Extracellular Protease as a Reversible Adhesion Regulator in Pseudomonas fluorescens

The investigation of growth dynamics and protein content in a batch Pseudomonas fluorescens culture grown in a synthetic medium with glucose as the sole source of carbon and energy showed that cells reversibly adhere to the walls of the cultivation flask during the first 2–3 h of growth. Over this time period, the total protein content of free and bound cells increased exponentially at a rate of 0.25 h^–1, the fraction of proteins in cells being almost the same (60–70%). The protein content in the medium increased from 3 to 50 mg/l, reaching about 30% of the total protein of the culture. The addition of the exponential culture liquid filtrate to the medium together with the inoculum led to the complete inhibition of cell adhesion and a drastic activation of proteolysis, with a concurrent release of more than 80% of cellular proteins into the medium. After 3–5 h of growth, the concentration of extracellular proteins decreased to the control level. Exogenously added proteinase K inhibited cell adhesion, the effect being more pronounced for R-type than for S-type cells. The hypothesis is discussed that the short-term reversible adhesion of cells is regulated with the involvement of a mixture of hydrocarbons, which inactivate the functional activity of bacterial adhesins, and proteases, which digest these adhesins.     

Characterization of a Protease from a Psychrotroph, Pseudomonas fluorescens 114

A psychrotrophic bacterium isolated from river sediment was identified as Pseudomonas fluorescens 114. It grew at 0°C and optimally at 20°C. The bacterium produced a protease with a molecular weight of 47,000, which was stable in the pH range of 5 to 9 and worked optimally between pH 6.5 and 10. Activity was optimal at 35°C and was lost immediately at 50°C and after 5 min at 45°C. At 0, 10, and 20°C, 24, 38, and 57% of optimal activity were observed, respectively.     

Lipase and Protease Double-Deletion Mutant of Pseudomonas fluorescens Suitable for Extracellular Protein Production

Pseudomonas fluorescens, a widespread Gram-negative bacterium, is an ideal protein manufacturing factory (PMF) because of its safety, robust growth, and high protein production. P. fluorescens possesses a type I secretion system (T1SS), which mediates secretion of a thermostable lipase (TliA) and a protease (PrtA) through its ATP-binding cassette (ABC) transporter. Recombinant proteins in P. fluorescens are attached to the C-terminal signal region of TliA for transport as fusion proteins to the extracellular medium. However, intrinsic TliA from the P. fluorescens genome interferes with detection of the recombinant protein and the secreted recombinant protein is hydrolyzed, due to intrinsic PrtA, resulting in decreased efficiency of the PMF. In this research, the lipase and protease genes of P. fluorescens SIK W1 were deleted using the targeted gene knockout method. Deletion mutant P. fluorescens ΔtliA ΔprtA secreted fusion proteins without TliA or protein degradation. Using wild-type P. fluorescens as an expression host, degradation of the recombinant protein varied depending on the type of culture media and aeration; however, degradation did not occur with the P. fluorescens ΔtliA ΔprtA double mutant irrespective of growth conditions. By homologous expression of tliA and the ABC transporter in a plasmid, TliA secreted from P. fluorescens ΔprtA and P. fluorescens ΔtliA ΔprtA cells was found to be intact, whereas that secreted from the wild-type P. fluorescens and P. fluorescens ΔtliA cells was found to be hydrolyzed. Our results demonstrate that the P. fluorescens ΔtliA ΔprtA deletion mutant is a promising T1SS-mediated PMF that enhances production and detection of recombinant proteins in extracellular media.     

Characterization of a Heat-Stable Protease of Pseudomonas fluorescens P26

A heat-stable, extracellular proteolytic enzyme was isolated from Pseudomonas fluorescens P26. Brain heart infusion broth (Fisher Scientific Co.), pH 7.5, and incubation at 21 C provided the optimal conditions for bacterial growth for enzyme production. The organism had a D value of 2.6 min at 62.8 C (145 F). The enzyme, however, was quite heat-stable, requiring 15 hr at 62.8 C, 8 hr at 71.4 C, and 9 min at 121 C for complete inactivation. Milk, whey, and casein each had a protective effect on the enzyme against heat inactivation. Purification was accomplished by growth of organisms in broth, centrifugation, sterilization by filtration, ammonium sulfate precipitation (55% saturation), dialysis (against six changes of water), and protein separation by passage through a Sephadex G-100 column. Ultracentrifugation revealed a single band with a sedimentation coefficient of 1.51 which suggested a molecular weight of approximately 23,000. As little as 0.2 unit of the purified enzyme caused detectable flavor defects during 30 days of storage at 4 C, and the Hull test for liberation of tyrosine compared favorably in sensitivity with the sensory method in milk.     

