Mesnage (Spring) Flashcards
What is cardinal temp?
- min and max temps that define limits of growth and dev of organism
How does bacterial growth change at temp changes?
- at min temp, membrane gelling, transport processes so slow that growth can’t occur
- growth increases w/ temp as enzymatic reactions occurring at increasing rates, until optimum reached
- at max temp, proteins denature, cyto membrane collapses and thermal lysis
What are the different classes of MOs in terms of adaption to temperature, from lowest optimum to highest?
- psychrophile
- mesophile
- thermophile
- extreme thermophile
What are the requirements for bacterial growth?
- temperature
- pH
- osmotic pressure
- nutrients
- oxygen
How are psychrophiles adapted to cold temps?
- increased membrane fluidity –> higher content of unsat, polyunsat and methyl branched FAs, shorter acyl chain length (limit membrane cohesion)
- prod of anti-freeze proteins (AFPs) –> bind to small ice crystals to inhibit growth by covering water accessible ice surfaces
- prod of cryoprotectants –> Trehalase and exopolysaccharides
- prod of cold adapted enzymes –> high prop of α helices, less weak bonds and interdomain interaction, giving greater flexibility
How are thermophiles adapted to high temps?
- genome protection –> stabilisation of DNA by DNA binding proteins, intro of supercoils by reverse DNA gyrases, resistance to denaturation favoured by high GC%
- mod of membrane composition –> ester linked phospholipids, single lipid layer (glycerol tetraethers)
- prod of thermostable proteins –> hydrophobic interactions, higher prop of ionic interactions
- existence of thermostable chaperonins –> thermosome
What are the metabolic adaptations of alkaliphiles?
- resp chains pump Na+ out
- H+/Na+ antiporters scavenge H+
- Na+ driven ATPases export Na+
- Na+ motive force powers motility, drives ATP synthesis and substrate symport
- decarboxylases secrete Na+
What are the metabolic adaptations of acidophiles?
- resp chains pump H+ out
- H+/Na+ antiporters maintain pH below external pH
- pmf powers motility, drives ATP synthesis and substrate symport
- K+/H+ antiporters excrete H+
How do bacteria respond to osmotic stress?
- reg of water movements by passive diffusion and aquaporins
- prod of compatible solutes (betaine, Pro, Glu)
- release of solutes by mechano-sensitive channels
What is the salt req in halophiles?
- stabilisation of S-layer glycoprotein by Na+
- accum of K+ as compatible solute
What are the different classes of MOs in terms of adaption to salt conc, from lowest optimum to highest?
- non-halophile
- halotolerant
- halophile
- extreme halophile
What nutrients do bacteria req?
- N
- S
- P
- some vitamins
- cofactors, eg. K+, Ca2+, Mg2+
- trace elements (Fe, Cu, Zn)
What are the toxic forms of ROS?
- superoxide (O2-)
- H2O2
- OH•
What is the overall reaction of O2 to form water?
- O2 + 4e- + 4H+ –> H2O
What enzymes can detoxify ROS?
- catalase/peroxidase –> convert H2O2 to H2O
- superoxide dismutase and catalase, and superoxide reductase and catalase –> convert O2- to H2O then H2O
Which bacteria req oxygen and why?
- obligate aerobes –> catalase and SOD, only use O2 for resp
- obligate anaerobes –> killed by normal atmospheric concs of O2
- facultative aerobes –> catalase and SOD, can use O2 for resp
- microaerophiles –> req O2 for resp
- aerotolerant anaerobes –> only SOD, don’t use O2 for resp
How can bacterial growth be directly measured and what are some assoc problems and solutions?
- microscopy –> counts live and dead cells, but can use dye to distinguish
- flow cytometry (FACS) –> measures cell size, identify and count diff pops of cells
- viable counts –> doesn’t reflect cell size or growth stage, usually underestimates no.
How can bacterial growth be indirectly measured and what are some assoc problems and solutions?
- measure dry weight –> easy but time consuming, req high cell densities
- OD –> differs between organisms, need standard curve, vol depends on growth stage, so cell no. estimate could be wrong, counts dead cells too
- metabolic prod/protein levels
What are the 4 stages of bacterial growth and what occurs in each one?
- lag –> metabolism starts but no division
- log –> exponential increase
- stationary –> deaths balance new cell prod
- death –> pop decreases
What is the composition, structure and role of the outermost structure (S-layer) of archaea?
- made of glycoprotein
- 2D crystalline array
- poss role in cell shape
What are the characteristics of the cell wall of archaea?
- pseudomurein
- not always present
- heteropolymer (disaccharide-peptides)
- similar to bacterial peptidoglycan
- resistant to lysozyme
- resistant to most antibiotics targeting bacterial peptidoglycan synthesis (penicillin)
What are the characteristics of cytoplasmic membrane of archaea, and how does this differ from bacteria?
- phospholipids contain no FA, isoprenes instead
- phospholipids ether, not ester linked
- membranes more stable than bacteria, present as monolayers or bilayers mix
How are chromosomes organised and rep in archaea?
- circular chromosome (and plasmids)
- histones
- multiple replication origins
- encode pol B (eukarya) and D (specific to archaea)
How does transcrip and RNA processing occur in archaea?
- single RNA pol exist, similar to euk RNA pol II
- presence of introns
- genes organised in operons
How does translation occur in archaea?
- coupled to transcrip
- involves several translation factors, like in eukarya
- ribosomes are 70S particles
What is the physiology of archaea like?
- v diverse, reflects wide range of habitats
What are the characteristics of (hyper)thermophiles?
