Production of active Serratia marcescens metalloprotease from Escherichia coli by alpha-hemolysin HlyB and HlyD

Abstract

Serratia marcescens produces an abundant extracellular metalloprotease. The gene for this protease had previously been cloned and expressed in Escherichia coli, in which no functional protease could be found.However, the protease gene carries the LXGGXGND repeat motif found in at-hemolysin and other proteins secreted by homologous systems. Using a dual-plasmid complementation system, we show that the a-hemolysin hIyB and hlyD transport determinants are sufficient to allow secretion and activation of a functional metalloprotease species from E. coli, as are the comparable protease secretion functions of Erwinia chrysanthemi.However, strains expressing protease with the hlyBD transport system are unstable and rapidly lose the ability to produce functional protease.A number of extracellular proteins are secreted into the medium by the gram-negative bacterium Serratia marcescens, including a nuclease, a chitinase, lipases, and proteases. Of these, the most abundant protein is a zinc metalloprotease (1). This enzyme is produced during the growth of a culture and reaches its maximal level as the cell enters stationary phase (5, 7). It is inducible by the addition of proteinaceous substrates to the medium, by certain amino acids such as leucine, or by a dense bacterial culture in which the concentration of proteins released by the cells is presumably sufficient to induce its expression (5). Its regulation, however, is not coordinated with that of other extracellular proteins of S. marcescens (3).The genes encoding most of these proteins have been cloned and sequenced. The nuclease (2), chitinase (18), phospholipase (12), and serine protease (30) genes are all expressed in Escherichia coli. However, the gene for the metalloprotease, which has also been cloned, does not express functional protease in E. coli; only a higher-molecular-weight zymogen can be found (6, 23).The major form of the metalloprotease is found as a species with a molecular weight of 51,000 (1, 5). A minor form with a molecular weight of 53,000 was also observed previously (25). The gene for metalloprotease from S. marcescens E-15 was sequenced previously (23), and a protein of 470 amino acids (Mr = 50,632) was predicted. A similar gene with 95% identity was also cloned and sequenced from the S. marcescens strain SM6 (6). The DNA sequence predicts a preprotein which could initiate at any one of three ATG start codons in frame with the peptide sequence. However, none of the three potential start sites leads to an amino-terminal domain which resembles a classical signal peptide. It is not clear which start codon is used in vivo.The B and C proteases of Erwinia chrysanthemi have also been cloned and sequenced previously (9). These proteases are about 60% related to the S. marcescens metalloprotease. Both groups of protease have a striking repeat motif of LXGGXGND. This motif has been found in a number of toxins, such as a-hemolysin, leukotoxin, and cyclasin (4), all of which are secreted extracellularly by similar transport systems. Secretion of the a-he...

SlpE is a calcium-dependent cytotoxic metalloprotease associated with clinical isolates of Serratia marcescens

“…When expressed in Escherichia coli , the SM6 protease was not secreted [6] unless the E. coli hemolysin transporter genes hlyB and hlyD were expressed in trans, indicating that the protease is secreted through a type I secretion system [30]; however, the native S. marcescens type I secretion system used to secrete the SM6 protease was not determined until this current study, which implicates LipBCD as the native secretion system (Fig. 3B).…”

Characterization of a Cytotoxic Factor in Culture Filtrates of Serratia marcescens

“…The denatured proteins were fractionated by SDS-12% PAGE and electrotransferred to a polyvinylidene difluoride (PVDF) membrane (Osmonics, Westborough, Mass.). The membrane was probed with antiserum raised against the S. marcescens 56-kDa metalloprotease (28,61) and then with alkaline phosphataseconjugated secondary antibodies (Sigma). The Western blots were visualized by chemiluminescence (Genor Technology, St. Louis, Mo.…”

“…The S. marcescens 56-kDa metalloprotease was expressed as a recombinant protein to determine whether a cytotoxic phenotype would be conferred on a nonpathogenic E. coli strain. E. coli MB568, which has plasmid pGSD6 that carries the genes encoding an ABC transporter necessary for activation and secretion of the metalloprotease, was transformed with a plasmid harboring the gene encoding the 56-kDa metalloprotease, resulting in a strain (MB2031) that secretes the 56-kDa metalloprotease (61). The same pGSD6-carrying E. coli strain transformed with only the parent plasmid (pUC19) was also prepared (MB2033).…”

Highly Solvent Tolerance in Serratia marcescens IBBPo15

“…For sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, extracellular proteins were extracted from the cell-free culture broths by ethanol precipitation and intracellular proteins were extracted from cell pellets by sonication 17 . Extracted proteins were dissolved in Laemmli buffer and denaturated at 96°C, for 10 min.…”