides •selenocysteine •pyrrolysine

Lipid Catabolism

-Triglycerides are common energy sources (glycerol core attached to 3 fatty acids) - can be hydrolyzed to glycerol and free fatty acids by lipases - glycerol degraded via the glycolytic pathway -fatty acids oxidized via the β-oxidation pathway typically used by chemoorganotrophic microorganisms

Constitutive genes

-are housekeeping genes that are expressed continuously by the cell

E. coli electron transport chain

-different cytochromes used than in mitochondrial branched pathway -upper branch, stationary phase and low aeration, less efficient -lower branch, log phase and high aeration, more efficient

tRNA and rRNA Genes

-genes coding for tRNA may code for more than a single tRNA molecule or type of tRNA -genes coding for rRNA are transcribed as single, large precursor -spacers between the coding regions of both are removed after transcription, some by the use of special ribonucleases called ribozymes

Codon

-genetic code word, 3 base pairs long -specifies an amino acid -anticodon on tRNA is complementary

Paracoccus denitrificans

-gram-negative, soil bacterium -facultative anaerobe can use several metabolic strategies under oxic conditions, uses aerobic respiration -similar electron carriers and transport mechanisms as mitochondria -protons transported to periplasmic space instead of the inner mitochondrial membrane -can use one carbon molecule instead of glucose

Metabolism regulation

-important for conservation of energy/materials -maintenance of metabolic balance despite changes in enviro 3 major mechanisms • metabolic channeling • regulation of synthesis of a particular enzyme (transcriptional/translational) • direct stimulation of inhibition of the activity of a critical enzyme -post translational

Electron Carriers

-located in plasma membranes of chemoorganotrophs in bacteria and archaeal cells -located in internal mitochondrial membranes of eukaryotic cells Cytochromes- use irons to transfer electrons (iron is part of a heme group) Nonheme iron-sulfur proteins- use iron to transport electrons (iron is not part of a heme group) i.e. NAD, NADP, FAD, FMN, CoQ (ubiquinone)

Energy sources

-many different energy sources are funneled into common degradative pathways -most pathways generate glucose or intermediates of the pathways used in glucose metabolism -few pathways greatly increase metabolic efficiency

Microbial death

-microorganisms are not killed instantly -population death usually occurs exponentially -measure of agent's killing efficiency --- decimal reduction time - time to kill 90% D value, minutes of exposure. i.e. Dsubscript80 = 2 means that the decimal reduction time at 80 degrees Celsius, is 2 minutes.

Protein secretion in bacteria

-movement of proteins from the cytoplasm to external environment •hydrolytic enzymes for nutrient break down •Sec-dependent pathway -the major pathway for all bacteria for transporting proteins across the plasma membrane •Gram-negative bacteria -may use Sec system -also must cross the outer membrane using Types I, II, III, IV, V systems •All pathways require energy

Chlorine

-oxidizes cell constituents -important in disinfection of water supplies and swimming pools, used in dairy/food industries, effective household disinfectant -destroys vegetative bacteria and fungi -chlorine gas is sporicidal (but needs moisture) -can react w/ organic matter to form carcinogenic compounds

Protein and Amino Acid Catabolism

-protease - an enzyme that hydrolyzes proteins to amino acids first, deamination: removal of amino group from amino acid then, resulting organic acids converted to pyruvate, acetyl-CoA, or TCA cycle intermediate • can be oxidized via TCA cycle, or used for biosynthesis • can occur through transamination (the transfer of an amino group from an amino acid to an α-keto acid acceptor, used for both degradation/synthesis of amino acids)

Type I Secretion System

-related to ABC transport systems -Gram-positive/Gram-negative bacteria, and Archaea -Secretion of toxins, proteases, other proteins

Type IV secretion system

-secrete proteins -secrete DNA from donor to recipient bacterium during conjugation -found in both Gram-positive and Gram- negative

Start codon

-start site for translation -always AUG

Gene structure

-the basic unit of genetic information -also defined as the nucleic acid sequence that codes for a polypeptide, tRNA or rRNA -linear sequence of nucleotides with a fixed start point and end point -codons are found in mRNA and code for single amino acids -promoter at the start of the gene

Generation (doubling time)

-time required for the population to double in size *refers to log phase** Nt = number of cells in bacterial culture at a given time t N0= initial number of cells n= number of generations Nt = N0 x 2^n log Nt = Log N0 + n x log 2 n = (log Nt - LogN0 )/ log 2 k = n/t (k = growth constant) G = t / n (G = generation time) i.e. generation time = 30 minutes, growing for 4 hours, ended at 9 pm. at 9 pm, the Nt = 10^6. What time was the population half of the Nt? 8:30 pm (generation time is 30 minutes, and it doubles every 30 minutes so 8:30 is half of what it is at 9)

How do enzymes lower Ea

1) by increasing concentrations of substrates at active site of enzyme 2) by orienting substrates properly with respect to each other in order to form the transition-state complex induced fit model for enzyme-substrate interaction

If a process kills 90% of the organisms per minute, how many minutes would it take to kill all organisms when starting with 100,000 organisms? a) 6 min b) 5 min c) 2 min d) 7 min e) 4 min

10^5 6 minutes

Why would you not use bleach to clean up organic materials (vomit/blood/urine etc)

1) organic material reacts w/ active ingredient, the chlorine ions 2) bleach reacting with organic material produces carcinogenic fumes

Post-translational regulation

2 reversible control measures • allosteric regulation • covalent modification

Tricarboxylic Acid Cycle (TCA)

= citric acid cycle = krebs cycle -common in aerobic bacteria, free-living protozoa, most algae and fungi -in cytoplasm (bacteria) or mitochondria (eukaryotes) • oxidizes acetyl coenzyme A to CO2 and generates NADH and FADH2 for oxidation in an ETC • supplies precursor metabolites for biosynthesis (4 carbon molecules produced) • must complete 2 full cycles to break down 2 pyruvate molecules completely for each acetyl-CoA molecule oxidized, TCA generates: - 2 molecules of CO2 - 3 molecules of NADH - 1 FADH2 - 1 GTP (one glucose molecule yields twice as much because it was broken down in to 2 pyruvates)

Sec-Dependent pathway

=general secretion pathway -highly conserved in all domains •Translocates proteins from cytoplasm across or into plasma membrane •Secreted proteins synthesized as preproteins having amino-terminal signal peptide -signal peptide delays protein folding -chaperone proteins (SecB) keep preproteins unfolded •secY, secE, and secG form a channel in the membrane •secA translocates preprotein through the plasma membrane •When preprotein emerges from plasma membrane a signal peptidase removes the signal peptide

Pentose Phosphate Pathway

=hexose monophosphate pathway -can be used aerobically or anaerobically -can be at the same time as the Embden -Meyerhof or Entner-Doudoroff pathways -in both eukaryotes and bacteria -not in archaea -amphibolic pathway NADP+ is electron acceptor, NADPH is produced -glucose 6-phosphate and 6-phosphogluconate are oxidized -reducing power in the form of NADPH -sugar transformation reactions catalyzed glucose-6-P + 12NADP+ + 7H2O --> 6CO2 + 12NADPH + 12H+ Pi

Continuous culture system

A culture system with constant environmental conditions maintained through continual provision of nutrients and removal of wastes. See also chemostat and turbidostat.

