FIG 7.
Genes of interest are expressed in vitro and do not significantly contribute to growth in LB broth or minimal medium. (A and B) Expression of 8 genes encoding candidate fitness factors of interest (tatC, clpP, ddlA, fliC, sopB, yscC, and yscI) and one housekeeping gene (recA) was assessed by quantitative reverse transcriptase PCR (qRT-PCR) for P. stuartii BE2467 cultured in LB broth or human urine for 2 h. (A) Raw quantification cycle (Cq) values for each gene of interest. (B) The Pfaffl method was utilized to generate a Relative Quantification (RQ) value for each gene (see Materials and Methods). Error bars represent means and standard deviations of results from three independent experiments performed with two replicates each. Statistical significance was determined relative to recA by Wilcoxon signed-rank test (**, P < 0.01). (C and D) Mutants were constructed in seven genes of interest, and growth was assessed in LB broth (C) and minimal medium (D) for comparison to wild-type P. stuartii BE2467. Each panel displays a representative growth curve on a log scale; error bars represent the means and standard deviations of results from four replicates.