Fig 1.
Norspermidine related processes in V. cholerae.
(A) Synthesis of norspermidine and vibriobactin. Norspermidine can be synthesized by the enzyme NspC through decarboxylation of carboxynorspermidine. Norspermidine is utilized by VibF to form the backbone of the siderophore vibriobactin. Norspermidine backbone outlined by the grey box.(B) Norspermidine synthesis, utilization, transport, and signaling pathways. Norspermidine can be synthesized by NspC and can also be imported from the environment presumably through the PotABCD1 ABC-type transporter. Vibriobactin, synthesized from norspermidine, is secreted into the environment and binds to iron. This ferric-vibriobactin complex is recognized by the outer membrane protein ViuA and transported into the periplasm, which is then imported into the cell by an ABC-type transporter (not shown). Exogenous norspermidine is sensed by the NspS/MbaA signaling complex, which leads to increased VPS production and biofilm formation, presumably through increasing c-di-GMP levels in the cell. Norspermidine is represented by a zig-zag and carboxynorspermidine is represented by a branched zig-zag. VPS, Vibrio polysaccharide. The PotABCD1 ABC-type transporter is denoted as A, B, C, and D1.
Table 1.
Bacterial strains and plasmids.
Table 2.
Primers.
Fig 2.
Effects of vibriobactin synthesis and utilization on biofilm formation in V. cholerae.
(A) Biofilm formation of ΔvibF and viuA::tetR mutants. (B) Biofilm formation of ΔvibF, nspC::kanR, and nspC::kanRΔvibF mutants. Biofilms were formed in borosilicate tubes in LB broth for 24 h at 27°C and quantified as described in Materials and Methods. EDDA was added to chelate iron to generate iron-deplete conditions. Error bars show standard deviations of three biological replicates. A star indicates a statistically significant difference between wild type and the mutants. A double star indicates a statistically significant difference between growth media conditions. A p-value <0.05 was considered significant. WT, wild type.
Fig 3.
Role of NspS and NspC on cellular polyamine content in V. cholerae. Polyamine composition of nspC::kanRΔnspS cells with and without exogenous norspermidine.
Polyamines were extracted from cells, derivatized by benzoylation and analyzed by HPLC as described in Materials and Methods. Labeled peaks on the chromatogram correspond to putrescine (put), diaminopropane (dap), cadaverine (cad), norspermidine (nspd), and spermidine (spd). AU254, absorbance units at 254 nm. Only 4–14 minutes of a 40-minute run are plotted for clarity.
Fig 4.
Role of NspS, MbaA, and NspC on biofilm formation in V. cholerae.
(A) Biofilm assay of ΔnspS, nspC::kanR, and nspC::kanRΔnspS mutations, with and without exogenous norspermidine. (B) Biofilm assay of nspC::kanR, ΔmbaA, and nspC::kanRΔmbaA mutations, with and without exogenous norspermidine. Biofilms were formed in borosilicate tubes in LB broth for 18 h at 27°C and quantified as described in Materials and Methods. Error bars show standard deviations of three biological replicates. A star indicates a statistically significant difference between wild type and the mutants. A double star indicates a statistically significant difference between growth media conditions. A p-value <0.05 was considered significant. WT, wild type.
Fig 5.
Effects of ΔpotD1, nspC::kanR, nspC::kanRΔpotD1, and nspC::kanRΔnspSΔpotD1 mutations, with and without exogenous norspermidine, on biofilm formation in V. cholerae.
Biofilms were formed in borosilicate tubes in LB broth for 18 h at 27°C and quantified as described in Materials and Methods. Error bars show standard deviations of three biological replicates. A star indicates a statistically significant difference between wild type and the mutants. A double star indicates a statistically significant difference between growth media conditions. A p-value <0.05 was considered significant. WT, wild type. The values for WT and nspC::kanR in Fig 4 are the same as these experiments were performed simultaneously.
Fig 6.
Artificially increasing c-di-GMP levels can overcome the nspS defect.
(A) Biofilm assay of ΔnspS with qrgB and ΔnspS with qrgBAADEF mutants. (B) Biofilm assay of nspC::kanRΔpotD1ΔnspS with qrgB and nspC::kanRΔpotD1ΔnspS with qrgBAADEF mutants. Biofilms were formed in borosilicate tubes in LB broth for 18 h at 37°C and quantified as described in Materials and Methods. Relative biomass was calculated using the following equation OD655 mutant/OD655 wild type (Y-axis). Error bars show standard deviations of three biological replicates. A star indicates a statistically significant difference between wild type and the mutants. A p-value <0.05 was considered significant. WT, wild type.
Fig 7.
Environmental norspermidine may primarily derive from endogenously-produced norspermidine that is released during cell lysis or exported to the periplasm by an unknown transporter. It may also be provided by nearby eukaryotic organisms. Norspermidine may also act as a quorum sensing molecule, allowing V. cholerae to detect this signal, recognize that it is in the presence of other Vibrios, and respond appropriately by forming the Vibrio polysaccharide. In this way, norspermidine may allow V. cholerae to persist in its biofilm form in its natural environment.