Characterization of a metalloprotease inhibitor protein (SmaPI) of Serratia marcescens.

As suggested by Y. Suh and M.J. Benedik (J. Bacteriol. 174: 2361-2366, 1992), Serratia marcescens ATCC 27117 produced very small amounts (0.8 U ml-1) of an inhibitor protein (SmaPI) that shows an inhibitory activity against extracellular 50-kDa metalloprotease (SMP) of S. marcescens and that is localized in the periplasm of cells at the optimal growth temperature of 25 degrees C. A recombinant S. marcescens harboring plasmid pSP2 encoding SMP and SmaPI genes produced 20 U of SmaPI ml-1 that is also localized in the periplasm of cells at 25 degrees C. However, a large amount of SmaPI (86 Uml-1) was extracellularly produced at the supraoptimal growth temperature 37 degrees C from the recombinant S. marcescens (pSP2). We purified SmaPI from the culture supernatant of S. marcescens (pSP2) grown at 37 degrees C, and some biochemical properties were characterized. SmaPI had a pI value of about 10.0 and was a monomeric protein with a molecular mass of 10,000. SmaPI was produced from a precursor SmaPI by cleavage of a signal peptide (26 amino acid residues). The inhibitor was stable in boiling water for up to 30 min. The thermostability of SmaPI can be attributed to its reversible denaturation. SmaPI inhibited SMP by formation of a noncovalent complex with a molar ratio of 1:1 and showed a high protease specificity, which inhibited only SMP among the various proteases we examined.     

Crystallization of recombinant chitobiase from Serratia marcescens.

We are currently investigating the biochemical and structural properties of both chitin degrading enzymes chitinase and chitobiase from Serratia marcescens. Previously we have reported the first crystallization and characterization of chitinase crystals (Vorgias et al., 1992). In this communication we present the first crystallization of chitobiase. The protein was synthesized in Escherichia coli and purified to homogeneity using cation exchange chromatography and fast protein liquid chromatography. The crystals have the shape of small prisms and the space group is P2(1) with beta = 101.0 degrees and unit cell dimensions a = 63.2 A, b = 133.2 A, c = 55.1 A. They diffract X-rays to about 2.5 A resolution and are suitable for three-dimensional structural analysis.     

The metalloprotease gene of Serratia marcescens strain SM6

Summary Utilizing the DNA sequence of the metalloprotease fromSerratia strain E-15, we isolated and sequenced the homologous gene fromSerratia strain SM6. These two genes are similar at both the DNA and protein sequence level. Expression of the protease gene inEscherichia coli was achieved by use of thelac promoter. This resulted in the production and excretion of an immunologically detectable but inactive protein of slightly higher molecular weight than that fromSerratia. We introduced the cloned gene into previously described protease mutants. The observed pattern of protease expression suggested that these mutations fall into three classes.     

Production, purification and characterization of a 50-kDa extracellular metalloprotease from Serratia marcescens

The extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRL B-23112 was purified and characterized. SMP 6.1 was purified from the culture supernatant by ammonium sulfate precipitation, acetone fractional precipitation, and preparative isoelectric focusing. SMP 6.1 has a molecular mass of approximately 50 900 Da by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The following substrates were hydrolyzed: casein, bovine serum albumin, and hide powder. SMP 6.1 has the characteristics of a metalloprotease, a pH optimum of 10.0, and a temperature optimum of 42° C. The isoelectric point of the protease is 6.1. Restoration of proteolytic activity by in-gel renaturation after SDS-PAGE indicates a single polypeptide chain. SMP 6.1 is inhibited by EDTA (9 μg/ml) and not inhibited by antipain dihydrochloride (120 μg/ml), aprotinin (4 μg/ml), bestatin (80 μg/ml), chymostatin (50 μg/ml), E-64 (20 μg/ml), leupeptin (4 μg/ml), Pefabloc SC (2000 μg/ml), pepstatin (4 μg/ml), phosphoramidon (660 μg/ml), or phenylmethylsulfonyl fluoride (400 μg/ml). SMP 6.1 retains full activity in the presence of SDS (1% w/v), Tween-20 (1% w/v), Triton X-100 (1% w/v), ethanol (5% v/v), and 2-mercaptoethanol (0.5% v/v). The extracellular metalloprotease SMP 6.1 differs from the serratiopeptidase (Sigma) produced by S. marcescens ATCC 27117 in the following characteristics: isoelectric point, peptide mapping and nematolytic properties. Received: 22 November 1996 / Received revision: 27 February 1997 / Accepted: 7 March 1997     

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

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 alpha-hemolysin and other proteins secreted by homologous systems. Using a dual-plasmid complementation system, we show that the alpha-hemolysin hlyB 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.     

Crystallization of recombinant chitinase from the cloned chiA gene of Serratia marcescens.

