In the described study, we observed that the FlhD mutants made quantitatively higher amounts of biofilms on numerous carbon sources. Interestingly, the parental strain did not form higher quantities of biofilm than the mutant on any of the tested carbon sources. These observations shed light into the ongoing controversial debate, elucidating the role of motility in biofilm formation. In certain bacterial species including Yersinia enterocolitica, the presence of motility has been shown to be beneficial for biofilm formation (Wang et al.,

2007) . Several previous studies from our lab demonstrate that the absence of motility enhances the ability of E. coli to form substantial amounts of biofilm. As one example, strains transformed with the FlhD expressing plasmid pXL27 showed diminished biofilm forming capabilities (Prufi et al., 2010). Additionally, ongoing studies carried out in the lab with E. coli O157:H7 and the E. coli K-12 strains MC1000 and AJW678 point in the same direction, exemplifying our belief that FlhD and motility are detrimental to biofilm formation for our bacterial strains and under the conditions of our experiments (Sule et al., unpublished data). As a second observation, carbon sources that supported maximal biofilm formation by either strain all fed into glycolysis eventually, and produced actetate. Although the carbon sources that promoted the highest biofilm amounts were different for the two strains, they still were in the same pathway. The previous high-throughput experiment that had pointed towards nutriition as instrumental in determining biofilm associated biomass had also postulated acetate metabolism as one of the key players in biofilm formation (Prufi et al.,

2010) . Phosphorylation of OmpR and RcsB by the activated acetate intermediate acetyl phosphate (Kenney et al., 1995) and acetylation of RcsB by acetyl-CoA (Thao et al., 2010) have been described in the past. These activated 2CSTS response regulators then affect the expression level of biofilm associated cell surface organelles, such as flagella, type I fimbriae, curli, and capsule (Ferrieres & Clarke, 2003; Francez-Charlot et al., 2003; Oshima et al., 2002; Prufi, 1998; Shin & Park, 1995) (Figure 6). The positive effect on biofilm amounts of carbon sources that lead to the production of acetate can be explained with the combined inhibitory effect of acetyl phosphate and acetyl-CoA on flagella through OmpR and RcsB and the above described disadvantage of flagella and motility during biofilm formation. We however do not state that acetate is the sole controlling mechanism as the complexity of the bacterial system cannot be explained based on a small number of signaling molecules.

The most striking observation obtained from our studies pertains to the pattern of growth and biofilm formation on sugar acids. It was observed that the FlhD mutants grew to lower optical densities on sugar acids, but formed much higher amounts of biofilm as compared to the parental strain. Previous work from the Prufi lab had shown similar defects in growth of flhD mutants on sugar acids (Prufi et al., 2003), biofilm formation was not tested in that study. The inverse effect of sugar acids on growth and biofilm amounts may have implications in the intestine. Mutants in flhD have an early disadvantage in colonization, but recover after prolonged incubation (Horne et al., 2009). They even take over the population after more than two weeks (Leatham et al., 2005). The initial lack of colonization could be explained by the inability of the flhD mutant to degrade the numerous sugar acids present in the intestine (Peekhaus & Conway, 1998). On the other hand, the ability to take over the bacterial population at a later stage may have to do with the lack of the flagellin, which is a potent cytokine inducer (McDermott et al., 2000). The here discovered ability to make an increased amount of biofilm may add to the long term survival of flhD mutants in the intestine. Bacteria deep within the biofilm will be protected from the immune system, while metabolizing very slowly and not needing much nutrition.

Among the carbon sources that were the least supportive of biofilm formation, the inability of the C5-sugars to support growth and/or biofilm formation was the most striking. Ribose supported growth by the parent strain, but yielded the lowest biofilm amount of all tested carbon sources. The flhD mutant did not even grow on ribose. According to Fabich and coworkers (Fabich et al., 2008), ribose is not among the carbon sources that the E. coli K-12 strain MG1655 utilizes when bacteria colonize the intestine. Our data are consistent with this observation. Since E. coli O157:H7 EDL933 does actually utilize ribose in the intestine, ribose utilization may constitute a mechanism by which pathogenic E. coli can find a niche in the intestine to co-exist with the commensal E. coli strains.

The inability to grow on lyxose is also consistent with previous observations, where only a mutation in the rha locus enabled the bacteria to grow on lyxose via the rhamnose pathway (Badia et al., 1991). Normally, E. coli are unable to grow on lyxose. Most interesting is the behavior of the two strains on xylose. The parent E. coli strain was unable to grow on xylose. The flhD mutant did grow, while producing moderately low amounts of biofilm. Co­utilization of glucose and xylose by E. coli strains is of upmost importance during the production of biofuels, since the fermented plant material contains both, cellulose (polymer of glucose) and hemicellulose (polymer of glucose and xylose), in addition to lignin. Much research is currently dedicated to the genetic modification of E. coli that enables the bacteria to utilize xylose more efficiently (Balderas-Hernandez et al., 2010; Hanly & Henson, 2010). It would be interesting to see whether a mixture of our parent strain and its isogenic flhD mutant would be able to co-utilize glucose and xylose, particularly since the mutant produced a moderate amount of biofilm which can also be beneficial to the production of biofuels.

4. Conclusion

In summary, we developed an assay system that quantifies biofilm biomass in the presence of distinct nutrients. The assay enables the user to screen a large number of such nutrients for their effect on biofilm amounts. Examples of metabolic analysis relate back to previous literature, as well as giving raise to new hypotheses. Yielding further evidence for the previous hypothesis that acetate metabolism was important in determining biofilm amounts can serve as a positive control that the assay actually yields data of biological significance. Particularly with respect to life in the intestine and the production of biofuels, the data open new avenues of research by providing testable hypotheses. Overall, there is no limit to extensions of the assay into different bacterial species or serving the development of high — throughput data mining algorithms that will computerize the statistic/ metabolic analysis that we started in this study.