For this study, a mutation was needed that would abolish one of the early cell surface organelles that contribute to the biofilm, while still permitting the formation of biofilms. We performed scanning electron microscopy (SEM) to determine the ability of the five bacterial strains (parental strain, flhD mutant, fliA mutant, fimA mutant, fimH mutant) to form biofilms. Biofilms were grown for 38 h at 37oC on glass cover slips with tryptone broth (TB; 1% tryptone, 0.5% NaCl) as a growth medium. Biofilms were fixed in 2.5% glutaraldehyde and prepared for SEM as described (Sule et al., 2009). Images were obtained with a JEOL JSM-6490 LV scanning electron microscopy (SEOL Ltd., Tokyo, Japan) at 3,000 fold magnification. 10 to 15 images were obtained per bacterial strain from at least three independent biological samples. One representative image is shown per bacterial strain.

1.2 Biofilm quantification with PM technology and the ATP assay

We used the PM1 plate of the BioLog PM system that contains 95 single carbon sources. When used with the tetrazolium dye that is provided by the manufacturer and indicative of respiration (Bochner et al., 2001), the PM system can be used for measuring growth of bacterial strains on single nutrients. We here describe a protocol for the determination of biofilm amounts (Figure 2).

As recommended by the manufacturer for the determination of growth phenotypes, the bacterial cultures were streaked from LB plates onto R2A plates (to deplete nutrient stores) and incubated at 37°C for 48 hours. Bacteria were removed from the plates with a flocked

swab (Copan, Murrieta CA), resuspended and then further diluted with IF-0a GN/ GP Base (BioLog, Hayward CA) inoculation fluid to an optical density (OD600) of 0.1. Leucine, methionine, threonine and thiamine were added at a final concentration of 20 pg/ml, the redox dye that is used for the determination of growth phenotypes was omitted for biofilm quantification. 100 pl of the inoculum was then dispensed into each of the 96 wells of the PM1 plates. The inoculated plates were wrapped with parafilm to minimize evaporation and incubated at 37°C for 48 hours. Biofilm amounts were quantified using the previously described ATP based technique (Sule et al., 2008, 2009). Briefly, the growth medium was carefully aspirated out of each well, minimizing loss of biofilm at the air liquid interface. The biofilms were then washed twice with phosphate buffered saline (PBS) in order to remove any residual media components. The biofilms were air dried and quantified using 100 pl BacTiter Glo™ reagent (Promega, Wisconsin, WI). The biofilms were incubated with the reagent for 10 min at room temperature and the bioluminescence was recorded using a TD 20/20 luminometer from Turner Design (Sunnyvale, CA). The bioluminescence was reported as relative lux units (RLU).

The determination of biofilm amounts in the presence of single nutrients was performed four times for each strain. In addition, growth on these carbon sources was determined in three independent replicate experiments, following the protocol that is described for the determination of growth phenotypes and including the redox dye (Bochner et al., 2001). Carbon sources on which both strains grew to an average OD600 of 0.5 or more were selected for the f-test analysis and carbon sources on which each strain grew to an average OD600 of

0.

5 or more were selected for the ANOVA/Duncan analysis of biofilm amounts (see below).

the fold variation was calculated, using the lowest experiment as a norm (1 fold). Data points in each experiment were divided by the respective fold variation. The normalized experimental data sets were subjected to two independent types of statistical analysis, all done statistically significant differences between the amounts of biofilm that were formed on a given carbon source between the two strains. Since this analysis yielded more carbon sources than we could comprehend on a physiological level, we then analyzed each strain individually and then compared biofilm amounts on individual carbon sources for specific nutrient categories of structurally related carbon sources. For this analysis, the normalized biofilm data from each strain were subjected to separate one way ANOVAs, followed up with Duncan’s multiple range tests. The tests compared the means of the amount of biofilm formed in the presence of each carbon source to all the other carbon sources within each strain. Carbon sources whose mean was different from the means of all the other carbon sources with statistical significance formed their own group in the Duncan’s test. Carbon sources whose mean difference from the other carbon sources was not statistically significant formed overlapping groups.

Performing Duncan’s test on the parent strain, two carbon sources formed groups A and B. Among the remaining carbon sources, we determined those that were structurally related to group A and B carbon sources. This was done after a determination of the respective chemical structures with the Kyoto Encyclopedia of Genes and Genomes (KEGG; Kanehisa & Goto, 2000; KEGG, 2006). Biofilm amounts formed by the flhD mutant were compared to the parent strain for all these carbon sources. In a second analysis, one carbon source formed group A in the Duncan’s test for the flhD mutant. Among the remaining carbon sources, we identified two carbon sources that were structurally related. Biofilm amounts for these three carbon sources were compared between the two strains. For both analyses, data were summarized in a Table (3 and 4).