Phosphatase activity of non-heme chloroperoxidase from the bacterium Serratia marcescens

Haloperoxidases are enzymes capable of formation of carbon-halogen bonds in the presence of hydrogen peroxide and halide ions. A mechanism of halogenation catalyzed by heme- and metal-independent bacterial haloperoxidases differs from other representatives of this group of enzymes. Here we report for the first time that bacterial non-heme haloperoxidases possess a phosphatase activity. Chloroperoxidase from Serratia marcescens W 250 purified up to homogeneity is shown to catalyze p-nitrophenylphosphate hydrolysis (K(m) value, 1.8+/-0.1 mM at pH 5.7). The reaction is activated by Mg(2+) and F(-), and is inhibited by WO(4)(2-), tartrate, acetate and phosphate anions. The irreversible inhibition by phenylmethanesulfonyl fluoride, modifier of serine residue in active site, decreases in the presence of phosphate ions. A mechanism of phosphoesters hydrolysis by non-heme haloperoxidases is proposed.     

Pili mediated agglutination of Serratia marcescens in human urine

Of 51 strains of Serratia marcescens isolated from patients with urinary or respiratory tract infections, 35 agglutinated in human urine. The agglutinating strains possessed numerous pili which were morphologically distinct from common pili or type I pili. The diameter of the pili was 3 nm and the average length was 0.3 micrometer. Electron microscopic examination showed that 80% or more of the cells of the agglutinating strains and 0 to 8% of the cells of the nonagglutinating strains were piliated. When an agglutinating strain was heated at 55 C for 10 min, it lost its agglutinating capacity and concomitantly its pili. These results suggest that the agglutination might occur because of interactions between the pili and some factors in human urine. The urinary slime appears to contain these agglutinating factors.     

Control of the bacterium Serratia marcescens in an insect host-parasite rearing program

Two occluded viruses of the Entomopoxvirus (D)/¹: ¹/¹: $\text{X}\text{X}\text{:}\text{I}\text{O}$ (= Vagoiavirus) group have been found in larvae of Dermolepida albohirtum (Scarabaeidae: Melolonthinae) and Aphodius tasmaniae (Scarabaeidae: Aphodiinae) from northern Queensland and northern Tasmania, Australia, respectively. Electron microscopical studies have been made of thin sections of occluded (mature) and nonoccluded virus particles within the fat body tissue of living diseased D. albohirtum larvae and of occluded virus particles within a dead, field-collected A. tasmaniae larva. The morphology and development of the known Australian entomopoxviruses are compared with previously known entompox or spheroidosis viruses from various insects.     

Structure of the O-specific polysaccharide from the lipopolysaccharide of Serratia marcescens O8

Structural studies have been carried out on the O-specific polysaccharide from the lipopolysaccharide of the reference strain (CDC 1604-55) for serogroup O8 of Serratia marcescens. The polymer has a branched, tetrasaccharide repeating unit of D-galactose(Gal),D-glucose(Glc), and 2-acetamido-2-deoxy-D-glucose(GlcNAc) with the following structure: (Formula: see text). The anomeric configuration assigned to the glucose residue differs from that (beta) previously proposed [Tarcsay, L., Wang, C. S., Li, S.-C. and Alaupovic, P. (1973) Biochemistry 12, 1948-1955]. The structure of the O8 polymer is identical with that of one of two polymers present in the cell envelope of a strain (CDC 1783-57) of S. marcescens O14.     

Oxidation of vanillin by Serratia marcescens

A Serratia marcescens strain quantitatively converted vanillin (0·1%, w/v) to vanillic acid, which exerted an inhibitory effect on bacterial growth. Vanillic acid producing activity was found in cell-free extracts of cultures grown in media with and without vanillin, when glucose was the substrate.     

Influence of growth temperature and lipopolysaccharide on hemolytic activity of Serratia marcescens.

Log-phase cells of Serratia marcescens cultured at 30 degrees C were approximately 10-fold more hemolytic than those grown at 37 degrees C. By using a cloned gene fusion of the promoter-proximal part of the hemolysin gene (shlA) to the Escherichia coli alkaline phosphatase gene (phoA), hemolysin gene expression as a function of alkaline phosphatase activity was measured at 30 and 37 degrees C. No difference in alkaline phosphatase activity was observed as a function of growth temperature, although more hemolysin was detectable immunologically in whole-cell extracts of cells grown at 30 degrees C. The influence of temperature was, however, growth phase dependent, because the hemolytic activities of cells cultured to early log phase at 30 and 37 degrees C were comparable. Given the outer membrane location of the hemolysin, lipopolysaccharide (LPS) was examined as a candidate for mediating the temperature effect on hemolytic activity. Silver staining of LPS in polyacrylamide gels revealed a shift towards shorter O-antigen molecules at 37 degrees C relative to 30 degrees C. Moreover, there was less binding of O-antigen-specific bacteriophage to S. marcescens with increasing growth temperature, a finding consistent with temperature-mediated changes in LPS structure. Smooth strains of S. marcescens were 20- to 30-fold more hemolytic than rough derivatives, a result confirming that changes in LPS structure can influence hemolytic activity. The alkaline phosphatase activity of rough strains harboring the shlA-phoA fusion was threefold lower than that of smooth strains harboring the fusion plasmids, a result consistent with a decrease in hemolysin gene expression in rough strains. The absence of a similar effect of temperature on gene expression may be related to less-marked changes in LPS structure as a function of temperature compared with a smooth-to-rough mutational change.     

