Last min flashcards

Question 1

FISH probe design

Answer 1

Length - 15-25
Mismatches - NO., quality, position (middle = ok)

Question 2

Probe mismatch qualities

Answer 2

Destablaising: A-A, A-C, T-T, T-C, C-C
Slightly destabalising: G-T, G-A, G-G
Need to be aware of GC stretches

Question 3

Synthetic analogues

Answer 3

LNA-FISH (locked) - increased specificity and sensitivity

PNA-FISH (peptide) - commonly used, not -vly charged, 15bp, less stringent hybridisation conds

CARD-FISH (catalysed reporter deposition) - signal increased because of HRP (horseradish-peroxide), more stringent, incubated with fluorecently labelled tyramide

CLASI-FISH (combinational labelling and spectral imaging) - combo for mixed population, 15 possible combos, BUT may be dificult due to mixed hybrid conds

For metabolism:
MAR-FISH (micro auto radiography) - uptakw of radiolabelled substance in food, detected by MAR in combo w FISH

ISRT-FISH (in situ reverse transcription) - specific mRNA amplified by qPCR to cDNA, targeted by flourescent + probes, used to study metabolically active groups + control gene expression in population BUT not as useful for mixed communties

Question 4

QS in P. aeruginosa

Answer 4

Relies on Las system:
LasR - regulator - master QS regulator (also effects Rhl system)
LasI - synthase - encodes odDHL (homoserine lactone)
odDHL (C12) upregulates expression of virulence genes + causes activation of second system: Rhl:
RhlR = regulator
RhlI = synthase - synthesises second homoserine lactone - BHL (C4)
RhII isnt just upregulated by Las syste,, requires pqs system

Question 5

PQS system

Answer 5

psudenomas quinalone signalling system - involved in iron metabolism
PqsA = autoinducer synthase - produce HHQ (precursor for pqs)
pqs = upregulates pqsA directly + via pqsR, promotes Aqs biosynthesis - gene involved in iron starvation response +virulence factor production
PqsE = -vely regulates pqsA

Question 6

PNMs/dynorphin A

Answer 6

PMNs produce dynorphin A - induces pqs + rhamnolipids - this makes up PMN shield

Question 7

Quorum quenching

Answer 7

Bacillus produce lactonase - cleaves lactone ring
acylase - cleaves middle
oxoreductase - modifies R groups

ways of inactivating p.aroginosa via odDHL and BHL

Question 8

Delisa pulchra

Answer 8

Red seweed
has halogenated furanone compounds- strong biological activity including anti-QS + antimicrobial
stop biofilm formation
lead to creating of synthetic analogues (C30+C56)

Question 9

Autoinducer 2 family

Answer 9

e.g. invibro bacteria:
precursor ——-> (methyltransferase) ribsylhomosystine ——-> (luxS) DPD —–> (cyclises into) ProAI-2 —–> (borate presence) AI-2 (S-THMF) (enters cells throughj LuxP receptor) - sets off phosphorelay luxQ-luxU-LuxO -> regulation of LuxR (negative reg)

in e.coli no borate use as not aquatic - make R-THMF - goes througj lsRB receptor and CsRABC transporter system ending in p of LuxR (-vely regulates)
Switch off these systems -> increase in QS genes due to increase in LUxR

Question 10

QS in Burkholdoria cenocepacia

Answer 10

4 systems:
1. CepIR, cciIR systems
2. burkoldiaria diffusable signal factor (BDSF)
3. non-ribosomal peptide synthase-lile cluster
4. CepI - interplays w AHL, cep..,

Question 11

Diffusable signal factors

Answer 11

Family - BDSF (Burkholderia C12), DSF (Xanthamonas capestris C11), CDA (p.arogenosa C10)
e.g. X-campestris
DSF interplays w sensory domain of RpfC
RpfC p’s RpfG
RpfG involved in reg of RpfB
RpFB involved w/ RpfF production of DSF
DSF leaves cell

DSF originates from leucine, BDSF carbs

Question 12

Gram positive QS

Answer 12

encoded by global reg locus Agr
Agr encodes 2 operons controlled by promoters P3+P3
P2 encodes 4 genes: AgrA,B,C,D
A= autoinducer peptide (AIP) (when phosphorylated)
B membrane translocating AgrA out of the cell
C = receptor tha brings Agr into cell

P3 encodes for reg of transcription of an RNA molecule RNAIII

Question 13

Features of the gram positive autoindicer peptide (AIP) AgrA

Answer 13

usually 8 AAs (7-9)
Cyclic
share common cystine 5 from N terminal
cystine forms catalytic thioester bond

Question 14

summary of AIs/AIP

Answer 14

AI1 - based on HSLs
AI2 - based on THMs (w or wo borate)
AIP - have specific receptors

Question 15

define rhizosphere

Answer 15

habitat below ground colonized by microbes

Question 16

Define phyllosphere

Answer 16

the areal plant habitat colonized by microbes

Question 17

Define phylloplane

Answer 17

the leaf surface where micribes love

Question 18

What are inhibitants of the phylloplane called?