The chiA gene encoding for the chitinase enzyme from Serratia marcescens was efficiently overexpressed under the pL promoter and the enzyme was secreted into the growth medium. The chitinase was purified to homogeneity using affinity chromatography on a Phenyl-Sepharose column and the protein was successfully crystallized. The crystals are presently in the form of small needles in space group C222(1) and have unit cell dimensions a = 204(+/- 0.5) A, b = 134(+/- 0.5) A, c = 60(+/- 0.5) A. The crystals diffract X-rays to about 3 A resolution and are suitable for three-dimensional structural analysis.     

beta-N-acetylhexosaminidases from Serratia marcescens.

Two forms of beta-N-acetylhexosaminidase from Serratia marcescens with an optimum pH of 5.0 and 6.5, respectively, to 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside were separated by DEAE-cellulose chromatography and Sephacryl S-200 chromatography. On the basis of their molecular weights, thermal stability, substrate specificity and isoelectric points, the form with an acidic pH optimum resembled hexosaminidase B, whereas the form with a neutral pH optimum resembled hexosaminidase C. Lectin binding studies showed that the acidic form does not bind to concanavalin-A-Sepharose, Tetragonolobus purpurea-agarose, wheat germ-agglutinin-Sepharose or Ricinus communis-agglutinin-agarose, whereas the neutral form binds to the last two lectin columns.     

Isolation and composition of oligosaccharide cores from endotoxins of two Serratia marcescens strains

The oligosaccharide cores isolated from the acetic acid hydrolysates of endotoxins from Serratia marcescens 08 and Serratia marcescens Bizio were analyzed for their sugar composition. The intact oligosaccharide core from S. marcescens 08 consisted of 2-keto-3-deoxyoctonate, d-glycero-d-mannoheptose, l-glycero-d-mannoheptose, d-glucose, d-galactose, and d-glucosamine in a molar ratio of 2:1:5:3:1:3 and that from S. marcescens Bizio consisted of the same sugar components in a molar ratio of 2:1:5:5:1:2. This result indicates that endotoxins from S. marcescens genus may contain more than one structural type of oligosaccharide core. Both oligosaccharide cores also differ in their chemical compositions from cores of other Enterobacteriaceae.     

Molecular characterization of polyphosphate kinase (ppk) gene from Serratia marcescens

To understand the mechanism of phosphate accumulation, a gene encoding polyphosphate kinase (PPK) was cloned from the genomic library of Serratia marcescens by Southern hybridization. From the nucleotide sequence of a 4 kb DNA fragment, an open reading frame of 2063 nucleotides was identified encoding a protein of 686 amino acids with molecular mass of 70 kDa. The potential CRP binding site and pho box sequence were found upstream of the putative promoter in the regulatory region. The expression of PPK resulted in the formation of inclusion bodies and the product was active at low temperature. The E. coli strain harboring plasmid pSPK5 with ppk gene increased enzyme activity of polyphosphate kinase, resulting in increased accumulation of polyphosphate in E. coli.     

Immobilization of Chloroperoxidase from Serratia marcescens

A bacterial non-heme chloroperoxidase from Serratia marcescensW 250 was immobilized in calcium alginate gel. Methods for stabilization of the immobilized enzyme were developed, and some kinetic parameters of the immobilized preparations were determined. The enzyme encapsulated into the gel granules in the presence of potassium ferricyanide followed by treatment with glutaraldehyde demonstrated the highest stability under the reaction conditions.     

Isolation and characterization of extracellular lipase preparations from wild-type (V-10) and mutant (M-1)Serratia marcescens strains

—The cultivation conditions of wild-type strain V-10 and mutant strain M-l (overproducer of endonuclease and chitinase) ofSerratia marcescens optimal for extracellular lipase biosynthesis were determined. The strain V-10 displayed the maximum lipase yield (840 AU/ml) after 10–12 h of cultivation; the strain M-l (330 AU/ml), after 25–30 h. The data showed that extracellular lipases from V-10 and M-1 can be precipitated in a weakly acidic medium (pH 5.0 and 4.5, respectively). This property was used to obtain partially purified lipase preparations. The effect of the ionic composition of the reaction mixture on the activities of these enzymatic preparations was studied. Both preparations displayed the highest activities in weakly alkaline media (pH 8.0); however, the wild-type strain lipase displayed higher thermal stability and stability at alkaline pH compared with M-1 lipase. Both lipases were activated by various anionic and nonionic surfactants and were inactive in the presence of cetyltrimethylammonium bromide.     

Immobilization of chloroperoxidase from Serratia marcescens

A bacterial non-heme chloroperoxidase from Serratia marcescens W 250 was immobilized in calcium-alginate gel. Methods for stabilization of the immobilized enzyme were developed, and some kinetic parameters of the immobilized preparations were determined. The enzyme encapsulated into the gel granules in the presence of potassium ferricyanide followed by treatment with glutaraldehyde demonstrated the highest stability under the reaction conditions.     

