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72 Cards in this Set
- Front
- Back
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PbrR |
Transcriptional regulator that binds to Pb(II) |
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PbrT |
Transmembrane protein that allows for uptake of Pb(II) from environment. |
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PbrA |
P-type ATPase that pumps Pb(II) out of the cell |
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PbrB/C |
Involved in precipitation of Pb(II) as Pb-phosphate, preventing its reentry into the cell. |
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Effect of mutations in pbr genes. |
Can cause increased transcription in the chromosomal encoded genes ZntA and Cad A (both homologs to PbrA - function to pump metal ions to periplasm) Not present in all mutants or in plasmid free. |
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Model of Pbr action |
1. When Pb(II) enters the cell, Pbr protein synthesis is initiated 2. PbrA (+ZntA and CadA) begin pumping metal ions to the periplasm where Pb(II) is precipitated with phosphates produced by PbrB. 3. Sequestration of lead discontinues the expression of the pbr operon, avoiding potentially harmful overexertion of PbrB *PbrC/D synthesis also initiated but not essential for resistance. |
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Is the Pbr operon lead specific? |
no - BUT contains lead specific element PbrB |
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What Pbr genes are necessary for full lead resistance? |
PbrRAB PbrA: divalent metal P1B-type ATPase PbrB: C55-PP Phosphatase |
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Common lead resistance mechanism |
active efflux followed by sequestration |
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Copper tolerance assay in P. putida |
Normal growth at or under 3 mM at 4 mM growth several reduced at 5 mM no growth |
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Heavy metal phenotype (P. putida) |
-Expressed in response to multiple heavy metals: strain shows growth in presence of nickel, copper, zinc and arsenic. -only susceptible to SbCl3 (antimony trichloride) |
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Genetic determinants of p. putted metal resistance |
CopA2: encodes P-type ATPase for importing copper CopA3: encodes P-type ATPase for exporting copper |
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How do Gallium based antimicrobials work? |
-Unlike iron, gallium cannot be physiologically reduced which inhibits biological actions (DNA/protein synthesis and energy production) -Reduces the expression of pyoverdine, resulting in increased gallium uptake and decreased iron consumption -mediated by transcriptional regulator pvdS |
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When is CueC expressed and at what levels? |
Low levels under aerobic and low to moderate copper concentrations. Higher levels under anaerobic, low copper concentration or high copper concentrations. Higher levels at low oxygen concentrations when CueR is knocked out |
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When is CopA expressed and at what levels? |
expressed at comparatively high level regardless of oxygen availability or copper concentration |
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When is CueO expressed and at what levels? |
-expressed at moderate concentration regardless of oxygen availability and copper concentration -increased activity in aerobic, high copper - expressed at high concentrations of copper in absence of oxygen |
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Why is CusC said to be expressed under "extreme stress" |
-only shows increased expression when stress is due to oxygen availability and copper concentration |
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How does the Cue system work and when does the Cus system come into play? |
-works because of higher toxicity of Cu(I) as opposed to Cu(II) -Oxidase (CueO) convers Cu(I) to Cu(II) -stops working in the absence of oxygen, causing Cu(I) accumulation, Cus is how to get rid of excess copper without having to oxidize it |
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MctB |
-mycobacterial Cu Transport Protein B (Cu(I) specific) -Outer membrane channel protein |
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MctB mutant |
-not more susceptible to reactive nitrogen or oxygen intermediates -susceptible to copper because of increased accumulation -mutant bacterial load is reduced > 100 fold compared to WT |
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Arsenate Reductase |
As(V) to As(III) |
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How many families of As reductases are there and how did they emerge? |
Three, through convergent evolution |
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Two forms of ArsC and explain differences |
-Spa ArsC: dependent on potassium and sulphate for stabilization, specific activity and substrate selection -BsArsC: activity independent of potassium and sulfate levels -differences due to aa difference in primary sequence that influence binding ability |
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What does arsenic exposure result in?
