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| Status |
Public on May 01, 2012 |
| Title |
Microcolony formation by the opportunistic pathogen Pseudomonas aeruginosa requires pyruvate and pyruvate fermentation. |
| Organism |
Pseudomonas aeruginosa |
| Experiment type |
Expression profiling by array
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| Summary |
A hallmark of the biofilm architecture is the presence of microcolonies. However, little is known about the underlying mechanisms governing microcolony formation. In the human pathogen Pseudomonas aeruginosa, microcolony formation is dependent on the two-component regulator MifR, with mifR mutant biofilms exhibiting an overall thin structure lacking microcolonies, and overexpression of mifR resulting in hyper-microcolony formation. Here, we made use of the distinct MifR-dependent phenotypes to elucidate mechanisms associated with microcolony formation. Using global transcriptomic and proteomic approaches, we demonstrate that cells located within microcolonies experience stressful, oxygen limited, and energy starving conditions, as indicated by the activation of stress response mechanisms and anaerobic and fermentative processes, in particular pyruvate fermentation. Inactivation of genes involved in pyruvate utilization including uspK, acnA and ldhA abrogated microcolony formation in a manner similar to mifR inactivation. Moreover, depletion of pyruvate from the growth medium impaired biofilm and microcolony formation, while addition of pyruvate significantly increased microcolony formation. Addition of pyruvate partly restored microcolony formation in ∆mifR biofilms. Moreover, addition of pyruvate to or expression of mifR in lactate dehydrogenase (ldhA) mutant biofilms did not restore microcolony formation. Consistent with the finding of denitrification genes not demonstrating distinct expression patterns in biofilms forming or lacking microcolonies, addition of nitrate did not alter microcolony formation. Our findings indicate the fermentative utilization of pyruvate to be a microcolony-specific adaptation to the oxygen limitation and energy starvation of the P. aeruginosa biofilm environment.
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| Overall design |
For biofilm growth experiments, three independent replicates of P. aeruginosa strains PAO1 and ΔmifR were grown as biofilms in a flow-through system using a once-through continuous flow tube reactor system for biofilm sample collection and in flow cells (BioSurface Technologies) for the analysis of biofilm architecture as previously described (Sauer et al., 2002, Sauer et al., 2004, Petrova & Sauer, 2009). Cells were treated with RNAprotect (Qiagen) and total RNA was extracted using an RNeasy mini purification kit (Qiagen) per the manufacturer’s instructions. RNA quality and the presence of residual DNA were checked on an Agilent Bioanalyzer 2100 electrophoretic system pre- and post-DNase treatment. Ten micrograms of total RNA was used for cDNA synthesis, fragmentation, and labeling according to the Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol.
Sauer, K., A. K. Camper, G. D. Ehrlich, J. W. Costerton & D. G. Davies, (2002) Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J. Bacteriol. 184: 1140-1154.
Sauer, K., M. C. Cullen, A. H. Rickard, L. A. H. Zeef, D. G. Davies & P. Gilbert, (2004) Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm. J. Bacteriol. 186: 7312-7326.
Petrova, O. E. & K. Sauer, (2009) A novel signaling network essential for regulating Pseudomonas aeruginosa biofilm development. PLoS Pathogens 5: e1000668.
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| Contributor(s) |
Petrova OE, Schurr JR, Schurr MJ, Sauer K |
| Citation(s) |
22931250 |
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| Submission date |
Jan 23, 2012 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Michael J Schurr |
| E-mail(s) |
[email protected]
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| Phone |
3037244221
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| Organization name |
University of Colorado School of Medicine
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| Department |
Microbiology
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| Street address |
12800 E 19th Ave
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| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
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| Platforms (1) |
| GPL84 |
[Pae_G1a] Affymetrix Pseudomonas aeruginosa Array |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA151585 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE35286_RAW.tar |
5.3 Mb |
(http)(custom) |
TAR (of CEL, CHP) |
| Processed data included within Sample table |
| Processed data provided as supplementary file |
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