Excretion of a protease by Serratia marcescens

Excretion of an extracellular protease of Serratia marcescens ATCC 25419 occurred during logarithmic growth and was highest (per cell) when cultures reached the stationary growth phase. Production of the extracellular protease was induced by leucine or casein in minimal medium or by growth in tryptone-yeast medium. In the late stationary phase an intracellular protease activity accumulated which was also observed in mutants with very low extracellular protease activity. The excreted protease was the dominant protein in the growth medium. The protease was purified to homogeneity by column chromatography on Bio-Gel P-100 and on DEAE-cellulose. Quantitative amino acid analysis revealed the absence of sulfurcontaining amino acids. The enzyme consists of one polypeptide chain. A molecular weight of 51,000 and 55,000 was estimated using polyacrylamide gel electrophoresis and chromatography on Bio-Gel P-100 respectively. The enzyme cleaved only N--benzoyl-DL-lysine-and-arginine-nitroanilides but not the corresponding leucine or tyrosine derivatives nor a set of diand tripeptides.Abbreviation SDS sodium dodecylsulfate     

Regulation of extracellular protease formation by Serratia marcescens.

Heavy cell suspensions of Serratia marcescens, when grown in gelatin-containing media, produce extracellular proteases which increase in specific activity in a linear fashion for 3 to 4h. During partial purification, a single peak of proteolytic activity was demonstrated by Sephadex G-100 chromatography. However, electrophoresis using 5% polyacrylamide gels discloses three proteolytically active bands. Evidence in favor of gelatin acting as an inducer of the 'proteolytic system' was provided by two observations. First, proteolytic activity is only present in media containing gelatin. Secondly, the addition of 10(-4) M rifampicin to cells growing in gelatin-containing medium plus an additional carbon source inhibits protease activity totally, but has no effect on growth. When glycerol is added to a growing cell suspension in gelatin-containing medium, growth increases, but protease specific activity decreases. This 'glycerol effect' is not due to an accumulation of active or inactive enzyme in association with the cell, nor to a decrease in the total number of proteases synthesized. Rather, glycerol, as other utilizable carbohydrates, exerts a repression which can be eliminated by 5 mM dibutyryl cyclic AMP.     

Interdomain cleavage of plasma fibronectin by zinc-metalloproteinase from Serratia marcescens

Limited proteolysis of porcine plasma fibronectin by the 56 kDa proteinase (56K proteinase) (EC 3.4.24.4) from Serratia marcescens released six polypeptides: a 27 kDa peptide, the heparin-binding domain which comprises the NH2-terminal end; a 50 kDa peptide, a mid-molecule that mediates binding to gelatin or collagen; a 160 kDa peptide, that contained the heparin-binding domain with cell-spreading activity; and a 140 and a 20 kDa peptide which released from the 160 kDa peptide. Each fragment was purified and characterized by its chemical and biological properties, and it was found that they were respectively different domains. Both the 160 and the 140 kDa peptide contained one cysteine per mole of peptide. The 160 kDa peptides were connected by a 6 kDa peptide, which was present at the COOH-terminal end of the molecule and was biologically inactive. Only 6 kDa peptide contained a disulfide bond and produced 3 kDa peptide after reduction, whereas other fragments did not change with or without reduction on SDS-polyacrylamide gel electrophoresis. NH2-terminal sequence analyses of the released peptides showed that the 56K proteinase cleaved the fibronectin between the Arg-Thr (located at two different sites), Leu-Ser and Gln-Glu bonds. Out of 118 Arg residues, there are nine sequences containing Arg-Thr, and two of them near or at an interdomain location (at Arg 259 and 2239) were cleaved. Out of 124 Leu residues, there are 11 Leu-Ser sequences and only one, at 687, was cleaved. The above fragments with functional domain activity could be aligned according to the previously reported amino-acid sequence of human or bovine plasma fibronectin. The treatment of fibroblast cells by the 56K proteinase resulted in loss of morphological integrity and extracellular matrix.     

A Function of 40 kDa Outer Membrane Protein in Serratia marcescens

The function of one of the outer membrane proteins of Serratia marcescens was investigated. S. marcescens with an abundant 40 kDa outer membrane protein was induced to form spheroplast at a high rate in an isotonic medium in the presence of calcium, although the spheroplasts were generally fragile in the isotonic environment. The degree of spheroplast induction was correlated to the amount of the 40 kDa protein present in the membrane. In the 40 kDa proteinless mutant strains, the spheroplast induction rate was remarkably decreased. Autoradiography of the outer membrane revealed the presence of a calcium-binding protein as a radioactive band whose position coincided with the 40 kDa protein. These results suggest that the 40 kDa protein has an important role in maintaining the structural integrity of the cell wall against osmotic shock.     