- inc Crenarchaeota and Euryarchaeota
- high growth temp (80-120ºC)
- most req elemental S for growth
- often acidophiles
What are the characteristics of halophiles?
- mostly Euryarchaeota (few bacteria and euks)
- found in evaporating ponds, Dead Sea etc.
- req up to 5M NaCl for growth
What are the characteristics of methanogens?
- Eukarchaeota
- found in anaerobic envs (rumen, gut, marine sediments)
- use acetate/fumarate/CO2 as e- acceptor
What are the methods of controlling bacterial growth?
- heat
- irradiation
- filtration
How can heat be used to control bacterial growth?
- moist heat (boiling water/autoclave) –> 15 mins at 121º, under pressure for spores
- dry heat (oven) –> direct flaming, incineration, >150ºC for 2 hours
- pasteurisation (mild heat/HTST/UHT) –> temps below 100ºC to avoid casein aggregation
How can irradiation be used to control bacterial growth and what is it used for?
- not widely used for food
- ionising –> food industry, med and lab equipment, DNA destruction via ROS
- non-ionising –> surface decontam, DNA damage (breaks/dimers)
How can filtration be used to control bacterial growth?
- used to sterilise gases (air) or liquids that can be damaged by heat
- porosity of filters can be chosen for specific apps (1mm-0.01μm)
- nucleopore/membrane/depth filter
What are the types of chemical antimicrobial control agents and what are they capable of?
- sterilants and disinfectants used to destroy MOs on objects
- sterilants completely eliminate all MO forms, inc spores, eg. ethylene oxide (g)
- disinfectants kill MOs, not necessarily endospores, eg. alcohol (60-85%)
- antiseptics and germicides applied on living tissue, inhibit growth or kill MOs, eg. handwash
How does a bacteriostatic chemical antimicrobial control agent work?
- stops reproduction of bacteria
How does a bactericidal chemical antimicrobial control agent work?
- kills bacteria, so only viable cell count decreases
How does a bacteriolytic chemical antimicrobial control agent work?
- bacteria destroyed, so no. cells counted reduced
How can antimicrobial activity be measured by the disc diffusion technique?
- inoculate plate w/ liquid culture of test organisms
- discs containing antimicrobial agents placed on surface
- incubate 1-2 days
- test organism shows susceptibility to some agents, indicated by inhibition of bacterial growth and disease (zones of inhibition)
What is the min inhibitory conc (MIC)?
- lowest conc of drug inhibiting visible growth of test organism after overnight incubation
What is the min bactericidal conc (MBC)?
- lowest conc of drug killing >99.9% of test organism after overnight incubation
What kind of chemicals can be used for antimicrobial control?
- phenolic compounds –> local anesthetic at low concs, antibac but toxic at high temps, disrupt cytoplasmic membrane and denature proteins
- alcohols –> active conc 60-85%, denature proteins, lipid solvent, disrupt cyto membrane
- aldehydes –> alkylating agents, modify proteins and DNA causing cell death
- 4º ammonium compounds –> interact w/ phospholipids of cyto membrane (cationic detergents)
- halogen releasing agents
What are the 2 types of halogen releasing agents? (chemicals for antimicrobial control)
- Cl releasing –> sodium hypochlorite (in water ionises to prod Na+ and OCl-, in equilibrium w/ HOCl), formation of chlorinated bases in DNA, ox of proteins
- I releasing (iodine/iodophors) –> v powerful, but stain, target DNA and proteins
What role did Emmerich and Loew play in antibiotic discovery?
- Pyocyanase shown to have antibac activity
- prod large scale and app to patients
- unstable, sporadic effectiveness and toxic
What role did Ehrlich play in antibiotic discovery?
- Salvarson
- synthetic dye linked to arsenic
- treated Syphilis
- important toxicity and painful side effects
What role did Domagk play in antibiotic discovery?
- Prontosil red
- dye tested in vitro/in vivo (systematic screen)
What role did Fleming play in antibiotic discovery?
- Penicillin
- prod by fungus
What are the major classes of antibiotics?
- cell wall synthesis
- DNA gyrase
- RNA elongation
- DNA directed RNA pol
- protein synthesis (50S and 30S inhibitors and tRNA)
- lipid biosynthesis
- cyto membrane structure and function
- folic acid metabolism
What are the causes of antibiotic resistance?
- misuse in human therapeutics
- farming –> animals fed at subtherapeutic doses, far more than human consumption, dose req increased
- agriculture –> treatment of tree and plant diseases
- aquaculture
- pets
What are the factors that must be considered for the ideal antibiotic?
- target –> selective toxicity and inhibition of essential process
- stability and effectiveness –> pharmacokinetics and pharmacodynamics
- cost
How do β-lactams work?
- inhibit peptidoglycan polymerisation mediated by D,D-transpeptidases
- structural analogs of D-Ala-D-Ala C terminal residues in peptide stem
- used by PBP as substrates and inactivate these enzymes irreversibly
What are the resistance mechanisms of β-lactams?
- inactivation by β-lactamases –> can cleave ring structure w/ nucleophilic attack by catalytic Ser, breaks ring and adds OH using water, preventing penicillin binding proteins from attaching
- mutation of target enzyme –> reduce PBP affinity for β-lactams, so not able to target as well, can also overexpress PBP, so not enough to inhibit all of them
- secretion of antibiotics (gram -ve bacteria) –> overprod diff systems to help reduce permeability of penicillin into cell, and to help push it out
- mod of synthetic pathway targeted by β-lactams –> some bacteria don’t use D-Ala-D-Ala, so proteins can’t be affected by β-lactams
