Synthesis of Protocatechuate Oxygenase by Pseudomonas fluorescens in the Presence of Exogenous Carbon Sources.

Kirkland, Jerry J. (Oklahoma State University, Stillwater), and Norman N. Durham. Synthesis of protocatechuate oxygenase by Pseudomonas fluorescens in the presence of exogenous carbon sources. J. Bacteriol. 90:15-22. 1965.-The addition of glucose, ribose, or fructose (0.45 or 45.0 mumoles/ml) simultaneously with protocatechuic acid shortens the lag period required for synthesis of protocatechuate oxygenase by a washed-cell suspension of Pseudomonas fluorescens. Glucose is readily oxidized and supports growth of P. fluorescens, whereas neither ribose nor fructose readily supports growth. High glucose concentrations (45.0 mumoles/ml) shorten the lag period but lower the total enzyme synthesis. The pH drops during glucose oxidation, and this is accompanied by a decrease in the rate of enzyme synthesis. High glucose concentrations, with adequate buffering, permitted "normal" enzyme synthesis. A decrease in the total enzyme synthesis was not observed in the presence of high concentrations of ribose or fructose. Succinate, pyruvate, acetate, or formate (0.45 mumole/ml) were readily oxidized, but did not shorten the lag period required for synthesis of the enzyme. The data suggest that glucose, ribose, or fructose may serve as a "specific" carbon source (such as ribose-5-phosphate or a similar precursor important in ribonucleic acid synthesis) functional in the synthesis of protocatechuate oxygenase.     

Culturability and Expression of Outer Membrane Proteins during Carbon, Nitrogen, or Phosphorus Starvation of Pseudomonas fluorescens DF57 and Pseudomonas putida DF14

Changes in culturability and outer membrane protein profiles were investigated in Pseudomonas fluorescens DF57 and Pseudomonas putida DF14 during starvation for carbon, nitrogen, and phosphorus. P. fluorescens DF57 remained fully culturable for 4 days in all starvation regimes. The cell mass increased during starvation for nitrogen and phosphorus, indicating the accumulation of storage compounds, whereas it decreased slightly in carbon-starved cells. P. putida DF14 lost culturability during phosphorus starvation, and the mass of phosphate-starved cells did not increase. Analysis of additional P. fluorescens and P. putida strains, however, showed that the ability to preserve culturability during phosphorus starvation was not species but strain dependent. In DF57, an outer membrane protein of 55 kDa appeared during starvation for phosphorus, while another protein of 63 kDa was seen during all starvation conditions. DF14 induced two outer membrane proteins of 28 and 29 kDa during starvation for carbon and nitrogen, but no phosphorus-specific starvation protein could be detected. Therefore, starvation-induced outer membrane proteins do not seem to be conserved among the fluorescent pseudomonads and a unique starvation response might be found in individual strains.     

产葡萄糖酸荧光假单胞菌的分离鉴定及解磷作用

从番茄根际土壤中分离到l株解磷荧光假单胞菌LAD6,并对其进行了形态特征、生理生化鉴定及16S rDNA同源性序列分析.高效毛细管电泳仪(HPCE)分析结果显示,LAD6能够产生葡萄糖酸.对LAD6的解磷能力的测定结果表明,培养液中的磷溶解量的变化与茵株LAD6的生长和生理代谢的变化相关,其最大磷溶解量达到了24.5μg/mL.     

Initiation of Yeast Sporulation by Partial Carbon, Nitrogen, or Phosphate Deprivation

