The effect of mafenide on dihydropteroate synthase.

Using intact bacterial cells, it was found that Pseudomonas aeruginosa was more susceptible to mafenide than Escherichia coli, that p-aminobenzoic acid (pABA) did not reverse or prevent inhibition by mafenide and that pABA itself was inhibitory. Under the experimental conditions used in these studies, pABA was more inhibitory to E. coli than to P. aeruginosa. It is proposed that pABA could be of use in the topical treatment of burn wounds. At the enzyme level, it was shown that mafenide did not inhibit dihydropteroate synthase. Thus, mafenide appeared not to exert its inhibitory effects in the same manner as the structurally related sulphonamides.

Phosphanilic acid inhibits dihydropteroate synthase.

Intact cells of Pseudomonas aeruginosa were more susceptible to phosphanilic acid (PA) than cells of Escherichia coli. In cell extracts, the dihydropteroate synthases of P. aeruginosa and E. coli were about equally susceptible to inhibition by PA. These results suggest that cells of P. aeruginosa are more permeable to PA than cells of E. coli. Although a weak inhibitor, PA acted on dihydropteroate synthase in the same manner as the sulfonamides with which PA is structurally related. Inhibition of E. coli by PA in a basal salts-glucose medium was prevented by p-aminobenzoic acid (pABA). However, pABA did not protect P. aeruginosa from PA under these conditions, possibly because pABA itself exhibited an inhibitory effect. PA also appeared to have a second mode of action. The mechanism was not elucidated.

The kinetics of dihydrostreptomycin uptake in Pseudomonas putida membrane vesicles: absence of inhibition by cations.

Dihydrostreptomycin was taken up in isolated cytoplasmic membrane vesicles of Pseudomonas putida by an active transport mechanism. Saturation kinetics were observed with an apparent Km and Vmax of 15 mM and 50 nmol/min/mg of protein respectively. The evidence suggested that the observed kinetics was that of the energy-dependent phase I component of dihydrostreptomycin uptake. Neither magnesium nor the polyamine, spermine, inhibited dihydrostreptomycin transport. Thus, the inhibition of aminoglycoside uptake in intact cells of Gram-negative bacteria and the increase in the minimal inhibitory concentration in the presence of multivalent cations and polyamines were interpreted to be effects that take place at the outer membrane level.

Comparison of kinetics of active tetracycline uptake and active tetracycline efflux in sensitive and plasmid RP4-containing Pseudomonas putida.

Membrane vesicles prepared from tetracycline-sensitive cells of Pseudomonas putida took up tetracycline by an active transport system with an apparent Km of 2.5 mM and a Vmax of 50 nmol min-1 mg protein-1. In contrast, resistance determinant RP4-containing P. putida had an active high-affinity efflux system for tetracycline with a Km of 2.0 to 3.54 microM and a Vmax of 0.15 nmol min-1 mg protein-1. Thus, the efflux system of tetracycline-resistant P. putida(RP4) had an average of 1,000-fold greater affinity for tetracycline than the influx system of tetracycline-sensitive cells. From these results, it is clear that a major mechanism of tetracycline resistance in RP4-containing P. putida is an active tetracycline efflux mechanism. There was also evidence for a second tetracycline efflux system with low affinity for tetracycline n P. putida(RP4). This efflux system had a Km of 0.25 mM and a Vmax of 1.45 nmol min-1 protein-1. Whether this low-affinity efflux system was also present in tetracycline-sensitive P. putida could not be discerned from these experiments.

Effect of dihydrostreptomycin on active transport in isolated bacterial membrane vesicles.

Membrane vesicles prepared from bacterial cells grown in the absence of dihydrostreptomycin but subsequently incubated in the presence of dihydrostreptomycin transported proline normally, but vesicles prepared from cells grown in media to which dihydrostreptomycin was added 30 min before harvesting had a greatly impaired ability to accumulate proline. The latter cells extruded protons normally but were unable to maintain a proton gradient as effectively as normal cells. These data indicated that metabolism was required for dihydrostreptomycin to exert an effect on the bacterial cell membrane.

