Breeding of a Cyclic Imide-Assimilating Bacterium,Pseudomonas putida s52, for High Efficiency Production of Pyruvate

A succinimide-assimilating bacterium, Pseudomonas putida s52, was found to be a potent producer of pyruvate from fumarate. Using washed cells from P. putida s52 as catalyst, 400 mM pyruvate was produced from 500 mM fumarate in a 36-h reaction. Bromopyruvate, a malic enzyme inhibitor, was used for the selection of mutants with higher pyruvate productivity. A bromopyruvate-resistant mutant, P. putida 15160, was found to be an effective catalyst for pyruvate production. Moreover, under batch bioreactor conditions, 767 mM of pyruvate was successfully produced from 1,000 mM fumarate in a 72-h reaction with washed cells from P. putida 15160 as catalyst.     

Production of Rhamnolipids by Pseudomonas chlororaphis, a Nonpathogenic Bacterium

Rhamnolipids, naturally occurring biosurfactants constructed of rhamnose sugar molecules and β-hydroxyalkanoic acids, have a wide range of potential commercial applications. In the course of a survey of 33 different bacterial isolates, we have identified, using a phenotypic assay for rhamnolipid production, a strain of the nonpathogenic bacterial species Pseudomonas chlororaphis that is capable of producing rhamnolipids. Rhamnolipid production by P. chlororaphis was achieved by growth at room temperature in static cultures of a mineral salts medium containing 2% glucose. We obtained yields of roughly 1 g/liter of rhamnolipids, an amount comparable to the production levels reported in Pseudomonas aeruginosa grown with glucose as the carbon source. The rhamnolipids produced by P. chlororaphis appear to be exclusively the mono-rhamnolipid form. The most prevalent molecular species had one monounsaturated hydroxy fatty acid of 12 carbons and one saturated hydroxy fatty acid of 10 carbons. P. chlororaphis, a nonpathogenic saprophyte of the soil, is currently employed as a biocontrol agent against certain types of plant fungal diseases. The pathogenic nature of all bacteria previously known to produce rhamnolipids has been a major obstacle to commercial production of rhamnolipids. The use of P. chlororaphis therefore greatly simplifies this matter by removing the need for containment systems and stringent separation processes in the production of rhamnolipids.     

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.     

Nematicidal Activity of a Nonpathogenic Biocontrol Bacterium, Pseudomonas chlororaphis O6

Bacterial culture filtrates of an aggressive rhizobacterium, Pseudomonas chlororaphis O6, displayed strong nematicidal activity. The nematicidal activity of P. chlororaphis O6 was markedly reduced in the gacS mutant of P. chlororaphis O6 grown in the presence of glycine, but no reduction of nematicidal activity in the gacS mutant was noted in the absence of glycine. The results of bioassay with P. chlororaphis O6 mutants showed that phenazine and pyrrolnitrin production was not a major factor, but the effects of glycine in the culture medium suggest that formation of hydrogen cyanide might be important. Assessments in greenhouse studies with tomatoes growing in nematode-infested soils confirmed that the application of P. chlororaphis O6 resulted in the control of the root-knot nematode. Our results demonstrated that P. chlororaphis O6 could be employed as a biocontrol agent for the control of the root-knot nematode, and the global regulator, GacS, functions as a positive regulator of the expression of nematicidal compounds and enzymes in P. chlororaphis O6.     

Genes Involved in Cyclic Lipopeptide Production Are Important for Seed and Straw Colonization by Pseudomonas sp. Strain DSS73

Survival in natural bulk soil and colonization of sugar beet seeds and barley straw residues were determined for Pseudomonas sp. strain DSS73 and Tn5 mutants in amsY (encoding a peptide synthetase involved in production of the cyclic lipopeptide amphisin) and gacS (encoding the sensory kinase of the two-component GacA/GacS regulatory system). No differences in survival or growth in response to carbon amendment (citrate) were observed in bulk soil. However, both mutants were impaired in their colonization of sugar beet seeds and barley straw residues by an inoculum established in the bulk soil. The two mutants had comparable colonization phenotypes, suggesting that amphisin production is more important for colonization than other gacS-controlled traits.     