Characterization of an Extracellular Protease from the Insect Pathogen Xenorhabdus luminescens

Xenorhabdus luminescens Hm cultured in gelatin broth produced a single extracellular protease. The protease was purified by a factor of 500 and characterized as a monomeric protein with an approximate molecular weight of 61,000. On the basis of inhibitor studies and its pH optimum, the protease was classified as an alkaline metalloprotease with a pH optimum near 8; the isoelectric point of the enzyme is 4.2 +/- 0.2. The protease may be a major factor in the ecology of X. luminescens, which is carried as a symbiom of some parasitic nematodes.     

Screening, Nucleotide Sequence, and Biochemical Characterization of an Esterase from Pseudomonas fluorescens with High Activity towards Lactones

A genomic library of Pseudomonas fluorescens DSM 50106 in a λRESIII phage vector was screened in Escherichia coli K-12 for esterase activity by using α-naphthyl acetate and Fast Blue RR. A 3.2-kb DNA fragment was subcloned from an esterase-positive clone and completely sequenced. Esterase EstF1 was encoded by a 999-bp open reading frame (ORF) and exhibited significant amino acid sequence identity with members of the serine hydrolase family. The deduced amino acid sequences of two other C-terminal truncated ORFs exhibited homology to a cyclohexanone monooxygenase and an alkane hydroxylase. However, esterase activity was not induced by growing of P. fluorescens DSM 50106 in the presence of several cyclic ketones. The esterase gene was fused to a His tag and expressed in E. coli. The gene product was purified by zinc ion affinity chromatography and characterized. Detergents had to be added for purification, indicating that the enzyme was membrane bound or membrane associated. The optimum pH of the purified enzyme was 7.5, and the optimum temperature was 43°C. The showed highest purified enzyme activities towards lactones. The activity increased from γ-butyrolactone (18.1 U/mg) to -caprolactone (21.8 U/mg) to δ-valerolactone (36.5 U/mg). The activities towards the aliphatic esters were significantly lower; the only exception was the activity toward ethyl caprylate, which was the preferred substrate.     

Characterization of hydantoinase from Pseudomonas fluorescens strain DSM 84

The hydantoinase (EC 3.5.2.2) from Pseudomonas fluorescens strain DSM 84 was purified either by hydrophobic interaction chromatography on phenyl-Sepharose or by salting out chromatography on Sepharose 4B, gel filtration on Sephacryl S-400, and preparative electrophoresis. Molecular weight values of 230,000 and 60,000 for the native enzyme and each of the four subunits were estimated for the hydantoin hydrolysing activity. The hydantoinase was stable at temperatures up to 40 degrees C but showed an optimal activity at 55 degrees C. The enzyme was markedly inhibited by copper, para-hydroxymercuribenzoate, 8-hydroxyquinoline, and 2,2'-dipyridyl but not by zinc, and poorly by EDTA and o-phenanthroline. The hydantoin-hydrolyzing activity could be reactivated by ferrous ions. Dihydrouracil was the most readily hydrolyzed substrate. The dihydropyrimidinase produced by strain DSM 84 could also hydrolyze 5-substituted hydantions such as isopropylhydantoin (valine derivative) continuously for 10 days in a membrane reactor at a conversion rate of 30%. The only identified end product was N-carbamyl-D-valine.     

