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Microbial Metabolic Regulation

by: Sunny Rau

Microbial Metabolic Regulation MB 714

Marketplace > North Carolina State University > Microbiology > MB 714 > Microbial Metabolic Regulation
Sunny Rau
GPA 3.65

Amy Grunden

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Amy Grunden
Class Notes
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This 8 page Class Notes was uploaded by Sunny Rau on Thursday October 15, 2015. The Class Notes belongs to MB 714 at North Carolina State University taught by Amy Grunden in Fall. Since its upload, it has received 21 views. For similar materials see /class/223843/mb-714-north-carolina-state-university in Microbiology at North Carolina State University.


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Date Created: 10/15/15
Microbial stress resp onselec 2 2 Slide 3 Cells enzymatically get rid of superoxide and hydrogen peroxide Superoxide is least reactant Will not directly damage protein or lipids or dna Does not have enough oxidation power to do this But it does interat with fes cluster in proteins and damages them Will release fe which can react with h202 which can spontaneously make hydroxyl radical which can damage ever macro molecule fenton rxn Will make replacement enzymes that do not contain fes clusters when cell realizes has rosin cells Also produce enzymes that repair damage In order for cells to turn on this expression has transcriptional regulators to do this Slide 4 Two different transcriptional regulators that response to different ros Nadph is primary source of reductant for repair Slide 5 How recognize oxidative stress and how respond to make it activated soxR initial responds to oxidative stress and goes on to turn on sox S which turns on host of genes that is turned on in presence of superoxide under normal nonoxidative expressions soxS is turned off Slide 6 Normally soxR has a two fe cluster in it 1 is in red form2 and other oxidized at 3 Binds to promoter region in front of soxS 35 and 10 promoter regions are on opposite sides Normally transcription of soxs is not done because of this when S0 enters cell the red fe center is oxidized and it chanes conformation of sox R which causes 35 and 010 region to swivel so on same sides of dna which allows map to bind and turn on soxS which activates all SO induced genes once SO gone nadph reduces the oxidated fe center so it rechanges conformation and turns off soxS expression slide 7 OxyR systems responds to h202 oxyR is transcriptional regulator Can hang out and bind promoter regions that are h202 responders under unstressed conditions I is hanging out but not promoting transcription Cannot help with open conformation it is stalled there but this is good because it allows map to bind quickly once oxidative stress is present oxidative stress causes oxyR conformation to change which allows dna to open and ngA is activated for h202 stress slide 8 normal nice reducing conditions of cell the cysteines are reduced Under oxidative stress conditons the cysteines are oxidized and form disulfide bridge Which can modify structure of proteins Modified struc of oxyR is able to interact with dna and change it to open conformation Slide 9 Needs to reset oxyR once stress gone Does this using GrXA protein can rereduce the oxyR by reducing the cysteine Slide 10 HSP in E coli Cells proteins have been denatured so other things can cause HSP that are not heat OH s solvents Chaperones are turned on at high numbers interact with proteins that are not folded properly And holdases that help refold misfolded Proteases for proteins that cannot be refolded Controlled by RpoH sigma 32 Not controlled at initiation level but what changes is whether or not message gets translated or not Will get turned over rapidly and degraded rapidly Antisig factor that controls Slide 11 Sigma 32has a lot ofpromoters in front ofit to control it 3 sigma 70 promoters housekeeping and a sigma E promoter responding to heat shock in periplamsic space in cells 1St clue that things are going badly for them level of rpoH transcript does not vary during times Rifampicin inhibts transcription heat shock and treat cells with rifamipicin see no change in heat shock bc rpoH around However ifuse one that inhibits protein syth do see problems with heat shock Motifs in a and b are important to affecting wheter or not transcript gets translated Slide 12 form a thermosensor under normal cond have 2ndary struc and SD seq is sequestered so will not get translated at high temp the 2ndary struc melts and the SD seq is available and can get translated Slide 13 at nonheat shock temps has some sig 32 that is translated but when not undergoing heat shock plenty of dnajk that will snag it Regions on sig 32 have high affinity for K and I escort sig 32 to ftsh which is a protease and is turned over Keeps hsp from being expressed when no heat shock under heat shock cond Gives rise to unfolded proteins so I and K are busy so sig32 binds rnap and activation of HSP occurs Slide 14 proteins that bind to dnaK fused in lacZ at different regions of rpoH gene Slide 1 1 Rpoh region is completely intact Get heat induction shut off heat induction and half life of sig 32 is short As soon as heat shock is over the 32 is turned over