Induction of rapid detachment in Shewanella oneidensis MR-1 biofilms - PubMed (original) (raw)

Induction of rapid detachment in Shewanella oneidensis MR-1 biofilms

Kai M Thormann et al. J Bacteriol. 2005 Feb.

Abstract

Active detachment of cells from microbial biofilms is a critical yet poorly understood step in biofilm development. We discovered that detachment of cells from biofilms of Shewanella oneidensis MR-1 can be induced by arresting the medium flow in a hydrodynamic biofilm system. Induction of detachment was rapid, and substantial biofilm dispersal started as soon as 5 min after the stop of flow. We developed a confocal laser scanning microscopy-based assay to quantify detachment. The extent of biomass loss was found to be dependent on the time interval of flow stop and on the thickness of the biofilm. Up to 80% of the biomass of 16-h-old biofilms could be induced to detach. High-resolution microscopy studies revealed that detachment was associated with an overall loosening of the biofilm structure and a release of individual cells or small cell clusters. Swimming motility was not required for detachment. Although the loosening of cells from the biofilm structure was observed evenly throughout thin biofilms, the most pronounced detachment in thicker biofilms occurred in regions exposed to the flow of medium, suggesting a metabolic control of detachability. Deconvolution of the factors associated with the stop of medium flow revealed that a sudden decrease in oxygen tension is the predominant trigger for initiating detachment of individual cells. In contrast, carbon limitation did not trigger any substantial detachment, suggesting a physiological link between oxygen sensing or metabolism and detachment. In-frame deletions were introduced into genes encoding the known and putative global transcriptional regulators ArcA, CRP, and EtrA (FNR), which respond to changes in oxygen tension in S. oneidensis MR-1. Biofilms of null mutants in arcA and crp were severely impacted in the stop-of-flow-induced detachment response, suggesting a role for these genes in regulation of detachment. In contrast, an DeltaetrA mutant displayed a variable detachment phenotype. From this genetic evidence we conclude that detachment is a biologically controlled process and that a rapid change in oxygen concentration is a critical factor in detachment and, consequently, in dispersal of S. oneidensis cells from biofilms. Similar mechanisms might also operate in other bacteria.

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Figures

FIG. 1.

FIG. 1.

Detachment induced by stop of flow. The experiment was conducted as a standard stop-of-flow assay as described in Materials and Methods. Images are shadow projections of 12-h-old CLSM files taken of AS93 biofilms before (A), 10 min after (B), and 30 min after (C) the initial stop of medium flow. The scale bar in each image represents 40 μm.

FIG. 2.

FIG. 2.

Effect of duration of stop of flow on extent of detachment of biofilms of different thicknesses. The graph represents quantified CLSM images generated by COMSTAT. Detachment assays were conducted as described in Materials and Methods, but the duration of stop of flow was varied as indicated by the x axis. ▴, 48-h-old biofilm; ▪, 18-h-old biofilm; and ⧫, 12-h-old biofilm. Each data point is the mean from six independent images taken from two channels. Error bars represent one standard deviation.

FIG. 3.

FIG. 3.

Induction of detachment in 12- and 48-h-old biofilms. Images display cross sections of stop-of-flow-induced detachment in 12-h-old (A to C) and 48-h-old (D and E) biofilms. Images A, B, and C were obtained immediately before, during (15 min), and after the stop of flow, respectively; images D and E were obtained before and after the stop of flow. Each scale bar represents 25 μm in A to C and 50 μm in D and E.

FIG. 4.

FIG. 4.

Induction of detachment by nutrient downshifts. The graph (A) represents quantified CLSM images generated by COMSTAT. Modified detachment assays were conducted as described in Materials and Methods. The nutrient downshift was accomplished either by switching to a medium containing buffer only or by removal of oxygen through applying vacuum to the medium inflow tubing. Each data point is the mean from at least three independent images. Error bars represent one standard deviation. Images B1 and B2 are biofilm cross sections 45 and 90 min after oxygen downshift, respectively. Each scale bar represents 25 μm.

FIG. 5.

FIG. 5.

Induced detachment in biofilms of strains with in-frame deletions in crp, arcA, and etrA. The graph represents quantified CLSM images generated by COMSTAT. Detachment assays were conducted as described in Materials and Methods on biofilms of strains with either null mutations (AS122, AS123, and AS124) or null mutations complemented with the corresponding wild-type allele in trans (AS126, AS125, and AS127). Values are the means from at least six independent images taken from two channels. Error bars represent one standard deviation.

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