Cyclic Lipopeptide Biosynthetic Genes and Products,and Inhibitory Activity of Plant-Associated Bacillus against Phytopathogenic Bacteria

The antibacterial activity against bacterial plant pathogens and its relationships with the presence of the cyclic lipopeptide (cLP) biosynthetic genes ituC (iturin), bmyB (bacillomycin), fenD (fengycin) and srfAA (surfactin), and their corresponding antimicrobial peptide products have been studied in a collection of 64 strains of Bacillus spp. isolated from plant environments. The most frequent antimicrobial peptide (AMP) genes were bmyB, srfAA and fenD (34-50% of isolates). Most isolates (98.4%) produced surfactin isoforms, 90.6% iturins and 79.7% fengycins. The antibacterial activity was very frequent and generally intense among the collection of strains because 75% of the isolates were active against at least 6 of the 8 bacterial plant pathogens tested. Hierarchical and correspondence analysis confirmed the presence of two clearly differentiated groups. One group consisted of Bacillus strains that showed a strong antibacterial activity, presented several cLPs genes and produced several isoforms of cLPs simultaneously, mainly composed of B. subtilis and B. amyloliquefaciens, although the last one was exclusive to this group. Another group was characterized by strains with very low or none antibacterial activity, that showed one or none of the cLP genes and produced a few or none of the corresponding cLPs, and was the most heterogenous group including B. subtilis, B. licheniformis, B. megaterium, B. pumilus, B. cereus and B. thuringiensis, although the last two were exclusive to this group. This work demonstrated that the antagonistic capacity of plant-associated Bacillus against plant pathogenic bacteria is related to the presence of cLP genes and to the production of the corresponding cLPs, and it is mainly associated to the species B. subtilis and B. amyloliquefaciens. Our findings would help to increase the yield and efficiency of screening methods to obtain candidate strains to biocontrol agents with a mechanism of action relaying on the production of antimicrobial cLPs.     

Hemolytic Activity of a Togavirus,Getah*

A purified toga-alphavirus, Getah (GET), showed optimal hemolytic activity for one-day-old chick red blood cells when incubated at 37 C for 120 min at pH 6.2. Experimental data obtained from various angles, such as pH dependency, inhibition by virus-specific antiserum and by concanavalin A, indicated that the hemolysis was a property of the virus particle itself. Although the mechanism of hemolysis by togaviruses has not been known, our results indicated that viral lipids may participate in this activity since the hemolytic activity was impaired by delipidation procedures.     

Heat Production by the Denitrifying Bacterium Pseudomonas fluorescens and the Dissimilatory Ammonium-Producing Bacterium Pseudomonas putrefaciens during Anaerobic Growth with Nitrate as the Electron Acceptor

The heat production rate and the simultaneous nitrate consumption and production and consumption of nitrite and nitrous oxide were monitored during the anaerobic growth of two types of dissimilatory nitrate reducers. Pseudomonas fluorescens, a denitrifier, consumed nitrate and accumulated small amounts of nitrite or nitrous oxide. The heat production rate increased steadily during the course of nitrate consumption and decreased rapidly concomitant with the depletion of the electron acceptors. A mean experimental enthalpy change value of −800 kJ/mol of nitrate and a mean growth yield value of 33 g (dry weight)/mol of nitrate consumed were obtained for different concentrations of nitrate. For Pseudomonas putrefaciens, a dissimilatory ammonium producer, the nitrate consumption resulted in an accumulation of nitrite and nitrous oxide. Nitrite consumption commenced after depletion of the nitrate; consequently, two phases were noted in the heat production rate curve during growth. A mean experimental enthalpy change value of −810 kJ/mol of nitrate was obtained for different concentrations of nitrate.     

