The fate of a genetically modified Pseudomonas strain and its transgene during the composting of poultry manure

The fate of the genetically modified (GM) Pseudomonas chlororaphis strain 3732 RN-L11 and its transgene (lacZ insert) during composting of chicken manure was studied using plate count and nested polymerase chain reaction (PCR) methods. The detection sensitivity of the nested PCR method was 165 copies of the modified gene per gram of moist compost or soil. Compost microcosms consisted of a 100-g mixture of chicken manure and peat, whereas soil microcosms were 100-g samples of sandy clay loam. Each microcosm was inoculated with 4 x 1010 CFU of P. chlororaphis RN-L11. In controlled temperature studies, neither P. chlororaphis RN-L11 nor its transgene could be detected in compost microcosms after incubation temperature was elevated to 45 degrees C or above for one or more days. In contrast, in the compost microcosms incubated at 23 degrees C, the target organism was not detected by the plate count method after 6 days, but its transgene was detectable for at least 45 days. In compost bins, the target organism was not recovered from compost microcosms or soil microcosms at different levels in the bins for 29 days. However, the transgene was detected in 8 of the 9 soil microcosms and in only 1 of the 9 compost microcosms. The compost microcosm in which transgene was detected was at the lower level of the bin where temperatures remained below 45 degrees C. The findings indicated that composting of organic wastes could be used to reduce or degrade heat sensitive GM microorganisms and their transgenes.     

Interactions of certain sapronotic infection pathogens in mixed cultures on a solid medium at different temperatures

Under experimental conditions within the time limit of 21-35 days the causative agents of sapronotic infections in binary cultures, grown on a solid medium at 37 degrees C, 25-27 degrees C and 6-8 degrees C, interacted with one another transbiotically and through contact, their interactions having the character of amensalism, commensalisms-amensalism, competitive equilibrium, antibiosis. Irrespective of the initial density, a change in the species composition was observed, one of them playing the dominating role. At 37 degrees C mutual antagonism of Yersinia pseudotuberculosis and Pseudomonas aeruginosa killed both cultures. P. aeruginosa cells were also killed when cultivated at 37 degrees C jointly with Listeria monocytogenes, the most resistant species under experimental conditions. While studying the character of microorganisms interactions the method of contacting cultures on a solid medium was shown to give more information in comparison with the "cross-strip" method. Possible interspecific relationships between the causative agents of sapronotic infections under natural conditions are discussed.     

Nature of Escherichia coli cell lysis by culture supernatants of Bacillus species.

Escherichia coli cells were found to be sensitive to lysis by the supernatants of a variety of protease-positive Bacillus species when treated at 45 degrees C but not when treated at 37 degrees C. Different E. coli strains manifested different lysis sensitivities when treated at 45 degrees C. When the lysis rates of E. coli cells at various stages of growth were investigated, post-exponential-phase cells were shown to be most sensitive to lysis. The lysis rate was inversely related to cell viability, and susceptibility appeared to be at least partly due to lysis of dead E. coli cells. A close relation was observed between levels of lysis activity and proteolytic activity. A Bacillus subtilis mutant lacking alkaline and neutral protease activity failed to lyse E. coli cells. It was concluded that Bacillus proteases played a major role in the observed E. coli lysis.     

Nature of Escherichia coli cell lysis by culture supernatants of Bacillus species

Escherichia coli cells were found to be sensitive to lysis by the supernatants of a variety of protease-positive Bacillus species when treated at 45 degrees C but not when treated at 37 degrees C. Different E. coli strains manifested different lysis sensitivities when treated at 45 degrees C. When the lysis rates of E. coli cells at various stages of growth were investigated, post-exponential-phase cells were shown to be most sensitive to lysis. The lysis rate was inversely related to cell viability, and susceptibility appeared to be at least partly due to lysis of dead E. coli cells. A close relation was observed between levels of lysis activity and proteolytic activity. A Bacillus subtilis mutant lacking alkaline and neutral protease activity failed to lyse E. coli cells. It was concluded that Bacillus proteases played a major role in the observed E. coli lysis.     

Utilization of monocrotophos as phosphorus source by Pseudomonas aeruginosa F10B and Clavibacter michiganense subsp. insidiosum SBL 11