Answer 18

epiphites

Question 19

compare colonization of S. enteria vs e.coli

Answer 19

S enteria - easily attacks and colonizes roots of alpha alpha and capable of colonizing seeds

E.coli - cant do this as easily

Question 20

commin human gut pathogens found on fresh produce

Answer 20

E.coli O157:H7, salmonella, Campylobacter spp, listeia, shigella

Question 21

attachment mechanisms to the phylloplane

Answer 21

• flagellum can anchor to surface
• fimbraie and pili typically longer and fewer
• salmonella are capable of producing cellulose
• H and ionic bonding aid attachment

Question 22

salmonella curli amyloid formation

Answer 22

RpoS controls expression of cgsD

cgsD (curli formation) is able to switch on adrA and bap

adrA partially controls celulose production by cyclic-di-GMP

Question 23

Which monitoring model does NOT have the disadvantage of having a flat coupon on curved surface

Answer 23

Chemostat model

Question 24

Which model allows you to alter water quality and shear stress

Answer 24

annular reactor

Question 25

which phage takes the capsid into the cell

Answer 25

dsRNA genome would be degraded if not
Phi6 - uses RNA-dependent RNA polymerase (RdRP) to transcribe dsRNA into positive sebse RNAs so can exit capsid

Question 26

Individual decisions and voting

Answer 26

phage lambda
1. phage infects cell, increasesCll
2. Cll leads to Cl, leads to lysogeny
if Cll gets turned off get lysis pathway as no repression of Cro

voting: when more than one phage infects, decision for lysogeny has to be unanamous

Question 27

Group decisions

Answer 27

e.g. phi3T
during infection, phages produce protein AimP
AimP cleaved to arbitrium
This leaves cell and is taken uop by other cells via OPP transporter

low levels of arbitrium - lytic cycle
high - lysogenic

Question 28

Molecular mechanisms of Arbitrium

Answer 28

Aim R and Aim P = early genes expressed immediatly upon infection
aimR activates aimX expression
AimX is an inhibator of lysogeny -directing cell to lytic cycle
AimP processed to produce arbitrium - internalised into other cells via OPP
AimR binds to arbitrum molecules and cannot activate expression of AimX therefote lyspgeny favoured

Question 29

Chronic state infections:

Answer 29

e.g. F1 uses bacterium machinery to replicate

Question 30

class 1 CRISPR-cas systems

Answer 30

Type I - cas 3, dsDNA
TypeIII - cas10 ssDNA, RNA
TypeIV - csf

Question 31

Class 2 CRISPR-as systems

Answer 31

Type II - cas9, dsDNA requires tracer RNA - RuvC domain

Type V - cas12, dsDNA, RuvC

Type VI - cas13, ssRNA

Question 32

CRISPR defence by collateral damage

Answer 32

Cas10 - HD domain DNA, Palm domain creates cOA, cOA activates csm6 (binds to CARF domain, activates HEPN domain)RNA, -ve reg by csm3 (DNA+RNA, dormancy +suicide)
Cas12a - RuvC domain, blocked by rec lobe till target recognition, DNA - suicide
Cas13a - HEPN domain RNA - dormancy

Question 33

target DNA vs RNA (CRISPR)

Answer 33

Cas3,9,12 DNA
Cas10,13 RNA

Question 34

Blunt vs staggered ends Cas systems

Answer 34

Blunt = Cas9
Staggered = Cas12a

Question 35

Single nucleotide editing

Answer 35

Cas9 nickase fused w cytidine deamainase

for increased editing efficiency - two uracil glycosylase inhibators (UGIs) fused

Question 36

Catalytically inactive Cas13

Answer 36

binds to target DNA but doesnt destroy
1. RNA visualisation + tracing
2. Adenosine deaminase acting on RNA (ADAR) fused w dcas13 or RNA A->I editing to correct disease related mutations

Question 37

CRISPR i vs a

Answer 37

i = transcription repression: dcas9 alone or fused w effectorsne.g. KRAB can be targeted to promoters, 5’ untranslated regions (UTRs) or enhancers
a = transcription activation: achieved by fusion of dcas9 to transcription activation domains to achieve expression of specific genes

Question 38

Evasion of host immunity using CRISPR cas: e.g. in Francisella novicida

Answer 38

Evasion of host immunity: Cas9 + tracerRNA + scaRNA altogether target an endogenous transcript encoding:

immunostimulatory bacterial lipoprotein (BLP)
leads to mRNA degradation and decreased transcript levels

in the absence of this regulation -> increased BLP -> activation of TLR2 dependent proinflammatory response -> attenuation of bacteria during infection

Question 39

Regulation of biofilm by CRISPR cas

Answer 39

P. aeroginosa - interacts to inhibit creation of biofilms upon being lysed

Salmonella - Cas3 gene doenregulates expression of genes involved in degradation of QS molecules and tehrfore promotes forn ation of biofilm

Question 40

Anti CRISPR-cas proteins

Answer 40

Acr
transcribed by Aca

Question 41

unique about type iV R-M systems

Answer 41

no methyl transferases only restriction endo, cleave methylated forign DNA

Question 42

Whivch has a higher level of stability out if REase and MTAse

Answer 42

REase - why host cell can become addicted

Question 43

Solo or orphan enzymes (methyltransferases)

Answer 43

DcM (5-sytosine) and Dam (6methyladenine)

Question 44

How do BREX ad Type IV R-M provide complementary protection

Answer 44

BREX prevents infection fron non-m ethylated phages
R-M IV (BrxU)frim methylated phages

Question 45

DISARM

Answer 45

Class 1 and 2 share drmABC
Class 1 - drmMI (andenine methylase) and drmD (heloicase domain)
Class2 - drmMII (cytosine methylase)

DNA recognised by autpinhibition of drmA/B when DNA substrates containg 5’ overhang cause dislodging of trigger loop

Question 46

CBASS

Answer 46

abortive infection counteracted by phages with Abc1 (e.g. from phage 4T)