Global regulation and virulence mediated by the histidine-responsive local transcription factor HutC in <i>Pseudomonas aeruginosa</i>

Host-derived nutritional substrates fuel infection in invading bacteria, yet their potential as signaling molecules for host perception remains largely unexplored. Here, we report the functional characterization of HutC, a transcriptional repressor of <i>hut</i> genes for the utilization of histidine and its derivative, urocanate, in the human pathogenic bacterium <i>Pseudomonas aeruginosa</i> PAO1. Using electrophoretic mobility shift assay (EMSA) and DNase I footprinting combined with site-directed mutagenesis, we demonstrate that HutC specifically binds with high affinity to the promoters of the two <i>hut</i> operons. This analysis led to the identification of a noncanonical HutC-binding site in the <i>hutF</i> promoter of a non-pathogenic <i>Pseudomonas</i> strain, which is absent in <i>P. aeruginosa</i>. A genome-wide search of the PAO1 genome using a probability matrix of the canonical HutC-binding motif identified 172 candidate sites, many associated with bacterial pathogenesis. Their predicted low-affinity binding was experimentally validated by EMSA for six selected targets, including the aminoglycoside response regulator (<i>arr</i>). Deletion of <i>hutC</i> resulted in increased tobramycin-induced biofilm formation and impaired production of pyoverdine, an iron-scavenging siderophore. Moreover, the <i>hutC</i> mutant exhibited altered motility and significantly reduced virulence in the <i>Caenorhabditis elegans</i> infection model. Finally, transcriptome sequencing of three genetically distinct <i>hutC</i> mutants provided further support for the HutC-mediated global regulation. Together, these findings highlight the functional significance of low-affinity DNA binding by this transcription factor and support the hypothesis that HutC mediates <i>P. aeruginosa</i> virulence, with histidine and urocanate as effectors. Thus, HutC may represent a potential therapeutic target within the bacterial host-perception system.IMPORTANCE<i>Pseudomonas aeruginosa</i> is a metabolically versatile environmental pathogen whose virulence relies on coordinated expression of catabolic genes, particularly the histidine utilization (<i>hut</i>) operon. Disruption of the <i>hut</i> operon reduces virulence, but the underlying mechanism remains rudimentary. Here, we genetically characterized the histidine-responsive transcriptional factor HutC in <i>P. aeruginosa</i> PAO1, alongside HutC in the non-pathogenic strain <i>Pseudomonas fluorescens</i> SBW25. Two important features emerged. First, HutC recognizes two distinct DNA-binding motifs with little sequence similarity; notably, a noncanonical-binding site was identified in the <i>hutF</i> promoter of SBW25 but was absent in PAO1. Second, HutC exhibits low-affinity binding to genes beyond histidine catabolism and contributes to the expression of multiple virulence traits. These findings identify HutC as a local regulator linking histidine catabolism with virulence and as a unique prokaryotic model for studying how noncanonical transcriptional factor-DNA interactions achieve binding specificity, a phenomenon so far investigated only in eukaryotes.