Monocrotophos (dimethyl (E)-1-methyl-2-(methylcarbamoyl) vinyl phosphate, or MCP), an organophosphorus insecticide, was used as a sole phosphorus source by the microorganisms isolated from the soil. None of the isolates could utilize MCP as a sole source of carbon. Two of the potential microbial isolates, Pseudomonas aeruginosa F10B and Clavibacter michiganense subsp. insidiosum SBL 11, could utilize MCP as a sole source of phosphorus. Pseudomonas aeruginosa F10B showed a lag phase of 4 h, while in the case of C. michiganense subsp. insidiosum SBL 11, it was 8 h when cultured in the presence of MCP. The generation time for both strains was increased in the medium containing MCP. It was 2.15 h for P. aeruginosa F10B in MCP medium as compared with 1.29 h in basal medium, while in case of C. michiganense subsp. insidiosum SBL 11 it was increased to 3.4 h in MCP medium as compared with 1.28 h in basal medium. These two strains were able to degrade technical MCP in shake-flask culture up to 98.9 and 86.9%, respectively, and pure MCP up to 79 and 80%, respectively, within 24 h at 37 degrees C. The optimal concentration of MCP required for the normal growth was 500 ppm. In the substrate preference study, Tris-p-nitrophenyl phosphate was the most preferred substrate followed by paraoxon. The enzyme responsible for the break down of MCP was phosphotriesterase, which was localized on the membrane-bound fraction of the disrupted cells. The gene responsible for the production of phosphotriesterase (opd) in P. aeruginosa F10B was plasmid-borne.     

Physical factors affecting the production of organic solvent-tolerant protease by Pseudomonas aeruginosa strain K

The physical factors affecting the production of an organic solvent-tolerant protease from Pseudomonas aeruginosa strain K was investigated. Growth and protease production were detected from 37 to 45 degrees C with 37 degrees C being the optimum temperature for P. aeruginosa. Maximum enzyme activity was achieved at static conditions with 4.0% (v/v) inoculum. Shifting the culture from stationary to shaking condition decreased the protease production (6.0-10.0% v/v). Extracellular organic solvent-tolerant protease was detected over a broad pH range from 6.0 to 9.0. However, the highest yield of protease was observed at pH 7.0. Neutral media increased the protease production compared to acidic or alkaline media.     

Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor

The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.     

Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor.

The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.     

Selective medium for Pseudomonas aeruginosa that uses 1,10-phenanthroline as the selective agent.

The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa.     

Selective medium for Pseudomonas aeruginosa that uses 1,10-phenanthroline as the selective agent

The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa.     

Fate of Escherichia coli O157:H7 in manure-amended soil

Escherichia coli O157:H7 cells survived for up to 77, >226, and 231 days in manure-amended autoclaved soil held at 5, 15, and 21 degrees C, respectively. Pathogen populations declined more rapidly in manure-amended unautoclaved soil under the same conditions, likely due to antagonistic interactions with indigenous soil microorganisms. E. coli O157:H7 cells were inactivated more rapidly in both autoclaved and unautoclaved soils amended with manure at a ratio of 1 part manure to 10 parts soil at 15 and 21 degrees C than in soil samples containing dilute amounts of manure. The manure-to-soil ratio, soil temperature, and indigenous microorganisms of the soil appear to be contributory factors to the pathogen's survival in manure-amended soil.     

Growth of Pseudomonas aeruginosa in tap water in relation to utilization of substrates at concentrations of a few micrograms per liter.

Five Pseudomonas aeruginosa strains were tested for the utilization of 47 low-molecular-weight compounds as their sole sources of carbon and energy for growth at a concentration of 2.5 g/liter. Of these compounds, 31 to 35 were consumed. Growth experiments in tap water at 15 degrees C were carried out with one particular strain (P1525) isolated from drinking water. This strain was tested for the utilization of 30 compounds supplied at a concentration of 25 microgram of C per liter. The growth rate (number of generations per hour) of strain P1525 in this tap water was approximately 0.005 h-1, and with 10 compounds it was larger than 0.03 h-1. An average yield of 6.2 x 10(9) colony-forming units per mg of C was obtained from the maximum colony counts (colony-forming units per milliliter). The average yield and maximum colony count of strain P1525 grown in tap water supplied with a mixture of 45 compounds, each at a concentration of 1 microgram of C per liter, enabled us to calculate that 28 compounds were utilized. Growth rates of two P. aeruginosa strains (including P1525) in various types of water at 15 degrees C were half of those of a fluorescent pseudomonad. The concentrations of assimilable organic carbon calculated from maximum colony counts and average yield values amounted to 0.1 to 0.7% of the total organic carbon concentrations in five types of tap water. The assimilable organic carbon percentages were about 10 times larger in river water and in water after ozonation.     

Purification and characterization of a novel cholesterol esterase from Pseudomonas aeruginosa,with its application to cleaning lipid-stained contact lenses

With the aim of developing a new cholesterol esterase for eliminating lipids on used contact lenses, microorganisms were screened for the enzyme activity. A Pseudomonas aeruginosa isolated from soil was found to produce a desirable enzyme. The enzyme had an isoelectric point of 3.2, and molecular mass of 58 kDa. The optimal temperature was around 53 degrees C at pH 7.0, and the optimal pH was from 5.5 to 9.5. The enzyme was stable between pH 5 and 10 for 19 h at 25 degrees C, and retained its activity up to 53 degrees C on 30 min of incubation at pH 7.0. The rates of hydrolysis of cholesteryl esters of different fatty acids were in the following order: linoleate > oleate > stearate > palmitate > caprylate > myristate > laurate, caprate > caproate > butyrate, acetate. Addition of (tauro)cholate to a final concentration of 100 mM markedly promoted the hydrolysis of triglycerides of short-, medium-, and long-chain fatty acids. When used with taurocholate, the enzyme acted as an effective cleaner for contact lenses stained with lipids consisting of cholesteryl oleate, tripalmitin, and stearyl stearate.     

