biostudies-arrayexpressAndrew BridgesVibrio choleraebcl2fastq; HISAT2; featureCountshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16165RNA-seq was performed to characterize transcriptional changes governed by the DbfRS two-component signaling system in Vibrio cholerae. The experiment compared global gene expression in wild-type and mutant strains lacking dbfS or dbfQ, as well as a phospho-dead dbfR(D51V) variant. Cultures were grown in M9 minimal medium to mid-log phase before RNA extraction. Libraries were sequenced and mapped using HISAT2, and gene quantification was performed with featureCounts followed by normalization and differential expression analysis using edgeR. The resulting data define the DbfR regulon and the transcriptional consequences of DbfRS pathway activation.biostudies-arrayexpressNucleic Acid Extraction - Total RNA was extracted from bacterial pellets using the Qiagen RNeasy Mini Kit following the manufacturer’s instructions. Samples were treated with TURBO DNase (Invitrogen) to remove residual genomic DNA. RNA concentration and purity were assessed using a NanoDrop spectrophotometer.Sample Collection - Vibrio cholerae strains (WT, dbfS, dbfQ, and dbfR(D51V)) were cultured in M9 minimal medium supplemented with dextrose and casamino acids at 30 °C with shaking until mid-log phase (OD₆₀₀ ≈ 0.1). Triplicate cultures were harvested by centrifugation and stabilized in RNAprotect (Qiagen) before RNA extraction.Library Construction - RNA integrity was verified, and sequencing libraries were prepared using Illumina TruSeq stranded mRNA or equivalent library preparation kits by the Microbial Genome Sequencing Center (MiGS). Libraries were constructed from high-quality RNA and converted to cDNA for paired-end sequencing.Sequencing - Sequencing was performed by the Microbial Genome Sequencing Center (MiGS) on an Illumina platform (paired-end 150 bp reads). Raw basecalls were converted and demultiplexed using bcl2fastq software. Read mapping was performed using HISAT2, and read quantification was performed using featureCounts.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Read count data were imported into R and processed using the edgeR package. Counts were normalized using the Trimmed Mean of M-values (TMM) method and converted to counts per million (CPM). Differential expression analysis was performed using edgeR’s quasi-likelihood F-test (QLF test). The resulting normalized CPM values are reported in the processed data files.UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000Two-component signaling pathways allow bacteria to sense and respond to environmental changes, yet the sensory mechanisms of many remain poorly understood. In the pathogen <i>Vibrio cholerae</i>, the DbfRS two-component system controls the biofilm lifecycle, a critical process for environmental persistence and host colonization. Here, we identified DbfQ, a small periplasmic protein encoded adjacent to <i>dbfRS</i>, as a direct modulator of pathway activity. DbfQ directly binds the sensory domain of the histidine kinase DbfS, shifting it toward phosphatase activity and promoting biofilm dispersal. In contrast, outer membrane perturbations, caused by mutations in lipopolysaccharide biosynthesis genes or membrane-damaging antimicrobials, activate phosphorylation of the response regulator DbfR. Transcriptomic analyses reveal that DbfR phosphorylation leads to broad transcriptional changes spanning genes involved in biofilm formation, central metabolism, peptidoglycan synthesis, and cellular stress responses. Constitutive DbfR phosphorylation imposes severe fitness costs in an infection model, highlighting this pathway as a potential target for anti-infective therapeutics. We find that <i>dbfQRS</i>-like genetic modules are widely present across bacterial phyla, underscoring their broad relevance in bacterial physiology. Collectively, these findings establish DbfQ as a new class of periplasmic regulator that influences two-component signaling and bacterial adaptation.RNA-seq of coding RNAVibrio choleraeA small periplasmic protein governs broad physiological adaptations in Vibrio cholerae via regulation of the DbfRS two-component systemAndrew BridgesEmmy Nguyen, Charles Agbavor, Anjali Steenhaut, M. R. Pratyush, N. Luisa Hiller, Laty A. Cahoon, Irina V. Mikheyeva, Wai-Leung Ng & Andrew A. BridgesfalseRNA-seq of Vibrio cholerae wild-type and dbfS, dbfQ, and dbfR(D51V) mutant strainsRNA-seq was performed to characterize transcriptional changes governed by the DbfRS two-component signaling system in Vibrio cholerae. The experiment compared global gene expression in wild-type and mutant strains lacking dbfS or dbfQ, as well as a phospho-dead dbfR(D51V) variant. Cultures were grown in M9 minimal medium to mid-log phase before RNA extraction. Libraries were sequenced and mapped using HISAT2, and gene quantification was performed with featureCounts followed by normalization and differential expression analysis using edgeR. The resulting data define the DbfR regulon and the transcriptional consequences of DbfRS pathway activation.2025-11-20T00:00:00Z2025-11-20T02:02:22.478Z2025-11-17T14:04:31.895ZE-MTAB-1616540196685EFO_0002944EFO_0004170EFO_0005518EFO_0003816EFO_0003738EFO_000418410.1101/2025.03.24.645060