D-Alanine substitution of teichoic acids as a modulator of protein folding and stability at the cytoplasmic membrane/cell wall interface of Bacillus subtilis

J Biol Chem. 2000 Sep 1;275(35):26696-703. doi: 10.1074/jbc.M003804200.

Abstract

The extracytoplasmic folding of secreted proteins in Gram-positive bacteria is influenced by the microenvironment of the compartment into which they are translocated, namely the negatively charged matrix of the cell wall polymers. In this compartment, the PrsA lipoprotein facilitates correct post-translocational folding or prevents misfolding of secreted proteins. In this study, a secretion mutant of B. subtilis (prsA3) encoding a defective PrsA protein was mutagenized and screened for restored secretion of the AmyQ alpha-amylase. One mini-Tn10 insertion, which partially suppressed the secretion deficiency, was found to interrupt dlt, the operon involved in the d-alanylation of teichoic acids. The inactivation of dlt rescued the mutant PrsA3 protein from degradation, and the increased amount of PrsA3 was shown to enhance the secretion of PrsA-dependent proteins. Heterologous or abnormal secreted proteins, which are prone to degradation after translocation, were also stabilized and secreted in increased quantities from a dlt prsA(+) strain. Furthermore, the dlt mutation partially suppressed the lethal effect of PrsA depletion, suggesting that the dlt deficiency also leads to stabilization of an essential cell wall protein(s). Our results suggest that main influence of the increased net negative charge of the wall caused by the absence of d-alanine is to increase the rate of post-translocational folding of exported proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alanine / chemistry*
  • Bacillus subtilis / enzymology
  • Bacillus subtilis / metabolism*
  • Bacterial Proteins*
  • Base Sequence
  • Cell Membrane / metabolism*
  • Cell Wall / metabolism*
  • DNA Primers
  • DNA Transposable Elements
  • Hydrogen-Ion Concentration
  • Lipoproteins / chemistry
  • Lipoproteins / metabolism
  • Membrane Proteins / chemistry
  • Membrane Proteins / metabolism
  • Mutation
  • Operon
  • Protein Folding
  • Teichoic Acids / chemistry
  • Teichoic Acids / metabolism*
  • alpha-Amylases / metabolism

Substances

  • Bacterial Proteins
  • DNA Primers
  • DNA Transposable Elements
  • Lipoproteins
  • Membrane Proteins
  • Teichoic Acids
  • prsA protein, bacteria
  • alpha-Amylases
  • Alanine