Fig. 4.

Exogenous nitrogen is not the driving force behind in vivo gdhA and glnA expression. (A) RNA was isolated from P. mirabilis HI4320 cultured in LB supplemented with either 500 mM NH₄Cl or 400 mM urea. No significant changes in gdhA or glnA were found by qRT-PCR in comparison to unsupplemented LB. However, addition of urea significantly upregulated expression of ureA. (B) Enzyme activities were determined for whole-cell lysates of P. mirabilis HI4320 cultured under identical conditions to those described for panel A. Glutamate dehydrogenase (GDH), total glutamine synthetase (GS), and urease (URE) enzyme activities (average of five technical replicates) were measured and expressed as fold change relative to preparations from P. mirabilis cultured in LB broth. (C) A ureC mutant was cultured in LB broth or pooled human urine with or without 100 mM NH₄Cl, and expression of gdhA and glnA was measured by qRT-PCR. (A and C) Expression was normalized to rpoA. At least three independent experiments were conducted for each condition tested. Error bars represent standard errors of the mean. *, P < 0.05.