The experiments performed here allow for a clear set of alternative hypotheses concerning the development of V. paradoxus EPS swarms. The availability of growth limiting substrates may be the key factor, or some particular nutrients may have a more direct effect BIBW2992 through specific signals.
This can be directly tested in growth experiments using combinations of nutrients, as well as by analysis of mutant population swarming characteristics. Experiments of both of these types are either planned or ongoing. Biofilm formation in M9 based medium was robust with succinate as carbon source, regardless of nitrogen source, over 24 and 48 hour batch culture. Dense biofilms were also present with several other carbon sources, notably d-sorbitol, glucose, malic acid, mannitol, and sucrose. The strongest biofilms by far, however, were formed with casamino acids as the source of carbon. This may be due to signaling considerations,
as amino acids are present in plant exudates , or energetic considerations, because these cultures have a lower anabolic load. It should be noted here that some components of the casein hydrolysate might be used as a nitrogen source in this instance. Simultaneous growth experiments suggest that maleic acid, maltose, sucrose, and sodium benzoate are poor growth substrates CFTRinh-172 cost in this particular format, although strong growth on these substrates was evident in well aerated culture tubes under identical nutrient conditions. This is the likely explanation for the low biofilm formation with these substrates (Fig 8B). In culture conditions under shear, filamentous forms were frequently observed, suggesting a developmental response to this physical stress. The larger scale structure of a biofilm under continuous nutrient flow developed similarly
in our two sheared bioreactors, with an early phase of “”pioneer”" cells attaching to the surface, and microcolony formation (Fig 9B, Fig 10A, B). As the film developed further with input of nutrients, the honeycomb structure frequently observed in other biofilms  through is apparent (Fig 10C, F). Our data support the notion of exopolysaccharide (eps) production as a primary consideration in biofilm productivity, with some potential staining of eps present in our static biofilm experiments (Fig 9A). This critical role of eps has been identified in numerous other systems (for review see ), and is reaffirmed in this work. This bacterium forms robust biofilms on abiotic surfaces under diverse culture conditions in the laboratory, consistent with the production of a profuse, sticky matrix. Further genetic work (Pehl et al, manuscript in preparation) has shown that putative LPS/eps synthesis genes are important in this phenotype. Conclusion In this work we have established culture techniques for studying coordinated surface behaviors in the ubiquitous soil bacterium Variovorax paradoxus.