Some properties of the extracellular protease produced by the psychotrophic bacterium Pseudomonas fluorescens strain AR-11.

The major extracellular protease from Pseudomonas fluorescens strain AR-11 has been partially purified by a factor of 300 by a combination of DEAE-cellulose ion-exchange chromatography and gel filtration. The enzyme had a molecular weight of 38 400 and exhibited optimum activity with isoelectrically precipitated casein substrate at pH 6.5 with Km - 0.13 mM. The protease was strongly inhibited by a number of heavy metal ions at the 10 mM level and also inhibited by thiol agents, while 10 mM EDTA led to slight activation. Optimum activity was retained, amounting to 33% of the maximum activity at 4 degrees C and 72% at 20 degrees C. Heat inactivation studies in which the isolated protease was heated at high temperature before subsequent incubation at 35 degrees C with substrate showed that for 50% inactivation 25 s heating at 130 degrees C or 17 s at 140 degrees C of 8.5 s at 150 degrees C was requried. The combination of high stability to heat treatments and retention of considerable activity at low incubation temperatures indicates that such a protease might have considerable significance in the processing and subsequent storage of food and other products.     

Localization of metabolites which are regulators of extracellular protease synthesis in Pseudomonas fluorescens bacteria

The effect of over forty low molecular weight substrates on the growth and synthesis of exocellular neutral proteases was studied in Pseudomonas fluorescens. Neutral exoproteases were found to be regulated enzymes. Glucose did not repress the synthesis of exocellular proteases; the regulation of their synthesis by amino acids involved the mechanism of induction. The data suggest that the primary intracellular inductors of the synthesis of exoproteases are formed for different groups of amino acids at different levels of their utilization by the cells, viz. at the level of transport and at the level of the first steps in the degradation of their carbon backbones. The paper discusses possible molecular mechanisms for integrating the signal of the primary intracellular inductors, which directly regulate the activity of the operon(s) of neutral exocellular proteases.     

Pseudomonas fluorescens and closely-related fluorescent pseudomonads as biocontrol agents of soil-borne phytopathogens

Many strains of Pseudomonas fluorescens show potential for biological control of phytopathogens especially root pathogens. In taxonomic terms, several of them are indeed P. fluorescens sensu stricto , while others belong in fact to neighbouring species of the ' P. fluorescens ' complex or to ill-defined related species within the fluorescent Pseudomonas spp. These bacteria have become prominent models for rhizosphere ecological studies and analysis of bacterial secondary metabolism, and in recent years knowledge on their plant-beneficial traits has been considerably enhanced by widening the focus beyond the case of phytopathogen-directed antagonism. Current genomic analyses of rhizosphere competence and biocontrol traits will likely lead to the development of novel tools for effective management of indigenous and inoculated P. fluorescens biocontrol agents and a better exploitation of their plant-beneficial properties for sustainable agriculture.     

Transformation by extracellular DNA produced by Pseudomonas aeruginosa

Most Pseudomonas aeruginosa strains are capable of producing extracellular DNA. Very closely linked chromosomal markers (leu+ and trp+) were co-transferred to P. aeruginosa PAO1819 (leu9001, trp9008) by the extracellular DNA produced by P. aeruginosa strains IFO3445 and PAO1 at a frequency of 10(-7) to 10(-8). Treatment of the extracellular DNA with DNase, heating at 95 C or sonication completely destroyed its transforming ability. The R plasmid in the extracellular DNA produced by P. aeruginosa IFO3445 (RP4) or PAO2142 (RLb679) could be transferred to Escherichia coli ML4901 or P. aeruginosa PAO1819. The resultant transformants showed identical resistance patterns in the respective donors, and the sizes of the DNAs of RLb679 and RP4 isolated from the transformants were the same as those in the respective donors. These results demonstrate that the extracellular DNA contains both chromosomal DNA and plasmid DNA, and that it exhibits transforming ability. This implies that transformation by the extracellular DNA produced by P. aeruginosa may occur in nature and this seems to be of clinical importance in view of the spread of R plasmids among pathogens.     