Progressively made fusion to earlier pointsonce fusion to aa 121 saw much greater half like for sig 3 2 even after shut off of heatshock So can mutate this region to have heat shock responses at all time but cells are sickly bc of this 5 same as before under normal cond rpoh transcript is ready to go but not translated bc of thermosensor sd seq when higher temp get melting of secondary struc and ribosome binds and translation competiion for sig32 bw rnap and dna K and I so under HS cond I and K bind unfolded proteinssig 32 binds rnp and heat shock genes turned on will see overlapses in stress response So if have large oxidative stress will kick on HSP so soxRS and oxyR is turned on slide 16 Slide 1 Slide 1 Slide 1 hs response for proteins in periplasmic space controlled by sigma e will turn on genes meant to func in periplasmic space 7 under non HS cond anti sig called rseA bound through membrane is sensing what Is occurring in periplasmic space So sigE is bound to rseA RseB caps rseA protects it from degradation Normally Under HS cond RseB has high affinity for degraded protein and will interact with it rather than capping rseA So RseA is exposed which is good target for degS which degrades it so sig E is released to turn on transcription ofits set ofperiplamsic HS proteins Repsone will happen quicker than repsone in cell bc these proteins are the front line defense Activation to repression 8 gram pos bacteria HrcACirCe systems Hrca binds circe elements infront of chaperone genes Non heat shock Hrca protein sits on promoter element circe and prevents expresson Ino rder for this to happen the hrca protein cant fold by itselfit requires chaperones to help fold it and the chaperones to help fold it are only available under nonheat shock cond So when folded properly is a func repressor Under heat shock hrca is still produced but will not be foled prop bc chaperones are busy elsewhere to bind misfolded proteins so promoters are not bound Repression control to activation 9 stringent response cells response to nutritional stress Spec aa starvation when cells undergo nut stress they make alarmmone guaninetetraphosphatewill create drop in ribosomal and expression of aa biosynth goes up expression of ribosomal rna trna goes downbuilds up Folds aa so it can go back to translation Uncharged tRNA sits in a stie and this signals stringent response Slide 20 relA 1 attached to ribosomehas an uncharged trna sit in a site will convert gtp and atp to pppGpp alarmone will redirect transcription in spot will make ppGpp takes gdp and make ppGpp Ifknock out relaspoT will not grow wo aa supplement Slide 21 rela bound to ribosome normally when uncharged trna sit in a site will start converting gtpgdp Keep making alarmone until charged trna bumps it out of a site ppGpp redirects rnaP away from making 16 23 and SS rnas so rrn operons have p1 and p2 as promoters rnap is being prevented from transcribingneg control pos control thought that ppGpp is potentially helping threonine or histidine biosyth operonsmakes binding tighter and changes to open conformation 23 under normal growth cond rnap on rrna operon but in high ppgpp the rnap is being activlty helped to bind aa operons 24 what has to happen when cells responding to stresses there must be a massive change from growth to stationary phase cell needs help transitioning from growth to stationary So uses alarmone dksA to help redirect transcriptional control Uses anti sig to bind sig 70 to ull off and relaced b other si factors depends on what is causing it to go into stationary phase that dependson what sig factor is bound 25 reprogramming transcriptional response of the cell 26 universal stress protein UspA cold chock uspA levels went down Is bc guanine tetraphosphate is very important in making uspA levels So in all other stresses it goes up and uspa goes upcold school ppgpp goes down and uspa levels go down when cells are growing normally in fatty acid synthetic modewhen starved use fatty acid to start growing and so FadR is turned off FadR on during normal and uspA is not expressed produced when cell experiencing dna damage Mutation in ftsK chromosomes safely in new cell damages dna and turns on recA which upregulates uspA uspA helps maintain DNAhelps facilitate repair of DNAupon phosphorylation it is turned on Bacterial biofilm formation Slide 1 Biofilms are propably natural state ofbacteria Everything else is a planktonic state Slide 4 Surface attached bacteria As result ofadhering to surface they make a glue out of sugar that cements them in place Grow and devide and make more glue and stay in place first documented from marine organisms normal for marine organissm to try and biofilm slide 5 medical microbiology perspective Making biofilms in parts ofbody not wanted Heart values bone biofilm surgeriesbiggest danger is opportunity for biofils to deelop in body from contanmination Dental cavities antibiotics issue with access Organisms are cemeted in layer and prevents access from antibiotics Are heterogenous physiologyso have org all in different growing states so some are able to deal with the antibodies Industrialized processes and water quality because they are able to foul pipes Can completely clog the pipes Ifit is supposed to sterile it will not be Slide 6 heterogenous in