Replisomes

A large protein complex that copies the DNA double helix to form two daughter chromosomes. -DnaA (initiator protein) -Helicase (DnaB) - breaks hydrogen bonds holding 2 strands together -Helicase loader (DnaC) - directs helicase -DNA gyrase (relieves supercoiling) -SSB proteins (bind SSDNA after strands separate) -Primase (DnaG) -sunthesis of RNA primer -DNA polymerase III (catalyzes msot of DNA synth. during replication) -DNA polymerase I (removes RNA primers) -DNA ligase (synthesizes phosphodiester bonds to join Okazaki fragments) -Tus (terminator) -Topoisomerase IV (separates daughter molec)

Feedback inhibition

A method of metabolic control in which the end product of a metabolic pathway acts as an inhibitor of an enzyme within that pathway. • also called end-product inhibition • inhibition of one or more critical enzymes in a pathway regulates the entire pathway i.e. pacemaker enzyme • catalyzes the slowest or rate-limiting reaction in the pathway • each end product regulates its own branch of the pathway • each end product regulates the initial pacemaker enzyme • isoenzymes --- different enzymes that catalyze same reaction

Chemolithotrophs

A microorganism that uses reduced inorganic compounds as a source of energy and electrons. -typical reduced inorganic compounds are hydrogen, reduced nitrogen, sulfur, and ferrous iron. -energy released from the energy source (inorganic) is transferred to the terminal electron acceptor (typically O2. but can be sulfate and nitrate as well) via ETC -ATP synthesized by oxidative phosphorylation much less energy is available from oxidation of inorganic molecules than glucose oxidation due to more positive redox potentials

23. The net gain of ATP per molecule of glucose metabolized anaerobically via the Embden-Meyerhoff pathway in prokaryotes is A. 2. B. 36. C. 38. D. 85.

A. 2.

DNA gyrase

A topoisomerase enzyme that relieves tension generated by the rapid unwinding of DNA during DNA replication or transcription. -introduces negative supercoiling to help compact bacterial chromosome

9. __________ alter repressor proteins thereby increasing the rate of transcription initiation. A. Inducers B. Corepressors C. Effectors D. Modulators

A. Inducers

60. Which of the following lacks sufficient penetrating power for bulk sterilization? A. Ultraviolet (UV) radiation at 260 nm. B. X rays. C. Gamma radiation. D. Alpha and beta particles.

A. Ultraviolet (UV) radiation at 260 nm.

11. In __________ ribosomes can attach to the mRNA and begin translation even though transcription has not been completed. A. bacteria B. humans C. fungi D. protozoa and some plants

A. bacteria

53. Organisms that are not drastically affected by increased pressure are called A. barotolerant. B. osmotolerant. C. barophilic. D. osmophilic.

A. barotolerant.

56. Membranes of thermophilic bacteria are stabilized by phospholipids with A. fatty acids that tend to be more saturated. B. fatty acids that have lower molecular weights. C. fatty acids that have more double bonds. D. all of the choices

A. fatty acids that tend to be more saturated.

5. In __________, ribosomes can attach to the mRNA and begin translation even though transcription has not been completed. A. prokaryotes B. eukaryotes C. fungi D. protozoa and some plants

A. prokaryotes

4. A growth medium that favors the growth of some microorganisms but inhibits the growth of other microorganisms is a __________ medium. A. selective B. differential C. selective and differential D. neither selective nor differential

A. selective

12. In order for the cell to be able to input energy into necessary endergonic reactions, energy-generating processes such as photosynthesis, fermentation, and respiration are used to produce __________.

ATP

Phosphate Transfer potential

ATP has a HIGH phosphate transfer potential • regularly donates AND accepts a phosphate group to other molecules • other molecules have higher phosphate transfer potential than ATP •ATP = ADP and the phosphate from those molecules

Anaerobic respiration

An energy-conserving process in which the terminal electron transport chain acceptor is a molecule other than oxygen. (most commonly nitrate, sulfate, and CO2) -generally yields less energy because E0 of electron acceptor is less positive than E0 of O2 dissimilatory nitrate reduction -use of nitrates as the terminal electron acceptor, making it unavailable to the cell for assimilation or uptake denitrifcation -reduction of nitrate to nitrogen gas -in soil, causes loss of soil fertility

Fermentation

An energy-yielding process in which an organic molecule is oxidized without an exogenous electron acceptor. -Usually, pyruvate or a pyruvate derivative serves as the electron acceptor (is endogenous, or present in the cell) -does not involve ETC -no proton motive force (PMF) -oxidation of NADH produced by glycolysis -substrate only partially oxidized -oxygen not needed -no oxidative phosphorylation instead, ATP is synthesized by substrate-level phosphorylation (a phosphate is transferred to ADP from a high-energy molecule generated by catabolism of the energy source)

Respiration

An energy-yielding process in which the energy substrate is oxidized using an exogenous or externally derived electron acceptor. 1) organic energy source oxidized 2) electrons released from step 1 are accepted by electron carriers (such as NAD+ and FAD). 3) Reduced electron carriers (NADH, FADH2) donate electrons to an electron transport chain 4) electrons pass through ETC to final electron acceptor generating a potential energy called proton motive force (PMF) 5a) final electron acceptor is oxygen in aerobic 5b) final electron acceptor is any other oxidized molecule in anaerobic such as... NO3-, SO42-, CO2, Fe3+, or SeO42- PMF is used to synthesize ATP from ADP and phosphate -oxidative phosphorylation.

ATP synthase

An enzyme that catalyzes synthesis of ATP from ADP and Pi, using energy derived from the proton motive force (down gradient) -functions like rotary engine (conformational changes)

Autotrophs

An organism that uses CO2 as its sole or principal source of carbon.

Photolithoautotrophs (photoautotrophs)

An organism that uses light energy, an inorganic electron source (e.g., H2O, H2, H2S), and CO2 as its carbon source. i.e. algae, cyanobacteria *most organisms

Chemoorganoheterotrophs (chemoheterotrophs)

An organism that uses organic compounds as sources of energy, electrons, and carbon for biosynthesis. i.e. most nonphotosynthetic microbes, most pathogens, fungi, and many protists are used industrially to make foods (yogurts, pickles, cheese), medical products (antibiotics), and beverages (beer and wine). nearly all pathogenic microorganisms are chemoheterotrophs

Heterotrophs

An organism that uses reduced, preformed organic molecules as its principal carbon source.

14. At 4:00 p.m. a closed flask of sterile broth is inoculated with 10,000 cells. The lag phase lasts 1 hour. At 9:00 p.m. the log phase culture has population of 65 million cells. The approximate number of generations that has occurred is A. 5. B. 13. C. 21. D. 27.

B. 13.

16. At 4:00 p.m. a closed flask of sterile broth is inoculated with 10,000 cells. The lag phase lasts 1 hour. At 9:00 p.m. the log phase culture has a population of 65 million cells. The mean generation time is approximately A. 10 minutes. B. 20 minutes. C. 30 minutes. D. 40 minutes.

B. 20 minutes.

8. __________ activate repressor proteins thereby decreasing the synthesis of certain enzymes. A. Inducers B. Corepressors C. Effectors D. Modulators

B. Corepressors

46. A culture in a closed vessel to which no additional medium is added and from which no waste products are removed is called a __________ culture. A. continuous B. batch C. fed-batch D. semicontinuous

B. batch

17. An open system in which the growth rate is maintained by adding a nutrient (present in limiting quantities) at the same rate that medium containing microorganisms is removed is called A. manostat. B. chemostat. C. turbidostat. D. culturostat.

