Quantifying spatio-temporal expression of the chemosensory system and the flagellum to elucidate their role in biofilms.

Many bacteria are found growing in protective slime on surfaces in communities of microbes known as biofilms. Biofilms are important medically as they cause chronic infections and colonise implants, in industry where they are found blocking pipes and causing metal to breakdown. To form a biofilm, individual cells, come into contact with a surface (usually by swimming to it), stick, grow, multiply and spread, gradually producing a mature 3-dimensional biofilm. This consists of mushroom shaped heaps of cells, embedded in slime, with water channels running through. The channels carry nutrients and oxygen into the biofilm, which diffuse to the cells however diffusion does not occur equally throughout the biofilm. So individual cells experience different environmental conditions and thus the biofilm structure is very variable. Changes in environmental conditions also cause the cells to rearrange themselves and thus the shape of the biofilm can change. In this project we will study bacterial movement in the biofilm, determining how the cells move in response to environmental changes an d when they are motile within the biofilm. Bacterial movement is controlled by a sensory system, the Che system, which senses changes in the concentrations of chemicals in the environment and controls swimming so that the cell can respond, so we will also investigate the role of this system in the biofilm. To do this we will study how the genes involved in motility and chemosensing are controlled in the biofilm. The control or expression of genes will be studied by coupling them with a fluorescent protein which we can see using microscopy and thus directly study the expression of genes deep within the biofilm.

Bacterial biofilms develop internal heterogeneities due to gradients formed by chemical diffusion, light absorption and metabolic activity. Cells respond to microenvironments by altering levels of gene expression, which leads to cell differentiation within the biofilm population. In addition biofilms are dynamic structures as the cells reposition in response to gradients causing restructuring of the biofilm. In planktonic cells motility i.e. flagellar rotation is directed by the Che system, but the role of chemotaxis and motility in the development of mature biofilms and their dynamic restructuring is currently not understood. We have found that the Che system and flagellar driven motility are responsible for determining the spatial arrangement within the mature 3-D biofilm structure of Rhodobacter sphaeroides WS8. The Che system in this organism does not have a role in the initial step of surface interaction, though active motility does. In planktonic R. sphaeroides the expression levels of the che Op2 and che Op3 are determined by oxygen and light levels. In this study we will map and quantify the spatio-temporal expression levels of the che operons and of the flagellum using GFP as a reporter of promoter activity. Confocal laser scanning microscopy will be used to study expression within the biofilm and during gradient changes to elucidate the role of the chemosensory system in determining biofilm structure. A reporter system will be developed to define the oxygen gradient within the biofilm using the cells as oxygen biosensors. FRAP will be applied to determine whether or not the reporter systems in cells at defined biofilm locations are active. Motility , per se, in the biofilm using activated PA-GFP to tag cells to enable their movement to be tracked. In addition the structure of the EPS matrix will be elucidated with fluorescent staining techniques to determine if it is altered during restructuring of the biofilm and cell dispersal.