Ethylenediaminetetraacetate-extractable protein-lipopolysaccharide complex of Pseudomonas aeruginosa: characterization of protein components.

Five major outer membrane proteins (D1, D2, E, G, and H1) of Pseudomonas aeruginosa, but not proteins F (porin), I (lipoprotein), and H2, were detected in high-molecular-weight protein-lipopolysaccharide complex(es) solubilized from sucrose-stabilized cells on exposure to ethylenediaminetetraacetate and tris(hydroxymethyl)aminomethane.

Inhibition, but not uncoupling, of respiratory energy coupling of three bacterial species by nitrite.

The effect of nitrite on respiratory energy coupling of three bacteria was studied in light of a recent report that nitrite acted as an uncoupling agent with Paracoccus denitrificans grown under denitrifying conditions. Our determinations of proton translocation stoichiometry of Pseudomonas putida (aerobically grown), Pseudomonas aeruginosa, and P. denitrificans (grown both aerobically and under denitrifying conditions) showed nitrite inhibition of proton-to-oxidant stoichiometry, but not uncoupling. Nitrite both reduced the H+/O ratio and decreased the rate of proton resorption. Increased proton resorption rates, characteristic of authentic uncoupling agents, were not observed. The lack of enhanced proton permeability due to nitrite was verified via passive proton permeability assays. The H+/O ratio of P. aeruginosa increased when growth conditions were changed from aerobic to denitrifying. This suggested the induction of an additional coupling site in the electron transport chain of denitrifying P. aeruginosa.

Bacterial inhibitory effects of nitrite: inhibition of active transport, but not of group translocation, and of intracellular enzymes.

Nitrite inhibited active transport of proline in Escherichia coli but not group translocation of sugar via the phosphoenolpyruvate:phosphotransferase system. These results were consistent with previous results that nitrite inhibits active transport, oxygen uptake, and oxidative phosphorylation in aerobic bacteria. Nitrite also inhibited aldolase (EC 4.1.2.13) from E. coli, Pseudomonas aeruginosa, Streptococcus faecalis, and rabbit muscle. Thus, these various data showed that nitrite has more than one site of attack in the bacterial cell. These data also indicated that nitrite is inhibitory to a wide range of physiological types of bacteria.

The effect of phenazine methosulfate-ascorbate on bacterial active transport and adenosine triphosphate formation: inhibition of Pseudomonas aeruginosa and stimulation of Escherichia coli.

The artificial electron-donor system, phenazine methosulfate (PMS) ascorbate, inhibited active transport of glucose by Pseudomonas aeruginosa irrespective of whether the incubation systems were in air, flushed with oxygen, or gassed with nitrogen under anaerobic denitrifying conditions. Active transport of glucose by P. aeruginosa was also inhibited by reduced 5-N-methyl-phenazonium-3-sulfonate, a membrane-impermeable electron donor. PMS-ascorbate caused rapid depletion of intracellular adenosine triphosphate (ATP) when added to respiring cell suspensions of P. aeruginosa either in the presence or absence of glucose or succinate as oxidizable energy sources. In contrast, under identical conditions, Escherichia coli formed ATP with PMS-ascorbate as the sole oxidizable energy source and ATP formation continued when glucose or succinate was present in addition to PMS-ascorbate in the incubation system.

Denitrifying Pseudomonas aeruginosa: some parameters of growth and active transport.

Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying conditions was obtained with 100 mM nitrate as the terminal electron acceptor, irrespective of the medium used. Nitrite as the terminal electron acceptor supported poor denitrifying growth when concentrations of less than 15 mM, but not higher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accumulated in the medium during early exponential phase when nitrate was the terminal electron acceptor and then decreased to extinction before midexponential phase. The maximal rate of glucose and gluconate transport was supported by 1 mM nitrate or nitrite as the terminal electron acceptor under anaerobic conditions. The transport rate was greater with nitrate than with nitrite as the terminal electron acceptor, but the greatest transport rate was observed under aerobic conditions with oxygen as the terminal electron acceptor. When P. aeruginosa was inoculated into a denitrifying environment, nitrate reductase was detected after 3 h of incubation, nitrite reductase was detected after another 4 h of incubation, and maximal nitrate and nitrite reductase activities peaked together during midexponential phase. The latter coincided with maximal glucose transport activity.

A cytochrome b-like pigment with a peak at 567 nm in Pseudomonas aeruginosa.

A cytochrome b - like pigment with an absorption peak at 567 nm was detected in Pseudomonas aeruginosa irrespective of whether the organism was grown aerobically or anaerobically under denitrifying conditions. This pigment has not been reported previously for P. aeruginosa but it has been detected in other denitrifying bacteria including closely related Pseudomonas species.

The inhibitory effect of the artificial electron donor system, phenazine methosulfate-ascorbate, on bacterial transport mechanisms.

The artificial electron donor system, phenazine methosulfate (PMS)-ascorbate, inhibited active transort of solutes in Pseudomonas aeruginosa irrespective of whether the active transport systems were shock sensitive or shock resistant. N,N,N',N'-tetramethylphenylenediamine could be substituted for PMS but a higher concentration was required. PMS-ascorbate also inhibited active transport in several other bacterial species with the exception of Escherichia coli and of a nonpigmented strain of Serratia marcescens. PMS-ascorbate previously has been shown to energize active transport in isolated membrane vesicles, even those prepared from the same bacterial species in whose intact cells active transport was inhibited. The apparent Km of glucose active transport in untreated cells of P. aeruginosa was 40 micron while the Km of glucose transport in cells incubated with PMS-ascorbate was 25 mM, and PMS-ascorbate had no effect on efflux of accumulated glucose. These results strongly suggested that facilitated diffusion resulted upon exposure of the cells to PMS- ascorbate. Thus, PMS-ascorbate appeared to have an uncoupler-like effect on cells of P. aeruginosa. The experimental data also pointed out that there are fundamental differences between the response of intact cells and membrane vesicles to exogenous electron donors.

A model system for studying protein-lipopolysaccharide synthesis, assembly, and insertion in the outer membrane of Pseudomonas aeruginosa.

Ethylenediaminetetraacetate (EDTA) has been previously shown to cause the release of a complex of protein and lipoplysaccharide (PrLPS) from the outer membrane of Pseudomonas aeruginosa. In this present work, cells of P. aeruginosa were incubated in a solution of EDTA, phosphate buffer, and hypertonic sucrose to prepare osmoplasts (osmotically sensitive cells). Osmoplasts were able to undergo self-repair of the outer-membrane damage resulting from EDTA treatment and to regain osmotic stability when incubated in growth medium/hypertonic sucrose for 2 h in the absence of exogenous PrLPS and in the absence of cell division. This repair process was inhibited by either chloramphenicol or KCN. Examination of freeze-etched preparations demonstrated that 40% of the PrLPS units in the outer membrane were removed by EDTA; after 2 h in growth medium/hypertonic sucrose, cells were able to repair this damage by synthesizing and inserting new PrLPS units into the outer membrane. Osmoplasts could also be restored to osmotic stability by suspension in osmoplast supernatant fluid containing PrLPS and Mg2+. This latter restoration process, which was not inhibited by chloramphenicol or KCN, was purely physical while the self-repair restoration process was metabolic. These data are consistent with the concept that the outer membrane proteins, especially the PrLPS units play a role in stabilizing the cell envelope and in maintaining the osmotic stability of P. aeruginosa.

Effect of sodium on the transport and utilization of citric acid by Aerobacter (Enterobacter) aerogenes.

