Mutation and diversification Flashcards
how are the colonies of P. aeruginosa DNA mismatch repair deficient strains different
– ∆mutS
– ∆mutL
P. aeruginosa mutator strains have:
* Large colonies
* Enhanced microcolony development
* Increased Biofilm biovolume is increased
what is himA?
integration host factor
what are mutL and micA?
mismatch repair proteins
what is recN
DNA repair protein
what is recQ?
DNA helicase
what is recR?
recombination protein
what is sss?
site specific recombinase
what is ung?
uracil DNA glycosylase
what is uvrC?
excinuclease subunit
what is xseA?
exodeoxyribonuclease
the more genetically unstable the strain the higher the….
…The more genetically unstable the strain the higher the propensity to form biofilms and microcolonies and structures.
what type of mutations were observed in pseudomonas aeruginosa biofilm?
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
what are non synoymous mutations
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.
how can we visualise where in biofilms mutations are occuring
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
what is the difference in mutation frequency between microcolony and non microcolony cells?
Increase in mutation frequency up to 100 - 1000 fold compared to non-microcolony cells
biofilm formation is analogous to what other biological process
Clonal succession and solid tumour development
what portion of the microcolony change their physiology to bceome motile and swim out
the interior portion
what is Pseudoalteromonas tunicata (D2)?
- 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.
what protein is repsonsible for the mechanism of cell death within the biofilm that appears wihtin 3 days beginning detachment?
in p.tunicata
AlpP
what happens if you mutate AlpP
you dont have cell death in the centre of colonies
what is the mode of action of AlpP?
how is this detected in the lab
- 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
AlpP occurs in several ……. organisms
AlpP occurs in several Gram negative organisms
what does AlpP and its homologs do ?
AlpP and its homologues produce H2O2 and induce lysis, dispersal, and phenotypic variation
what gene triggers dispersal in p.aeruginosa
nirS - nitrite reductase
what is the p.aeruginosa response to low levels of NO
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)
Nitric oxide induces ____ of
P. aeruginosa biofilms
Nitric oxide induces dispersal of
P. aeruginosa biofilms
high cyclic-di-gmp gives what phyicology
biofilm
low cyclic-di-gmp levels lead to what physiology
planktonic
what are the conserved domains for cyclic di gmp turnover
GGDEF - guanylate cyclase
EAL - phosphodiesterase
what binds and responds to NO in both eukaryotes and prokaryotes
PAS or H-NOX
what causes cystic fibrosis
- Autosomal recessive mutations in CF transmembrane conductance regulator (CFTR)
- Increased mucus production from goblet cell hyperplasia & poor ciliary clearance = ideal environment for bacterial colonisation
whats the primary cause of morbitity and mortality in cystic fibrosis
onset of chronic and persistent bacterial infections
delivery of low dose NO or NO donors to the CF lung can do what?
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
what features of biofilms contribute to antimicrobial tolerance
- 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
what methods did the PseudoCAP project use to deep sequence the pseudomonas biofilm population to observe genome evolution
- 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
which genes are affected by non-syn mutations?
- 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
what did the GFP +1 Reversion system pMDGFP reveal about where mutatiosn occur
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
microcolonies are foci for….
Microcolonies are foci for genetic mutation and evolution
microcolony growth seems to involve ______…______ processes and ______…______ selection
seems to involve competitive processes and mutation selection
describe a model for microcolony development
based on cloncal succession
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
what is alpP?
how big is it and when can it be detected?
The P.Tunicata autolytic protein AlpP
Size: 190 KDa
Detected in the waste effluent of biofilms ≥ 3 days old.
what is the the red fluorescent oxidation product of amplex red with H2O2?
resuforin