The description of an esculin-positive biovar of Pseudomonas aeruginosa

In the study of 280 P. aeruginosa strains isolated in different hospitals of St. Petersburg for the first time 48 strains capable of hydrolyzing esculin have been detected. The hydrolysis of esculin is determined in plates with the use of the microvolume techniques the results were evaluated after 3-hour incubation at 37 degrees C. The data confirming the existence of the exculin-positive biovar of P. aeruginosa have been obtained; these data show the wide spread of esculin-positive strains in hospitals of different specialization (17.1 +/- 5.1% of P. aeruginosa strains), the characteristic combination of the sign of esculin hydrolysis with such signs as the absence of the smell of trimethylamine and the phenomenon of "iridescent lysis" of the colonies, the stability of the sign of esculin hydrolysis in strains, repeatedly isolated from patients, after the storage of the cultures and their treatment with plasmid-eliminating preparation. The name "esculinolytica" has been proposed for this biovar. The typing strain of biovar esculinolytica has been deposited in the culture collection of the Russian Research Institute of Agricultural Microbiology as P. aeruginosa ARRIAM 64-A. This biovar been found to be most widely spread in urological hospitals, where esculin-positive strains are isolated 3 times more frequently (32.2 +/- 5.1% of P. aeruginosa strains) than in surgical hospitals (10.7 +/- 2.2%).     

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: DNA-gp3 intermediate in in vivo and in vitro assembly

The assembly of phage phi 29 occurs by a single pathway, and DNA-protein (DNA-gp3) has been shown to be an intermediate on the assembly pathway by a highly efficient in vitro complementation. At 30 degrees C, about one-half of the viral DNA synthesized was assembled into mature phage, and the absolute plating efficiency of phi 29 approached unity. DNA packaging at 45 degrees C was comparable to that at 30 degrees C, but the burst size was reduced by one-third. When cells infected with mutant ts3(132) at 30 degrees C to permit DNA synthesis were shifted to 45 degrees C before phage assembly, DNA synthesis ceased and no phage were produced. However, a variable amount of DNA packaging occurred. Superinfection by wild-type phage reinitiated ts3(132) DNA synthesis at 45 degrees C, and if native gp3 was covalently linked to this DNA during superinfection replication, it was effectively packaged and assembled. Treatment of the DNA-gp3 complex with trypsin prevented in vitro maturation of phi 29, although substantial DNA packaging occurred. A functional gp3 linked to the 5' termini of phi 29 DNA is a requirement for effective phage assembly in vivo and in vitro.     

Pseudomonas aeruginosa PAO1 ceases to express serotype-specific lipopolysaccharide at 45 degrees C.

Most Pseudomonas aeruginosa strains are able to produce two distinct lipopolysaccharide (LPS) O-polysaccharide types, A-band (common-antigen) and B-band (serotype-specific) LPSs. The relative expression levels of these two LPS types in P. aeruginosa PAO1 (O5 serotype) at various growth temperatures were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining or Western blotting (immunoblotting) with monoclonal antibodies specific for each O polysaccharide. A-band and B-band LPSs were expressed concurrently when the cells grew at 15, 25, and 35 degrees C; however, growth at 45 degrees C resulted in a surface deficiency in B-band LPS as determined by immunoblotting and agglutination with B-band-specific monoclonal antibody. Transfer of these cells (expressing A-band LPS but deficient in B-band LPS) [A+B-]) to a lower temperature (at which the division time was comparable) resulted in a rapid resumption of normal A-band and B-band expression. B-band LPS was detectable by immunoblotting before measurable growth of the culture had occurred.     

