Novel function of single-target regulator NorR involved in swarming motility and biofilm formation revealed in Vibrio alginolyticus

Abstract

NorR, as a single-target regulator, has been demonstrated to be involved in NO detoxification in bacteria under anaerobic conditions. Here, the norR gene was identified and deleted in the genome of Vibrio alginolyticus. The results showed that deletion of norR in Vibrio alginolyticus led to lower swarming motility and more biofilm formation on aerobic condition. Moreover, we proved that NorR from E. coli had a similar function in controlling motility. NorR overexpression led to increased resistance to oxidative stress and tetracycline. We also observed a reduced ability of the NorR-overexpressing strain to adapt to iron limitation condition. Transcriptome analysis showed that the genes responsible for bacterial motility and biofilm formation were affected by NorR. The expressions of several sigma factors (RpoS, RpoN, and RpoH) and response regulators (LuxR and MarR) were also controlled by NorR. Furthermore, Chip-qPCR showed that there is a direct binding between NorR and the promoter of rpoS. Based on these results, NorR appears to be a central regulator involved in biofilm formation and swarming motility in Vibrio alginolyticus.

Keywords: Vibrio alginolyticus; Bacterial motility; Biofilm; NorR; Sigma factor.

Fig. 1

Comparison of NorR between Vibrio alginolyticus and Escherichia coli. A Alignment of protein sequence of NorR. The conserved amino acids were shown in red color. The domain is positioned at the appropriate position. B The location of NorR gene

Fig. 2

qRT-PCR analysis of transcriptional levels of genes from cells under NO stress conditions. The error bars indicate the standard deviation from the mean of biological triplicates with 2 technical replicates. A The relative mRNA levels of NO stress-related genes were detected in ΔnorR compared with that in wild type under NO stress. B The relative mRNA levels of NO stress-related genes were detected in wild type under NO stress compared with that without stress. The results are normalized to the control gene 16S rDNA using the 2^−ΔΔCt method. ** indicates a highly significant change (P ≤ 0.01) according to Students t-test, * indicates a significant change (P ≤ 0.05) according to Students t-test. ns P > 0.05, t-test

Fig. 3

Phenotype analysis. The growth of different strains under normal condition (A) and iron deficiency stress condition (B) 10 μmol of sodium nitroprusside dihydrate was used to make NO stress condition. The oxidative stress resistance (C), tetracycline resistance (D), and ECPase production (E) of wild type, ΔnorR, C-ΔnorR, and Val/pMMB207-norRVal strains. 1 μmol of 30% H₂O₂ generate oxidative stress and 5 mg/mL tetracycline was spotted onto the plate. ** indicates a highly significant change (P ≤ 0.01) according to Students t-test, *P ≤ 0.05, ns P > 0.05

Fig. 4

NorR negatively regulated swarming motility of Vibrio alginolyticus and Escherichia coli. A Analysis of swarming motility of Vibrio alginolyticus. B Assays of swarming motility of NorR overexpression in Vibrio alginolyticus and Escherichia coli. *** indicates a highly significant change (P ≤ 0.001) according to Students t-test. ** P ≤ 0.01 *P ≤ 0.05, ns P > 0.05

Fig. 5

NorR positively regulates biofilm formation. A Assays of biofilm formation of wild type, ΔnorR, C-ΔnorR, and Val/pMMB207-norR_Val strains. Biofilm formation in petri dish contain LB medium after 48 h of culturing was assayed. *** indicates a highly significant change (P ≤ 0.01) according to Students t-test. *P ≤ 0.05, ns P > 0.05. B Scanning electron micrographs. Wild type and ΔnorR (× 50,000, × 8000, × 2000 magnification)

Fig. 6

The regulon of NorR assessed using RNA-seq. A A volcano plot was generated to visualize the differentially expressed genes in Val/pMMB207-norR_Val. The x-axis represents the log2 of the fold change plotted against the − log10 of the adjusted false discovery rate. Red and green points indicate the up- and downregulated genes, respectively. B Map of gene expression difference. Orange and blue circle indicate the up- and downregulated genes, respectively

Fig. 7

CHIP experiments were followed qPCR to determine the relative enrichment of promoters bound to NorR. The results were calculated by ΔΔC_T method. *P ≤ 0.05, ***P ≤ 0.001, ns P > 0.05, t-test

Fig. 8

The regulatory network of NorR in Vibrio alginolyticus. NorR affects the expression of sigma factors and transcriptional regulators to control the swarming motility and biofilm formation of Vibrio alginolyticus