Biofilms- Tiffany

Quorum Sensing: Biofilms: 

Quorum Sensing: Biofilms Tiffany Doan 4/20/10

Last week…: 

Last week… Hfq and 4 sRNAs ( qrr1-4 ) destabilize the hapR transcript, leading to repression of hapR (also probably for luxR ) qrr1-4 are activated by LuxO-σ 54 qrr1-4 are redundant Bioinformatics shows that V. parahaemolyticus and V. vulnificus have 5 probable sRNAs (and probably V. cholerae , too)

Slide 3: 

Biofilms can be beneficial to humans and the environment Hydrocarbonoclastic bacteria (HCB) Sewage treatment Primary line of defense against pathogens Biofilms can be harmful, too Catheters and prostheses (may lead to systemic infection) Dental disease (Brush your teeth!) Otitis media, inflammatory bowel disease, prostatitis, sinusitis, etc.

Slide 4: 

Biofilm formation on a contact lens Biofilm formation on a spider

Slide 5: 

Crystalline biofilm blocking a urinary catheter

Slide 6: 

All biofilms have a ‘matrix’ (EPS) to protect against the environment and hold colonies in place Different species, different biofilm development Mixes of polysaccharides, proteins, and nucleic acids

Biofilm Diversity: 

Growth in biofilms generates genetic diversity Nutrient gradients form within biofilms  MUTANTS How is this beneficial to bacteria in biofilms? The population can adapt better to sudden environmental changes than a genetically homogenous population Biofilm Diversity

The Lifestyle Switch: 

The Lifestyle Switch How do cells know when to switch from being nomadic to sedentary? Planktonic cells: low c-di-GMP c-di-GMP level rises when cells switch to sedentary lifestyle

The Lifestyle Switch: 

The Lifestyle Switch Mechanism by which c-di-GMP acts remains unknown Integrate external signal sensed by GGDEF and EAL? Fluctuations of c-di-GMP in cellular ‘compartments’? Post-translational activity and gene transcription control

How are biofilms studied in the lab?: 

How are biofilms studied in the lab? Single-species vs. Multi-species Transparent flow cells Bacteria attach to glass and nutrients flow through the cell Confocal microscopy

Slide 11: 

The Involvement of Cell-to-Cell Signals in the Development of a Bacterial Biofilm David G. Davies, Matthew R. Parsek, James P. Pearson, Barbara H. Iglewski, J. W. Costerton, E.P. Greenburg

Cell-to-Cell Signaling in P. aeruginosa: 

Cell-to-Cell Signaling in P. aeruginosa Two signaling systems: lasR-lasI rhlR-rhlI (or vsmR-vsmI ) lasI gene product directs synthesis of 3OC 12 -HSL lasR gene product is a transcriptional regulator (requires 3OC 12 -HSL)

Slide 13: 

rhlI product directs synthesis of N-buytryl-L-homoserine lactone rhlR gene product regulates expression of RpoS (stationary-phase σ factor)

Are these signals involved in biofilm differentiation?: 

Are these signals involved in biofilm differentiation? Monitor biofilm formation of WT P. aeruginosa and lasR-rhlI double mutant Both adhered to and proliferated on the glass surface Mutant biofilm was thin (continuous sheets) and more densely packed compared to WT WT formed microcolonies separated by water channels

Are lasI, rhlI, or both required for normal biofilm development?: 

Are lasI , rhlI , or both required for normal biofilm development? Measure average thickness of biofilms and cell packing Further compare WT and lasI mutant using GFP expression

Slide 16: 

Fig. 1

Conclusions: 

Conclusions QS signal 3OC-HSL required for normal biofilm differentiation Gradients of the signal are not necessary for differentiation

Next experiment…: 

Next experiment… Examine EPS levels by measuring total carbohydrates and uronic acids

Glycocalyx Distribution: 

Planktonic cells have compressed, incomplete glycocalyx Biofilm cells cemented to one another by the EPS matrix What do these results suggest?  lasI mutants develop biofilm normally in the initial stages, but differentiation into biofilm bacteria does not proceed. Glycocalyx Distribution

Are mutant biofilms sensitive to biocides?: 

Are mutant biofilms sensitive to biocides? Tested biocides that do not disrupt WT biofilms on lasI mutant SDS