Secretion of extracellular proteins by Pseudomonas aeruginosa

Pseudomonas aeruginosa is a bacterial species of commercial value secreting numerous extracellular proteins, involved in pathogenesis. Most strains produce at least a lipase, a phospholipase, an alkaline phosphatase, an exotoxin and 2 proteases (elastase and alkaline protease). Various mechanisms for secretion of exoproteins appear to exist in P aeruginosa. Genetic analysis has led to the identification of 2 secretion pathways: i) a "general" secretion pathway, defined by the xcp mutations, which mediates secretion of most extracellular proteins, and; ii) an independent secretion pathway specific for alkaline protease. Our present knowledge on the pathways and components of the secretion machinery in P aeruginosa is reviewed in this article.     

Human monoclonal antibodies to Pseudomonas aeruginosa produced by EBV-transformed cells

Numerous lymphoblastoid cell lines (LCLs) which secreted antibodies against Pseudomonas aeruginosa (all Fisher's immunotypes and Homma's immunotype 1) were established by Epstein-Barr virus (EBV)-transformation of lymphocytes. Five LCLs were established as long-term culture lines and their properties were determined. These LCLs produced monoclonal antibodies to Fisher's immunotype 1 and 4 and Homma's immunotype 1, and their immunoglobulin classes were IgM, IgG, and IgA. We found that three monoclonal antibodies (G3-1, H7-2, and E10-1) among them successfully protected mice from the corresponding immunotype of P. aeruginosa infection. Their protective dose (PD50) values were 0.5, 2.6, and 3.1 micrograms immunoglobulin/mouse. These human monoclonal antibodies against P. aeruginosa prepared by EBV-transformation method will be a valuable aid for the treatment for severe P. aeruginosa infections.     

Porins of Pseudomonas fluorescens MFO as fibronectin-binding proteins

Gene araA encoding an L-arabinose isomerase (AraA) from the hyperthermophile, Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 496 residues with a calculated molecular mass of 56677 Da. The deduced amino acid sequence has 94.8% identical amino acids compared with the residues in a putative L-arabinose isomerase of Thermotoga maritima. The recombinant enzyme expressed in E. coli was purified to homogeneity by heat treatment, ion exchange chromatography and gel filtration. The thermophilic enzyme had a maximum activity of L-arabinose isomerization and D-galactose isomerization at 85 degrees C, and required divalent cations such as Co(2+) and Mn(2+) for its activity and thermostability. The apparent K(m) values of the enzyme for L-arabinose and D-galactose were 116 mM (v(max), 119 micromol min(-1) mg(-1)) and 250 mM (v(max), 14.3 micromol min(-1) mg(-1)), respectively, that were determined in the presence of both 1 mM Co(2+) and 1 mM Mn(2+). A 68% conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80 degrees C.     

Defense responses of Fusarium oxysporum to 2,4-diacetylphloroglucinol, a broad-spectrum antibiotic produced by Pseudomonas fluorescens

A collection of 76 plant-pathogenic and 41 saprophytic Fusarium oxysporum strains was screened for sensitivity to 2,4-diacetylphloroglucinol (2,4-DAPG), a broad-spectrum antibiotic produced by multiple strains of antagonistic Pseudomonas fluorescens. Approximately 17% of the F. oxysporum strains were relatively tolerant to high 2,4-DAPG concentrations. Tolerance to 2,4-DAPG did not correlate with the geographic origin of the strains, formae speciales, intergenic spacer (IGS) group, or fusaric acid production levels. Biochemical analysis showed that 18 of 20 tolerant F. oxysporum strains were capable of metabolizing 2,4-DAPG. For two tolerant strains, analysis by mass spectrometry indicated that deacetylation of 2,4-DAPG to the less fungitoxic derivatives monoacetylphloroglucinol and phloroglucinol is among the initial mechanisms of 2,4-DAPG degradation. Production of fusaric acid, a known inhibitor of 2,4-DAPG biosynthesis in P. fluorescens, differed considerably among both 2,4-DAPG-sensitive and -tolerant F. oxysporum strains, indicating that fusaric acid production may be as important for 2,4-DAPG-sensitive as for -tolerant F. oxysporum strains. Whether 2,4-DAPG triggers fusaric acid production was studied for six F. oxysporum strains; 2,4-DAPG had no significant effect on fusaric acid production in four strains. In two strains, however, sublethal concentrations of 2,4-DAPG either enhanced or significantly decreased fusaric acid production. The implications of 2,4-DAPG degradation, the distribution of this trait within F. oxysporum and other plant-pathogenic fungi, and the consequences for the efficacy of biological control are discussed.     