Production of Pseudomonas Cells Having a High Fumarate Hydratase Activity

An extracellular 45 kDa endochitosanase was purified and characterized from the culture supernatant of Bacillus sp. P16. The purified enzyme showed an optimum pH of 5.5 and optimum temperature of 60°C, and was stable between pH 4.5-10.0 and under 50°C. The K _m and V _max were measured with a chitosan of a D.A. of 20.2% as 0.52 mg/ml and 7.71×10^?6 mol/sec/mg protein, respectively. The enzyme did not degrade chitin, cellulose, or starch. The chitosanase digested partially N-acetylated chitosans, with maximum activity for 15-30% and lesser activity for 0-15% acetylated chitosan. The chitosanase rapidly reduced the viscosity of chitosan solutions at a very early stage of reaction, suggesting the endotype of cleavage in polymeric chitosan chains. The chitosanase hydrolyzed (GlcN)₇ in an endo-splitting manner producing a mixture of (GlcN)_2-5. Time course studies showed a decrease in the rate of substrate degradation from (GlcN)₇ to (GlcN)₆ to (GlcN)₅, as indicated by the apparent first order rate constants, k ₁ values, of 4.98×10^?4, 2.3×10^?4, and 9.3×10^?6 sec^?1, respectively. The enzyme hardly catalyzed degradation of chitooligomers smaller than the pentamer.     

Heavy-Metal Resistance of a France Vineyard Soil Bacterium,Pseudomonas mendocina Strain S5.2, Revealed by Whole-Genome Sequencing

Here we present the draft genome of Pseudomonas mendocina strain S5.2, possessing tolerance to a high concentration of copper. In addition to being copper resistant, the genome of P. mendocina strain S5.2 contains a number of heavy-metal-resistant genes known to confer resistance to multiple heavy-metal ions.     

L-Serine Production by Temperature-sensitive Mutants of Methanol-utilizing Bacterium Pseudomonas MS 31

Temperature-sensitive mutants producing L-serine efficiently from glycine were obtained from the facultative methylotroph Pseudomonas MS 31. Forty-five mutant strains showed adequate growth on methanol at 30°C but little or no growth at 37°C. Fourteen of these mutants produced L- serine more efficiently than the wild-type strain. The typical mutant strain ts 162 showed a high conversion rate in glycine-to-L-serine when the cultivation temperature was changed from a permissive (30°C) to non-permissive state (38?42°C) together with the addition of glycine and methanol after adequate growth. The mutant strain accumulated 6.8 mg L-serine from 12 mg glycine per ml culture under optimum conditions. The reduction of L-serine degrading activity in the mutant strain seemed to contribute to the high productivity of L-serine.     

Production of Cyclic Lipopeptides by Pseudomonas fluorescens Strains in Bulk Soil and in the Sugar Beet Rhizosphere

The production of cyclic lipopeptides (CLPs) with antifungal and biosurfactant properties by Pseudomonas fluorescens strains was investigated in bulk soil and in the sugar beet rhizosphere. Purified CLPs (viscosinamide, tensin, and amphisin) were first shown to remain highly stable and extractable (90%) when applied (ca. 5 μg g⁻¹) to sterile soil, whereas all three compounds were degraded over 1 to 3 weeks in nonsterile soil. When a whole-cell inoculum of P. fluorescens strain DR54 containing a cell-bound pool of viscosinamide was added to the nonsterile soil, declining CLP concentrations were observed over a week. By comparison, addition of the strains 96.578 and DSS73 without cell-bound CLP pools did not result in detectable tensin or amphisin in the soil. In contrast, when sugar beet seeds were coated with the CLP-producing strains and subsequently germinated in nonsterile soil, strain DR54 maintained a high and constant viscosinamide level in the young rhizosphere for ~2 days while strains 96.578 and DSS73 exhibited significant production (net accumulation) of tensin or amphisin, reaching a maximum level after 2 days. All three CLPs remained detectable for several days in the rhizosphere. Subsequent tests of five other CLP-producing P. fluorescens strains also demonstrated significant production in the young rhizosphere. The results thus provide evidence that production of different CLPs is a common trait among many P. fluorescens strains in the soil environment, and further, that the production is taking place only in specific habitats like the rhizosphere of germinating sugar beet seeds rather than in the bulk soil.     

海洋芽胞杆菌B-9987产新型抗菌环脂肽Marinhysin A的培养基优化

Marinhysin A(MA)是由海洋芽胞杆菌B-9987产生的具有抗真菌活性的新结构环脂肽类化合物,该化合物不仅抑菌谱广而且盆栽防效良好。采用响应面分析法(Response Surface Methnology)对海洋芽胞杆菌B-9987产脂肽MA的培养基进行了优化。Plackett-Burman (PB)实验表明,蔗糖和酵母粉是显著影响MA产量的因素。中心组合实验(Central Composite Design)分析表明,当培养基中蔗糖和酵母粉的含量分别为42.0g/L和42.3g/L时,MA的浓度最大可达68.19mg/L,与实验值75.74mg/L相近。优化后,MA产量(250ml摇瓶发酵)较优化前的54.12mg/L提高了28.5%。用优化后培养基进行5L罐发酵,MA浓度可达182mg/L,较优化前的130mg/L 提高了28.4%。     