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ROS production and thiol redox reactions |
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As(V) efflux system |
-As(V) converted to As(III) -Mediated by two operons ArsRDABC and ArsRBC -Having both operons enhances As(V) resistance |
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Arsenic Operons |
ArsRDABC and ArsRBC
-ArsR: regulates transcription/expression -ArsC: converts As(V) to As(III) -ArsB: extrudes As(III) -ArsD: metallo-chaperone, delivers As(III) to ArsA ATPase |
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What metabolism does LSCJ7 use under arsenic exposure stress |
anaerobic nitrate respiration |
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LSJC7 arsenic exposure and reactive oxygen species |
-superoxide dismutase gene expression not increased -other genes encoding antioxidant enzymes down regulated indicates alternative methods against intracellular ROS |
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Important takeaway from LSJC7 transcriptomic analysis |
LSCJ7 evolved to utilize multiple strategies to combar arsenic exposure stress and its effects -implication: relation between antibiotic and metal resistance |
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Chromium impact on the cell |
-Promotes intracellular ROS -causes oxidative damage that then results in 8-oxo G lesions |
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Response of Sigma B to chromium stress |
upregulates: -KatA*, KatB, SodA (antioxidants) -oxidized guanine excision repair (MutM, MutY, MutT) -Efflux genes *KatA is the main protein responsible for antioxidant activity |
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Emerging resistance to antimony |
-thiols -all resistant strains had significantly higher levels of intracellular thiols |
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Resistance mechanisms to antimony |
-reduced uptake via AQP1 down-regulation -Increased efflux via P-type ATPase activity, P-gp pumps -Sequestration via thiols and MPRA |
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Mechanisms of Lichen Resistance to Metallic Pollution |
-metal rich particulate entrapment on the lichen surface and in intercellular spaces of fungal filaments -intracellular complexation to metallothioneins -extracellular complexation to functional groups of fungal macromolecules from the cell walls. -extracellular complexation to functional groups of fungal macromolecules from the cell walls extracellular complexation to organic acids (like oxalate) or lichen substances such as parietinic acid. |
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D. muscorum resistance to metallic pollution |
-hyper-accumulation of both Pb and Zn occurs via complexation with oxalate -2x higher concentration of contaminated D. mushroom than non-contaminated -undetermined if Pb and Zn oxalate salts secreted or kept inside cells but hypothesized that secreted |
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X. parietina resistance to metallic pollution |
-Pb resistance comes from complexation to the cell wall -Did not indicate which cell wall component Pb complexes to making it a weak finding, used alternative fungi known to complex with Pb at cell wall to confirm |
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What is Lichen |
Algae/cyanobacteria +fungus |
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Czc efflux systems |
widely present divalent cation antiporter |
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What is a riboswitch? |
non-coding RNA that binds to metabolite and regulates genes associated with pathways involving that metabolite |
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How does the NiCo Riboswitch work? |
If Co binds, the terminator cannot and genes are expressed. |
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Cooperative binding in NiCo Riboswitch |
-mutation of nickel binding sites reduces but does not completes remove its ability to bind cobalt |
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Genomic comparisons and X-ray crystallography of the NiCo Riboswitch |
-little consensus sequence but secondary structure highly conserved -P2 stem mutations result in loss of binding -Co and Ni are the only metals that induce structural change in cxc motif -P4 stem most variable - associates with cobalt binding sites by stabilizing anti-terminator region |
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Uranium contamination - bioremediation strategies? |
G. sulfurreducens and UVI radiation |
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Pili or Cytochromes implicated in U(VI) reduction? |
-no pili, no problem. -unlike iron, long range electron conduction through pili is not necessary for reduction of U(VI) -type c cytochromes are used, the number is proportional to the rate of reduction |
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Competence in Streptococcus pneumonia |
-Competence stimulating peptide (CSP) not required for basal expression of comCDE -ComD/E necessary for expression -Competence is coupled to growth rate |
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What is the importance of read through for competence in strep pneumonia |
-strong terminator upstream of CEbs results in a significant (4-5 fold) reduction in basal expression of comCDE and in spontaneous competence -read through allows maintenance of ComD and Com E |
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PhoP/PhoQ system |
-controls expression of SsrB/SpiR 2CRS -Ssrb (response regulator) controlled directly by PhoP binding to its promoter -SpiR (sensor) expression activated indirectly by PhoP post-translational modification |
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Ssrb/SpiR |
