Microbial Genetics Flashcards
mechanism of polymixin
causes loss of selective permeability of the cell membrane
mechanism of Quinolones (Cipro)
inhibits replication and transcription by inhibiting gyrase (unwinding enzyme) in humans this is topiosomerase
Mechanism of rifampin
inhibits RNA polymerase
Mechanism of macrolides (erythromycin)
inhibits 50s
Mechanism of aminoglycosides
inhibits 30s
Which 4 inhibit 50s
chloramphenicol, erythromycin, clindamycin, streptogramin
4 that inhibit 30s
aminoglycoside, gentamycin, streptomycin, tetracycline
Inhibit metabolism of bacteria
sulfonamides, trimethoprim
Ab with the broadest spectrum
Tetracylcines
Ab with Gram+ and Gram- coverage
carbapenems, sulfonamides, cephalosporins, tetracyclines
Mycobacteria coverage ( Tuberculosis)
isoniazid, streptomycin, less coverage tobramycin, polymixin
Mostly gram positive, some gram -, some chlamydias
Penicillin
Coverage for rocky mountain fever (rickettsias)
only tetracyclines
S aureus is resistant to penicillin by
cleaving the beta lactam ring inactivating the drug
Aminoglycoside point mutation
changes shape of the receptor the drug binds, drug cannot enter the cell
Pseudomonas/E coli resistance mechanism
MDR efflux pumps, pump drugs out of the cell
Erythromycin resistance
change in shape of the 50s ribosome, drug has no effect on protein synthesis
Sulfonamide resistance
change mechanism of folic acid production
What are the end products of Glycolisis
(2) 3 carbon pyruvates, 2 ATP, 2 NADH
What is the purpose of gluconeogenesis
maintains blood glucose levels providing intermediates needed for Citric acid cycle, clears products of metabolism such as lactate (muscles) and glycerol (adipose)
3 irreversible steps of glycolysis
- glucose to glucose 6 phosphate
via hexokinase/glucokinase, 1 ATP used - Rate limiting step: fructose 6 phosphate to Fructose 1,6 bisphosphate
via PFK1, 1 ATP used - phosphoenolpyruvate (PEP) to pyruvate
via pyruvate kinase, 2 ATP produced
Energy requiring phase
After transamination or deamination glucogenic amino acids yield
pyruvate - citric acid cycle intermediates
What is pyruvate converted into to enter the citric acid cycle
acetyl coA
one molecule of glucose is ____, one molecule of pyruvate is ____
C6H12O6
C3H3O3
Where does glycolysis occur
the cytosol
Where does the citric acid cycle occur,
the matrix of the mitochondria
What steps in glycolysis release energy
- Glyceraldehyde 3 phosphate (G3P) —-> 1,3 BPG
via G3P dehydrogenase, +2 NADH - 1,3, BPG —> 3 phosphoglycerate
via phosphoglycerate kinase, +2 ATP - phosphoenolpyruvate (PEP) —-> Pyruvate
via pyruvate kinase, +2ATP
RBCs are completely reliant on what for energy?
lack mitochondria, reliant on glucose and anerobic glycolysis
Glycolysis under anerobic conditions
pyruvate is reduced to lactate producing only 2 ATP
Net products of glycolysis
2 ATP, 2 NADH
What process can occur in critically ill patients in response to injury/infection and contributes to hyperglycemia
Gluconeogenesis
the citric acid cycle is the catabolism of what molecules
carbohydrates, lipids, and proteins
what begins the citric acid cycle
acetyl portions of acetyl coA
What type of reactions occur in the CAC
redox reactions
What happens in the electron transport chain
NADH and FADH2 go through oxidative phosphorylation to generate most of the ATP produced in cellular respiration
What are the 3 irreversible reactions (control points) in the CAC
- acetyl coA –> Citrate
via citrate synthase - isocitrate —>a-ketoglutarate + NADH
via isocitrate dehydrogenase - a-ketoglutarate —-> succinyl-coA + NADH
via oxidative decarboxylation
The citric acid cycles provides electrons that fuel the process of _____ ___________
oxidative phosphorylation
How much energy is produced in the CAC?