makeup various types of organisms Organisms would be mixed together Different organisms will aggregate together based on shared metabolisms One org making metabolite other can use and so forth anaerobic layer is probably most stressed layer and getting acids and other waste products from aerobic layer Not receiving nutrients more than likely Slide 7 v cholerae see uroscne each ind cell creates Bluemonolayer of cell yellow greencongegating Biofilming slide Glass box that has a pump assoc with it pumps liquid across surface of slide Takes atleast 24 hours to create a classic mounding of biofilm Slide 8 in native biofilm will see heterotrophs on surface and then see org dealing with lower levels of nutrients autotrophs will be inner surface Esp true of anaerobes will be deeper slide9 deadlive stain Greenlive reddead yellowlive on top of dead cells vicer versa S gordonii produces hydrogen peroxide forms foundation All of these are good to have Under periodontal disease some of the engative bact form biofilms in mouthcampylobacter and fungi Slide 1 slide 1 Slide 1 Slide 1 Smokers have different oral biofilms that nonsmokers 0 why they biofilm more than likely combo of all four 1 when in biofilsm they are more resistant to shear forces GI tract ora bladder ora can deal with changes in pH and oxidative damage much better are they protected bc of the eps layer or are the cells in biofilm expressing genes that can deal with the stresses better Believed bc cells are more poised to respond to stress Cells in biofilms grow more slowly than planktonic cells Bc access to nutrients are limited by having eps layer and fact that cells in bio m can have host ofphysiologies Are they exhibiting stress genes bc in biofilm or going into biofilm bc they are stressed 2 when biofilms are nutritionally deprived the cells will move out of the biofilm and become planktonic Regulators responding to nutrient availability 3 when cells biofilm they do typically become competent to transfer dna Plasmid exchange conjugation exchange chromosomal or fplasmid conjugation important bc using sex pilus it can persist long enough to do dna exchange altruism cell will opt to die when nutrients become limited and other cells can use these nutrients Programmed cell death Capsacisenzyme that does apoptosis Toxinantitoxin compounds to poison themselves Evolutionary biologists say its crap bc goes against selfpreservation slide 14 slide 1 Slide 1 if cell sees a surface it will stick to surface as long as nutrients available in environment Within a lab we grow them in ways that retards biofilms 5 psuedomona start with plankotonic cells What is the signal for biofilms Maybe have diff organisms waiting on different mechanisms Maybe has to do with nutrient avaialbe on surface Others say seeing stress genes active The biofilm is a stress mechanism when first infect org if cell planktonic will be more at mercy of our immune system Biofilm helps protect if from immune systems prob combo of two 2 101 agella is madecells are lonely and then cells see others and start mounding Once mounded they signal to make eps and are then able to cement itself 6 use agella to get to surface to make monolayer type 4 pili become imp at this point Allows ind vcells to start moving on top of each other Do not use agella bc they move too fast when they use them Then start getting gmound formation After mound get cell signaling for pseudo to make sugar algniate Only need agella and type 4 pili for early biofilm Slide 18 pila mutant Get monolayer but do not get charastically mounding that normally see in wild type Not as resistance to antimicrobials than wt potentially use down regulation ofpili to treat pseudo infections slide 19 e coli needs agella then uses type 1 pili for mounding om proteim Ag43 req for biofilm grown in minimal media not in rich media expression controlled by oxyRsenses h202 now sure how overlaps dam methylation controlled insynce with cell regulation slide 20 uses agella and then type 4 pili can make mutation in u ili and w ml ill still 39 et mature vibiro biofilm slide 21 all maturation for all organisms The cells start making copious amounts of eps Actual sugar is diff for each org slide 23 ecidence can get massive biofilms if get cells to overexpress eps slide 24 all initially use agella and all need to use pili vibiro is only one that needs eps at early formation slide 25 what prompts cells to leave biofilms nutrient depravation slide 26 most antibtioic req active metabolism in order to func Metabolically inert cell will not be affected by antibiotic Persister cells stay in biofilms and something causes them to become active and sees nutrients Switches them to metabolically active and have biofilm still Slide 27 biofilms chock full of microbes with different physiologies if evaluating gene expression in biofilms and looking at surface cellsthe gene expression will be diff from cells within inner parts of biofilm Aerobic vs anaerobic nutrient limitiations toxic cmpds from aerobic parts to anaerobic iflook at literature only 2 genes in common have been shown to give same expression exponse e coli but what type ofplankotonic cell using Stationary phase or exponential phasesurface exponenetial innerstationary


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