粘质沙雷氏菌脂肪酶基因的克隆表达和酶学性质的研究

目的:克隆粘质沙雷氏菌脂肪酶基因(lipA)使其在大肠杆菌B121(DE3)中实现高效表达,并对重组酶进行酶学性质研究.方法:以产脂肪酶粘质沙雷氏菌总DNA为模板,PCR扩增脂肪酶基因lipA,构建重组表达载体pET-lipA,并将其导入大肠杆菌进行诱导表达,对表达产物进行SDS-PAGE和酶学性质的测定.结果:经过优化培养条件,脂肪酶活力最高能达到104U/mL.重组脂肪酶的最适反应温度为40～45℃,最适pH为7.0～7.5,在50℃保温1h下仍能保持80%的酶活力,Ca2+、Sr2+、Mn2+和Mg2+对脂肪酶酶活有较强的激活作用,尤其是Ca2+使脂肪酶酶活提高了1倍多,而Ni2+、Fe2+、Fe3+、Cu2+、Zn2+和Al3+对酶活具有较强的抑制作用,尤其是Zn2+和Al3+使酶活力几乎完全丧失.该酶对一些有机溶剂有较好的耐受性,与50%甲醇混合24h,仍能保持84%的酶活力.结论:该脂肪酶具有较好的热稳定性和甲醇耐受力,作为生产生物柴油的催化剂具有很大的应用价值,为基因工程酶法生产生物柴油打下良好的基础.     

Characterization of Serratia marcescens surviving in disinfecting footbaths

AIM: To determine if disinfecting footbaths in the food industry were contaminated with bacteria and to characterize some of the bacteria present. METHODS AND RESULTS: Bacterial strains were isolated from disinfecting footbaths containing TEGO 103G (amphoteric disinfectant) or TP-99 (alkyl amino acetate-based disinfectant) in five of six dairy factories. Fourteen strains identified as Cedecea spp. by their fatty acid composition were further characterized. Results from Rapid ID 32 E API analysis and 16S-rDNA-sequencing showed that all strains were Serratia marcescens. Unlike S. marcescens ATCC 13880, the isolates from disinfecting footbaths were not killed (<5 log10 reduction) by the recommended in-use concentration of TEGO 103G, TEGO 51 or benzalkonium chloride. Survival and multiplication in tap water with an in-use concentration of TEGO 103G was demonstrated for one of the strains. All strains were killed by the in-use concentrations of commercial disinfectants based on peracetic acid, hypochlorite, quaternary ammonium compounds and alkyl amino acetate (TP-99). There were no indications of cross-resistance between disinfectants and antibiotics. CONCLUSION: Serratia marcescens may survive and multiply in disinfecting footbaths containing TEGO 103G or alkyl amino acetate because of disinfectant resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: Disinfecting footbaths may act as contamination sources in food factories and should not be used without regular hygienic monitoring.     

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.     

Genetic analysis of extracellular proteins of Serratia marcescens.

Serratia marcescens, a gram-negative enteric bacterium, is capable of secreting a number of proteins extracellularly. The types of activity found in the growth media include proteases, chitinases, a nuclease, and a lipase. Genetic studies have been undertaken to investigate the mechanisms used for the extracellular secretion of these exoproteins by S. marcescens. Many independent mutations affecting the extracellular enzymes were isolated after chemical and transposon mutagenesis. Using indicator media, we have identified loci involved in the production or excretion of extracellular protease, nuclease, or chitinase by S. marcescens. None of the mutations represented general extracellular-excretion mutants; in no case was the production or excretion of multiple exoproteins affected. A variety of loci were identified, including regulatory mutations affecting nuclease and chitinase expression. A number of phenotypically different protease mutants arose. Some of them may represent different gene products required for the production and excretion of the major metalloprotease, a process more complex than that for the other S. marcescens exoproteins characterized to date.     

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.     

Overproduction of Serratia marcescens metalloprotease (SMP) from the recombinant Serratia marcescens strains

Summary To overproduce Serratia marcescens metalloprotease(SMP), various recombinant plasmids encoding SMP gene were constructed and the SMP productivities from the recombinant S. marcescens strains were examined. The recombinant S. marcescens strains indispensably required proteinaceous substrates such as casein for the extracellular production of SMP. We obtained maximum 9,100U/ml of SMP from the culture supernatant of S. marcescens ATCC27117 containing a regulatory plasmid pTSP2 encoding SMP gene fused with a strong trc99a promoter and its repressor gene lacI^q, which is about 23 times higher than that of wild type strain. SMP produced from the recombinant S. marcescens(pTSP2) was 88.3% of total extracellular proteins.     

