FIG 2 .
pESI is maintained in a single copy number and does not confer a significant metabolic burden in vitro. (A) Ten-fold serial dilutions of genomic DNA extracted from S. Infantis 335-3 harboring pESI were subjected to real-time PCR using specific primers of the pESI-carried gene pilV and the chromosome-carried gene rpoD. Bars represent the mean threshold cycle (CT) values and the standard deviation (SD) from three biological repeats. The CT values of each gene at the different dilutions were used to create a standard curve. The amplicon size (in base pairs), amplification efficiency (percentage), the slope of the standard curve, and the coefficient of determination (R2), showing the linearity of the standard curve, are indicated in the bottom table. (B) Competitive index of S. Infantis 335-3 carrying pESI and its isogenic strain lacking pESI. Both strains were grown together in LB and N-minimal medium for 24 h at 27°C. Competitive index values were determined as (335-3-pESI/335-3)output/(335-3-pESI/335-3)input. Dots represent the results of single (out of 12) independent competition experiments; the mean and SD are shown by the horizontal line and error bars, respectively. A D’Agostino and Pearson omnibus normality test confirmed Gaussian distributions, and a one-sample t test against a theoretical mean of 1 was used to determine statistical significance of the mean. ns, not significant (P > 0.05).