Isolation from urine of two Serratia marcescens strains excreting a diffusible yellow pigment

Two bacterial strains excreting a yellow pigment were isolated from human urine and identified as Serratia marcescens. The pigment was produced in the late exponential and early stationary phases of growth. Minimal media supplemented with tyrosine, phenylalanine, 3,4-dihydroxyphenylacetate or tryptophan, as well as complex media, induced pigment production. UV-visible spectra of the extracted pigment had peaks characteristic of 2-hydroxy-5-carboxymethylmuconate semialdehyde, produced from meta-cleavage of 3,4-dihydroxyphenylacetate by the enzyme 3,4-dihydroxyphenylacetate 2,3-dioxygenase (EC 1.13.11.15). This enzyme was active when the bacteria were grown under conditions promoting pigment production. The kinetics and factors affecting pigment production are also reported.     

Molecular cloning and purification of an endochitinase from Serratia marcescens (Nima)

An endochitinase gene from the Serratia marcescens Nima strain (chiA Nima) was cloned, sequenced, and expressed in Escherichia coli DH5αF′, and the recombinant protein (ChiA Nima) was purified by hydrophobic interaction chromatography. chiA Nima contains an open reading frame (ORF) that encodes an endochitinase with a deduced molecular weight and an isoelectric point of 61 kDa and 6.84, respectively. A sequence at the 5′-end was identified as a signal peptide, recognized by Gram-negative bacteria transport mechanism. Comparison of ChiA Nima with other chitinases revealed a modular structure formed by the catalytic domain and a putative chitin-binding domain. The purified chitinase was able to hydrolyze both trimeric and tetrameric fluorogenic substrates, but not a chitobiose analog substrate. ChiA Nima showed high enzymatic activity within a broad pH range (pH 4.0–10.0), with a peak activity at pH 5.5. The optimal temperature for enzymatic activity was detected at 55°C.     

Purification of Cytosine Deaminase from Serratia marcescens

Cytosine deaminase was purified about 900-fold from the cell-free extract of Serratia marcescens. The purification procedure included heat treatment, ammonium sulfate fractionation, ethyl alcohol fractionation, DEAE-cellulose and hydroxylapatite column chromatography, and Sephadex G–200 gel filtration.

The enzyme was homogeneous by the criteria of ultracentrifugation and acrylamide gel electrophoresis. The molecular weight was determined to be approximately 580,000 and the molecule was composed of equimolecular weight of 8 subunits.

The enzyme catalyzed the stoichiometric conversion of cytosine into uracil and ammonia.     

New R-plasmids in strains of Serratia marcescens with multiple drug resistance

In 4 S. marcescens polyresistant strains isolated from patients conjugative plasmids transferred to Escherichia coli have been detected. Two of these strains carry each one plasmid which codes resistance to 10 different antibiotics, including aminoglycosides which rarely occur in our country, and belongs to group IncC. The third strain is the host of 2 plasmids. One of them is similar to the above-mentioned 2 plasmids with respect to the incompatibility group and a set of markers, but additionally codes resistance to cephalosporins; the second plasmid has been determined as belonging to group IncM, unstable and capable of rendering the cells highly resistant only to aminoglycosides. And, finally, the fourth strain also carries 2 plasmids: one of them is unstable and belongs, supposedly, to group IncI alpha, and the second plasmid is stable and belongs to group IncM. The plasmid of group IncI alpha differs from all other plasmids of our Serratia by its capacity of rendering the cells highly resistant to chloramphenicol.     

Utilization of ATP-binding cassette exporter for hyperproduction of an exoprotein: construction of lipase-hyperproducing recombinant strains of Serratia marcescens

The Serratia marcescens extracellular lipase (LipA) is an enzyme applicable to enantioselective hydrolysis of racemic substrates. The enzyme is secreted through an ATP-binding cassette (ABC) exporter, the Lip system, encoded by the lipBCD genes. The S. marcescens recombinant carrying pLIPE121, which encodes the lipA gene in pUC19, exhibited a higher LipA production level than the wild-type strain. However, the level was lower than expected, and secretion was suggested to be a bottleneck. lipBCD plasmids were introduced into S. marcescens recombinants harboring lipA plasmids and the effectiveness of the lipBCD plasmids in elevating LipA productivity was investigated. S. marcescens strains harboring both lipA and lipBCD plasmids showed sevenfold greater extracellular LipA activity than the strain harboring the lipA plasmid alone. A high level of extracellular LipA production (1,300 kU/ml) and high plasmid stability (enough to carry out large-scale cultivation) were observed under non-selective conditions. Addition of L-proline and Tween 80 was effective in increasing cell growth of the recombinant, which led to high LipA production. In batch cultivation using a 30-l jar fermentor, LipA production was achieved at a high level of 5,200 kU/ml. This is the first report describing utilization of ABC exporter for the overproduction of an industrially important extracellular protein.