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Preliminary investigation into the impact of GGDEF family gene deletion on the phenotype of Aeromonas hydrophila

ABSTRACT: This study took Aeromonas hydrophila as an example to study the GGDEF family gene and explore its biological function in Aeromonas hydrophila and its influence on c-di-GMP concentration. In order to systematically study the biological function of GGDEF family genes, 36 strains (54 strains screened by whole genome sequencing) with gene deletion mutation were first constructed, and then phenotypes such as biofilm, crawling and extracellular protease activity were detected. The concentration of c-di-GMP in aeromonas hydrophila was determined by HPLC. At the same time, the deletion strains with GGDEF domain that had a significant effect on the physiological function of Aeromonas hydrophila were screened for mass spectrometry identification, so as to explore the possible molecular mechanism of GGDEF domain protein affecting the physiological function.

ORGANISM(S): Aeromonas Hydrophila Subsp. Hydrophila Atcc 7966

SUBMITTER: Xiangmin Lin

PROVIDER: PXD059486 | iProX | Mon Jan 06 00:00:00 GMT 2025

REPOSITORIES: iProX

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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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Preliminary investigation into the impact of GGDEF family gene deletion on the phenotype of Aeromonas hydrophila

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