Mutation and diversification of biofilms

Question 1

Antimicrobial tolerance in bacterial biofilms

Answer 1

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 2

Biofilm associated growth creates..

Answer 2

heritable genetic change throughout the genome.

Role of genetic in the context of biofilm arc not been explored

Also very common to isolate mutator phenotype from biofilms,therefore mutation + genet hetro appears to play an important role in biofilm develop

Question 3

Mutant strains were selected by querying the Pseudomonas Genome Database for genes associated with the P.aeruginosa Community Annotation Project (PseudoCAP) functional classification “DNA replication, recombination, modification and repair”

Ten strains were obtained for this study with mutations in:

Answer 3

himA - integration host factor
micA - mismatch repair protein
mutL - mismatch repair protein
recN - DNA repair protein
recQ - DNA helicase
recR - recombination protein
sss - site-specific recombinase
ung - uracil-DNA glycosylase
uvrC - excinuclease subunit
xseA - exodeoxyribonuclease

Question 4

A strong positive correlation exists between mutation frequency (resistance to rifampicin) and …

Answer 4

microcolony growth for a range of P. aeruginosa DNA error repair mutants.

Question 4

Predominant mode of mutation in biofilm/ dispersal populations is based on

Answer 4

point mutations (e.g. via downregulation of mismatch repair?)

Question 5

Which genes have been affected by non-syn. mutations?

Answer 5

Regulatory proteins: PA434,PA2897, PA0928,PA2492,MexT

Energy generations: PA263 NADH dehydrogenase I, PA0106 cytochrome c oxidase, PA4811 nitrate-inducible formate dehydrogenase, 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 6

reporter system to enable detection of frameshift events in real time

Answer 6

GFP-based mutation detection

GFP +1 Reversion system pMDGFP (PCR Site Directed Mutagenesis)

visualised with epif or confoc microscopy

Question 7

GFP reversion occurs in the microcolonies and not in ____

Answer 7

the non differentiated biomass

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

Question 8

Darwinian processes in biofilm development

Answer 8

Microcolonies as foci for genetic mutation and evolution

Microcolony growth may involve mutation selection

Question 9

Model for Microcolony Development

Answer 9

1. Primary mutation
2. Clonal expansion
3. Secondary mutation

NB: Selection for growth on a surface is strong, Stress responses

Question 10

Pseudoalteromonas tunicata (D2)

Answer 10

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 11

The P. tunicata autolytic protein, AlpP

Answer 11

Size: 190 KDa

Detected in the waste effluent of biofilms ≥ 3 days old.

Question 12

AlpP mediates…

Answer 12

cell death in Pseudoalteromonas tunicata biofilms

Question 13

AlpP mode of action

Answer 13

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 14

AlpP is required for..

Answer 14

biofilm dispersal in P. tunicata

Question 15

Does AlpP occurs in several Gram negative organisms

Answer 15

YES

Question 16

AlpP and its homologues produce ____ and induce _____

Answer 16

H2O2

lysis, dispersal, and phenotypic variation

Question 17

Microcolony differentiation of Pseudomonas aeruginosa is linked to…

Answer 17

production of reactive oxygen species

e.g. peroxynitrite (ONOO-)

Question 18

What is nitric oxide involved in in P. aeruginosa

Answer 18

cell death and dispersal events (inducing dispersal)

Question 19

P. aeruginosa response to low levels of NO:

Answer 19

Motility
↑ flgG, pilAGHIMOQZ

Adhesion and biofilm relevant
↓ cupP, cupC, EPS Pel genes (PA3058 – 3064), alg genes.

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 21

What regulates biofilm v. planktonic lifestyle across a broad range of bacteria, give examples of bacteria where is is the case

Answer 21

Cyclic-di-GMP

High levels promote biofilm lifestyle, low levels planktonic

e.g. Pseudomonas spp. , Xanthomonas campestris, Salmonella spp., Vibrio cholerae, E. coli, Caulobacter crescentus

Question 22

Conserved domains involved in Cyclic-di-GMP turnover

Answer 22

GGDEF - guanylate cyclase
EAL - phosphodiesterase

other domain = PAS/H-NOX - bind and respond to NO in both eukaryotes and prokaryotes

Question 23

Low concentrations of NO induce….

Answer 23

overall decrease in c-di-GMP levels, turn off biofilm traits and trigger dispersal in P. aeruginosa

Question 24

Features of CF

Answer 24

Most common lethal, hereditary disease (1 in 2,500 live births in UK)

Autosomal recessive mutations in CF transmembrane conductance regulator (CFTR)

Increased mucus production from goblet cell hyperplasia & poor ciliary clearance = ideal environment for bacterial colonisation

Morbidity and mortality primarily associated with onset of chronic and persistent bacterial infection of which P. aeruginosa biofilms are recognised as a key factor

Question 26

Adjunctive NO in cystic fibrosis - Hypothesis

Answer 26

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