Cell-bound and secreted proteases of Serratia marcescens

Abstract

Exoprotease of Serratia marcescens ATCC 25419 is exceptional among members of the family Enterobacteriaceae in that it is secreted in large amounts by viable cells into the culture medium. Labeling of cells with radioactive amino acids revealed no intracellular protein that could be precipitated with antibodies raised against purified exoproteases. With substances known to interfere with the excretion of some proteinstosyl-L-lysine chloromethyl ketone, phenethyl alcohol, procaine, and sodium azide-and with rifampin, an intracellular form (apparent molecular weight, 52,000) larger than the major exoform (molecular weight, 51,000) was identified. Moreover, the 52,000-molecular-weight form was the main protein in immunoprecipitates of a cysteine-auxotrophic mutant starved for cysteine. Beside the major exoform, protease I, two additional exoproteases, termed II and III, appeared in the medium of stationary cultures. They were precipitated by antibodies against protease I, were identical in the Ouchterlony double-diffusion assay, and exhibited only a small difference, if any at all, in the peptide pattern after partial hydrolysis with protease V8 of Staphylococcus aureus. The amino-and carboxy-terminal amino acid sequences of protease I and II were determined and found t4 be identical, NH2-Ala-Ala-Thr-Gly-Gly-Tyr-Asp-Ala-Val-Asp and Phe-Ile-Val-* Corresponding author. 25419, isolated by F. Engelbrecht of this institute. The strains were grown at 30°C in TY medium (10 g of tryptone [Difco Laboratories, Detroit, Mich.], 5 g of yeast extract, and 5 g of NaCl per liter) or in minimal salt medium containing 15.8 g of glycerol per liter as carbon source (4). In some experiments this medium was supplemented with 20 mg of each L-amino acid per liter. For starvation of cysteine, strain EN6 was grown for 2.5 h in the minimal medium plus all amino acids except cysteine.Radioactive labeling and fractionation procedures. Cells were labeled with either 1 1±Ci of [35S]cysteine or [35S]methionine per ml or 20 ,uCi of a mixture of '4C-amino acids per ml. Incorporation was stopped by the addition of unlabeled amino acids (0.33 mg/ml) and of chloramphenicol (0.05 mglml). In chase experiments the solution of unlabeled amino acids was prewarmed to 30°C. Synthesis was terminated by pouring the mixture on ice. Cells were sedimented by centrifugation for 4 min, washed three times with ice-cold 10 mM Tris-hydrochloride (pH 8), and then dissolved by heating for 5 min in 10 mM Tris-hydrochloride, (pH 8) that contained 1% sodium dodecyl sulfate (SDS) and 1 mM EDTA. The SDS solutions (50 ,ul) were diluted with 1.3 ml of 50 mM Tris-hydrochloride (pH 8.0) which contained 2% Triton X-100, 150 mM NaCl, and 1 mM EDTA (30). Undissolved remnants were removed by centrifugation before protease was precipitated with antibodies. The supernatant fraction of labeled cell cultures was immunoprecipitated as described above. Labeled cells were fractionated after disruption by treatment with lysozyme-EDTA. Cells (4 x 109 in 10 ml of culture) were sedimented by c...

The extracellular proteases ofMyxococcus xanthus

“…They may arise from separate genes, by post-translational modification or, as seems likely for some bands, by partial proteolysis. A similar problem exists in Serratia marcescens, where the 3 extracellular proteases have been shown to be closely related by sequence and serological studies [10].…”

Separation of the Prodigiosin‐Localizing Crude Vesicles Which Retain the Activity of Protease and Nuclease in Serratia marcescens

“…It was reported that a clinical isolate, kums 3958 strain, produced four types of protease (11) and two types of nuclease (15). Another reported that there were two forms of protease, i.e., intracellular form and exoform (14), though the types of the protease and the nuclease in the forms of the vesicles were not investigated. The extent of the participation of the vesicles in the secretion of the two enzymes seemed to be different from each other, because the highest activity of the protease was found in the supernatant fraction whereas that of the nuclease was found in the vesicle fraction (Fig.…”

Secretion of an Aeromonas hydrophila aerolysin by a mutant strain of Escherichia coli

“…Secretion of proteins to the external medium is an unusual process in E. coll. The a-haemolysin produced by some strains seems to be one of the few exceptions [1]. When heterospecific secretable proteins are cloned in E. coli, they are mostly not secreted, and remain in the periplasmic space [1][2][3][4][5][6].…”

“…The a-haemolysin produced by some strains seems to be one of the few exceptions [1]. When heterospecific secretable proteins are cloned in E. coli, they are mostly not secreted, and remain in the periplasmic space [1][2][3][4][5][6]. A few cases have recently been reported in which foreign proteins appear to be secreted by E. coli [7][8][9][10].…”