-controls expression of spi/ssa genes (type 3 secretion system) -essential in salmonella intramacrophage survival |
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mechanisms of antimicrobial peptides |
-membrane depolarization -membrane micellization |
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Important results from Investigation of the cytotoxicity of eukaryotic and prokaryotic antimicrobial peptides in intestinal cells in vitro |
-Gallidermin: most potent, least toxic. -Melittin: multiple mechanisms of toxicity |
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Class I Bacteriocins |
-substantial post-translational modifications -split into type A and B based on charge and mechanism of action |
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Class II Bacteriocins |
-not many post-translational modifications |
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Why is Clavibacter Michiganensis special? |
-only species in its genus, produces CmmAMP-1 -Michigianin A: Type I, Class B Lantibiotic -CmmAMP1 much larger, only 1 homolog in target bacteria -laughably premature b/c unknown structure and mechanism of action -may be viable as biocontrol agent - only Cms strains showed response to very low [] |
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How are AMPs harvested |
-natural extraction -chemical synthesis -genetic engineering |
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Why is prokaryotic expression of AMPs difficult? |
-they are toxic to their hosts and are susceptible to proteolytic degradation |
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What is the potential solution to difficult expression of AMPs in prokaryotes? |
Fusion tags - ubiquitin and SUMO Sumo = small ubiquitin related modifier (human derived) |
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What are fusion tags? |
-fusion tag is a short peptide or protein that can be fused to a target protein of interest to create a fusion protein |
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Give an example of an effective fusion tag production of an AMP |
-A20L (interacts with liposomes?) - both Ub and SUMO effective but Ub more so -high antibacterial specificity with negligible hemolytic activity against human erythrocytes -forms alpha-helical structure under hydrophobic conditions (such as within membranes) |
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Stringent response and biofilm development key results |
-ppGpp and RelA/SpoT -Peptide 1018 (synthetic IDR) targets (p)ppGpp and marks for degradation/actively degrades -broad spectrum activity indicates stringent response is widely used to signal biofilm development |
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Alvinella pompejana |
-forms alvinellacin, AMP, cause membrane permeabilization in the epibiont -Acts against gram (-) bacteria: epibiont is gram - -controls symbiotic microbes, not pathogens - selectively kills most dominant filamentous bacteria to prevent over proliferation -induced by archaea (thermococcus) |
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Epibiont |
-detoxifies reactive heavy metals and free hydrogen sulfide -interactions mediate host immunity, which shapes microbiota composition - but host must protect itself from inappropriate colonization and replication of symbiotic flora |
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Alvinellacin Structure |
-conserved BRICHOS domain: hydrophobic region (signal peptide) and C-terminal beta sheet (AMP) never seen in other AMP precursors, involved in post-translational processing -2 disulfide bonds -evolutionarily driven adaptation of worms to correctly fold AMP under extreme conditions |
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Methanogenesis in Ruminants |
-thermoplasmata undergo methylotrophic methanogenesis using methylamines as energy sources -Rapeseed Oil (RSO) supplementation targets thermoplasmata for methane mitigation (reduces methane emission and decreases Methyl coenzyme M reductase (MCR) mRNA) |
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SusD |
-Sensing -functions that are dependent and independent of the binding site -homologs highly conserved in bacteroides app. and with SusC may provide fitness in the gut |
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Sus C |
-transport -along with susD provides fitness in the gut |
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SusG |
-hydrolysis |
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SusE/F |
-help in attachment of starches but role is less critical for growth |
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Purpose of Sus system |
The Sus includes the requisite proteins for binding and degrading starch at the surface of the cell preceding oligosaccharide transport across the outer membrane for further depolymerization to glucose in the periplasm SusD, C, G, R critical for growth |
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Bioinformatic tool used to catalogue and define transcription factor families |
Predicted Prokaryotic Transcription Factors (P2TF) |
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P2TF Process |
-proteomes screened for presence of conserved domains using SMART and Pfam domain profiles -proteins with hits to appropriate domain profiles are then assigned to specific categories and subcategories of TFs and annotated with domain architecture and classification results -Results of TF analysis can then be viewed as an interactive webpage or exported in a user-defined format |
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P2TF Categories |
-TRs: transcriptional regulators -OCSs: one-component systems -RRs: response regulators -SFs: sigma factors |
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Broad Response AMP |
targets ppGpp and RelA/SpoT pathway and marks for degradation/actively degrades stringent response - biofilm development |