B. chemostat.

19. A nonprotein component of an enzyme that is loosely attached to the protein component is referred to as a(n) A. apoenzyme. B. coenzyme. C. holoenzyme. D. prosthetic group.

B. coenzyme.

3. The genetic code is said to be __________ because more than one codon will specify a particular amino acid. A. ambiguous B. degenerate C. multiplicative D. repetitious

B. degenerate

65. The two most important alcohol germicides are __________ and __________. A. ethanol; methanol B. ethanol; isopropanol C. methanol; butanol D. methanol; isopropanol

B. ethanol; isopropanol

11. Microorganisms are most nearly uniform in terms of chemical and physiological properties during __________ phase. A. lag B. exponential C. stationary D. decline

B. exponential

21. In metabolic channeling, the regulation of metabolic pathways is controlled by the __________ of metabolites and enzymes involved in the pathway. A. amount B. location C. activity D. all of the choices

B. location

10. The disinfectant action of phenol and phenolic derivatives mainly is due to A. its inherent detergent action. B. membrane damage and protein denaturation. C. oxidation of disulfide bonds in proteins. D. extraction of lipids from membranes. E. damage to nucleic acids and proteins caused by free radicals.

B. membrane damage and protein denaturation.

15. The __________ is the electron acceptor in a redox reaction. A. reductant B. oxidant C. enzyme D. product

B. oxidant =oxidizing agent

16. The standard reduction potential of a redox reaction is a measure of the tendency of the __________ to __________ electrons. A. reductant; gain B. reductant; lose C. oxidant; gain D. oxidant; lose

B. reductant; lose

61. When comparing dry heat and moist heat sterilization, dry heat is A. faster. B. slower. C. equally as fast. D. sometimes faster but sometimes slower.

B. slower.

10. Given a log phase bacterial culture with 1 x 106 cells per ml and a generation time of 30 minutes, how long does it take the culture to reach a density of 6.4 x 10^7 cells per ml? A. 1 hour B. 2 hours C. 3 hours D. 4 hours

C. 3 hours

25. The most common pathway for conversion of glucose to pyruvate is A. Entner-Doudoroff. B. Pentose phosphate. C. Embden-Meyerhoff. D. mixed acid fermentation.

C. Embden-Meyerhoff.

15. Which of these methods can be used to determine the number of viable microorganisms in a sample? A. Light scattering in a spectrophotometer. B. Measuring total cell mass. C. Measuring colony forming units per ml. D. Counting a known volume of cells in a hemocytometer.

C. Measuring colony forming units per ml.

10. The __________ is a factor-independent termination site found in the leader region of certain operons, which, under the influence of ribosome behavior, controls the continued transcription of that operon. A. modulator B. operator C. attenuator D. terminator

C. attenuator

1. Many of the enzymes found in virus particles are A. required for viral attachment to host cells. B. involved in viral entry into host cells. C. involved in the replication of viral nucleic acid. D. all of the choices

C. involved in the replication of viral nucleic acid.

2. The Pribnow box of E. coli A. is centered approximately 35 bp upstream of the start site of transcription B. is centered between 7 and 13 bp upstream of the start codon C. is centered approximately 10 bp upstream of the start site of transcription D. is located at the site of addition of poly (A) in eukaryotic mRNAs

C. is centered approximately 10 bp upstream of the start site of transcription

8. Gamma radiation A. is a type of nonionizing radiation. B. has poor penetrating power. C. is used to sterilize some food products. D. has a longer wavelength than UV radiation.

C. is used to sterilize some food products.

63. Joseph Lister A. pioneered the use of gamma rays for sterilizing food. B. demonstrated that microorganisms are carried on dust particles in the air. C. revolutionized surgery by introducing phenol as a disinfectant. D. designed and built the first autoclave.

C. revolutionized surgery by introducing phenol as a disinfectant.

24. The synthesis of ATP from ADP and Pi, when coupled with an exergonic chemical reaction is called __________ phosphorylation. A. chemiosmotic B. oxidative C. substrate-level D. conformational change

C. substrate-level

9. The limitation on microbial growth rate at high nutrient levels is A. the rate of protein synthesis. B. the rate of DNA replication. C. the saturation of the transport proteins for nutrient uptake. D. all of the choices.

C. the saturation of the transport proteins for nutrient uptake.

Steam sterilization

Carried out above 100 degrees celsius which requires saturated steam under pressure -uses an autoclave -effective against all types of microorganisms (including spores!) -quality control - includes strips with Geobacillus stearothermophilus

Work

Chemical work- • synthesis of complex molecules Transport work- • take up of nutrients, elimination of wastes, and maintenance of ion balance Mechanical work- • cell motility and movement of structures within cells

Other trophs...

Chemolithoautotrophs A microorganism that oxidizes reduced inorganic compounds to derive both energy and electrons; CO2 is the carbon source. i.e. sulfur-oxidizing bacteria, methanogens, iron-oxidizing bacteria Photoorganoheterotroph A microorganism that uses light energy, organic electron sources, and organic molecules as a carbon source. i.e. purple nonsulfur bacteria, green nonsulfur bacteria. commonly found in polluted lakes/streams Chemolithoheterotroph A microorganism that uses reduced inorganic compounds to derive both energy and electrons; organic molecules are used as the carbon source. i.e. some sulfur-oxidizing bacteria

Stationary phase

Closed system population growth eventually ceases, total number of viable cells remains constant -active cells stop reproducing or the reproductive rate is balanced by the death rate time when cells are most dissimilar -entry into stationary phase due to starvation and other stressful conditions - may have morphological changes (endospore formation), decrease in size, protoplast shrinkage, nucleoid condensation -RpoS protein assists RNA polymerase in transcribing genes for starvation proteins

3. Microscopic or macroscopic degenerative changes or abnormalities in infected host cells and tissues are called __________ __________.

Cytopathic effects

Which is true about DNA replication? A) It is semiconservative where each daughter cell receives 2 new strands of DNA. B) DNA polymerase I synthesizes the leading and lagging strand. C) It is unidirectional. D) It requires RNA primers and DNA ligase. E) All of the choices are correct.

D) It requires RNA primers and DNA ligase.

The 3' end of DNA A) has a hydroxyl group attached to the number 3 carbon of ribose. B) attaches to the 3' phosphate group of the incoming nucleotide. C) attaches to the 5' phosphate group of the purine or pyrimidine base . D) has a hydroxyl group attached to the number 3 carbon of deoxyribose. E) None of the above correct

D) has a hydroxyl group attached to the number 3 carbon of deoxyribose.

