Figure 1.
(A) Coomassie-brilliant-blue-stained SDS-PAGE gel. (B) Western blot detecting RbmC (upper panel); proteins on the Western blot membrane were stained with Coomassie-brilliant-blue and shown as loading control (lower panel). Supernatant samples were prepared from bacteria cultivated in LB medium at 30°C. Lane 1, A1552 (wild type); lane 2, DNY7 (ΔvrrA); lane 3, DNY11 (ΔvrrA+pvrrA); lane 4, DNY12 (ΔvrrA+pMMB66HE); lane 5, DNY8 (Δhfq); lane 6, DNY9 (ΔhfqΔvrrA); lane 7, DNY16 (ΔhfqΔvrrA+pvrrA); lane 8, DNY17 (ΔhfqΔvrrA+pMMB66HE); lane 9, DHS196 (ΔrbmC). LaProtein marker sizes (lane M) are given to the left in kDa. The asterisk indicates the protein band that was excised from the gel and subjected to mass spectrometry analysis.
Figure 2.
VrrA sequesters the 5′-UTR of rbmC by an antisense mechanism.
(A) Graphical presentation of the proposed interaction of VrrA sRNA with the 5′-UTR of rbmC sequence, and of compensatory base-pair changes. Numbering for rbmC is relative to start codon AUG (A is +1), and that for VrrA is relative to the +1 transcription start site. The predicted SD sequence of rbmC (AGGGAGU) is underlined. Vertical arrows denote nucleotides introduced into rbmC and VrrA for compensatory base-pair change experiment. (B) Sequences of wild-type VrrA (pTS2) and its nucleotide substitution mutants. Nucleotides that were substituted are underlined.
Figure 3.
Detection of VrrA and its mutant variants by Northern blot analysis.
(A) Wild-type VrrA is expressed from plasmid pTS2. Mutant variants VrrAM7 to VrrAM10 are expressed from corresponding basepair-substituted plasmids pTS2-M7 to pTS2-M10. All plasmids were transformed into V. cholerae strain DNY7 (ΔvrrA). pJV300 is used as plasmid control for pTS2. The 5S rRNA was probed as an internal control for Northern blot analysis. (B). Potential structures of VrrA variants. RNA folding was performed using the Mfold algorithm [40].
Figure 4.
Western blot analyses of RbmC levels in the culture supernatants of the wild type V. cholerae strain A1552 and vrrA mutant derivatives: (A) Detection of RbmC (upper panel) in the supernatants from DNY7 (ΔvrrA) carrying different plasmids expressing either the wild-type VrrA (from plasmid pTS2) or mutant variants VrrAM1 to VrrAM6 are expressed from plasmids pTS2-M1 to pTS2-M6. A SDS-PAGE Coomassie blue stained gel is shown as the sample loading control (lower panel). (B) Western blot analysis of the RbmC levels in supernatants isolated from DNY7 (ΔvrrA) and DNY189 (ΔvrrA rbmC*) carrying different plasmids (upper panel). A SDS-PAGE gel stained with Coomassie blue is shown as loading (lower panel). Protein marker sizes are given to the left in kDa.
Figure 5.
Nucleotide substitutions at residues 69–78 in VrrA do not affect repression on RbmC.
Western blot analysis of supernatants from DNY7 (ΔvrrA) carrying the indicated plasmids (upper panel). A SDS-PAGE Coomassie blue staind gel was shown as a sample loading control (lower panel).
Figure 6.
Impact of vrrA on biofilm formation (A) Confocal laser scanning microscopy images of horizontal (xy) and vertical (xz) projections of biofilm structures formed by wild-type strain (WT) carrying the vector or pBAD-vrrA.
Cells were grown for 2(a and d), 24 h (b and e); 48 h (c and f) in 2% LB medium in the presence of ampicillin and 0.05% arabinose at room temperature. (B) COMSTAT analysis of biofilms formed by wild-type strains harboring a plasmid vector or vrrA over-expression plasmid.
Table 1.
Oligonucleotides used in this study.
Table 2.
Bacterial strains and plasmids used in this study.