A mutagenic screen reveals NspS residues important for regulation of Vibrio cholerae biofilm formation

Microbiology (Reading). 2021 Mar;167(3):001023. doi: 10.1099/mic.0.001023. Epub 2021 Jan 27.

Abstract

Biofilm formation in the human intestinal pathogen Vibrio cholerae is in part regulated by norspermidine, spermidine and spermine. V. cholerae senses these polyamines through a signalling pathway consisting of the periplasmic protein, NspS, and the integral membrane c-di-GMP phosphodiesterase MbaA. NspS and MbaA belong to a proposed class of novel signalling systems composed of periplasmic ligand-binding proteins and membrane-bound c-di-GMP phosphodiesterases containing both GGDEF and EAL domains. In this signal transduction pathway, NspS is hypothesized to interact with MbaA in the periplasm to regulate its phosphodiesterase activity. Polyamine binding to NspS likely alters this interaction, leading to the activation or inhibition of biofilm formation depending on the polyamine. The purpose of this study was to determine the amino acids important for NspS function. We performed random mutagenesis of the nspS gene, identified mutant clones deficient in biofilm formation, determined their responsiveness to norspermidine and mapped the location of these residues onto NspS homology models. Single mutants clustered on two lobes of the NspS model, but the majority were found on a single lobe that appeared to be more mobile upon norspermidine binding. We also identified residues in the putative ligand-binding site that may be important for norspermidine binding and interactions with MbaA. Ultimately, our results provide new insights into this novel signalling pathway in V. cholerae and highlight differences between periplasmic binding proteins involved in transport versus signal transduction.

Keywords: Vibrio cholerae; biofilm; homology modelling; norspermidine; polyamine.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Biofilms*
  • Gene Expression Regulation, Bacterial
  • Mutagenesis
  • Periplasm / genetics
  • Periplasm / metabolism
  • Protein Domains
  • Sequence Alignment
  • Signal Transduction
  • Vibrio cholerae / chemistry
  • Vibrio cholerae / genetics*
  • Vibrio cholerae / physiology

Substances

  • Bacterial Proteins