13. At 4:00 p.m. a closed flask of sterile broth is inoculated with 10,000 cells. The lag phase lasts 1 hour. At 9:00 p.m. the culture enters stationary phase with a population of 65 million cells. At what time is the population half maximal? A. 6:30 p.m B. 7:20 p.m C. 8:20 p.m D. 8:40 p.m

D. 8:40 p.m

6. At 4:00 p.m. a closed flask of sterile broth is inoculated with 10,000 cells. The lag phase lasts 1 hour. At 9:00 p.m. the culture enters stationary phase with a population of 65 million cells. At what time is the population half maximal? A. 6:30 p.m B. 7:20 p.m C. 8:20 p.m D. 8:40 p.m

D. 8:40 p.m

17. Which of the following is used as an electron carrier by living organisms? A. NAD+ B. NADP+ C. ubiquinone D. all of the choices

D. all of the choices

13. Semiconservative replication refers to A. each base bonding at the 1' position of the sugar. B. a purine always bonding to a pyrimidine. C. one helix strand that runs from the 5' to 3' direction and the other strand that runs from the 3' to 5' direction. D. an original parent DNA strand and one newly synthesized DNA strand comprising a new DNA molecule.

D. an original parent DNA strand and one newly synthesized DNA strand comprising a new DNA molecule.

7. The reduction of the microbial population to levels that are considered safe by public health standards is called A. disinfection. B. antisepsis. C. sterilization. D. sanitization.

D. sanitization.

DNA synthesis

DNA polymerase synthesizes in 5' to 3' direction only. -lagging strand is synthesized in short fragments called Okazaki fragments -new primer is needed for the synthesis of each Okazaki fragment

Metabolic channeling

Differential localization of enzymes and metabolites compartmentation • differential distribution of enzymes and metabolites among separate cell structures or organelles • can generate marked variations in metabolite concentrations

Chemical control agents

Disinfection Antisepsis Sterilization

Electron Transport Chain (ETC)

Electron carriers organized into ETC -First electron carrier having the most E'0 -the potential energy stored in first redox couple is released and used to form ATP -first carrier is reduced and electrons moved to the next carrier and so on -series of electron carriers operating together to transfer electrons from NADH and FADH2 to a terminal electron acceptor, O2 -each carrier is reduced and then reoxidized -carriers constantly recycled -in euk, electron transport chain carriers are in the inner mitochondrial membrane, connected by coenzyme Q and cytochrome c -electron transfer accompanied by proton movement across inner mitochondrial membrane -bacterial and archaeal ETCs are on plasma membrane, some resemble mitochondrial ETC but can be different ATP synthesis occurs on membrane

First law of thermodynamics

Energy can neither be created nor destroyed Total energy in universe remains constant • however, energy may be redistributed either within a system or between the system and its surroundings

How enzymes speed up reations

Enzymes increase the rates of reactions but do not alter their equilibrium constants activation energy - energy required to form transition-state complex enzyme speeds up reaction by lowering Ea

Topoisomerases

Enzymes that change the topology of DNA molecules by transiently breaking one or both strands. Prevents supercoiling

Helicase

Enzymes that use ATP energy to unwind DNA ahead of the replication fork.

Standard Redox potential (E0)

Equilibrium constant for an oxidation-reduction rxn. • Measure of tendency of the reducing agent to lose electrons • more negative E0 => better electron donor • more positive E0 => better electron acceptor The greater the difference between the E0 of the donor and the E0 of the acceptor, the more negative the delta G

Second law of thermodynamics

Every energy transfer or transformation increases the entropy of the universe. Entropy - the measure of randomness in a system •The greater the disorder, the greater the entropy. •Physical and chemical processes proceed in such a way so that the disorder of the universe increases to the maximum possible.

12. During the lag phase of microbial growth, the cells are metabolically inactive. True False

FALSE

18. Enzymes increase the rate of a reaction by increasing molecular motion, thereby providing kinetic energy to drive the reaction. True False

False

20. One important ribozyme located in mitochondria is responsible for catalyzing peptide bond formation during protein synthesis. True False

False

22. Some aerobic bacteria lack the Entner-Doudoroff and Embden-Meyerhoff pathways and instead use the pentose phosphate pathway for glycolysis. True False

False

4. The stop codons are translated codons; that is, they specify amino acids to be inserted into the last position of a growing polypeptide chain. True or False

False

5. When a young, vigorously growing culture of bacteria is transferred to fresh medium of different composition, the lag phase is usually short or absent. True False

False

Holoenzyme

Functional enzyme. apoenzyme + cofactor apoenzyme protein component of an enzyme cofactor nonprotein component of an enzyme - prosthetic group- firmly attached -coenzyme - loosely attached, can act as carriers/shuttles

Ionizing Radiation

Gamma radiation penetrates deep into objects -destroys bacterial endospores; not always effective against viruses -used for sterilization and pasteurization of antibiotics, hormones, sutures, plastic disposable supplies, and good.

49. The __________ __________ is the length of time it takes for a population of microorganisms to double in number.

Generation time

DNA as Genetic Material

Hershey and Chase, 1952 -used bacteriophage T2 infection as model -DNA labeled with 32P; protein coat labeled with 35S -only DNA entered cell but both new DNA and protein coats synthesized in new viruses indicating DNA had genetic information for both of these viral components

Transforming Principle

Griffith, 1928 -observed change of non-virulent organisms into virulent ones as a result of "transformation" MacLeod and McCarty, 1944 -showed that DNA was the transforming principle

Batch culture System

Growth of microorganisms in a closed culture vessel without adding fresh or removing old (spent) medium.

9. The type of filter used in a laminar flow biological safety cabinet is called a(n) __________ filter.

HEPA

Single stranded binding proteins (SSB)

Keep DNA strands apart once they have been separated so that replication can occur.

Dry heat sterilization

Less effective than moist heat sterilization, requiring higher temperatures and longer exposure times - items subjected 160 to 170 degrees C for 2 to 3 hours -oxidizes cell constituents and denatures proteins i.e. bench top incinerations used to sterilize inoculating loops in microbio labs

Viable counting methods

Membrane filler technique -bacteria from aquatic samples are trapped on membranes -membrane soaked in culture media -colonies grow on membrane -colony count determines # of bacteria in sample CFU = (# of colonies X reciprocal of dilution) / aliquot if microbe can not be cultured, serial dilutions, and see which solution has turbidity

57. Organisms that grow best at pH levels above 10 are called __________ __________.

extreme alkaliphiles

Anabolism

Metabolic pathways that construct molecules, requiring energy. -synthesis of complex organic molecules from simpler ones -requires energy from fueling reactions

Catabolic pathways

Metabolic pathways that release energy by breaking down complex molecules into simpler compounds. -enzyme catalyzed reactions whereby the product of one reaction serves as the substrate for the next -pathways provide materials for biosynthesis

14. The __________ __________ is the substrate concentration needed for an enzyme to achieve half maximal velocity.

Michaelis constant (Km)

2. Some viruses cause abnormal growth of cells rather than destruction. This is called __________.

Neoplasia

Chemotrophs

Organisms obtain energy from the oxidation of chemical compounds. (either organic or inorganic)

Phototrophs

Organisms that use light as their energy source.

Organotrophs

Organisms that use reduced organic compounds as their electron source.

11. The disinfectant screening method that is known as the ________ _________ test, which is used to measure the potency of a disinfectant.

Phenol coefficiency

64. The disinfecting properties of Lysol, a common household disinfectant, depend upon the presence of __________.

Phenolics

UV radiation

Physical control 260 most bactericidal (DNA absorbs) -causes thymine dimers preventing replication and transcription -UV limited to surface sterilization because it does not penetrate glass, dirt films, water, and other substances

Phenol Coefficient Test

Potency of a disinfectant is compared to that of phenol -useful in screening, may be misleading If Phenol coefficient >1, admissible.