3 NADH = 9 ATP
1 FADH = 2 ATP
1 GTP = 1 ATP
12 Total x2 = 24 per glucose molecule
triacylglycerols are hydrolyzed by lipases to form….
fatty acids and monoacylglycerol which are taken up through the small intestine
stored fat is hydrolyzed to ____ and ____ when needed as fuel
glycerol and fatty acids
How is energy extracted from glycerol
glycolysis
the pathway used by cells to obtain energy from fatty acids is called
beta oxidation
the major storage of chemical energy is
triacylglycerol’s
purpose of beta oxidation
break down fatty acid to generate acetyl coA to enter the citric acid cycle and produce NADH and FADH2
When sugar is plentiful fatty acid oxidation is inhibited ad fats are stored
in muscle tissue or adipose tissue
How ketone bodys are formed
low glucose = acetylcoA build up
Acetoacetate is hydrolyzed then reduced to form B-hydroxybutyrate and acetone which are known as ketone bodies
serve as fuel for the brain during starvations
ketone bodies
What happens as a result of ketoacidosis
blood pH drops = Na+ decreased in interstitial fluid drawing out K+, water and glucose lost in the urine decreasing BP
Central Dogma
transcription - DNA to mRNA
Translation - tRNA interprets mRNA in to sequence of amino acids (proteins)
Vertical gene transfer
copy of DNA to offspring
Horizontal transfer
donor to recipient, contributes to genetic diversity if forms a RECOMBINANT donor DNA is incorporated into recipient DNA
Transformation
recipient cell is able to take up donor DNA from a lysed bacteria cell that dies and releases DNA into the environment
Transduction
A phage accidentally incorporates bacterial DNA during replication and assembly, the phage then infects another bacteria and inserts the bacterial DNA which is incorporated into the bacteria
Conjugation
donor creates a pilis and passes a plasmid to neighboring bacteria, plasmids often contain multiple resistance genes, plasmid is incorporated in recipients DNA
kirby bauer/ AST testing
incubating bacteria with antibiotics to find out which are effective
Advantages/Disadvantages of PCR
A: culture not required, specific & sensitive, rapid, accurate, reduced risk of contamination multiplex can ID many pathogens simultaneously
D: highly precise thermal cycler needed
trained laboratory personnel needed to perform the test
Advantages and disadvantages of DNA sequencing
A: 16s and 18s rDNA are gold standard, can ID fastidious and uncultivable organisms
D: trained laboratory personnel and powerful software required, expensive, not suitable for routine clinical use
Temps and steps for PCR
Denaturation 94-96C
Annealing 68C
Elongation 72C
Can melt DNA apart and utilize polymerase that functions at high temperatures
How do you target DNA you want to amplify
design specific primers that are added to the OH 3 prime end
what direction does DNA polymerase work
3 prime to 5 prime
what subunit is used in PCR amplification
the 16s subunit which is part of the 30s subunit
What area is targeted by the primer
the conserved region of 16s, same for all prokaryotes
What region is used for identification in PCR
The variable region of the 16s allows for identification by comparing it with the database
What do you need for a PCR reaction
dNTP - nucleotides needed to build DNA
DNA Ligase
Taq Polymerase
Two primers
DNA Template
reaction buffer
When is PCR used in medicine
- Detect disease and cancer
- Personalized medicine
- Pathogen testing
- efficacy of drug therapy
- to produce drugs – insulin
- directed evolution - enzymatic drug treatments
- organ transplant screening
AST =
antibiotic susceptibility testing
NAAT
Nucleic Acid Amplification Test, or NAAT, is a type of viral diagnostic test for SARS-CoV-2, the virus that causes COVID-19. NAATs detect genetic material (nucleic acids). NAATs for SARS-CoV-2 specifically identify the RNA (ribonucleic acid) sequences that comprise the genetic material of the virus
qPCR
qPCR is a technique for the selective amplification and quantitative detection of regions of DNA or complimentary DNA (cDNA). Oligonucleotide primers flanking a region of interest are used to amplify the sequence utilizing a DNA polymerase enzyme.1 Repeated cycling of the amplification process leads to exponential expansion of the number of copies of the target region which is tracked either using an intercalating dye or sequence-specific probe whose fluorescence is then detected in the qPCR machine and plotted on an output graph.
