Two Genes Encoding Serine Protease Homologues in Serratia marcescens and Characterization of Their Products in Escherichia coli

Abstract

A serine protease (SSP) of Serratia marcescens is one of the extracellular enzymes secreted from this Gram-negative bacterium. SSP is produced as a large precursor and converted to a mature protein by cleavages removing an NH2-terminal signal sequence and a COOH-terminal pro-region. This COOH-terminal pro-region is integrated into the outer membrane and has a functional role for the export of the mature protein across the outer membrane. Southern hybridization analysis with a DNA fragment encoding the COOH-terminal pro-region as the probe showed a wide distribution of nucleotide sequences encoding SSP exporter-like proteins among Serratia species. Moreover, S. marcescens IFO 3046, from which the ssp gene had been cloned, was found to contain two ssp homologues (ssp-h1 and ssp-h2). They were cloned and their nucleotide sequences were determined. The two ssp homologues were found to exist in tandem on the genome and their amino acid sequences showed 81% identity to each other. Both of them showed 55% identity in amino acid sequence to preproSSP. In addition, both showed end-to-end similarity to the 100 kDa serotype-specific antigen (Ssa1) of Pasteurella haemolytica. Escherichia coli JM105 containing ssp-h1 gene produced a 53 kDa protein corresponding to the NH2-terminal portion and a 49 kDa protein corresponding to the COOH-terminal portion, both of which were rigidly integrated in the outer membrane. Consistent with the significant similarity of the COOH-terminal portions of the homologues to that of SSP, they showed the ability to translocate the mature SSP part across the outer membrane into the medium. Furthermore, the NH2-terminal portion of the homologue was not translocated into the outer membrane without its COOH-terminal part. All of these data show that the SSP homologues are outer membrane proteins that are translocated into the outer membrane with the aid of the translocator function of their COOH-terminal part.

The aprX–lipA operon of Pseudomonas fluorescens B52: a molecular analysis of metalloprotease and lipase production The GenBank accession numbers for the sequences reported in this paper are AF216700, AF216701 and AF216702.

“…We conclude, firstly, that prtB ::cam has a polar effect on lipA expression and that the lipA promoter is upstream of prtB. Secondly, although, by these criteria, PrtB does not possess detectable protease activity it may have specificity for an unknown substrate ; alternatively, it, and prtA, may be outer-membrane proteins, as suggested in the case of homologues in S. marcescens (Ohnishi et al, 1997), with a primary role other than proteolysis of exogenous substrates. The aprX : : pJPkan mutant, derived by a single crossover event (see Methods) was phenotypically proteasenegative on skimmed milk media and had visibly lower lipase activity (Fig.…”

Type V Protein Secretion Pathway: the Autotransporter Story

“…Moreover, S. marcescens IFO 3046, from which the ssp gene had been cloned, was found to contain two ssp homologues, called ssp-h1 and ssp-h2 (360). ssp-h1 and ssp-h2 showed 55% identity in the amino acid sequence to Ssp.…”

“…ssp-h1 and ssp-h2 showed 55% identity in the amino acid sequence to Ssp. Furthermore, both genes are located in tandem on the genome, and the amino acid sequences of their products showed 81% identity to each other, suggesting that one of them was generated by gene duplication during evolution (360). E. coli JM105 containing the ssp-h1 gene produced a 53-kDa protein corresponding to the N-terminal portion and a 49-kDa protein corresponding to the C-terminal portion, both of which were rigidly integrated in the outer membrane.…”

Autotransported Serine Protease A of Neisseria meningitidis : an Immunogenic, Surface-Exposed Outer Membrane, and Secreted Protein

“…The characteristic structure of autotransporter proteins and the secretion mechanism have been reviewed elsewhere (15,17). Autotransporter proteins, possessing a diverse array of N-terminal "functional" domains, have been reported in many gram-negative organisms (1,2,5,6,9,10,12,13,(23)(24)(25)(29)(30)(31). Typically, these proteins exhibit virulence-associated functions, such as adhesion, cytotoxicity, serum resistance, and proteolysis (14).…”