Starvation responses

Production of starvation proteins -increase cross-linking in cell wall -Dpps protects DNA -chaperone proteins prevent protein damage Cells are called persister cells -long-term survival -increased virulence

Chemostat

Rate of incoming medium = rate of removal of medium from vessel -an essential nutrient is in limiting quantities

Iodine

Skin antiseptic -oxidizes cell constituents and iodinates proteins -at high concentrations, may kill spores -skin damage, staining, and allergies can be a problem -iodophor ••• iodine complexed w/ organic carrier ••• released slowly to minimize skin burns

Ribozymes

Some RNA molecules can also catalyze reactions (Thomas cech & sidney altman) i.e. catalyze peptide bond formation, self-splicing, self-replication

Primase

Synthesizes short complementary strands of RNA (~10 nucleotides) to serve as primers needed by DNA polymerase

Allosteric Regulation

The binding of a regulatory molecule to a protein at one site that affects the function of the protein at a different site. -most regulatory enzymes -activity altered by small molecule allosteric effector -binds non-covalently at regulatory site -changes shape of enzyme and alters activity of catalytic site -positive effector increases enzyme activity -negative effector inhibits the enzyme

Chemiosmotic Hypothesis

The hypothesis that proton and electrochemical gradients are generated by electron transport and then used to drive ATP synthesis by oxidative phosphorylation or photophosphorylation. -Peter Mitchell (1920-1992) -protons move outward from the mitochondrial matrix as electrons are transported down the chain -protein expulsion during electron transport results in formation of a concentration gradient of protons and a charge gradient -combined chemical and electrical potential difference makes up the proton motive force (PMF)

Death phase (senescence)

The number of cells that are multiplying are fewer than those that are not. 1) Cells are viable, but not culturable (they're alive, but dormant) 2) programmed cell death leads to apparent 5th phase... prolonged decline in growth -bacterial population continually evolves -successive waves of genetically distinct variants -natural selection occurs

Oxidative phosphorylation

The production of ATP using energy derived from the redox reactions of an electron transport chain; is the third major stage of cellular respiration. -4 ATP molecules synthesized directly from the oxidation of glucose to CO2 -most ATP is made when NADH and FADH2 (formed as glucose degraded) are oxidized in the electron transport chain (ETC) -PMF used to perform work, proton flow back across the membrane down concentration/charge gradients, phosphorylates ADP to ATP. diffusion of protons back across membrane (down gradient) drives formation of ATP explained by the Chemiosmotic Hypothesis

Metabolism

The total of all chemical reactions in the cell •• catabolism •• anabolism in microbes, very versatile. have representation in all 5 major nutritional types, some cycling reactions only by microbes. • obey laws of thermodynamics • use ATP to store energy • metabolic reactions are organized into pathways • enzymes and ribozymes involves • biochem pathways/enzymes subject to regulation •oxidation-reduction rxn is hallmark i.e. microbes can fix molecular nitrogen into ammonia, or convert ammonia into nitrate, etc

Phenotype

collection of observable characteristics

13. A molecule that binds noncovalently to an enzyme at the active site and thereby prevents a substrate from binding and reacting is a(n) __________ inhibitor.

competitive

12. Some microbial proteins are spliced after translation in a reaction that results in removal of one or more internal intervening sequences called inteins. True False

True

54. DNA of thermophiles is stabilized by binding of special nucleoid-associated proteins. True False

True

7. Small RNAs that regulate translation by binding to the leader region of mRNAs are called antisense RNAs. True or False

True

The minus strand of DNA serves as the template for RNA production and is complementary to the anticodon on tRNA True or False

True

The production of ATP via the electron transport system, in eukaryotes and prokaryotes always requires the use of a membrane True False

True

The rate of killing by an antimicrobial agent may slow when the microbial population has been greatly reduced because the remaining population may have a high proportion of resistant organisms. True or False

True

Sterilizing Gases

Used to sterilize heat-sensitive materials -microbicidal and sporicidal -ethylene oxide sterilization is carried out in equipment resembling an autoclave -combine w/ and inactivate DNA and proteins i.e. Ethylene oxide, Betapropiolactone, Hydrogen peroxide

Genome

all DNA present in a cell or virus -bacteria and archaea generally have one set (haploid) -eukaryotes have two sets (diploid)

Microorganisms fueling reactions

all have same basic needs -ATP as energy currency -reducing power to supply electrons for chemical reactions -precursor metabolites for biosynthesis (small organic molecules that provide carbon skeletons needed for biosynthesis of monomers such as amino acids) microorganisms may change nutritional type -metabolic flexibility based on environmental requirements. -provides an advantage if enviro conditions change frequently

Alcohols

among most widely used disinfectants and antiseptics -bactericidal, fungicidal, but not sporicidal -denatures proteins, destabilizes membranes by dissolving some membrane lipids -inactivates some viruses (enveloped) i.e. Ethanol, Isopropanol

Halogens

any of the five elements: fluorine, chlorine, bromine, iodine, and astatine ALL oxidize cell constituents i.e. bleach has chlorine i.e. water treatments include some bromine, chlorine, fluorine

Which of the statements about catabolism and anabolism is TRUE? a) they refer only to the reactions involved in the metabolism of lipids b) the energy gathered during catabolism is used during anabolism c) they refer only to the reactions involved in carbohydrate synthesis d) the energy gathered during anabolism is used during catabolism

b) the energy gathered during catabolism is used during anabolism

59. An agent that kills bacteria is referred to as __________.

bactericidal

50. If all cell components are synthesized at constant rates relative to one another, the culture is said to be in __________ growth; however, if the rates of synthesis of some components change relative to the rates of synthesis of other components, the culture is said to be in __________ growth.

balanced; unbalanced

A pure culture in exponential growth phase has a bacterial concentration of 6.4 x 10^8 cells/ml. If the bacterium has a generation time of 1 hr, how long ago was the cell concentration 8.0 x 10^7 cells/ml? a) 1 hr b) 2 hr c) 3 hr d) 4hr e) 24 hr

c) 3 hr

Catabolism of carbohydrates

can be supplied externally or from internal reserves goal: convert carb into a monomer that can be fed into one of the glycolytic pathways -monosaccharides are converted into other sugars that enter the glycolytic pathway disaccharides and polysaccharides are cleaved by hydrolysis and phosphorolysis starch and glycogen are excess carbon stores from when there was a great supply, and they are used as energy sources in absence of external nutrients -cleaved by phosphorylases, glucose-1-P enters glycolytic pathway i.e. poly-hydroxybutyrate becomes acetly-CoA

enzymes

carry out reactions at physiological conditions so they proceed in a timely manner -enzymes (biological catalysts) speed up the rate at which a reaction proceeds toward its final equilibrium protein catalysts -high specificity for the reaction catalyzed and the molecules acted on -substance that increases the rate of a rxn w/out being permanently altered substrates = reacting molecules products = substances formed by reaction some enzymes are composed solely of one or more polypeptides some enzymes are composed of one or more polypeptides and nonprotein components

Antimicrobial agents

chemotherapy- chemicals use to kill or inhibit growth of microorganisms within host issue -cide indicating that agent kills germicide - kills pathogens and many nonpathogens but not necessarily endospores i.e. germicide, bactericide, fungicide, viricide -static suffix indicating that agent inhibits growth i.e. bacteriostatic and fungistatic

enzyme inhibition

competitive inhibitor -directly competes w/ binding of substrate to active site noncompetitive inhibitor -binds enzyme at site other than active site -changes enzyme's shape so that it becomes less active

DNA Replication

complex process involving numerous proteins which ensure accuracy - 2 strands separate, each serving as a template for synthesis of a complementary strand -synthesis is semi-conservative; each daughter cell obtains one old and one new strand replication begins at a single point, the origin of replication. the DNA helix is unwound at the y-shaped replication fork, and two replication forks move outward so bidirectional replication can take place. replicon - A unit of the genome that contains an origin of replication and in which DNA is replicated.

