Biofilms

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Biofilms

• Biofilms are everywhere
• 1) plaque on out teeth
• 2) slime on river stones
• 3)gunge in water pipes
• Microbes mostly live in biofilm communities
• Most information on biofilms is derived from
  studies of planktonic cells
• Most microbial species can and do form biofilms

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• Formation of biofilms involves a lifestyle change
• - nomadic to community
• Biofilms become more and more sophisticated as
  they grow.
• Individual cells begin to take on specific tasks

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• Natural biofilms almost always have many
  different species of bacteria (ex. your teeth)
• Diverse nature of species is helpful when
  confronted with a changing environment
• There is still strain diversification in signal
  species biofilms.
• How?

P. Aeurginosa on a tooth
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Diversification in same species Biofilms

• Access to nutrients varies because of gradients
  that form in the biofilm.
• Results is the formation of microniches.
• Random mutations in bacteria result in bacteria
  that are able to exploit these niches.
• Leads to the multiple genotypes that are present
  in single strain biofilms

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Growing and Observing

• Transparent Flow Cells (constantly feed fresh
  nutrients)
• Confocal Laser Microscopy

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Formation (Matrix)

• Different species form different biofilms
• All biofilms secrete a matrix that acts to hold
  them together and in place
• P. aeruginosa matrix
• Aglutinate, Pel, and Psl

P. Aeurginosa biofilm in the respiratory tract of
an infected person
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Mobile to Non-mobile

• Bacteria give up their ability to move around in
  order to form biofilms.
• Ex. Bacillus subitis
• Flagellar component synthesis is traded for
  matrix synthesis.

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The Lifestyle Switch

• Bis-(3-5)-cyclic dimeric guanosine
  monophosphate (c-di-GMP)
• Low c-di-GMP in planktonic cells
• High c-di-GMP in biofilm cells
• c-di-GMP seems to control synthesis of motility
  components as well as matrix components

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c-di-GMP

• GGDEF and EAL are proteins that synthesize and
  degrade c-di-GMP.
• Most bacterial species have these proteins
• c-di-GMP is thought to regulate gene expression
  as well as to activate an enzyme that synthesizes
  extracellular cellulose
• The GGDEF and EAL proteins are associated with
  environment-sensing domains

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• Once biofilm formation is underway,
  Quorum-sensing signals become involved with
  biofilm development
• The signals control 100s of genes
• Biofilm formation is governed by genetic and
  environmental factors
• Big question is if signaling occurs between
  species. If they can, then how do they interact?

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The Involvement of Cell-to-Cell Signals in the
Development of Bacterial Biofilm

• David G. Davis, Matthew R. Parsek, James P.
  Pearson, Barbara H. Iglewski, J. W. Costerton, E.
  P. Greenberg

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• Study of the role of intercellular signals in P.
  aeruginosa biofilm development
• Focused on two cell-to-cell signaling systems
• lasR-lasI
• rhlR-rhlI

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lasR-lasI and rhlR-rhlI

• lasI gene product directs the synthesis of
  n-(3-oxododecanoyl)-L-homoserine lactone
  (3OC12-HSL)
• Extracellular signal
• lasR gene product is a transcriptional regulator
  that requires 3OC12-HSL to be active
• Activates virulence genes, rhlR-rhlI system, also
  lasI
• rhlI gene product directs synthesis of
  N-buytryl-L-homoserine lactone
• N-buytryl-L-homoserine lactone required for
  activation of virulence genes and the expression
  of the stationary sigma factor RpoS.
• Expression of RpoS is via the rhlR gene product.

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lasR-lasI and rhlR-rhlI
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Hypothesis

• These signals would not be expected to perform
  initial stages of biofilm formation, attachment,
  and proliferation.
• May be involved in differentiation.
• To test, a mutant was formed from WT P.
  aeruginosa PAO1 was observed alongside the WT.
• It was a lasI-rhlI double mutant.

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• WT and double mutant strains were grown on
  reaction chamber surfaces
• lasI-rhlI mutant biofilm was thinner than WT
  biofilm but its cells were more densely packed.

White boxes depth of the biofilm Black boxes
cell packing
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• Double mutant grew as continuous sheets on the
  glass
• Supported hypothesis that the cell-to-cell signal
  systems are not needed for attachment but they
  are needed for differentiation, or at least one
  is.

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lasI, rhlI, or both?

• To determine which of the genes were required for
  biofilm development single mutants were made from
  the WT strain
• rhlI-
• lasI-
• Thickness and cell density were measured for all
  strains

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White boxes depth of the biofilm Black boxes
cell packing

• rhlI mutant formed biofilms similar to WT.
• lasI mutant formed biofilms similar to those of
  the lasI-rhlI mutant.
• 3OC12HSL was run through the reaction chamber of
  the lasI mutant. Normal biofilm formation
  returned.

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Further Comparison

• Plasmid coding for green fluorescent protein
  inserted into WT and lasI mutant
• WT and lasI mutant contain GFP expression vector
  pMRP9-1
• Epifluorescence and scanning confocal microscopy
  used to observe

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Addition of 3OC12-HSL autoinducer returned lasI
mutant to normal biofilm differentiation. (Also
shown in 1A)
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• Based on the experiment, 3OC12-HSL was concluded
  to be the quorum-sensing signal required for
  biofilm differentiation.
• Also, gradients for the signal are not necessary
  for differentiation

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EPS Matrix

• EPS matrix cements cells together in biofilms.
• WT P. aeruginosa is embedded in an EPS matrix.

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Monitoring EPS levels

• Uronic acid levels were measured so as to monitor
  levels of EPS in WT and lasI mutant.
• No difference in EPS levels was found.
• Distribution of EPS is different between
  planktonic and biofilm cells.

Black Bars total carbohydrate Open bars total
uronic acid
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Hypothesis

• Planktonic glycocylax vs. Biofilm glycocylax
• Differentiation in WT is triggered when the cell
  produces a large amount of 3OC12-HSL.
• lasI mutant cells believed to have similar
  physical characteristics to planktonic cells
  rather than to biofilm cells
• Tested via detergent exposure

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Detergent exposure

• WT and lasI mutant exposed to sodium dodecyl
  sulfate
• No effect on WT
• After 5 min, most mutant bacteria detached and
  dispersed
• lasI mutant was also exposed to autoinducer which
  returned WT like resistance to the detergent

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Conclusions

• Cell-to-Cell signaling is required for
  differentiation of individual cells to
  multicellular structures.
• Mutations that block production of signal
  molecules hinder cell differentiation.