Relative contributions of norspermidine synthesis and signaling pathways to the regulation of Vibrio cholerae biofilm formation

Abstract

The polyamine norspermidine is one of the major polyamines synthesized by Vibrionales and has also been found in various aquatic organisms. Norspermidine is among the environmental signals that positively regulate Vibrio cholerae biofilm formation. The NspS/MbaA signaling complex detects extracellular norspermidine and mediates the response to this polyamine. Norspermidine binding to the NspS periplasmic binding protein is thought to inhibit the phosphodiesterase activity of MbaA, increasing levels of the biofilm-promoting second messenger cyclic diguanylate monophosphate, thus enhancing biofilm formation. V. cholerae can also synthesize norspermidine using the enzyme NspC as well as import it from the environment. Deletion of the nspC gene was shown to reduce accumulation of bacteria in biofilms, leading to the conclusion that intracellular norspermidine is also a positive regulator of biofilm formation. Because V. cholerae uses norspermidine to synthesize the siderophore vibriobactin it is possible that intracellular norspermidine is required to obtain sufficient amounts of iron, which is also necessary for robust biofilm formation. The objective of this study was to assess the relative contributions of intracellular and extracellular norspermidine to the regulation of biofilm formation in V. cholerae. We show the biofilm defect of norspermidine synthesis mutants does not result from an inability to produce vibriobactin as vibriobactin synthesis mutants do not have diminished biofilm forming abilities. Furthermore, our work shows that extracellular, but not intracellular norspermidine, is mainly responsible for promoting biofilm formation. We establish that the NspS/MbaA signaling complex is the dominant mediator of biofilm formation in response to extracellular norspermidine, rather than norspermidine synthesized by NspC or imported into the cell.

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.

Fig 2. Effects of vibriobactin synthesis and utilization on biofilm formation in V. cholerae.

(A) Biofilm formation of ΔvibF and viuA::tet^R mutants. (B) Biofilm formation of ΔvibF, nspC::kan^R, and nspC::kan^RΔvibF 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::kan^RΔnspS 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::kan^R, and nspC::kan^RΔnspS mutations, with and without exogenous norspermidine. (B) Biofilm assay of nspC::kan^R, ΔmbaA, and nspC::kan^RΔmbaA 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::kan^R, nspC::kan^RΔpotD1, and nspC::kan^RΔnspSΔpotD1 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::kan^R 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 qrgB_AADEF mutants. (B) Biofilm assay of nspC::kan^RΔpotD1ΔnspS with qrgB and nspC::kan^RΔpotD1ΔnspS with qrgB_AADEF 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. Proposed environmental model.

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.