Entner-Doudoroff Pathway

converts glucose to pyruvate and glyceraldehyde 3-phosphate -gram-negative bacteria, esp. those in soil. -not used by eukaryotes. -glycolytic -replaces 6 carbon phase of Embden-meyerhof pathway for every 1 glucose, -1 ATP -1 NADPH -1 NADH

Direct cell counts

counting chambers -easy, inexpensive, and quick. useful for euk. and prok. cannot distinguish living from dead cells flow cytometry - electronic counters -microbial susp. forced through small orifice w/ a laser light beam -disruption of current are counted -specific antibodies can be used to determine size and internal complexity counter on membrane -cells filtered through special membrane that provides dark background for observing cells -cells stained w/ fluorescent dyes -useful for counting bacteria -with certain dyes, can distinguish living from dead cells

ATP (Adenosine 5'-triphosphate)

currency and storehouse of energy. exergonic breakdown of high energy ATP is coupled with endergonic reactions to make them more favorable. ATP + H20 -> ADP + Pi + H+ Guanosine 5'-triphosphate, cytosine 5'-triphosphate and uridine 5'-triphosphate also supply some energy.

A sterilized item is always expected to be free of a) bacteria, viruses, AND prions b) bacteria, endospores AND viruses c) bacteria, endospores, AND prions d) bacteria AND endospores only

d) bacteria AND endospores only

Moist heat

destroys viruses, fungi, and bacteria- boiling will not destroy spores and does not sterilize -degrades nucleic acids, denatures proteins, and disrupts membranes

Sterilization

destruction or removal of all viable organisms

Quaternary Ammonium Compounds

detergents that have antimicrobial activity and are effective disinfectants -amphipathic organic cleansing agents -cationic detergents are effective • kill most bacteria, but not tuberculosis or endospores •safe and easy to use, inactivated by hard water and soap -used in toothpastes, dishwashing liquids, hand soaps, etc i.e. Cetylpyridinium, Benzalkonium

Dilution Test

determines rate at which selected bacteria are destroyed by various chemical agents

Pasteruization

does not sterilize controlled heating at temperatures well below boiling -used for milk, beer, and other beverages -kills pathogens present and slow spoilage by reducing total load of organisms present

Deoxyribonucleic acid (DNA)

double-helix polymer of deoxyribonucleotides connected by phosphodiester bonds -constitutes genetic material of all cellular organisms -bases: adenine, guanine, cytosine, and thymine -sugar: deoxyribose -phosphate esterified to sugar carbon sugar phosphate backbone -covalent bonds b/w the 3' hydroxyl of one sugar and a 5'-phosphate attached to an adjacent sugar

measurement of cell mass

dry weight -time consuming not very sensitive quantity of a particular cell constituent i.e. how much DNA, ATP, or chlorophyll -useful if amount of substance in each cell is constant turbidimetric measures (light scattering) -quick, easy, sensitive

pH and temperature on enzymes

each enzyme has a specific pH and temp. optima outside of that range, denaturation -loss of enzyme's structure and activity when temp and pH rise too much above optima

Heavy metals

effective disinfectant, but usually toxic -combines w/ and inactivates proteins; may also precipitate proteins -silver is still widely used today, silver nitrate drops given to newborn babies to protect from sexually transmitted diseases i.e. ions of mercury, silver, asernic, zinc, copper • silver arsphenamine was one of the first antimicrobials used to treat syphilis

Biological control of microorganism

emerging field, natural control mechanisms -uses microorganisms to kill other microorganisms •• predation by Bdellovibrio •• viral-mediated lysis using pathogen specific bacteriophage lysins •• toxin-mediated killing using bacteriocins

Biochemical pathways

enzymes can be linked together to form pathways - can be linear, cyclic, branching pathways often overlap/feed into each other -complex networks, dynamic pathways can be used to monitor changes in metabolite levels (flux)

6. In the presence of both glucose and lactose, the lactose repressor is not bound to the operator and the genes of the lactose operon are expressed. True or False

false, e.coli prefers glucose and CAP regulates it so

Phenolics

first antiseptic was Phenol (used by Joseph Lister) Phenolics are derivatives of Phenol, -commonly used as laboratory and hospital disinfectants -act by denaturing proteins and disrupting cell membranes -tuberculocidal, effective in presence of organic material, and long lasting -bad odor, can irritate skin i.e. Phenol, Orthocresol, Triclosan, Hexachlorophene

Lag phase

first phase - cell synthesizing new components - very active (acclimatizing, adjusting) - replenish spent materials, adapt to new mediums - varies in length i.e. e.coli is front loading so it gets ready and can divide quickly

55. Organisms that require high levels of sodium chloride in order to grow are called __________ organisms.

halophilic

Physical control of microorganisms

heat and radiation

Bacterial growth

increase in cell number refers to population growth rather than the growth of individual cells

Disinfection

killing, inhibiting, or removal of disease-causing (pathogenic) organisms -disinfectants (agents, usually chemical, usually on inanimate objects) -must be effective against wide variety of infectious agents at low concentrations -must be effective in the presence of organic matter; should be stable in storage

Growth curve

lag phase log phase stationary phase death phase

Oxidation-Reduction (Redox) Reactions

many metabolic processes involve oxidation-reduction reactions (electron transfers) •electron carriers are often used to transfer electrons from electron donor to an electron acceptor • transfer of electrons from a donor to an acceptor.. - can result in energy release, which can be conserved and used to form ATP -the more electrons a molecule has, the more energy rich it is. Oxidizing- electron donating, loss of hydrogen, adding oxygen Reducing - electron receiving, gain of hydrogen, loss of oxygen conjugate redox pair - acceptor and donor

Catabolism

metabolic process that breaks down large molecules into smaller ones, provides energy -fueling reactions -energy conserving reactions -provide ready source of reducing power (electrons_ -generate precursors for biosynthesis

67. The higher the phenol coefficient value, the _______ effective the disinfectant under the test conditions.

more

Embden-Meyerhof Pathway

most common pathway for glucose degradation to pyruvate, stage two of aerobic respiration -in all major groups of microorganisms, plants, animals -functions in the presence or absence of O2 -provides precursor metabolites, NADH and ATP, for the cell -glycolytic pathway initial 6 carbon phase (6 C phase) -"primes the pump" by using ATP to do 2 phosphorylation reactions, yielding fructose 1,6-biphosphate. energy-conserving 3 carbon phase (3 C phase) -cleaves 1,6-biphosphate in to two 3-C molecules and then catabolized to pyruvate in a five-step process. -NADH and ATP are produced -example of substrate-level phosphorylation glucose + 2ADP + 2Pi + 2NAD+ --> 2 pyruvate + 2ATP + 2NADH + 2H+