Oxidation of aromatic aldehydes by Serratia marcescens

Cell suspensions of Serratia marcescens catalyzed the oxidation of aromatic aldehydes into the corresponding acids in high yield under mild conditions.     

粘质沙雷氏菌发酵生产D-乳酸的研究

本文对粘质沙雷氏菌发酵生产D-乳酸进行了研究。以粘质沙雷氏菌G1(Serratia marcescens G1)为出发菌种,摇瓶试验确定了发酵培养方式:前12 h为菌体生长阶段,有氧培养,温度28℃,pH值7.0;后36 h为D-乳酸合成积累阶段,无氧培养,温度44℃,pH值6.0。且发现使用葡萄糖为碳源时更有利于D-乳酸的合成积累。采用缺失2,3-丁二醇合成能力的基因工程菌株R1为出发株,经筛选后得到耐受较高浓度乳酸盐的菌株R150,以R150为发酵菌种,在3.7 L发酵罐上采用两阶段发酵法,并通过增加起始菌体浓度的方法,发酵生成的D-乳酸浓度达到83.5 g/L,光学纯度达到98.9%。本研究成果为使用粘质沙雷氏菌发酵生产D-乳酸的深入研究打下了基础。     

Oxidation of Aromatic Aldehydes by Serratia marcescens

Cell suspensions of Serratia marcescens catalyzed the oxidation of aromatic aldehydes into the corresponding acids in high yield under mild conditions.     

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     

Variants of Serratia marcescens Induced by Freeze-Drying

Unusually high numbers of pigmentless variants and sectored colonies in cultures of lyophylized Serratia marcescens are reported. Clonal analyses of sectored colonies show the presence of unstable bacteria that continue to sector again when plated. Further analysis of pigmentless variants suggest that pigment formation, oxygen uptake, and the production of an inducible protease are affected.     

Degradation of 2,4,6-trinitrotoluene by Serratia marcescens

A strain of Serratia marcescens, isolated from the soil of a contaminated site, degraded 2,4,6-trinitrotoluene (TNT) as the sole source of carbon and energy. At an initial concentration of 50mg , TNT was totally degraded in 48h under aerobic conditions in a minimal salt medium. Reduction intermediates (4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene) were observed. The presence of a surfactant (Tween 80) is essential to facilitate rapid degradation.     

Microbial oxidation of alpha-pinene by Serratia marcescens

Summary A strain of the bacterium Serratia marcescens, isolated from sewage sludge, can oxidise the terpene hydrocarbon -pinene to produce rans-verbenol as the major product, with verbeone and trans-sobrerol as minor products. A change in nitrogen source and inclusion of glucose as a second carbon source caused the bacterium to produce -terpineol as the major oxidation product. Products were identified by gas liquid chromatography and mass spectrometry.     

Preservation of Serratia marcescens by High-Vacuum Lyophilization

Water-washed Serratia marcescens (ATCC strain 14041) cells were lyophilized in an all-glass system capable of evacuation to pressures of less than 5 x 10(-6) torr. Lyophilization at the lowest pressures resulted in 50 to 65% survival for unstabilized washed organisms compared with 10 to 20% when the cells were lyophilized at pressures of about 2.5 x 10(-2) torr. At the latter pressures, 45 to 65% survival was obtained when NaCl or Naylor-Smith stabilizer was added to the cell suspensions before lyophilization. However, the stabilizers failed to increase significantly the levels of survival compared with water suspension when cells were lyophilized at pressures less than 10(-5) torr. The high survival rates obtained by the high-vacuum technique may be attributed to the reduction of traces of molecular oxygen which has been reported to be destructive of the dried bacteria. Survival of unstabilized dried S. marcescens after 1-day storage increased markedly with decreasing sealing pressure. Under the highest vacuum attained, survival of the dried bacteria was not impaired by storage for up to 1 month at Dry Ice temperatures; at higher temperatures, viability losses occurred. Exposure of the dried unstabilized bacteria to dry air resulted in rapid viability loss. The inactivation could be stopped almost immediately by evacuation to pressures of less than 10(-5) torr, but the evacuation failed to reverse the viability losses that occurred during exposure.     

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.     

Resistance of Serratia marcescens to Hydrogen Peroxide

Irradiation with ultraviolet (u.v.) light (71 J/m²) reduced the viable count of suspenrsions of Serratia marcescens , grown in a glycerol-salts defined medium, to five in 10⁴ cells. Subsequent incubation of irradiated cells in hydrogen peroxide failed to decrease the survivors, but u.v. irradiation in the presence of hydrogen peroxide reduced the viable count to fewer than two in 10⁶ cells. Cells grown in defined medium with added iron had more measurable catalase activity and were more resistant to hydrogen peroxide alone and to simultaneous treatment with u.v. irradiation and hydrogen peroxide. Cells grown in a non-defined medium contained little iron and measurable catalase activity but were more resistant to hydrogen peroxide. Treatment with toluene, heat killing or sonication increased the catalase activity detected in all cell suspensions and showed that resistance to hydrogen peroxide and to u.v. irradiation in hydrogen peroxide was related to the total catalase activity within cells.