Biochemical, Genetic, and Zoosporicidal Properties of Cyclic Lipopeptide Surfactants Produced by Pseudomonas fluorescens

Zoospores play an important role in the infection of plant and animal hosts by oomycetes and other zoosporic fungi. In this study, six fluorescent Pseudomonas isolates with zoosporicidal activities were obtained from the wheat rhizosphere. Zoospores of multiple oomycetes, including Pythium species, Albugo candida, and Phytophthora infestans, were rendered immotile within 30 s of exposure to cell suspensions or cell culture supernatants of the six isolates, and subsequent lysis occurred within 60 s. The representative strain SS101, identified as Pseudomonas fluorescens biovar II, reduced the surface tension of water from 73 to 30 mN m⁻¹. The application of cell suspensions of strain SS101 to soil or hyacinth bulbs provided significant protection against root rot caused by Pythium intermedium. Five Tn5 mutants of strain SS101lacked the abilities to reduce the surface tension of water and to cause lysis of zoospores. Genetic characterization of two surfactant-deficient mutants showed that the transposons had integrated into condensation domains of peptide synthetases. A partially purified extract from strain SS101 reduced the surface tension of water to 30 mN m⁻¹ and reached the critical micelle concentration at 25 μg ml⁻¹. Reverse-phase high-performance liquid chromatography yielded eight different fractions, five of which had surface activity and caused lysis of zoospores. Mass spectrometry and nuclear magnetic resonance analyses allowed the identification of the main constituent as a cyclic lipopeptide (1,139 Da) containing nine amino acids and a 10-carbon hydroxy fatty acid. The other four zoosporicidal fractions were closely related to the main constituent, with molecular massesranging from 1,111 to 1,169 Da.     

Extracellular Protease of Pseudomonas fluorescens CHA0, a Biocontrol Factor with Activity against the Root-Knot Nematode Meloidogyne incognita

In Pseudomonas fluorescens CHA0, mutation of the GacA-controlled aprA gene (encoding the major extracellular protease) or the gacA regulatory gene resulted in reduced biocontrol activity against the root-knot nematode Meloidogyne incognita during tomato and soybean infection. Culture supernatants of strain CHA0 inhibited egg hatching and induced mortality of M. incognita juveniles more strongly than did supernatants of aprA and gacA mutants, suggesting that AprA protease contributes to biocontrol.     

Development of an Adhesion Assay and Characterization of an Adhesion-Deficient Mutant of Pseudomonas fluorescens

A sand column adhesion assay was developed which distinguishes the adhesion abilities of a number of pseudomonads isolated from fine sandy loam. Pseudomonas fluorescens Pf0-1 which adhered at >90% of the total cells added was subjected to transposon Tn5 insertion mutagenesis. From 2,500 Pf0-1::Tn5 mutants examined in the sand column assay, two adhesion-deficient Pf0-1 mutants showing <50% attachment were isolated. Marker exchange analysis of one of these mutants, Pf0-5, confirmed that the decreased adhesion was linked to the Tn5 insertion in the chromosome. The growth rate of Pf0-5 in enriched media and sterile soil was similar to that of the wild type; in minimal medium, however, Pf0-5 grew faster. In a soil column assay, less Pf0-5 than wild-type bacteria were recovered, suggesting a decreased ability to persist in soil. A 34-kilodalton major outer membrane protein present in the wild type was missing in Pf0-5. Transmission electron microscopy of the cell surface revealed that the wild-type possessed polar flagella which were absent in the mutant.     

Purification and Characterization of an Extracellular Protease from Penicillium chrysogenum Pg222 Active against Meat Proteins

An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60°C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45°C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.     

Purification and Characterization of an Extracellular Alkaline Serine Protease from Aspergillus terreus (IJIRA 6.2)

An extracellular alkaline serine protease has been purified from Aspergillus terreus (IJIRA 6.2). The purification procedure involved chromatography on DEAE-Sephadex A25, phosphocellulose, hydroxyapatite, casein-Sepharose, gel filtration on Sephacryl-S-300 and by glycerol density gradient centrifugation. The enzyme was further purified to apparent homogeneity through a combination of electrophoresis in polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) with or without protease substrate (gelatin) and subsequent regeneration of its activity in situ by removal of SDS. The active enzyme was visualized in a zymogram or on the basis of protease activity exhibited on an X-ray film. The protein in the unstained segment of the gel was electroeluted. The eluted protein with protease activity exhibited a molecular mass of 37,000-daltons on electrophoresis in SDS-polyacrylamide gel. A sedimentation coefficient of 3.2S was obtained by glycerol density gradient contrifugation. Maximum activity of protease was observed at pH 8.5 and at 37°C. Purified protease was active between pH 5.5 and 9.5 and was found to be stable up to 60°C. With Na-caseinate, the K _m of the purified protease was found to be 0.055 mM. Antipain, phenylmethane sulfonyl fluoride, and chymostatin served as non-competitive inhibitors. Substrate specificity was determined by using a synthetic chromogenic peptide containing N-P-Tosyl-Gly-Pro-Arg-p-nitroanilide. Results showed that the protease cleaved the peptide on the -COOH end of arginine residue. Received: 8 October 1999 / Accepted: 3 November 1999     