Aldehydes

mostly used in labs, or for sterilizing substances that can not go in an autoclave -highly reactive molecules -sporicidal and can be used as chemical sterilants -sterilize endoscopes -combine w/ and inactivate nucleic acids and proteins i.e. Formaldehyde, Glutaraldehyde

Ribonucleic Acid (RNA)

normally single-stranded polymer of ribonucleotides connected by phosphodiester bonds -bases: adenine, guanine, cytosine, and uracil (instead of thymine) -sugar: ribose -phosphodiester bonds -messenger RNA (mRNA) -ribosomal RNA (rRNA) -transfer RNA (tRNA)

58. Quorum sensing in Gram-positive bacteria generally involves the use of ____________ as the external signaling molecules.

oligopeptides

Protein Structure

polymers of amino acids linked by peptide bonds -amino acids are defined by a central carbon attached to a carboxyl group (C-terminal), an amino group (N-terminal), and a side chain. -normally, 20 amino acids used to form proteins -amino acids can be polar, non-polar, or charged depending on side chains -fold to 3-dimensional structures

Conditions influencing effectiveness of antimicrobial agent activity

population size -larger populations take longer to kill than smaller populations population composition -in log phase, will die faster. -if endospores are miked in, will take longer to kill -some organisms are more resistant (i.e. Pseudomonas vs e.coli) concentration of antimcrobial agent -usually higher concentrations kill more rapidly, but, in alcohol (which kills via denaturation/destruction of membrane) it does not get more potent after 80% >? duration of exposure -longer exposure, more organisms killed temperature -higher temperatures, usually increase killing local environment -pH, viscosity, and concentration of organic matter -biofilms are less susceptible to many antimicrobial agents

Antisepsis

prevention of infection of living tissue by microorganisms i.e. hand sanitizers antiseptics are chemical agents that kill or inhibit growth of microorganisms when applied to tissue -overuse of antiseptics such as triclosan has selected for triclosan resistant bacteria and possibly abx resistance

aerobic respiration

process that can completely catabolize an organic energy source to CO2 using -glycolytic pathways -TCA cycle -ETC w/ oxygen as final electron acceptor produces ATP (mostly indirectly via ETC) and high energy electron carriers

Alcoholic fermentation

produces ethanol and CO2 from sugars -pyruvate decarboxylated to acetaldehyde, reduced to ethanol by alcohol dehydrogenase w/ NADH as electron donor. produces ethanol used to make beer/ale/wine

1. The non-transcribed region of DNA to which RNA polymerase binds to initiate transcription is called the A. promoter. B. operator. C. operon. D. exon. E. intron.

promoter

Dilution rate

rate at which medium flows through vessel relative to vessel size note: cell density maintained at wide range of dilution rates and chemostat operates best at low dilution rate

effects of substrate concentration on enzyme activity

rate increases as concentration of substrate increases -no further increase occurs after all enzyme molecules are saturated with substrate Michaels constant = the substrate concentration required ..... the smaller the michaels constant, the greater the affinity the enzyme has.

Exponential (log) phase

rate of growth and division is constant and maximal -population is most uniform in terms of chemical and physical properties during this phase balanced growth -cellular constituents manufactures at constant rates relative to each other unbalanced growth -rates of synthesis vary relative to each other -change in nutrient levels, change in environmental conditions during log phase... -organisms are most vulnerable to microbial -maximum yield of biomass, bioproducts

Filtration

reduces microbial population or sterilizes solution of heat-sensitive materials by removing microorganisms liquids- membrane filters -porous membranes with defined pore sizes that remove microorganisms primarily by physical screening depth filtration -fibrous or granular materials that have bonded into a thick later w/ narrow, twisting channels. solution is sucked through this layer under vacuum air- -surgical masks -cotton plugs on culture vessels -HEPA (high efficiency particulate air) filters

Sanitization

reduction of microbial population to levels deemed safe (based on public health standards)

Lactic acid fermenters

reduction of pyruvate to lactate -bacteria, protists, animal skeletal muscle homolactic fermenters -use Embden-meyerhof pathway and directly reduce almost all pyruvate to lactate w/ enzyme lactate dehydrogenase heterolactic fermenters -use pentose phosphate pathway, form products other than lactate (also ethanol and CO2) lactic acid bacteria used to make yogurt, cheese

Evaluation of Antimicrobial Agent Effectiveness

regulated by US federal agencies ••Environmental Protection Agency (EPA) -disinfectants, what is used outside of the body ••Food and Drug Administration (FDA) -what is used inside/ or on the surface of the body

Turbidostat

regulates the flow rate of media through vessel to maintain a predetermined turbidity or cell density -dilution rate varies -no limiting nutrients -operates best at high dilution rates

Genotype

specific set of genes an organism possessses

Environmental effects on enzyme activity

substrate concentration pH temperature

DNA polymerase

synthesizes new DNA using a parental nucleic acid strand as a template -5' to 3' direction, adds deoxyribonucleoside triphosphate (dNTP), forming phosphodiester bonds -enzyme requires • template - directs the synthesis of complementary strands • primer - DNA or RNA strand • dNTPs (dATP, DTTP, DCTP, dGTP) e.coli has 5 different DNA polymerases, 2 core enzymes in each polymerase... to catalyze DNA synthesis and proofread

Normal In-use testing

testing done using conditions that approximate normal use of disinfectant

Sense codons

the 61 codons that specify amino acids

Stop (nonsense) codons

the three codons used as translation termination signals do not encode amino acids

Code degeneracy

up to six different codons can code for a single amino acid

ATP yield during aerobic respiration

• maximum ATP yield can be calculated - includes P/O ratios of NADH and FADH2 - ATP produced by substrate level phosphorylation • the theoretical maximum total yield of ATP during aerobic respiration is 38 - the actual number closer to 30 than 38 estimates 10 protons moved across the membrane due to NADH oxidation, and 6 electrons transferred when FADH2 oxidation initiates electron flow to oxygen ATP yield may be affected by: -shorter ETCS (such as bacteria), have lower P/O ratios -environmental conditions -PMF generated by ETC can be used for functions other than ATP synthesis (i.e. flagella rotation) -precursor metabolite may be used for biosynthesis

Covalent modification of enzymes

• reversible on and off switch • addition or removal of a chemical group (phosphate, methyl, adenyl) • advantages of this method -respond to more stimuli in varied/sophisticated ways -regulation of enzymes that catalyze covalent modification adds second level of control

Breakdown of Glucose to Pyruvate

• three common routes - Embden-Meyerhof pathway - pentose phosphate pathway - Entner-Duodoroff pathway

Riboswitches (sensory RNAs)

•A specialized form of transcription attenuation •Folding of mRNA leader sequence (the riboswitch) determines if transcription will continue/terminate •Folding pattern altered in response to mRNA binding of an effector molecule •Riboswitches in Gram-positive bacteria function in transcriptional termination

Transcription Elongation

•After binding, RNA polymerase unwinds the DNA •Transcription bubble produced -moves with the polymerase as it transcribes mRNA from template strand •within the bubble a temporary RNA:DNA hybrid is formed

Positive control

•Binding of a regulatory protein (activator protein) at a regulatory region on DNA (activator binding sites) promotes transcription initiation -mRNA synthesis is increased •Activation -inactive protein is activated by inducer (activator protein) -active protein is inactivated by inhibitor

