Purification and Characterization of a
            <i>Serratia marcescens</i>
            Metalloprotease

Lyerly, David M.; Kreger, Arnold S.

doi:10.1128/iai.24.2.411-421.1979

Cited by 59 publications

(24 citation statements)

References 49 publications

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“…Thus, the more plausible explanation for the small apparent increase in specific activity during enzyme purification is that the step 1 preparation contained relatively small amounts of contaminating proteins capable of reacting with the Bradford reagent and, therefore, their removal did not cause a large increase In specific activity during enzyme isolation. A similar situation has been observed during the purification of a Serrutia marcescens protease (Lyerly & Kreger, 1979) The step 3 protease preparation was homogenous by crossed immunoelectrophoresis (Fig. 1 a, b) and analytical thin-layer isolectric focusing (Fig.…”

Section: Purification Of Pro Teasesupporting

confidence: 73%

Purification and Characterization of an Elastolytic Protease of Vibrio vulnificus

Kothary¹,

Kreger²

1987

Microbiology

117

138

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Large amounts of a highly purified, extracellular elastolytic protease of Vibrio vulnificus were obtained by sequential ammonium sulphate precipitation and hydrophobic interaction chromatography with phenyl-Sepharose CL-4B. The protease had an Mr of about 50,500 (estimated by SDS-PAGE), a pI of 5.7, and a temperature optimum range of 55 to 60 degrees C. The pH optimum and the results of inactivation studies suggested that the enzyme was a neutral metalloprotease. The protease had about 429 amino acid residues, and the first 20 amino-terminal amino acid residues were Ala-Gln-Ala-Asn-Gly-Thr-Gly-Pro-Gly-Gly-Asn-Ser-Lys-Thr-Gly-Arg-Tyr-Glu- Phe-Gly . The purified protease was toxic for mice (about 1.5 mg kg-1 and 4.5 mg kg-1, intraperitoneal and intravenous LD50 values, respectively), and subcutaneous injection of the enzyme elicited rapid and extensive dermonecrosis.

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Section: Purification Of Pro Teasesupporting

confidence: 73%

Purification and Characterization of an Elastolytic Protease of Vibrio vulnificus

Kothary¹,

Kreger²

1987

Microbiology

117

138

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“…PrtA, also named serralysin or PrtS (7), is a 50.2-kDa repeats-in-toxin (RTX) alkaline zinc metalloprotease that has been well characterized structurally (8) and was shown to depend on the type I LipBCD system for secretion to the extracellular milieu (9,10). Similarly to proteases that belong to the serralysin family in other bacteria, such as Photorhabdus, Erwinia, or Pseudomonas, S. marcescens PrtA has been implicated in cytotoxicity, immunomodulation, or virulence traits using different experimental models, including nematodes, insects, and mice (11)(12)(13)(14)(15)(16)(17). In addition, the potential application of the enzymatic properties of PrtA, for instance, as a detergent additive, has attracted industrial interest, and different purification strategies and the optimization of its catalytic activity have been reported (15,(18)(19)(20)(21)(22).…”

mentioning

confidence: 99%

“…Similarly to proteases that belong to the serralysin family in other bacteria, such as Photorhabdus, Erwinia, or Pseudomonas, S. marcescens PrtA has been implicated in cytotoxicity, immunomodulation, or virulence traits using different experimental models, including nematodes, insects, and mice (11)(12)(13)(14)(15)(16)(17). In addition, the potential application of the enzymatic properties of PrtA, for instance, as a detergent additive, has attracted industrial interest, and different purification strategies and the optimization of its catalytic activity have been reported (15,(18)(19)(20)(21)(22). In several countries, purified serralysins are also commercially available as potent analgesic and anti-inflammatory drugs (23).…”

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confidence: 99%

CpxR-Dependent Thermoregulation of Serratia marcescens PrtA Metalloprotease Expression and Its Contribution to Bacterial Biofilm Formation

et al. 2018

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PrtA is the major secreted metalloprotease of Previous reports implicate PrtA in the pathogenic capacity of this bacterium. PrtA is also clinically used as a potent analgesic and anti-inflammatory drug, and its catalytic properties attract industrial interest. Comparatively, there is scarce knowledge about the mechanisms that physiologically govern PrtA expression in In this work, we demonstrate that PrtA production is derepressed when the bacterial growth temperature decreases from 37°C to 30°C. We show that this thermoregulation occurs at the transcriptional level. We determined that upstream of , there is a conserved motif that is directly recognized by the CpxR transcriptional regulator. This feature is found along strains irrespective of their isolation source, suggesting an evolutionary conservation of CpxR-dependent regulation of PrtA expression. We found that in s, the CpxAR system is more active at 37°C than at 30°C. In good agreement with these results, in a mutant background, is derepressed at 37°C, while overexpression of the NlpE lipoprotein, a well-known CpxAR-inducing condition, inhibits PrtA expression, suggesting that the levels of the activated form of CpxR are increased at 37°C over those at 30°C. In addition, we establish that PrtA is involved in the ability of to develop biofilm. In accordance, CpxR influences the biofilm phenotype only when bacteria are grown at 37°C. In sum, our findings shed light on regulatory mechanisms that fine-tune PrtA expression and reveal a novel role for PrtA in the lifestyle of We demonstrate that metalloprotease PrtA expression is transcriptionally thermoregulated. While strongly activated below 30°C, its expression is downregulated at 37°C. We found that in , the CpxAR signal transduction system, which responds to envelope stress and bacterial surface adhesion, is activated at 37°C and able to downregulate PrtA expression by direct interaction of CpxR with a binding motif located upstream of the gene. Moreover, we reveal that PrtA expression favors the ability of to develop biofilm, irrespective of the bacterial growth temperature. In this context, thermoregulation along with a highly conserved CpxR-dependent modulation mechanism gives clues about the relevance of PrtA as a factor implicated in the persistence of on abiotic surfaces and in bacterial host colonization capacity.

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“…Electrophoretic heterogeneity of serratia exoproteases has been observed before, but the proteins were not studied previously by chemical methods (12,24). Determination of the amino acid composition did not disclose a methionine residue (4,27), but labeling with [35S]methionine and cleavage with cyanogen bromide clearly indicated the presence of methionine, probably one residue, as derived before from amino acid analysis (8).…”

Section: Discussionmentioning

confidence: 83%

Cell-bound and secreted proteases of Serratia marcescens

Schmitz¹,

Braun²

1985

J Bacteriol

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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...

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Purification and Characterization of a Serratia marcescens Metalloprotease

Cited by 59 publications

References 49 publications

Purification and Characterization of an Elastolytic Protease of Vibrio vulnificus

Purification and Characterization of an Elastolytic Protease of Vibrio vulnificus

CpxR-Dependent Thermoregulation of Serratia marcescens PrtA Metalloprotease Expression and Its Contribution to Bacterial Biofilm Formation

Cell-bound and secreted proteases of Serratia marcescens

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