Fig 1.
Graphical illustration of the overall ICA workflow.
(A) Individual gene expression profiles from biofilm and planktonic cells under a total of 161 conditions were combined into the large-scale transcriptome data. Red and blue represent the high and low expression levels of the genes, respectively. (B) ICA decomposed the transcriptome data into 83 independent components and outputted two matrices, ‘Gene coefficient’ and ‘Activity’. The ‘Gene coefficient’ matrix displayed the varied gene coefficients of 4,264 genes constituting the 83 independent components (S3 Dataset). The ‘Activity’ matrix indicated the activities of each independent component along the 161 conditions (S4 Dataset). (C) The independent component-01 was converted into the iModulon-01 by selecting the genes with significant gene coefficients. The same procedure was applied to the remaining 82 independent components.
Fig 2.
Categorization of the iModulons by their biological functions.
72 iModulons identified by ICA were grouped into 10 categories according to their biological functions as illustrated with different colors. The number of genes in each iModulon is indicated by the size of the box.
Fig 3.
Validation of the BrpT-iModulon.
(A) A histogram of the gene coefficients in the BrpT-iModulon. The gray dashed lines show the cutoff values of the gene coefficients. Newly identified members of the BrpT regulon, VV1_3061 and VV2_1694, are presented with red bars. (B to D) Scatter plots of the expression levels of brpT and the activities of the BrpT-iModulon (B), the expression levels of VV1_3061 (C) and VV2_1694 (D). The expression levels are represented as log-TMM, log-transformed read counts after normalization using the trimmed mean of M-values (TMM) method. The Pearson R values between the two variables are denoted in the boxes. Each dot of the plots represents a single biological replicate. The red lines represent each regression line of the plots.
Table 1.
The element genes included in the BrpT-iModulon.
Fig 4.
Predicted protein domains and structures of CabA, CabH, BrpL, and BrpN.
(A) Protein repeats and domains of CabA, CabH, BrpL, and BrpN predicted by using InterPro. The numerical values represent the amino acid numbers of each protein. IPR001343, RTX calcium-binding nonapeptide repeat; IPR002656, acyltransferase-3 domain. (B, C) Superimpositions of the predicted protein structures of CabA (green) and CabH (magenta) (B), and BrpL (green) and BrpN (magenta) (C). Protein structures were predicted by using the Alphafold2 algorithm and the superimpositions of the proteins were performed by using PyMOL. RMSD, root-mean-square deviation.
Fig 5.
Direct regulation of the cabH and brpN expression by BrpT.
(A, B) Effects of BrpT on the expression of cabH and brpN. Total RNAs were isolated from the V. vulnificus strains grown in LB supplemented with 0.01% (w/v) arabinose (A) or 0.03% arabinose, 100 μg/ml kanamycin, and 100 μg/ml ampicillin for complementation experiments (B). The expression of brpT, cabH, and brpN was determined by qRT-PCR analysis, and the expression of each gene in the parent strain was set as 1 (n = 3). Error bars represent the SD. Statistical significance was determined by the Student’s t test (**, P < 0.005). ND, not detected. Parent and Parent (pJK1113), parent strain; ΔbrpT and ΔbrpT (pJK1113), brpT mutant; ΔbrpT (pJK1113::brpT), complemented strain. (C, D) Direct binding of BrpT to upstream regions of cabH and brpN. The 6-FAM labeled DNA fragments (10 nM) of the upstream regions of cabH (C) and brpN (D) were incubated with increasing amounts of BrpT as indicated. For competition analysis, the same but unlabeled DNA fragments were used as self-competitors. Various amounts of self-competitors were added as indicated to the reaction mixtures before the addition of BrpT (4 μM). Each gel representing the mean result from at least three independent experiments was photographed using ChemiDoc Touch Imaging System (Bio-Rad). B, bound DNA; F, free DNA.
Fig 6.
Biofilm formation of the cabH and brpN mutants.
