Mutation and diversification

Question 1

how are the colonies of P. aeruginosa DNA mismatch repair deficient strains different

– ∆mutS
– ∆mutL

Answer 1

P. aeruginosa mutator strains have:
* Large colonies
* Enhanced microcolony development
* Increased Biofilm biovolume is increased

Question 2

what is himA?

Answer 2

integration host factor

Question 3

what are mutL and micA?

Answer 3

mismatch repair proteins

Question 4

what is recN

Answer 4

DNA repair protein

Question 5

what is recQ?

Answer 5

DNA helicase

Question 6

what is recR?

Answer 6

recombination protein

Question 7

what is sss?

Answer 7

site specific recombinase

Question 8

what is ung?

Answer 8

uracil DNA glycosylase

Question 9

what is uvrC?

Answer 9

excinuclease subunit

Question 10

what is xseA?

Answer 10

exodeoxyribonuclease

Question 11

the more genetically unstable the strain the higher the….

Answer 11

…The more genetically unstable the strain the higher the propensity to form biofilms and microcolonies and structures.

Question 12

what type of mutations were observed in pseudomonas aeruginosa biofilm?

Answer 12

only single nucleotide mutations (SNPs)

non-coding/ intergenic regions
- coding regions
- silent/ synonymous (e.g. third codon position)
- missense (aa change or stop codons)

no apparent recombinations or large deletions/insertions

Question 13

what are non synoymous mutations

Answer 13

Nonsynonymous mutations have a much greater effect on an individual than a synonymous mutation. In a nonsynonymous mutation, there is usually an insertion or deletion of a single nucleotide in the sequence during transcription when the messenger RNA is copying the DNA.

Question 14

how can we visualise where in biofilms mutations are occuring

Answer 14

Mutation detection in-situ
GFP +1 Reversion system pMDGFP – frameshift mutation in the GFP of the gene, then added this form of the gene back into the pseudomonas aeruginosa. Then form a biofilm and using this strain (harbouring non fluorescent GFP then if a mutation occurs in the region of the gfp gene that knocks it back into frame cause it to fluoresce again.
This is a reporter system to enable detection of frameshift events in real time that were visualised with epif or confoc microscopy

Question 15

what is the difference in mutation frequency between microcolony and non microcolony cells?

Answer 15

Increase in mutation frequency up to 100 - 1000 fold compared to non-microcolony cells

Question 16

biofilm formation is analogous to what other biological process

Answer 16

Clonal succession and solid tumour development

Question 17

what portion of the microcolony change their physiology to bceome motile and swim out

Answer 17

the interior portion

Question 18

what is Pseudoalteromonas tunicata (D2)?

Answer 18

• Obligate marine bacterium
• Colonizes marine living surfaces (e.g. Ulva lactuca)
• Produces a range of bioactive compounds that inhibit marine fouling organisms (e.g. invertebrate larvae, algal spores)
• Forms microcolony-based biofilms in the laboratory and in-vivo.

Question 19

what protein is repsonsible for the mechanism of cell death within the biofilm that appears wihtin 3 days beginning detachment?

in p.tunicata

Answer 19

AlpP

Question 20

what happens if you mutate AlpP

Answer 20

you dont have cell death in the centre of colonies

Question 21

what is the mode of action of AlpP?

how is this detected in the lab

Answer 21

• Production of hydrogen peroxide from L-lysine
• L-lysine + O2 + H2O  6-amino-2-oxo-hexanoate + NH3 + H2O2

• Amplex Red reagent reacts with H2O2 in a 1:1 stoichiometry to produce the red fluorescent oxidation product resuforin

Question 22

AlpP occurs in several ……. organisms

Answer 22

AlpP occurs in several Gram negative organisms

Question 23

what does AlpP and its homologs do ?

Answer 23

AlpP and its homologues produce H2O2 and induce lysis, dispersal, and phenotypic variation

Question 24

what gene triggers dispersal in p.aeruginosa

Answer 24

nirS - nitrite reductase

Question 25

what is the p.aeruginosa response to low levels of NO

Answer 25

Motility increase flgG, pilAGHIMOQZ Adhesion and biofilm relevant decrease cupP, cupC, EPS Pel genes (PA3058 – 3064), alg genes. **Low concentrations of NO induce an overall decrease in c-di-GMP levels, turn off biofilm traits and trigger dispersal in P. aeruginosa** increase Genes containing GGDEF or EAL domains – cyclic-di-GMP tunover? PA1181 (GGDEF/EAL), bdlA (EAL), PA0575, PA2072 (GGDEF/EAL), arr (EAL), morA (EAL), PA0290 (GGDEF), PA5442 (GGDEF/EAL)

