Unknown,Transcriptomics,Genomics,Proteomics

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Increased mutability of staphylococci in biofilms as a consequence of oxidative stress

ABSTRACT: In this work we have demonstrated increased mutability of Staphylococcus aureus and S. epidermidis in biofilms and have explored the mechanisms underlying the enhanced mutability. A novel static biofilm model, utilising cellulose filter disks, was developed to support the formation of mature biofilms with sufficiently high cell densities to permit determination of mutation frequencies. The mutability of biofilm cultures increased up to 60 fold and 4 fold for S. aureus and S. epidermidis, respectively, compared with planktonic cultures. Incorporation of antioxidants into S. aureus biofilms reduced mutation frequencies, indicating that increased oxidative stress underlies increased mutability in the biofilm. Transcriptional profiling revealed upregulation of the superoxide dismutase gene, sodA, in early biofilm cultures, also suggesting enhanced oxidative stress in these cultures. However, loss of the genes encoding superoxide dismutases or peroxidases did not specifically exacerabate biofilm mutability. In S. aureus SH1000, hydrogen peroxide was found to contribute to biofilm mutability. Three growth conditions (18 hr planktonic growth, 48 hr biofilm growth and 144 biofilm growth) of which there are three biological replicates of each

ORGANISM(S): Staphylococcus aureus

SUBMITTER: Victoria Ryder

PROVIDER: E-GEOD-35837 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The physiological and transcriptional response of Nitrosomonas europaea biofilms to phenol and toluene was examined and compared to suspended cells. Biofilms were grown in Drip Flow Biofilm Reactors under continuous flow conditions of growth medium containing ammonia as growth substrate. The responses of N. europaea biofilms to the aromatic hydrocarbons phenol and toluene were determined during short-term (3 h) additions of each compound to the biofilms. Ammonia oxidation in the biofilms was inhibited 50% by 60 uM phenol and 100 uM toluene. These concentrations were chosen for microarray analysis of phenol- and toluene-exposed N. europaea biofilms. Liquid batch cultures of exponentially growing N. europaea cells were harvested alongside the biofilms to determine differential gene expression between attached and suspended growth of N. europaea. Four sample groups of N. europaea cells were used in this study, with biological triplicates of each group. Groups were: Control (untreated) biofilms, phenol-exposed biofilms, toluene-exposed biofilms, and exponentially growing suspended cells. Biofilms were grown in Drip Flow Biofilm Reactors containing 4 independent growth channels and subject to 2 hour inhibition tests. During each experiment, 2 biofilm channels served as control with no inhibitor present and the other 2 biofilm channels were exposed to either 60 uM phenol or 100 uM toluene. Nitrite production was monitored throughout the experiment, and the given concentrations of phenol and toluene resulted in 50% inhibition of ammonia oxidation by the biofilms. Suspended cells were grown in batch reactors. Three 4-plex NimbleGen microarray chips were used, and each chip contained one sample from each experimental group. QC of samples was determined by spectrophotometric methods and using Agilent bioanalyzer traces to determine purity and integrity of RNA and cDNA. A sample tracking report was used to verify the correct hybridization of each sample to the intended array.

Project description: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 M-bM-^HM-^FmifR 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. For biofilm growth experiments, three independent replicates of P. aeruginosa strains PAO1 and M-NM-^TmifR 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 manufacturerM-bM-^@M-^Ys 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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Increased mutability of staphylococci in biofilms as a consequence of oxidative stress

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