Comparison of temperature effects on soil respiration and bacterial and fungal growth rates

Temperature is an important factor regulating microbial activity and shaping the soil microbial community. Little is known, however, on how temperature affects the most important groups of the soil microorganisms, the bacteria and the fungi, in situ. We have therefore measured the instantaneous total activity (respiration rate), bacterial activity (growth rate as thymidine incorporation rate) and fungal activity (growth rate as acetate-in-ergosterol incorporation rate) in soil at different temperatures (0-45 degrees C). Two soils were compared: one was an agricultural soil low in organic matter and with high pH, and the other was a forest humus soil with high organic matter content and low pH. Fungal and bacterial growth rates had optimum temperatures around 25-30 degrees C, while at higher temperatures lower values were found. This decrease was more drastic for fungi than for bacteria, resulting in an increase in the ratio of bacterial to fungal growth rate at higher temperatures. A tendency towards the opposite effect was observed at low temperatures, indicating that fungi were more adapted to low-temperature conditions than bacteria. The temperature dependence of all three activities was well modelled by the square root (Ratkowsky) model below the optimum temperature for fungal and bacterial growth. The respiration rate increased over almost the whole temperature range, showing the highest value at around 45 degrees C. Thus, at temperatures above 30 degrees C there was an uncoupling between the instantaneous respiration rate and bacterial and fungal activity. At these high temperatures, the respiration rate closely followed the Arrhenius temperature relationship.     

Heat-induced disturbances of intracellular movement and the consistency of the aqueous cytoplasm in HeLa S-3 cells: a laser-Doppler and proton NMR study.

Intracellular particle movements, of both saltatory and streaming types, in HeLa S-3 cells were simultaneously interrupted after 1 h exposure of cells to 43 degrees C, within 10 min at 44 degrees C and within 5 min at 45 degrees C. Intracellular movement inhibited after 15 min at 44 degrees C and 10 min at 45 degrees C was not reversible in cells rescued at 37 degrees C. Brownian motion was not observed in heat-treated cells while they were maintained at elevated temperatures, but became pronounced in blebbing which occurred shortly after they were returned to 37 degrees C. Returning these cells to 45 degrees C intensified the Brownian activity inside blebs, and rapidly induced cell lysis. The same heat-treated cells were simultaneously studied by laser-Doppler microscopy, which confirmed: a) that flow (cytoplasmic streaming) is completely arrested at 44 degrees C within 10 min, b) flow recovered in 10-15 min in cells rescued after 10-15 min at 44 degrees C, c) submicroscopic particles down to the size of water molecules had faster self-diffusion coefficients at 44 degrees C than at 37 degrees C. Proton nmr studies on cells exposed from 4 to 45 degrees C gave corrected relaxation times T1 and T2 which rose with temperature in a predictable manner. Inhibition of cellular movement at elevated temperatures was not specifically attributable to the depletion of intracellular ATP levels.     

Factors that influence the permeability assay of the outer membrane of Pseudomonas aeruginosa

Brief exposure of Pseudomonas aeruginosa to a temperature of 10 degrees C or lower caused a significant leakage of the periplasmic beta-lactamase into the medium. The extent of leakage increased as the incubation temperature was lowered to 4 degrees C and reached a maximum at 0 degrees C. Cells grown in the presence of beta-lactamase inducers were unsuitable for the permeability assay. It was found that the diffusion rates of beta-lactams through the outer membrane of P. aeruginosa were much lower than those previously reported, as assayed under refined conditions. The diffusion rates of beta-lactams in one of the mutants tested were an order of magnitude lower than those of the other strains, despite the fact that the outer membrane protein profile of the strain appeared to be indistinguishable from those of the others. These results suggest that beta-lactam antibiotics diffuse through the outer membrane of P. aeruginosa, at least partly, through a non-porin pathway.     

Aerobic biodegradation of biphenyl and polychlorinated biphenyls by Arctic soil microorganisms.

We examined the degradation of biphenyl and the commercial polychlorinated biphenyl (PCB) mixture Aroclor 1221 by indigenous Arctic soil microorganisms to assess both the response of the soil microflora to PCB pollution and the potential of the microflora for bioremediation. In soil slurries, Arctic soil microflora and temperate-soil microflora had similar potentials to mineralize [14C]biphenyl. Mineralization began sooner and was more extensive in slurries of PCB-contaminated Arctic soils than in slurries of uncontaminated Arctic soils. The maximum mineralization rates at 30 and 7 degrees C were typically 1.2 to 1.4 and 0.52 to 1.0 mg of biphenyl g of dry soil-1 day-1, respectively. Slurries of PCB-contaminated Arctic soils degraded Aroclor 1221 more extensively at 30 degrees C (71 to 76% removal) than at 7 degrees C (14 to 40% removal). We isolated from Arctic soils organisms that were capable of psychrotolerant (growing at 7 to 30 degrees C) or psychrophilic (growing at 7 to 15 degrees C) growth on biphenyl. Two psychrotolerant isolates extensively degraded Aroclor 1221 at 7 degrees C (54 to 60% removal). The soil microflora and psychrotolerant isolates degraded all mono-, most di-, and some trichlorobiphenyl congeners. The results suggest that PCB pollution selected for biphenyl-mineralizing microorganisms in Arctic soils. While low temperatures severely limited Aroclor 1221 removal in slurries of Arctic soils, results with pure cultures suggest that more effective PCB biodegradation is possible under appropriate conditions.