Question 26

Nitric oxide induces ____ of P. aeruginosa biofilms

Answer 26

Nitric oxide induces dispersal of P. aeruginosa biofilms

Question 27

high cyclic-di-gmp gives what phyicology

Answer 27

biofilm

Question 28

low cyclic-di-gmp levels lead to what physiology

Answer 28

planktonic

Question 29

what are the conserved domains for cyclic di gmp turnover

Answer 29

GGDEF - guanylate cyclase EAL - phosphodiesterase

Question 30

what binds and responds to NO in both eukaryotes and prokaryotes

Answer 30

PAS or H-NOX

Question 31

what causes cystic fibrosis

Answer 31

* Autosomal recessive mutations in CF transmembrane conductance regulator (CFTR) * Increased mucus production from goblet cell hyperplasia & poor ciliary clearance = ideal environment for bacterial colonisation

Question 32

whats the primary cause of morbitity and mortality in cystic fibrosis

Answer 32

onset of chronic and persistent bacterial infections

Question 33

delivery of low dose NO or NO donors to the CF lung can do what?

Answer 33

Delivery of low dose NO or NO donors to the CF lung will significantly reduce carriage of Pseudomonas aeruginosa by reducing antibiotic tolerance of the biofilms. This may: * enhance efficacy of standard antibiotic regimes, especially ceftazidime/tobramycin * reduce infective treatment of exacerbations & overall antibiotic burden * improve respiratory function * improve quality of life

Question 34

what features of biofilms contribute to antimicrobial tolerance

Answer 34

* **Matrix impedence** e.g. gent * **Enzymes** e.g. β-lactamase * **Biochemical changes**, e.g. periplasmic glucans * **Metabolic gradients** e.g. Ciprofloxacin (replication) & tobramycin (translation) only kill cells in metabolically active top layer * **Physiological subpopulations**, e.g. persisters

Question 35

what methods did the PseudoCAP project use to deep sequence the pseudomonas biofilm population to observe genome evolution

Answer 35

- Harvest cells of 8/9 day -old PA01 biofilm - Extract total DNA from cell pellet - High throughput PYROSEQUECING – 50x coverage - Use alignment software software to crossmatch against Pseudomonas reference genome - Automated detection and manual check of differences - Mutations compared to predicted gene functions to predict impact

Question 36

which genes are affected by non-syn mutations?

Answer 36

* **Regulatory proteins**: PA4341 probable transcriptional regulator; PA2897 probable transcriptional regulator; PA0928 sensor/response regulator hybrid; PA2492 transcriptional regulator MexT * **Energy generations**: PA263 NADH dehydrogenase I chain C,D; PA0106 cytochrome c oxidase, subunit I; PA4811 nitrate-inducible formate dehydrogenase, beta subunit; PA1549 probable cation-transporting P-type ATPase * **Transport**: PA5434 tryptophan permease; PA0138 probable permease of ABC transporter; PA2042 probable transporter membrane subunit; A5287 ammonium transporter AmtB; PA3193 glucokinase, * Others: PA0176 aerotaxis transducer Aer2; ftsH cell division protein FtsH + 7 hypothetical proteins

Question 37

what did the GFP +1 Reversion system pMDGFP reveal about where mutatiosn occur

Answer 37

the GFP based mutation detection showed that these mutations occu much more frequently in microcolonies **Shows that GFP reversion occurs in the microcolonies and not in the non differentiated biomass**

Question 38

microcolonies are foci for....

Answer 38

Microcolonies are foci for genetic mutation and evolution

Question 39

microcolony growth seems to involve ______...______ processes and ______...______ selection

Answer 39

seems to involve competitive processes and mutation selection

Question 40

describe a model for microcolony development | based on cloncal succession

Answer 40

Bacteria on surface, one acquires mutation that confers advantage, this bacteria clonally expands. Then gets another mutation and the same thing happens, and this occurs in waves of clonal expansion

Question 41

what is alpP? | how big is it and when can it be detected?

Answer 41

The P.Tunicata autolytic protein AlpP Size: 190 KDa Detected in the waste effluent of biofilms ≥ 3 days old.

Question 42

what is the the red fluorescent oxidation product of amplex red with H2O2?

Answer 42

resuforin