TsrA regulates gene expression of horizontally acquired elements in Vibrio cholerae via both H-NS dependent and independent mechanisms [IPOD-HR]

Vibrio cholerae, the causative agent of the diarrheal disease cholera, poses an ongoing health threat due to its wide repertoire of horizontally acquired elements (HAEs) and virulence factors. New clinical isolates of the bacterium with improved fitness abilities, often associated with HAEs, frequently emerge. The appropriate control and expression of such genetic elements is critical for the bacteria to thrive in different environments. H-NS, the histone-like nucleoid structuring protein, is the best studied xenogeneic silencer of HAEs in gamma-proteobacteria. Although H-NS and other highly abundant nucleoid-associated proteins (NAPs) have been shown to play important roles in regulating HAEs and virulence in model bacteria, we still lack a comprehensive understanding of how different NAPs modulate transcription in V. cholerae. By obtaining genome-wide measurements of protein occupancy and active transcription in a clinical isolate of V. cholerae, harboring recently-discovered HAEs encoding for phage defense systems, we show that lack of H-NS causes a robust increase on the expression of genes found in many HAEs. We further found that TsrA, a protein with partial homology to H-NS, regulates virulence genes primarily through modulation of H-NS activity, but we also found a few sites that are affected by TsrA independently of H-NS. Our results demonstrate how the combinatorial activity of NAPs is employed by a clinical isolate of an important pathogen to regulate recently-discovered HAEs. IPOD-HR (in vivo protein occupancy display at high resolution (see method below or manuscript for details of the method), which consists of three sample types (IPOD, INPUT and RNA polymerase ChIP-seq) per sample) of V. cholerae strains: 4 replicates (reps.) of clinical isolate KDS1, and mutant strains: 2 reps. ∆hns::frt-spec-frt, 2 reps. ∆ihfA, 3 reps. ∆tsrA::frt-kan-frt, 3 reps. ∆tsrA::frt-kan-frt +∆hns::frt-spec-frt, and 2 reps. ∆vctA as a control. Additionally, IPOD-HR was performed on wild type KDS1 with Mitomycin C treatment in 3 replicates. RNA-sequencing was performed on wild type of KDS1 in 4 reps., 3 reps. of ∆ihfA, 4 reps. of ∆hns::frt-spec-frt, 4 reps. of ∆tsrA::frt-kan-frt, 4 reps. of ∆tsrA::frt-kan-frt +∆hns::frt-spec-frt.