Conclusions: 

Conclusions Cell-to-cell signal required for biofilm differentiation Mutation that blocks synthesis of signaling molecule hinders differentiation

Slide 23: 

Rhamnolipid Surfactant Production Affects Biofilm Architecture in Pseudomonas aeruginosa PAO1 Mary E. Davey, Nicky C. Caiazza, and George A. O’Toole

Why do biofilms have void spaces?: 

Why do biofilms have void spaces? Allows fluid flow throughout the biofilm to distribute nutrients and oxygen Also may play a role in removing metabolic waste products

Hypothesis: 

Hypothesis Rhamnolipids (surfactants) are not required for the formation of macrocolonies and channels, but participate in the maintenance of channels once they are formed

Rhamnolipids are required to maintain biofilm architecture.: 

Rhamnolipids are required to maintain biofilm architecture. Rhamnolipid synthesis is controlled by lasR-lasI system rhlA gene codes for a rhamnosyltransferase

Slide 27: 

Results suggest that rhlA mutant is more proficient at early colonization than is the wild-type strain. Fig. 1

Slide 28: 

Fig. 1 Once-through flow cell system provides continuous supply of fresh nutrients to the biofilm Result: rhlA mutant can make channels, but unable to maintain them

Quantitative analysis of biofilm architecture: 

Quantitative analysis of biofilm architecture rhlA mutant had higher average mean thickness and substratum coverage than wild-type Roughness coefficient lower in rhlA mutant Surface/Volume lower in mutant

Partial rescue of the rhlA biofilm architecture defect by the WT strain: 

Partial rescue of the rhlA biofilm architecture defect by the WT strain Spent supernatants of P. aeruginosa cultures did not rescue rhlA mutants Flow cell mixing experiment with WT and rhlA mutant cells

Partial rescue of the rhlA biofilm architecture defect by the WT strain: 

Partial rescue of the rhlA biofilm architecture defect by the WT strain Fig. 2

Expression of rhlA during biofilm development: 

Expression of rhlA during biofilm development When and where are rhamnolipids synthesized? Use transcriptional fusion of rhlA promoter and GFP Synthesis only observed in macrocolonies

Expression of rhlA during biofilm development: 

Expression of rhlA during biofilm development Fig. 3 High cell density  rhlA expression and rhamnolipid synthesis

Overproduction of rhamnolipids inhibits biofilm development: 

Overproduction of rhamnolipids inhibits biofilm development Could rhamnolipids affect the interactions of bacteria with surfaces and each other? Induce rhamnolipid synthesis with phosphate-limited medium

Overproduction of rhamnolipids inhibits biofilm development: 

Overproduction of rhamnolipids inhibits biofilm development High levels of rhamnolipids impedes biofilm formation Fig. 4

Rhamnolipids disrupt both cell-to-cell and cell-to-surface interactions.: 

Rhamnolipids disrupt both cell-to-cell and cell-to-surface interactions. Addition of rhamnolipids inhibited biofilm formation. Fig. 5

Invasion of preformed biofilm: 

Invasion of preformed biofilm Biofilm invasion assay: introduce GFP-tagged P. aeruginosa cells to 5-day-old biofilm Fig. 6 Planktonic cells do not attach to the preformed biofilm

Conclusions: 

Conclusions Rhamnolipids do not affect initial development of biofilms rhlA mutants are unable to maintain open channels Surfactant prevents planktonic cells from attaching to biofilm Rhamnolipids may cause detachment of cells from the biofilm Exposing rhamnolipids to preformed biofilm does not effect the architecture

Conclusions: 

Conclusions How the production of rhamnolipids maintain the open channels during biofilm development remains unclear! Rhamnolipids may prevent other planktonic microbes trying to take up residence within the channels Open-channel maintenance is not stochastic—it’s an active process

Promising biofilm technologies: 

Promising biofilm technologies Microbial fuel cells Biofilm barrier for groundwater denitrification

Viruses vs. Biofilms: 

Viruses vs. Biofilms Can viruses be engineered to kill bacteria in disease-causing biofilms? E. coli biofilms sensitive to viral infection Most biofilms are multi-species Fear of provoking a strong immune response