Cloning and Characterization of a Plasmid Encoded ACC Deaminase from an Indigenous Pseudomonas fluorescens FY32

In addition to the characterized mechanisms responsible for many direct effects of plant growth promoting bacteria (PGPB) on plants, it has been suggested that a number of PGPB contain the enzyme ACC deaminase that catalyzes degradation of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, into α-ketobutyrate and ammonia. As part of an effort to obtain an ACC deaminase encoding gene from a collection of soil samples, only one bacterial isolate, Pseudomonas fluorescens FY32 was capable of growing on ACC as a sole source of nitrogen. The ACC deaminase gene was amplified from the above isolate by polymerase chain reaction (PCR) giving an expected DNA fragment, 1017 bp. Sequence analysis of the fragment showed that it was highly homologous (94% and 98% identities at nucleotide and amino acid levels, respectively) to the previously characterized acdS gene from Pseudomonas sp. 6G5. Furthermore, fusion of the ACC deaminase ORF with lacZ gene resulted in the expression of active enzyme in Escherichia coli. In addition, further analyses revealed that the acdS gene was plasmid-encoded so that a large plasmid (pFY32) with almost 50 kb in size was identified from this bacterium. Furthermore, transfer of pFY32 into E. coli DH5α proved its ACC deaminase activity. This result was in accordance with previous reports suggesting horizontal transfer of the acdS gene. However, it needs more investigation to identify whether this pFY32 plasmid has undergone lateral gene transfer during the evolutionary process.     

Extracellular protease as a reversible adhesion regulator in Pseudomonas fluorescens

The investigation of growth dynamics and protein content in a batch Pseudomonas fluorescens culture grown in a synthetic medium with glucose as the sole source of carbon and energy showed that cells reversibly adhere to the walls of the cultivation flask during the first 2-3 h of growth. Over this time period, the total protein content of free and bound cells increased exponentially at a rate of 0.25 h-1, the fraction of proteins in cells being almost the same (60-70%). The protein content in the medium increased from 3 to 50 mg/l, reaching about 30% of the total protein of the culture. The addition of the exponential culture liquid filtrate to the medium together with the inoculum led to the complete inhibition of cell adhesion and a drastic activation of proteolysis, with a concurrent release of more than 80% of cellular proteins into the medium. After 3-5 h of growth, the concentration of extracellular proteins decreased to the control level. Exogenously added proteinase K inhibited cell adhesion, the effect being more pronounced for R-type than for S-type cells. The hypothesis is discussed that the short-term reversible adhesion of cells is regulated with the involvement of a mixture of hydrocarbons, which inactivate the functional activity of bacterial adhesins, and proteases, which digest these adhesins.     

Extracellular polysaccharides from Desulfovibrio desulfuricans and Pseudomonas fluorescens in the presence of mild and stainless steel

Summary This communication reports the presence of polysaccharides in biofilms formed by pure and mixed cultures of Desulfovibrio desulfuricans and Pseudomonas fluorescens on mild and stainless steel surfaces. The results of colorimetric assays, indicating significant differences between the amounts of neutral sugars present in these biofilms, were supported by gas chromatographic (GC)-mass spectrophotometric and GC-flame ionisation detection analyses. Neutral sugars in biofilms grown on mild steel surfaces were identified and quantified, revealing glucose as a major carbohydrate followed by mannose and galactose in all types of biofilm. Extracellular polymeric substances (EPS) precipitated from bacterial cultures grown with and without steel surfaces were also analysed for their carbohydrate content. The influence of the surfaces present in the cultures on the amount and type of sugars released into the bulk phase was established. There was significantly more carbohydrate in EPS harvested from pure and mixed cultures of D. desulfuricans incubated mild and stainless steel coupons than in EPS obtained from coupon-free cultures. No significant difference in sugar quantities was observed in EPS precipitated from cultures of P. fluorescens grown under different conditions (absence or presence of steel surfaces). The main carbohydrates identified in all types of EPS samples were mannose, glucose and galactose in order of prevalence. Offprint requests to: I. B. Beech     

