Cloning and expression in Escherichia coli of the Serratia marcescens metalloprotease gene: secretion of the protease from E. coli in the presence of the Erwinia chrysanthemi protease secretion functions

Abstract

The Serratia marcescens extraceliular protease SM is secreted by a signal peptide-independent pathway. When the prtSM gene was cloned and expressed in Escherichia coli, the cells did not secrete protease SM. The lack of secretion could be very efficiently complemented by the Erwinia chrysanthemi protease B secretion apparatus constituted by the PrtD, PrtE, and PrtF proteins. As with protease B and a.-hemolysin, the secretion signal was located within the last 80 amino acids of the protease. These results indicate that the mechanism of S. marcescens protease SM secretion is analogous to the mechanisms of protease B and hemolysin secretion.Serratia marcescens secretes a 50-kDa metalloprotease (SM protease) in large amounts into the culture medium (4). The gene encoding the exoprotease has been cloned and expressed in Escherichia coli, and its nucleotide sequence has been reported (32). It was shown that the mature protein is preceded by a propeptide of 16 amino acids which does not have the characteristics of a signal peptide, indicating that the secretion pathway is independent of the cleavage of an N-terminal signal peptide. In this regard, the SM protease is similar to the Erwinia chrysanthemi proteases B and C (7) and to E. coli hemolysin (12), which contain a C-terminal secretion signal (8, 34) and have partial amino acid sequence homology in a repeated glycine-rich nonapeptide located close to the C terminus (7). The SM protease also contains the same repeats. Moreover, the SM and B proteases share 60% identity (7).When the E. chrysanthemi genes involved in the synthesis and secretion of proteases B and C are expressed in E. coli, the proteases are made and secreted from this foreign host (45). This secretion requires three specific functions (27). On the other hand, when expressed in E. coli, the SM protease was not secreted but accumulated inside the cells as an inactive precursor of higher molecular mass (51 kDa), consistent with the existence of a 16-amino-acid propeptide (32). The absence of SM protease secretion in E. coli might indicate the absence of the genes required for secretion on the recombinant plasmid that carries the prtSM gene.On the basis of the high degree of homology between the two proteases, we hypothesized that the SM protease could be secreted by the E. chrysanthemi protease secretion system; proceeding from this hypothesis, we have introduced an S. marcescens cosmid DNA library into an E. coli strain expressing the E. chrysanthemi protease secretion functions. Proteolytic E. coli recombinant clones were found and characterized in this work. We have shown that the SM protease is efficiently produced in E. coli and secreted only in the presence of the E. chrysanthemi secretion functions. The data reported here suggest that the molecular mechanisms for the secretion of SM protease and protease B are similar. It therefore follows that the SM protease is a * Corresponding author.

The 32-kilobase exp gene cluster of Rhizobium meliloti directing the biosynthesis of galactoglucan: genetic organization and properties of the encoded gene products

“…ExpE1 contained 15 copies of the nonapeptide repeat (X[I/L]X[A/G]GXGXD) which was proposed to be implicated in the binding of Ca 2ϩ (51,83). These repeats were also found in NodO (12 copies) and other secreted proteins such as hemolysin (15 copies) and several proteases (4 copies) (20,26,49). After the last nonapeptide repeat, two highly conserved positions containing the aromatic residue tyrosine or phenylalanine were present in ExpE1 and the related proteins (Fig.…”

Iron acquisition from heme and hemoglobin by a Serratia marcescens extracellular protein.

“…We previously cloned a S. marcescens 4.2-kb DNA fragment coding for two inner membrane proteins-PrtDSM (a membrane ATPase) and PrtEsM, both required for the secretion of the S. marcescens metalloprotease (PrtSM) in E. coli via a signal peptide-independent pathway (6,16). In addition, this 4.2-kb insert encodes a third protein, HasA, of apparent molecular mass 19 kDa, which was secreted in large amounts (Fig.…”

“…Antibodies were raised in a rabbit against pure HasA protein and used in Western blot analysis (13) at a 1:2000 dilution. The other rabbit sera used have been described (14)(15)(16). For growth experiments, the sera were heated at 56TC for 10 min to inactivate complement.…”

The three genes lipB, lipC, and lipD involved in the extracellular secretion of the Serratia marcescens lipase which lacks an N-terminal signal peptide

“…The genes encoding the secretion device for the extracellular protein lacking the signal sequence have been reported in several bacteria: the E. coli hlyBC and tolC genes (2,27,45,48) for hemolysin, the prtDEF EC genes (21) for the metalloprotease in E. chrysanthemi, the aprDEF genes for Pseudomonas aeruginosa alkaline protease (12), the cyaBDE genes for cyclolysin of B. pertussis (10), and the lktBD genes for the P. haemolytica leukotoxin (42). It has been reported that the cells carrying the hlyBC and tolC or prtDEF EC genes secrete some of the extracellular proteins described above (3,8,22,28,39) and that the genes encoding the secretory apparatus for these proteins are similar to the hlyBC and tolC or prtDEF EC genes. Recently, the hasD and hasE genes coding for two components HasD and HasE, a secretion device for the S. marcescens metalloprotease, have been cloned, respectively (23).…”