Purification and Characterization of Metallo-β-Lactamase from Serratia marcescens

Carbapenem-hydrolyzing β-lactamase from Serratia marcescens FHSM4055 was purified 926-fold by means of carboxylmethyl Sephadex C-50, Sephacryl S-200, and Mono S column chromatography. The molecular weight was 30,000 by SDS-PAGE and the isoelectric point was 8.7. The enzyme activity was inhibited by EDTA, and restored by adding zinc (II) or manganese (II). It was inhibited by p-chloromercuribenzoate and iodine as well as the heavy metals, Hg (II), Fe (II), Fe (III), and Cu (II). These results indicate that the enzyme is a metallo-β-lactamase and that the SH-group of only one cysteine residue probably binds to the metal ion, thus contributing to the stability of the enzyme active center. The specific constant (k_cat/K_m) showed that the enzyme hydrolyzed various β-lactam antibiotics such as carbapenems, cephalosporins, moxalactam, cephamycins, and penicillins other than monobactams. Ampicillin and piperacillin with respective amino- and imino-groups, ceftazidime with a carboxypropyloxyimino-group, and cefclidin with a carbamoylquinuclidine-group were poor substrates among the β-lactam antibiotics other than the monobactams tested. The plots of the turnover number (k_cat) against pH for the hydrolysis of cephaloridine gave an asymmetrical curve with the ‘tail’ on the acid side (pK₁, 5.9; pK₂, 9.0; pK₃, 10.8), whereas those of k_cat/K_m gave a bell-shaped curve (pK₁, 5.8; pK₂, 9.8). Both results suggest that two ionic forms of an intermediate yield the same product at different rates and that the enzyme is stable under alkaline conditions. Since the N-terminal amino acid sequence of 27 residues determined was consistent with that of the metalloenzyme (Antimicrob. Agents Chemother., 1994, 38: 71-78), the above enzymatic characteristics seem to coincide.     

Expression of Serratia marcescens extracellular proteins requires recA.

A previously described regulatory mutation which abolishes expression of the extracellular nuclease of Serratia marcescens is shown to be a mutation of the Serratia recA gene. The defect in nuclease expression could be restored by introducing a plasmid carrying the recA gene of Escherichia coli. The DNA sequence of the Serratia gene is very similar to that of the E. coli gene. The putative LexA-binding site of the Serratia recA gene is almost identical to that of E. coli, along with the promoter. A similar LexA-binding site can also be found upstream of the nuclease gene. As expected from this finding, we show that nuclease expression can be induced by SOS-inducing agents such as mitomycin C. Although inducible in S. marcescens, the nuclease was expressed only at the uninduced levels in E. coli and could not be induced by mitomycin C. The extracellular chitinase and lipase were similarly affected by the mutations altering nuclease expression and were also induced by mitomycin 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.     

Purification and properties of L-asparaginase from Serratia marcescens

The purification and properties of a tumor inhibitory l-asparaginase from Serratia marcescens are described. The following properties of the enzyme were examined: kinetics of the enzyme reaction, catalytic activity as a function of pH, boundary sedimentation velocity, electrophoresis on polyacrylamide gel, immuno-electrophoresis against homologous and heterologous antisera, immunodiffusion, blood clearance rate in mice, and inhibition of the 6C3HED lymphoma in C3H mice. Complete regression of this tumor was obtained with a smaller dose of the enzyme from S. marcescens than with enzyme from Escherichia coli. The reason for this difference was not evident from a comparison of several properties of the two enzymes.     

The effect of nuclease on transformation efficiency in Serratia marcescens

No differences in the efficiency of transformation were observed from both plasmid and chromosomal DNA in Serratia marcescens 2170 and an extracellular nuclease defective isogenic strain. The efficiency of transformation was the same for Escherichia coli 5K and E. coli containing a recombinant plasmid conferring the ability to synthesize a S. marcescens nuclease. From these results we conclude that the extracellular nuclease of S. marcescens 2170 is not the main cause of the low efficiency of transformation observed in this bacterium.     

The extracellular nuclease gene of Serratia marcescens and its secretion from Escherichia coli

We are studying exoproteins of the enteric bacterium Serratia marcescens as a model system for the release of extracellular proteins from the cell. In this work we report the cloning of the gene for a secreted nuclease from S. marcescens and its complete nucleotide sequence. Following expression of the nuclease gene in both S. marcescens and Escherichia coli we were able to demonstrate the presence of the nuclease extracellularly in both organisms. Cell lysis did not occur and there was no concurrent release of cytoplasmic or periplasmic proteins. No accessory genes appeared to be required for extracellular secretion of the nuclease from E. coli. We can conclude that E. coli is capable of secreting certain proteins extracellularly, and may be a suitable host organism for the genetic analysis of extracellular protein secretion when provided with a suitable protein to export.