Transcriptional regulation by BfmRS assessed by qRT-PCR and reporter analysis.

<p>(A) RNA from the indicated strains was reverse-transcribed and probed via qPCR with primers specific for genes encoding proteins linked to the indicated functional category. Shown is the mean of the fold change in transcript levels (vs WT) ± s.d. (n = 3). For each gene, P < 0.02 in unpaired t-tests comparing mutant vs WT. 17180 denotes ACX60_RS17180, a predicted lytic transglycosylase. (B-E) Analysis of BfmRS control of gene expression by using transcriptional reporter fusions. Strains were grown to early post-exponential phase, and reporter activity measured in microtiter format (Materials and methods). (D) <i>A</i>. <i>baumannii</i> strains 17978 (WT), EGA195 (Δ<i>bfmS</i>), or EGA495 (Δ<i>bfmRS</i>) contained a derivative of plasmid pEGE245 encoding GFP transcriptional fusions to the promoter regions upstream of the indicated gene, as diagramed in panel <i>B</i>. 18040, 13710, and 09685 denote ACX60_RS18040, ACX60_RS13710, and ACX60_RS09685, respectively. (E) <i>E</i>. <i>coli</i> DH5α harbored pEGE245 reporter derivatives as well as compatible plasmid pJB1801 containing no additional gene (-) or the indicated <i>bfmRS</i> allele, as diagramed in panel <i>C</i>. Reporter fluorescence was calculated as the fluorescence units/A₆₀₀ of the reporter bacteria minus the fluorescence units/A₆₀₀ of control bacteria containing vector alone. Bars show mean ± s.d. (n = 3). In unpaired t-tests comparing mutant vs WT (D) or <i>bfmRS</i> allele vs vector alone (E), P < 0.04 for each comparison except for: <i>adc</i>p [Δ<i>bfmS</i> vs WT], <i>oxa51</i>p [<i>bfmRS</i> vs vector and <i>bfmRS</i>(G494V) vs vector], and 09685p [<i>bfmRS</i>(G494V) vs vector] (P >0.5).