Cosecretion of protease inhibitor stabilizes antibodies produced by plant roots

A plant-based system for continuous production of monoclonal antibodies based on the secretion of immunoglobulin complexes from plant roots into a hydroponic medium (rhizosecretion) was engineered to produce high levels of single-chain and full-size immunoglobulins. Replacing the original signal peptides of monoclonal antibodies with a plant-derived calreticulin signal increased the levels of antibody yield 2-fold. Cosecretion of Bowman-Birk Ser protease inhibitor reduced degradation of the immunoglobulin complexes in the default secretion pathway and further increased antibody production to 36.4 microg/g root dry weight per day for single-chain IgG1 and 21.8 microg/g root dry weight per day for full-size IgG4 antibodies. These results suggest that constitutive cosecretion of a protease inhibitor combined with the use of the plant signal peptide and the antibiotic marker-free transformation system offers a novel strategy to achieve high yields of complex therapeutic proteins secreted from plant roots.     

Analysis of extracellular matrix proteins produced by cultured cells

The extracellular matrix (ECM) is a highly organized multimolecular structure essential for the vital functions of any organism. Although much of the data of extracellular matrix components has been accumulated, the isolation of an entire set of these proteins remains a complex procedure due to the high content of fibrillar proteins and proteoglycans, which form multidomain, netlike structures. In the study presented, we developed a method for isolating ECM proteins from cell cultures. Human epidermoid carcinoma cells A431 and fibroblasts obtained from normal and scar human skin were used. We showed that EDTA solution removed cells from culture plates without destroying the cell membranes. Subsequent treatment of remaining ECM proteins with acetic acid in order to dissociate collagen fibers significantly improved the fractioning of ECM proteins. The extraction of remaining proteins from the surface of the culture plate was preformed by a buffer developed based on Laemmli probe buffer. Using this method, we isolated ECM proteins synthesized by cultured cells, and the extracted proteins were suitable for future analysis by SDS PAGE and two-dimentional electrophoresis, as well as for identifying individual proteins by mass spectrometry. This study may allow us to compare assortments of ECM proteins isolated from different sources, and elucidate impact of various proteins on structure and property of extracellular matrix of investigated cells.     

Isolation and characterization of a protease from Pseudomonas fluorescens RO98

Pseudomonas fluorescens RO98, a raw milk isolate, was inoculated into McKellar's minimal salts medium and incubated at 25 degrees C for 48 h to allow production of protease. A zinc-metalloacid protease was purified from the cell-free concentrate by anion exchange and gel filtration chromatography. The purified protease was active between 15 and 55 degrees C, and pH 4.5 and 9.0, and was stable to pasteurization. The enzyme had pH and temperature optima for activity of 5.0 and 35 degrees C, respectively. It was heat stable with a D55 of 41 min and a D62.5 of 18 h. Molecular weight of the enzyme was estimated to be 52 kDa by SDS PAGE and size exclusion chromatography. Values for kM of 144.28, 18.73, 110.20 and 35.23 micromol were obtained for whole, alpha-, beta- and kappa-casein, with a Vmax of 8.26, 0.09, 0.42 and 0.70 micromol mg-1 min-1, respectively. The enzyme hydrolysed kappa-casein preferentially when incubated with artificial casein micelles.     

