Metabolic and genetic processes in microbial biofilms
Microbial biofilms are of great environmental and medical importance, and research over the last years has shown that diverse molecular mechanisms exits to control biofilm formation. Stability and dissolution of microbial biofilms is governed by the local physical-chemical and cellular environment of the inhabiting microbes. In order to obtain molecular and energetic insight into biofilm stability, we investigated whether maintenance of biofilm stability is a metabolic energy-dependent process and whether transcription and/or translation are required for biofilm detachment. We found that in 12-hour-old Shewanella oneidensis MR-1 biofilms, a reduction in cellular ATP concentration, induced either by oxygen deprivation or addition of the inhibitors of oxidative phosphorylation CCCP, DNP or CN- resulted in massive detachment. Concomitant to the ATP decrease, the concentration of the cellular second messenger c-di-GMP, which was previously shown to positively modulate biofilm stability, also decreased, suggesting a link between energy metabolism and c-di-GMP signaling. In older biofilms, the extent of uncoupler-induced cell loss was strongly attenuated, indicating that integrity of older biofilms is maintained by means other than those operating in younger biofilms. The transcriptional and translational inhibitors rifampicin, tetracycline, and erythromycin were found to be ineffective in preventing energy-starvation-induced detachment. Biofilms of Vibrio cholerae were also induced to dissolve upon CCCP addition to a similar extent as in S. oneidensis. However, Pseudomonas aeruginosa and P. putida biofilms remained insensitive to CCCP addition. Collectively, our data show that metabolic energy is directly or indirectly required for maintaining cell attachment, and represents a novel, common but not ubiquitous mechanism for controlling stability of microbial biofilms.
We have previously shown that type IV pili and a putative EPS biosynthetic gene cluster (mxdABCD) are implicated in biofilm formation in Shewanella oneidensis MR-1. We found that the mannose-sensitive hemagglutinin (MSHA) pilus mediates a reversible, D-mannose-sensitive association of cells to the substratum surface or to other cells that is critical within the first 5 μm of the biofilm from the substratum. The presence of the MSHA pilus alone is insufficient to confer biofilm-forming capacity; its activity, as mediated by the putative pilus retraction motor protein, PilT, is also required. Deletion of pilD, encoding the type IV pili prepilin peptidase, revealed that additional PilD substrate(s) may be involved in biofilm formation beyond the major structural pilin of the MSHA pilus. The MSHA pilus and mxd genes encode for a complementary set of molecular machineries that constitute the dominant mechanisms enabling biofilm formation in this microorganism under hydrodynamic conditions.
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