Biochemical and Structural Characterization of SplD Protease from Staphylococcus aureus

Staphylococcus aureus is a dangerous human pathogen. A number of the proteins secreted by this bacterium are implicated in its virulence, but many of the components of its secretome are poorly characterized. Strains of S. aureus can produce up to six homologous extracellular serine proteases grouped in a single spl operon. Although the SplA, SplB, and SplC proteases have been thoroughly characterized, the properties of the other three enzymes have not yet been investigated. Here, we describe the biochemical and structural characteristics of the SplD protease. The active enzyme was produced in an Escherichia coli recombinant system and purified to homogeneity. P1 substrate specificity was determined using a combinatorial library of synthetic peptide substrates showing exclusive preference for threonine, serine, leucine, isoleucine, alanine, and valine. To further determine the specificity of SplD, we used high-throughput synthetic peptide and cell surface protein display methods. The results not only confirmed SplD preference for a P1 residue, but also provided insight into the specificity of individual primed- and non-primed substrate-binding subsites. The analyses revealed a surprisingly narrow specificity of the protease, which recognized five consecutive residues (P4-P3-P2-P1-P1’) with a consensus motif of R-(Y/W)-(P/L)-(T/L/I/V)↓S. To understand the molecular basis of the strict substrate specificity, we crystallized the enzyme in two different conditions, and refined the structures at resolutions of 1.56 Å and 2.1 Å. Molecular modeling and mutagenesis studies allowed us to define a consensus model of substrate binding, and illustrated the molecular mechanism of protease specificity.     

General Biochemical Characterization of Thermostable Extracellular beta-Amylase from Clostridium thermosulfurogenes

Clostridium thermosulfurogenes, an anaerobic bacterium which ferments starch into ethanol at 62 degrees C, produced an active extracellular amylase and contained intracellular glucoamylase but not pullulanase activity. The extracellular amylase was purified 2.4-fold, and its general physicochemical and catalytic properties were examined. The extracellular amylase was characterized as a beta-amylase (1,4-alpha-d-glucan maltohydrolase) based on demonstration of exocleavage activity and the production of maltose with a beta-anomeric configuration from starch. The beta-amylase activity was stable and optimally active at 80 and 75 degrees C, respectively. The pH optimum for activity and the pH stability range was 5.5 to 6 and 3.5 to 6.5, respectively. The apparent [S](0.5V) and V(max) for beta-amylase activity on starch was 1 mg/ml and 60 U/mg of protein. Similar to described beta-amylase, the enzyme was inhibited by p-chloromercuribenzoate, Cu, and Hg; however, alpha- and beta-cyclodextrins were not competitive inhibitors. The beta-amylase was active and stable in the presence of air or 10% (vol/vol) ethanol. The beta-amylase and glucoamylase activities enabled the organism to actively ferment raw starch in the absence of significant pullulanase or alpha-amylase activity.     

Lon Protease Influences Antibiotic Production and UV Tolerance of Pseudomonas fluorescens Pf-5

Pseudomonas fluorescens Pf-5 is a soil bacterium that suppresses plant pathogens due in part to its production of the antibiotic pyoluteorin. Previous characterization of Pf-5 revealed three global regulators, including the stationary-phase sigma factor ς^S and the two-component regulators GacA and GacS, that influence both antibiotic production and stress response. In this report, we describe the serine protease Lon as a fourth global regulator influencing these phenotypes in Pf-5. lon mutants overproduced pyoluteorin, transcribed pyoluteorin biosynthesis genes at enhanced levels, and were more sensitive to UV exposure than Pf-5. The lon gene was preceded by sequences that resembled promoters recognized by the heat shock sigma factor ς³² (ς^H) of Escherichia coli, and Lon accumulation by Pf-5 increased after heat shock. Therefore, ς^H represents the third sigma factor (with ς^S and ς⁷⁰) implicated in the regulation of antibiotic production by P. fluorescens. Lon protein levels were similar in stationary-phase and exponentially growing cultures of Pf-5 and were not positively affected by the global regulator ς^S or GacS. The association of antibiotic production and stress response has practical implications for the success of disease suppression in the soil environment, where biological control organisms such as Pf-5 are likely to encounter environmental stresses.