Bioremediation of Sterile Agricultural Soils Polluted with Crude Petroleum by Application of the Soil Bacterium, Pseudomonas putida, with Inorganic Nutrient Supplementations

The effects of bioremediation program of sterile agricultural soils contaminated with crude petroleum were determined with a view to developing a suitable technique for rehabilitation of similar environments upon pollution by oil spillage. Sterile soils inoculated with the soil bacterium, Pseudomonas putida (PP), with inorganic nutrients monitoring and supplementation constituted the experimental set-ups (ESU). The control set-ups (CSU) contained all the materials present in ESU except that they were not inoculated with PP. In ESU at week 9, the oil pollutant was completely biodegraded, and the inorganic nutrient ions, particularly PO₄ ⁻³ and NO₃ ⁻¹, were significantly utilized. In contrast, there were no significant changes in the concentrations of oil and inorganic nutrients in CSU. Also, the percentage germination and growth profiles of cress seeds (Lepidium sp.) planted as evidence of the recovery of the oil-impacted soils were poor in CSU (27.5%) with pronounced abnormal morphology when compared with the results obtained for ESU (98.8%). Inoculation of PP with addition of appropriate inorganic nutrients may be a suitable method for a rapid rehabilitation of agricultural land upon pollution with crude petroleum. Received: 28 June 2000 / Accepted: 5 September 2000     

Enzyme Activity of Lectins from the Nitrogen-Fixing Soil Bacterium Bacillus polymyxa

Lectins LI and LII, localized on the surface of the nitrogen-fixing soil bacterium Bacillus polymyxa 1460, were shown to possess proteolytic activity. A relationship was found between the proteolytic and hemagglutinating activities of the lectins. Blocking of hemagglutinating activity with specific carbohydrate haptens led to significant changes in the enzyme activity of both lectins. When lectin activity was blocked with glucuronic acid and fructose-1,6-diphosphate, the proteolytic activity of both LI and LII declined, whereas incubation with d-galactosamine and d-glucosamine promoted increases in the proteolytic activity of LII. This study proposes that the molecules of the B. polymyxa lectins may have two centers on their surfaces: one responsible for lectin activity and the other for proteolytic activity. Received: 27 March 2000 / Accepted: 26 April 2000     

Genome Sequence of a Cold-Adaptable Sulfamethoxazole-Degrading Bacterium,Pseudomonas psychrophila HA-4

Pseudomonas psychrophila HA-4 is a cold-adaptable, sulfamethoxazole-degrading bacterium. The genes related to its cold adaptation mechanism and sulfamethoxazole metabolism were unknown. We present the draft genome of strain HA-4. It could provide further insight into the sulfamethoxazole-degrading mechanism of strain HA-4.     

A Truncated Form of SpoT,Including the ACT Domain,Inhibits the Production of Cyclic Lipopeptide Arthrofactin,and Is Associated with Moderate Elevation of Guanosine 3′,5′-Bispyrophosphate Level in Pseudomonas sp. MIS38

Arthrofactin is a biosurfactant produced by Pseudomonas sp. MIS38. We have reported that transposon insertion into spoT (spoT::Tn5) causes moderate accumulation of guanosine 3′,5′-bispyrophosphate (ppGpp) and abrogates arthrofactin production. To analyze the linkage of SpoT function and ablation of arthrofactin production, we examined the spoT::Tn5 mutation. The results showed that spoT::Tn5 is not a null mutation, but encodes separate segments of SpoT. Deletion of the 3′ region of spoT increased the level of arthrofactin production, suggesting that the C-terminal region of SpoT plays a suppressive role. We evaluated the expression of a distinct segment of SpoT. Forced expression of the C-terminal region that contains the ACT domain resulted in the accumulation of ppGpp and abrogated arthrofactin production. Expression of the C-terminal segment also reduced MIS38 swarming and resulted in extensive biofilm formation, which constitutes the phenocopy of the spoT::Tn5 mutant.