Sodium inhibited citrate uptake by two of the four strains of Aerobacter (Enterobacter) aerogenes used in these studies, had no effect on one strain, and stimulated citrate uptake by one strain. Two of the four strains grew well anaerobically on citrate in the presence of Na(+), one grew poorly, and one grew not at all either in the presence or absence of Na(+). Na(+) stimulated the aerobic growth of one strain on citrate, increased the total growth but not the rate of growth of one strain, and prolonged the lag phase but not the rate of growth or total growth of two strains. The experimental data reported herein, therefore, indicate that there are appreciable physiological differences among strains of A. aerogenes.

Transport of glucose, gluconate, and methyl alpha-D-glucoside by Pseudomonas aeruginosa.

Glucose transport by Pseudomonas aeruginosa was studied. These studies were enhanced by the use of a mutant, strain PAO 57, which was unable to grow on glucose but which formed the inducible glucose transport system when grown in media containing glucose or other inducers such as 2-deoxy-d-glucose. Both PAO 57 and parental strain PAO transported glucose with an apparent K(m) of 7 muM. Free glucose was concentrated intracellularly by P. aeruginosa PAO 57 over 200-fold above the external level. These data constitute direct evidence that glucose is transported via active transport by P. aeruginosa. Various experimental data clearly indicated that P. aeruginosa PAO transported methyl alpha-d-glucose (alpha-MeGlc) via the glucose transport system. The apparent K(m) of alpha-MeGlc transport was 7 mM which indicated a 1,000-fold lower affinity of the glucose transport system for alpha-MeGlc than for glucose. While only unchanged alpha-MeGlc was detected intracellularly in P. aeruginosa, alpha-MeGlc was actually concentrated intracellularly less than 2-fold over the external level. Membrane vesicles of P. aeruginosa PAO retained transport activity for gluconate. This solute was concentrated intravesicularly several-fold over the external level. A component of the glucose transport system is believed to have been lost during vesicle preparation since glucose per se was not transported. Instead; glucose was converted to gluconate by membrane-associated glucose dehydrogenase and gluconate was then transported into the vesicles. Although this may constitute an alternate system for glucose transport, it is not a necessary prerequisite for glucose transport by intact cells since P. aeruginosa PAO 57, which lacks glucose dehydrogenase, was able to transport glucose at a rate equal to the parental strain.

Ultrastructural and chemical alteration of the cell envelope of Pseudomonas aeruginosa, associated with resistance to ethylenediaminetetraacetate resulting from growth in a Mg2+-deficient medium.

Cells of Pseudomonas aeruginosa became resistant to the lytic effect of ethylenediametetraacetate (EDTA) when grown in a Mg(2+)-deficient medium. To correlate ultrastructural changes in the cell wall associated with the shift to EDTA-resistance, a freeze-etch study was performed. Upon fracturing, the outer cell wall membrane split down the hydrophobic center to reveal the outer (concave) and inner (convex) layers. The concave cell wall layer of EDTA-sensitive cells grown in Mg(2+)-sufficient medium contained spherical units resting on an underlying smooth support layer. Upon EDTA treatment, approximately one-half of these spherical units were extracted. Cells grown in Mg(2+)-deficient medium were resistant to EDTA. The concave cell wall layer of EDTA-resistant cells had increased numbers of highly compacted spherical units, giving this layer a disorganized appearance. The highly compacted appearance of this layer was unaltered by EDTA treatment. Thus, growth in Mg(2+)-deficient medium resulted in cells which were resistant to EDTA and which possessed an ultrastructurally altered outer layer of the outer cell wall membrane. Cell envelopes from EDTA-resistant cells were found to possess 18% less phosphorus, 16.4% more total carbohydrate, and 13.3% more 2-keto-3-deoxyoctonate than cell envelopes from EDTA-sensitive cells. There were also qualitative, but not quantitative, differences in the protein content of cell envelopes from EDTA-resistant and EDTA-sensitive cells.