Possible role of calcium in the formation of active extracellular proteinase by Pseudomonas fluorescens

The requirement for calcium during synthesis of extracellular proteinase by Pseudomonas fluorescens B52 was examined. Synthesis was monitored using cells resuspended at high density in fresh growth medium. Optimum enzyme production was found with cells grown to mid-logarithmic phase in mineral salts medium containing calcium chloride (1.0 mmol/l). Inhibition of synthesis by EDTA addition was rapid, similar to the effect produced by chloramphenicol, an inhibitor of translation. Appearance of enzyme initiated by calcium addition to depleted cells was rapid and was dependent on de novo protein synthesis. Sephadex G-100 chromatography of L-[4,5-3H]leucine-labelled cell-free supernatant liquids revealed that, in the absence of calcium, a low molecular weight (12000-14000 daltons) irreversibly inactive 'precursor' of the proteinase was formed. The results are consistent with the hypothesis that calcium is required for structural integrity of the proteinase as well as for activity.     

Possible role of calcium in the formation of active extracellular proteinase by Pseudomonas fluorescens

The requirement for calcium during synthesis of extracellular proteinase by Pseudomonas fluorescens B52 was examined. Synthesis was monitored using cells resuspended at high density in fresh growth medium. Optimum enzyme production was found with cells grown to mid-logarithmic phase in mineral salts medium containing calcium chloride (1.0 mmol/l). Inhibition of synthesis by EDTA addition was rapid, similar to the effect produced by chloramphenicol, an inhibitor of translation. Appearance of enzyme initiated by calcium addition to depleted cells was rapid and was dependent on de novo protein synthesis. Sephadex G-100 chromatography of L-[4,5-³H]leucine-labelled cell-free supernatant liquids revealed that, in the absence of calcium, a low molecular weight (12000–14000 daltons) irreversibly inactive 'precursor' of the proteinase was formed. The results are consistent with the hypothesis that calcium is required for structural integrity of the proteinase as well as for activity.     

Trypanosoma cruzi: ultrastructural changes produced by an anti-trypanosomal factor from Pseudomonas fluorescens

Trypomastigotes and amastigotes of Trypanosoma cruzi exhibited distinct ultrastructural alterations when treated with an extracellular lytic substance (anti-trypanosomal factor) produced by Pseudomonas fluorescens. Marked swelling of the parasites and detachment of the plasma membrane from the subjacent cytoplasm were observed after 15 min of treatment. After 3 hr, the nucleus was extensively damaged, the kinetoplast was indistinguishable, the mitochondrion was markedly swollen, the cytoplasm was disrupted, and the plasma membrane showed extensive blebbing and focal loss of subpellicular microtubules. These changes were progressive, as shown by the occurrence of parasite ghosts after 10 hr. Amastigotes exhibited an extremely swollen mitochondrion with disrupted internal structure, widening of the perinuclear space, and blebbing of the external nuclear membrane. The kinetoplast, however, remained clearly discernible. The drugs used today in controlling Chagas' disease are toxic. Therefore, there is a need for new anti-trypanosomal agents such as the Pseudomonas fluorescens antibiotics. The observations described in this study indicate the potential chemotherapeutic usefulness of these compounds for this disease.     

铜绿假单胞菌分泌性蛋白的抗肿瘤效应

目的 探讨3株不同来源的铜绿假单胞菌分泌性蛋白的抗肿瘤活性。方法 将3株不同来源的铜绿假单胞菌经LB培养基对其过夜静置培养,离心获得上清液,采用硫酸铵盐析沉淀蛋白质,再经PBS透析除盐。然后将蛋白作用于人肝癌细胞Hep-G2、宫颈癌细胞HeLa、肺癌细胞A549和人永生化表皮细胞HaCaT,CCK-8检测其对细胞的毒性作用,吉姆萨染色观察凋亡细胞形态变化。结果 SDS-PAGE证实成功获得不同分子量的分泌蛋白,CCK-8结果显示混合蛋白对3种不同肿瘤细胞的生长均有不同程度的抑制作用,且其抑制作用存在一定的时间和浓度依赖性,但对人正常细胞无明显抑制作用。吉姆萨染色初步显示细胞形态为凋亡状态。结论 初步证实该3株铜绿假单胞菌产生的分泌性蛋白具有不同程度的抗肿瘤作用,为进一步分离纯化具有抗肿瘤活性的单一蛋白质及研究其抗肿瘤机制提供依据。