BETA-LACTAMASE TOPOLOGY PROBE ANALYSIS OF THE OUTO NMEPHE PEPTIDASE, AND 6 OTHER OUT PROTEIN-COMPONENTS OF THE ERWINIA-CAROTOVORA GENERAL SECRETION PATHWAY APPARATUS

The out gene cluster of Erwinia spp. encodes the proteins of the general secretory pathway (GSP) apparatus that is required for pectinase and cellulase secretion. We have used fusions between Erwinia carotovora subsp. carotovora (Ecc) out genes and the topology probe blaM to assess the ability of Out protein regions to export BlaM across the cytoplasmic membrane in Escherichia coli and Ecc. For the outO gene product (an NMePhe peptidase), seven transmembrane regions have been identified and one more is predicted. The region of OutO with the highest level of hydrophilicity is likely to exist as a large cytoplasmic loop, located between two hydrophobic domains, and is positioned towards the N-terminus of the protein. When BlaM was fused on the C-terminal side of the last hydrophobic stretch of OutO, the resulting hybrid protein transferred the BlaM moiety to the periplasm whilst retaining OutO activity. Removal of a portion of this hydrophobic stretch resulted in the loss of OutO activity, suggesting that there are tight constraints on the topological integrity of OutO far maintaining catalytic function. When outG, -H, -I, -J, -K and -N were fused to blaM, the resulting phenotype suggested that the majority of each protein was targeted to the periplasm. Our results indicate that these six Out proteins, when produced by E. coli or Ecc, each adopt, at least temporarily, a type II bitopic conformation in the cytoplasmic membrane. For OutG, -H, -I and -J this probably represents the membrane topology prior to processing by OutO in Ecc. When produced in vivo from a T7 gene 10 promoter construct, the outG product was processed in Ecc whereas the outO mutant RJP249 failed to process pre-OutG. BlaM fusions positioned on the C-terminal side of the hydrophobic stretches of pre-OutG, -H, -I, and -J were processed by wild-type Ecc but not RJP249 or E. coli DH1. Thus the periplasmic domains of these proteins play no role in the peptidase cleavage reaction. An OutG-BlaM fusion construct was used to demonstrate NMePhe peptidase activity in other bacterial strains including E. carotovora subsp. carotovora (ATCC39048), E. carotovora subsp. atroseptica (SCRI1043) and Erwinia chrysanthemi (3937).