We found that vpsL mRNA levels were approximately 15-fold higher in the strain with increased NspC levels (Table S1). These results indicate that increased NspC levels affect biofilms through a vps-dependent mechanism. Moreover, because the measurements in these 17-AAG assays are normalized to cell density and an internal standard, respectively, they support the conclusion that the effect of NspC on biofilms is in addition to and independent of its effect on growth. In most cases, biofilm formation and motility are inversely regulated

such that signals or mutations that lead to increases in biofilm formation lead to decreases in motility (Watnick et al., 2001; Moorthy & Watnick, 2005). To determine the effect of increased NspC levels on motility, check details we performed motility assays using semisolid agar plates. Increased NspC levels led to a twofold decrease in the swarm diameter (Fig. 1d), indicating that increased NspC levels affect biofilms and motility in an inverse manner. Because

decreases in intracellular norspermidine levels lead to a decrease in biofilm formation in V. cholerae O1 El Tor, we hypothesized that the increase in biofilm formation may be a consequence of increased levels of norspermidine in these cells. To test this hypothesis, we extracted and quantified the cellular polyamines from shaking cultures grown to log phase (Fig. 2a and b). Norspermidine levels did not increase in the strain overexpressing nspC. Under the conditions of our experiment, V. cholerae Galactosylceramidase also contains significant amounts of putrescine, diaminopropane, spermidine, and a small amount of cadaverine. The levels of these polyamines were also not different between the two strains. Next, we quantified polyamines in the planktonic cells and biofilm-associated cells of static biofilm cultures. Again, we observed no differences in the levels of the various polyamines in the two strains (Fig. 2c and d). However, cadaverine levels were increased in both the planktonic and biofilm-associated cells as compared

to shaking cultures. These results indicate that the increased biofilm levels seen in this strain did not result from increased levels of norspermidine or changes to levels of other polyamines in the cells. We next hypothesized that cells could indeed produce increased amounts of norspermidine as a result of increased NspC levels; however, the excess norspermidine might be exported to maintain norspermidine homeostasis in the cell. To test this hypothesis, we quantified the polyamines in the spent media of these strains as well as sterile LB medium. We did not detect any norspermidine in LB or the spent media. In addition, we found that the spent medium of these strains contained putrescine, diaminopropane, cadaverine, and spermidine; however, only putrescine levels were higher in the spent media of either of the strains as compared to LB, in shaking cultures (Fig. 3).