Dataset Information

A dual RNA regulator balancing carbon uptake and 1 central metabolism in Vibrio cholerae

ABSTRACT: Bacterial small RNAs (sRNAs) are well-known to modulate gene expression by base-pairing with trans-coded transcripts and are typically considered to be non-coding. However, several sRNAs have been reported to also contain an open reading frame and thus are considered dual regulators. In this study, we discovered a dual regulator from Vibrio cholerae, called VcdRP, harboring a 29 amino acid protein (VcdP), as well as a base-pairing sequence. Using a forward genetic screen, we identified VcdRP as a repressor of cholera toxin production and we link this phenotype to inhibition of carbon transport by the base-pairing segment of the regulator. By contrast, we demonstrate that VcdP acts downstream of carbon transport by binding to citrate synthase (GltA), the first enzyme of the citric cycle. Interaction of VcdP with GltA results in increased enzyme activity and together VcdR and VcdP reroute carbon metabolism. We further show that transcription of vcdRP is repressed by CRP allowing us to provide a model in which VcdRP employs two different molecular mechanisms to synchronize sugar uptake and metabolism in V. cholerae.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Vibrio Cholerae Serotype O1 (strain Atcc 39315 / El Tor Inaba N16961)

SUBMITTER: Andreas Tholey

LAB HEAD: Prof. Dr.Andreas Tholey

PROVIDER: PXD025275 | Pride | 2021-07-22

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Blank_KV01_1_200416194801.raw	Raw
	Blank_KV01_2_200416205020.raw	Raw
	Blank_KV04_1_200417064116.raw	Raw
	Blank_KV04_2_200417074336.raw	Raw
	KV_01_Ctrl_1.pdResult	Other

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Project description:The cholera disease bacterium V. cholerae, can adopt planktonic or biofilm lifestyles depending on the intracellular concentration of the second messenger cyclic diguanylic acid (c-di-GMP). Biofilm formation protects Vibrios from stressful conditions and facilitates disease transmission by enhancing infectivity. The histone-like nucleoid structuring protein (H-NS) is a global regulator of genes associated with pathogenicity and responses to environmental stresses. H-NS represses the transcription of genes vpsT, vpsA and vpsL, which are required for the biosynthesis of the biofilm exopolysacchide matrix. Here we demonstrate that the c-di-GMP-binding protein VpsT disrupts H-NS nucleoprotein complexes at the vpsA and vpsL promoters and that this effect is enhanced by c-di-GMP. We used ChIP coupled with Next Generation Sequencing (ChIP-Seq) and transcriptome analysis (RNA-Seq) to identify additional loci repressed by H-NS affecting biofilm formation. This study showed that H-NS directly represses the transcription of genes encoding proteins present in the biofilm matrix such as the rbmA-F cluster, hemolysin and chitinase. Similar to vpsA and vpsL, the promoter region of vpsU, rbmA and rbmF exhibited overlapping H-NS and VpsT binding motifs. Deletion of vpsT increased H-NS occupancy at the vpsU, vpsA, vpsL, rbmA and rbmF promoters. Conversely, artificially increasing the c-di-GMP pool diminished H-NS occupancy at the above promoters. Deletion of vpsT did not affect H-NS occupancy at its own promoter. However, deletion of genes encoding the regulators AphA and VpsR significantly increased H-NS occupancy at the vpsT promoter. In sum, our study shows that c-di-GMP enhances biofilm formation by acting through VpsT to activate an H-NS anti-repression cascade. The Binding profile of V. cholerae H-NS to the genome was determined by ChIP followed by Next Generation Sequencing (ChIP-Seq) using the Illumina HiSeq2000 platform. V. cholerae C7258 cells expressing H-NS-FLAG fusion protein from the hns transcription and translation signals were collected from LB cultures grown to mid-exponential phase (OD600 0.5). An anti-FLAG Immunoprecipitation (IP) and an Input samples were used for the analysis.

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A dual RNA regulator balancing carbon uptake and 1 central metabolism in Vibrio cholerae

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