In this paper we show that partial deprivation of a carbon source, a nitrogen source, or phosphate in the presence of all other nutrients needed for growth initiates meiosis and sporulation of Saccharomyces cerevisiae homothallic strain Y55. For carbon deprivation experiments, cells were grown in synthetic medium (pH 5.5) containing an excess of one carbon source and then transferred to the same medium containing different concentrations of the same carbon source. In the case of transfer to different acetate concentrations, the log optical density at 600 nm increased at the previous rate until the cells had used up all of the acetate, whereupon the cells entered a stationary phase and did not sporulate. The same was observed with ethanol. In contrast, at different concentrations of dihydroxy-acetone or pyruvate, cells grew at different rates and sporulated optimally at intermediate concentrations (50 to 75 mM). The response to galactose was similar but reflected the presence of a low-affinity galactose transport system and the induction of a high-affinity galactose transport system. Cells could also sporulate when a glucose medium ran out of glucose, apparently because they initiated sporulation during the subsequent lag period and then used the produced ethanol as a carbon source. For phosphate deprivation experiments, cells growing with excess ethanol or pyruvate and phosphate were transferred to the same medium containing limiting amounts of phosphate. First, they used up the intracellular phosphate reserves for rapid growth, and then they sporulated optimally when an intermediate concentration (30 μM) of phosphate had been added to the medium. For nitrogen deprivation experiments, cells grown with excess acetate, ethanol, or pyruvate and NH₄⁺ were transferred to the same medium from which all nitrogen had been removed. These cells sporulated well in acetate medium but poorly in ethanol and pyruvate media. However, the sporulation frequency in the latter media could be increased greatly by adding intermediate concentrations (1 mM) of the slowly metabolizable amino acids glycine, histidine, or phenylalanine. If one assumes that the sporulation response to partial deprivation of carbon-, nitrogen-, or phosphorus-containing compounds reflects control by a single metabolite, the intracellular concentration of this metabolite may decide at the START position (G1 phase) of the cell cycle whether a/α cells enter mitosis or meiosis.     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

Effects of Temperature and Nutrients on Proteinase Production by Pseudomonas fluorescens and Ps. aeruginosa in Broth and Milk

Temperature and the composition of the medium influenced the production of proteinase by Pseudomonas fluorescens and Pseudomonas aeruginosa isolated from raw milk. Many isolates of Ps. fluorescens digested litmus milk at 10° but not at 5° or 2°. With Ps. fluorescens proteinase production per unit of growth in a Peptone–Yeast Extract broth declined progressively as the incubation temperature was reduced from 20° to 5°. At 30° there was heavy growth in the same medium but only slight proteinase production whereas enzyme production by Ps. aeruginosa was maximal at this temperature. Proteinase production by both species in semi-defined media was essentially a function of the organic nitrogen content of the medium; there was evidence of catabolite repression by glucose and, to a lesser extent, lactate. In milks seeded with these pseudomonads, the extent of proteolysis was either increased markedly or slightly decreased when glucose was included.     

Biochemical and Genetic Characterization of an Extracellular Protease from Pseudomonas fluorescens CY091

Pseudomonas fluorescens CY091 cultures produce an extracellular protease with an estimated molecular mass of 50 kDa. Production of this enzyme (designated AprX) was observed in media containing CaCl₂ or SrCl₂ but not in media containing ZnCl₂, MgCl₂, or MnCl₂. The requirement of Ca²⁺ (or Sr²⁺) for enzyme production was concentration dependent, and the optimal concentration for production was determined to be 0.35 mM. Following ammonium sulfate precipitation and ion-exchange chromatography, the AprX in the culture supernatant was purified to near electrophoretic homogeneity. Over 20% of the enzyme activity was retained in the AprX sample which had been heated in boiling water for 10 min, indicating that the enzyme is highly resistant to heat inactivation. The enzyme activity was almost completely inhibited in the presence of 1 mM 1,10-phenanthroline, but only 30% of the activity was inhibited in the presence of 1 mM EGTA. The gene encoding AprX was cloned from the genome of P. fluorescens CY091 by isolating cosmid clones capable of restoring the protease production in a nonproteolytic mutant of strain CY091. The genomic region of strain CY091 containing the aprX gene was located within a 7.3-kb DNA fragment. Analysis of the complete nucleotide sequence of this 7.3-kb fragment revealed the presence of a cluster of genes required for the production of extracellular AprX in P. fluorescens and Escherichia coli. The AprX protein showed 50 to 60% identity in amino acid sequence to the related proteases produced by Pseudomonas aeruginosa and Erwinia chrysanthemi. Two conserved sequence domains possibly associated with Ca²⁺ and Zn²⁺ binding were identified. Immediately adjacent to the aprX structural gene, a gene (inh) encoding a putative protease inhibitor and three genes (aprD, aprE, and aprF), possibly required for the transport of AprX, were also identified. The organization of the gene cluster involved in the synthesis and secretion of AprX in P. fluorescens CY091 appears to be somewhat different from that previously demonstrated in P. aeruginosa and E. chrysanthemi.     