(A, B) For quantitative analysis of the biofilm, biofilms of the V. vulnificus strains were grown in VFMG supplemented with 0.01% arabinose (A) or 0.01% arabinose, 100 μg/ml kanamycin, and 100 μg/ml ampicillin for complementation experiments (B) in 96-well polystyrene microtiter plates for 24 h. Then supernatants were removed from the wells and the remaining biofilms were stained with 1% crystal violet. The crystal violet was eluted and its A570 was determined to quantify the biofilms (n = 3). Error bars represent the SD. Statistical significance was determined by the Student’s t test (*, P < 0.05; **, P < 0.005). Parent and Parent (pJK1113), parent strain; ΔcabH and ΔcabH (pJK1113), cabH mutant; ΔbrpN and ΔbrpN (pJK1113), brpN mutant; ΔbrpT, brpT mutant; ΔcabH (pJK1113::cabH) and ΔbrpN (pJK1113::brpN), complemented strains.
Fig 7.
Colony morphology of the cabH and brpN mutants.
(A, B) The V. vulnificus strains were spotted onto VFMG agar supplemented with 0.02% arabinose (A) or 0.02% arabinose, 100 μg/ml kanamycin, and 100 μg/ml ampicillin for complementation experiments (B) and incubated for 24 h. Each colony representing the mean rugosity from at least three independent experiments was visualized using a stereomicroscope (Stemi 305, Zeiss). All images are shown at the same scale, and 1-mm scale bars are shown on the images of the parent strain. Parent and Parent (pJK1113), parent strain; ΔcabH and ΔcabH (pJK1113), cabH mutant; ΔbrpN and ΔbrpN (pJK1113), brpN mutant; ΔbrpT, brpT mutant; ΔcabH (pJK1113::cabH) and ΔbrpN (pJK1113::brpN), complemented strains.
Fig 8.
Surface attachment and biofilm formation of the cabH, brpN, and cabA mutants.
(A, B) Biofilms of the V. vulnificus strains were grown in VFMG supplemented with 0.01% arabinose in polystyrene (A) or glass (B) test tubes for 24 h. Then supernatants were removed from the tubes and the remaining biofilms were stained with 1% crystal violet. The stained biofilms were then washed with vibration in PBS to remove loosely attached cells. To evaluate the attachment to the test tube surface, each test tube representing the mean result from at least three independent experiments was photographed using a mobile camera. Stained, photographed after the removal of the crystal violet solution; Washed, photographed after being washed with PBS. (C, D) After being washed with PBS, biofilms in the polystyrene (C) or glass (D) test tubes were quantified by eluting the crystal violet and measuring its A570 (n = 3). Error bars represent the SD. Statistical significance was determined by the Student’s t test (*, P < 0.05; **, P < 0.005; ns, not significant). Parent, parent strain; ΔcabH, cabH mutant; ΔbrpN, brpN mutant; ΔcabA, cabA mutant.
Fig 9.
EPS production of the brpN, brpL, and brpN brpL double mutants.
(A) EPS extracts were prepared from the V. vulnificus strains and then resolved on a 4% polyacrylamide gel by SDS-PAGE. The gel was stained with Stains-All and the gel representing the mean result from at least three independent experiments was photographed using a mobile camera. (B) EPS production of the mutants was quantified from the intensity of each lane, and the production of the parent strain was set as 100% in each experiment (n = 3). Error bars represent the SD. Statistical significance was determined by the Student’s t test (*, P < 0.05; **, P < 0.005). Parent, parent strain; ΔbrpN, brpN mutant; ΔbrpL, brpL mutant; ΔbrpN ΔbrpL, brpN brpL double mutant.
Fig 10.
The transcriptional networks of BrpT.
BrpT-iModulon contained cabH and brpN, the newly discovered genes in this study, in addition to brpS, cabABC, and the brp locus. Transcriptional regulator BrpT activates the expression of cabH, brpN, brpS, cabABC, and the brp locus. Transcriptional regulator BrpS activates the expression of cabABC and represses the expression of brpT. CabH is involved in the surface attachment and CabA is crucial for biofilm structural development. BrpN and the proteins expressed from the brp locus participate in the EPS biosynthesis. This sophisticated regulatory network elaborately controls robust biofilm and rugose colony development in V. vulnificus.