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Quantitative proteomics analysis reveals the transcriptional regulator UidR in Aeromonas hydrophila play important on the bacterial biofilm formation

ABSTRACT: Biofilm is a well-known characteristic that plays important roles on diverse physiological functions, whereas the current intrinsic regulatory mechanism of its formation is still largely unknown. In this study, we found the deletion of a TetR transcriptional regulator gene uidR significantly increased the biofilm formation in Aeromonas hydrophila. To further understanding its role on biofilm formation, a label-free based quantitative proteomics technology was used to compare the differentially expression proteins between ΔuidR and the wild-type strain in the biofilm state, and showed a total of 220 proteins altered, including 120 increased and 100 proteins decreased abundances. Bioinformatics analysis suggests that uidR may affect bacterial biofilm formation by regulating glyoxylic acid and dicarboxylic acid pathway related proteins. The varieties of selected proteins involved in this pathway were further confirmed by q-PCR assay. Moreover, three related genes, including AHA_3063, AHA_3062, and aceB were deleted respectively, and resulting a significant decrease in the biofilm formation ability. These results further validated the role of glyoxylic acid and dicarboxylic acid pathway on bacterial biofilm formation. In summary, our data indicate that uidR involved in regulatory of bacterial biofilm formation, and that may provide new clue for the prevention and treatment of pathogenic A. hydrophila in the future.

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

SUBMITTER: Xiangmin Lin

PROVIDER: PXD034710 | iProX | Sun Jun 19 00:00:00 BST 2022

REPOSITORIES: iProX

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Project description:The Escherichia coli quorum-sensing regulator, SdiA, belongs to the LuxR family of transcriptional regulators and is responsible for detecting signals from other bacteria. Previously we found that SdiA is necessary for E. coli to control its biofilm formation with indole just as SdiA is necessary for E. coli to alter its biofilm formation in the presence of N-acylhomoserine lactones (AHLs). Here we engineered SdiA by random mutagenesis to further control biofilm formation in the presence of indole and AHLs. After screening of 477?? mutants with indole and two AHLs (N-butyryl-DL-homoserine lactone, and N-(3-oxooctanoyl)-L-homoserine lactone, C6HSL), five SdiA variants were obtained that altered biofilm with and without signals of indole and AHLs. Two truncation variants (1E11 and 14C3) lacking the C-terminal DNA-binding domain of SdiA showed the reduction of biofilm formation by 5-fold and 10-fold in LB and LB glu, respectively. DNA microarrays show that the evolved SdiA (1E11) compared to wild-type SdiA influences indole synthesis genes, AI-2 uptake genes, acid-resistance genes, motility related genes, cold-shock protein genes, and several regulatory protein genes. Corroborating DNA microarrays, SdiA variants produced various amounts of indole which led to different survivals in low pH and influenced swimming motility and final cell density. Also, an AHL sensitive variant (2D10) 2-fold increased biofilm formation in the presence of C6HSL, while another variant (6B12) lowered biofilm formation in the presence of C6HSL. Hence, the results clearly showed that mutation of SdiA itself directly controls biofilm formation and SdiA variants could be further recognized by the inter-species signal AHLs. This is the first random protein engineering to control biofilm formation. Experiment Overall Design: For the microarray experiments, 10 g glass wool (Corning Glass Works, Corning, N.Y.) were used to form biofilms in 250 mL LB ( 1 mM IPTG + Cm30) in 1 L Erlenmeyer shake flasks which were inoculated with overnight cultures diluted that were 1:100. The cells were shaken at 250 rpm and 30C for 12 hours to form biofilms on the glass wool, and RNA was isolated from the the biofilm cells.

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Quantitative proteomics analysis reveals the transcriptional regulator UidR in Aeromonas hydrophila play important on the bacterial biofilm formation

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