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.     

Extracellular factors affecting the adhesion of Pseudomonas fluorescens cells to glass surface

Two factors affecting the adhesion of Pseudomonas fluorescens to glass surfaces were revealed in the culture liquid (CL) of this bacterium. One of these factors, adhesin, which is responsible for cell adhesion, was found to be a protein substance located both at the cell surface and in the CL. Bacterial cells grown in rich LB medium were less adhesive than cells grown in minimal M9 medium. The adhesive capacity of cells was independent of the growth phase. The other factor, anti-adhesion (AA), which reduces cell adhesion, was found only in the CL. AA concentration in the CL increased with the culture age.     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa.

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

Fluorimetric Assay of the Activity of Extracellular Lipases of Pseudomonas fluorescens and Serratia marcescens

A fluorimetric assay was carried out on the activity of extracellular lipase concentrations obtained from Serratia marcescens and Pseudomonas fluorescens using as substrates fatty acyl esters of 4-methylumbelliferone (4-methylumbelliferone elaidate, 4-methylumbelliferone nonanoate, 4-methylumbelliferone butyrate, 4-methylumbelliferone palmitate and 4-methylumbelliferone oleate) at pH 4.0, 6.0, 8.0 and 10.0. The Ser. marcescens and Ps. fluorescens were cultured in Pope and Skerman's basal medium (Skerman 1957) supplemented with 0.5% (w/v) of a commercial medium. The extracellular lipases were isolated and purified by ammonium sulphate precipitation. The assay was carried out by relating the fluorescent intensity emitted by two lipase concentrations on five substrates against four standard curves. These standard curves were prepared by estimating the intensity of fluorescence given by varying dilutions of 4-methylumbelliferone at the four pH levels. The results indicated that the oleic ester of 4-methylumbelliferone was a suitable substrate at pH 8.0 and pH 10.0. These pH values were also optimum for fluorescent intensity on substrates of 4-methylumbelliferone elaidate, 4-methylumbelliferone butyrate and 4-methylumbelliferone palmitate. However, on substrate 4-methylumbelliferone nonanoate, the optimum pH was 4.0.     

Synthesis of cinnamyl acetate catalysed by highly reusable cotton-immobilized Pseudomonas fluorescens lipase

Cinnamyl acetate as an important fragrance ingredient could be synthesized by lipase-catalysed transesterification in organic systems, but enzyme proteins tended to denature and inactivate for no water lubrication. To improve the non-aqueous stability of lipases, absorbent cotton was taken as an alternative “water” phase to stabilize enzyme proteins. In a mass ratio of 1:1, Pseudomonas fluorescens lipase was immobilized on cotton fibres by physical absorption in a column glass bottle, forming a facile cotton-lipase bioreactor in which the transesterification between cinnamyl alcohol and vinyl acetate processed efficiently. From the molar conversions after reaction for 2?h at 37?°C and 160?rpm, the ability of cotton-lipase to transform substrate was more than 5-folds of native lipase. And even in static state and at 4?°C, the conversion of reaction catalysed by cotton-PFL had 11-fold increase relative to native lipase after 8?h. Recycles showed that the cotton-lipase had an extra-long half-life of activity (t_1/2?=?693?h) and a negligible decay rate in the ability to transform substrate (D_r?=?0.08% h^?1). All these showed that this lipase had been effectively activated and stabilized by cotton fibres for the numerous hydroxyl groups and fluffy structure.     

The Requirements of Pseudomonas aeruginosa Dissociants for Carbon,Nitrogen, and Phosphorus

Quantitative data on the nutritional requirements of microorganisms are necessary to predict the behavior of bacterial populations and to control their cultivation. The requirements of the R, S, and M dissociants of P. aeruginosa for carbon, nitrogen, and phosphorus were derived from the results of 88 cultivation experiments. For each of the dissociants, we derived a coefficient that relates the optical density and the number of cells in the dissociant culture, determined the time when the cultures entered the stationary growth phase, studied cultural changes induced by transfer to the stationary phase, and determined what nutrients limit the growth of particular dissociants. The nutritional requirements of the dissociants are discussed in relation to our earlier data.     

