1a); however, under these conditions, we were not able to detect NspC in cells that did not overexpress this protein. To detect wild-type levels of NspC, we had to use a more sensitive detection system, which allowed us to visualize the NspC protein in cells that did not contain the pnspC plasmid. This result ensured that NspC was being expressed from its chromosomal location under our experimental conditions (Supporting Information, Fig. S1). We then assayed the effect of elevated NspC levels on various aspects of V. cholerae physiology. The presence of pnspC altered growth characteristics of the cells such that the lag time was much shorter, the growth rate 1.5-fold higher, and the cell density
experiment. Biofilm assays showed that increased production of NspC resulted in an approximately fivefold increase in biofilm cell density (Fig. 1b). This result is in contrast to a previous study which reported an inhibitory effect of ectopic expression of nspC on biofilms formed by V. cholerae O1 El Tor (Lee et al., 2009). The reasons for this disagreement are not known but can potentially be a result of different genetic backgrounds or plasmid systems used in these experiments. Planktonic cell density showed a very small but statistically significant reduction in the strain containing the pnspC plasmid. In most cases, strains that have a high propensity to form biofilms show reduced densities of planktonic cells. The fact that we did not see a large STK38 reduction in planktonic cells overexpressing nspC may be accounted
for by the fact that this strain can grow slightly faster and to higher cell densities. Formation of biofilms usually requires the presence of an exopolysaccharide in the biofilm matrix whose synthesis and export is achieved by proteins encoded by the vps genes (Watnick & Kolter, 1999; Yildiz & Schoolnik, 1999). Under most conditions, increases in biofilm formation are accompanied by increases in vps gene transcription. These genes are found on the V. cholerae large chromosome in two operons: vpsA-K and vpsL-Q (Watnick & Kolter, 1999; Yildiz & Schoolnik, 1999). To test whether increased nspC gene expression also leads to an increase in vps gene transcription, we assayed the activity of the vpsL promoter, making use of a chromosomal vpsLp-lacZ fusion in our strains (Haugo & Watnick, 2002). This insertion does not change the physiological characteristics of the wild-type bacteria such as growth, motility, and biofilm formation under the conditions of our experiments. Increased levels of the NspC protein resulted in a threefold and an eightfold increase in β-galactosidase activity in exponential and stationary-phase cells, respectively (Fig. 1c).