Negative transcriptional control

•Binding of regulatory protein (repressor) at DNA regulatory site (operator) inhibits initiation of transcription -mRNA expression is reduced •Repressor proteins -exist in active and inactive forms -inducers (substrates) and corepressors (enzymatic products) alter activity of repressor by binding

Proofreading

•Carried out by DNA polymerase III •Removal of mismatched base from 3' end of growing strand by exonuclease activity of enzyme •This activity is not 100% efficient

The Tryptophan (trp) Operon

•Consists of 5 structural genes which code for enzymes needed to synthesize tryptophan •Negative transcriptional control of repressible genes by trp repressor •Operon only functions in the absence of tryptophan -- •In addition to transcription initiation control, transcription continuation is also controlled in this operon •Attenuation is termination of transcription within the leader region (leader peptide) •Occurs through stem-loop structures in the mRNA depending on trp level

Linking Okazaki Fragments

•DNA ligase forms a phosphodiester bond between 3'-hydroxyl of the growing strand and the 5'-phosphate of an Okazaki fragment

Replication fork in e. coli

•DnaA proteins bind oriC (origin of replication) causing bending and separation of strands •DnaB and other helicases separate strands, SSB attach •Primase synthesizes RNA primer •Lagging and leading strand is synthesized •DNA polymerase I removes RNA primers, fills gaps with DNA •Okazaki fragments are joined by DNA ligase

elongation (translation)

•Elongation cycle -sequential addition of amino acids to growing polypeptide -consists of three phases •aminoacyl-tRNA binding •transpeptidation reaction •translocation -involves several elongation factors (EFs)

"Decision" process in gene expression

•Enzymes of a catabolite pathway are only needed (increased mRNA synthesis) when the preferred substrate is available •Enzymes not synthesized when substrate absent •Efficient use of energy and materials

Initiation of Protein Synthesis

•In bacteria initiation begins when -initiator codon binds 16S rRNA in 30S subunit -Shine Dalgarno sequence of mRNA is aligned with 16S rRNA •3 initiation factors (IFs) in bacteria -required for formation of the initiation complex GTP catalyzes

Negative control of Lactose (Lac) Operon

•Inducible genes -three structural genes coding for lactose uptake and metabolism -lac repressor (lacI) binds operator •inhibits transcription •Enzymes normally not produced unless lactose present lac repressor •Tetramers of repressor form and bind to three operator sites (O1, O2, O3) •Bends DNA, prevents RNA polymerase from accessing promoter •Presence of allolactose binds repressor - no longer binds operator

Regulatory proteins

•Induction and repression occur because of the activity of regulatory proteins and DNA binding domains •These proteins either inhibit transcription (negative control) or promote transcription (positive control)

Wobble

•Loose base pairing -3rd position of codon less important than 1st or 2nd •Eliminates need for unique tRNA for each codon

Transcription Termination

•Occurs when core RNA polymerase dissociates from template DNA •DNA sequences mark the end of gene in the trailer and the terminator •Some terminators require the aid of the rho factor for termination

Transcription initiation

•Only a short segment of DNA is transcribed •Promoter -site where RNA polymerase binds to initiate transcription -is not transcribed -has specific sequence before transcription starting point and a Pribnow box which contains consensus sequence

Protein Maturation

•Protein function depends on 3-D shape •Occurs as post translational event -requires folding -association with other proteins -delivered to proper subcellular or extracellular site Molecular chaperones proteins that aid the folding of nascent polypeptides protect cells from thermal damage e.g., heat-shock proteins aid in transport of proteins across membranes

Transcription

•RNA synthesis under the direction of DNA -RNA produced has complementary sequence to the template DNA -three types of RNA are produced •mRNA carries the message for protein synthesis •tRNA carries amino acids during protein synthesis •rRNA molecules are components of ribosomes in bacteria •Polycistronic mRNA often found in bacteria and archaea •Contains directions for >1 polypeptide catalyzed by a single RNA polymerase -reaction similar to that catalyzed by DNA polymerase •Most bacterial RNA polymerases -core enzyme composed of 5 chains and catalyzes RNA synthesis -the sigma factor has no catalytic activity but helps the core enzyme recognize the start of genes -holoenzyme = core enzyme + sigma factor •only the holoenzyme can begin transcription

Positive Control of the lac Operon

•Regulated by catabolite activator protein (CAP) -regulates in response to presence or absence of glucose -allows for preferential use of glucose

Protein Splicing

•Removal of part of polypeptide before folding •Inteins - removed portion •Exteins - portions that remain in protein

Termination of replication in E.coli

•Replication stops when replisome reaches termination site (ter) on DNA •Catenanes form when the two circular daughter chromosomes do not separate •Topoisomerases temporarily break the DNA molecules so the stands can separate

Protein Secretion in Gram-Negative bacteria

•Six proteins secretion systems identified •Types I and IV also in Gram-positives •Types II, III, and V are unique to Gram-negatives most secrete virulence factors •Type I pathways transport proteins across outer membrane that were first translocated across plasma membrane by Sec-dependent pathway •Types III is sec independent -forms injectisomes -transports virulence factors and other proteins •Type V are sec-dependent -autotransporters - transport themselves out •Type VI are similar to bacteriophage genome injection systems

Translation

•Synthesis of polypeptide directed by sequence of nucleotides in mRNA -direction of synthesis N terminal -> C-terminal •Ribosome = site of translation -coupled transcription/translation in Bacteria/Archaea •polyribosome - complex of mRNA with several ribosomes •Transfer RNA bears the anticodon (complementary to the codon on the mRNA

Termination of Protein Synthesis

•Takes place at any one of three codons -nonsense (stop) codons - UAA, UAG, and UGA •Release factors (RFs) -aid in recognition of stop codons -3 RFs function in prokaryotes -only 1 RF active in eukaryotes GTP hydrolysis required

Protein-coding genes

•Template strand of DNA directs RNA synthesis -is read in the 3' to 5' direction •Complementary DNA strand -is coding strand, same nucleotide sequence as mRNA (except in thymine)

Replication of linear chromosomes

•The "end" replication problem -shortening of chromosomes after each round of replication -solved in eukaryotes by telomerase enzyme -solved in bacteria by disguising the ends of the linear chromosome

Translocation (final phase of elongation cycle)

•Three simultaneous events -peptidyl-tRNA moves from A site to P site -ribosome moves down one codon -empty tRNA leaves P site •Requires GTP hydrolysis in bacteria, -movement of proteins from cytoplasm to plasma membrane or periplasmic space •include transport proteins, ETC proteins, proteins involved in chemotaxis and cell wall synthesis, enzymes

Regulation of Transcription Elongation

•Transcription can also be regulated by controlling transcription termination •This type of regulation, called attenuation, was first demonstrated with trp operon •More recently riboswitches have been demonstrated to also play a regulatory role

The Arabinose (ara) Operon

•Transcriptional control by a protein (AraC) that acts both positively and negatively -activity depends on environmental conditions -inactive when arabinose present -active when arabinose absent

Inducible genes

•are genes that code for inducible enzymes needed only in certain environments -such as b-Galactosidase •Inducible enzyme functions in a catabolic pathway •Inducible enzymes are present only when their substrate (inducer - effector molecule) is available •β-galactosidase reaction catalyzed is lactose hydrolysis into galactose and glucose