Isolation of alginate-producing mutants of Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas mendocina

Spontaneous alginate-producing (muc) variants were isolated from strains of Pseudomonas fluorescens, P. putida and P. mendocina at a frequency of 1 in 10(8) by selecting for carbenicillin resistance. The infrared spectrum of the bacterial exopolysaccharide was typical of an acetylated alginate similar to that previously described in Azotobacter vinelandii and in mucoid variants of P. aeruginosa. Mucoid variants were not isolated from P. stutzeri, P. pseudoalcaligenes, P. testosteroni, P. diminuta, P. acidovorans, P. cepacia or P. maltophilia.     

Regulation of Pectate Lyase Synthesis in Pseudomonas fluorescens and Erwinia carotovora

Inducible synthesis of extracellular pectate lyase occurs in Erwinia carotovora, a bacterial soft-rot pathogen of plants, and, to a lesser extent, in a nonpathogenic isolate of Pseudomonas fluorescens. A combination of pectin and a heat-labile factor in fresh potato tissue or acetone powders of the tissue provided the best carbon source for induction. Yields of inducible pectate lyase were much greater than those usually reported. The pathogen, but not the saprophyte, produced a small amount of constitutive enzyme when grown on glucose. The relatively low level or absence of constitutive synthesis in these bacteria did not result from catabolite repression. Attempts were made to relieve any existing catabolite repression by restricting growth through slow feeding of glucose or by growing the organisms on glycerol. These conditions did not significantly alter the differential rate of lyase synthesis compared with changes observed in the presence of inducers. Previous growth history did not affect induction in the pathogen. However, P. fluorescens previously cultured on glucose required 10 to 20 generations of growth on inducing medium before appreciable lyase synthesis occurred. Differences between the pathogen and nonpathogen suggest that regulation of pectate lyase synthesis is related to pathogenicity of soft-rot bacteria.     

Nitrite inhibition of denitrification by Pseudomonas fluorescens

Using a pure culture of Pseudomonas fluorescens as a model system nitrite inhibition of denitrification was studies. A mineral media with acetate and nitrate as sole electron donor and acceptor, respectively, was used. Results obtained in continuous stirred-tank reactors (CSTR) operated at pH values between 6.6 and 7.8 showed that growth inhibition depended only on the nitrite undissociated fraction concentration (nitrous acid). A mathematical model to describe this dependence is put forward. The maximum nitrous acid concentration compatible with cell growth and denitrification activity was found to be 66 mug N/L. Denitrification activity was partially associated with growth, as described by the Luedeking-Piret equation. However, when the freshly inoculated reactor was operated discontinuosly, nitrite accumulation caused growth uncoupling from denitrification activity. The authors suggest that these results can be interpreted considering that (a) nitrous acid acts as a proton uncoupler; and (b) cultures continuoulsy exposed to nitrous acid prevent the uncoupling effect but not the growth inhibition. Examination of the growth dependence on nitrite concentration at pH 7.0 showed that adapted cultures (grown on CSTR) are less sensitive to nitrous acid inhibition than the ones cultivated in batch. (c) 1995 John Wiley & Sons, Inc.     

Requirements of Pseudomonas aeruginosaDissociants in Glucose, Nitrate, and Phosphate. Limiting Concentrations of the Nutrients during Batch Cultivation

Requirements of R-, S-, and M-dissociants of Pseudomonas aeruginosaK-2 strain in glucose, nitrate, and phosphate, as well as the error of determination, have been calculated from the data of 250 experiments on their batch cultivation on the media with the various initial content of these resources. The resource requirements of R- and S-dissociants appear indistinguishable within the error limits. The requirements of M-dissociant significantly differ from those of R- and S-forms; however, due to specific growth of the M-form, the confidence intervals of the measured values can overlap with those of R-space and S-forms. The obtained requirement values allow an evaluation of the studied resources concentration limiting the culture growth.