Gentamicin-lipopolysaccharide interactions inPseudomonas aeruginosa

Gentamicin was found to bind toPseudomonas aeruginosa lipopolysaccharide. The interaction was partially ionic in nature and probably involved the core region of the lipopolysaccharide molecule. A comparison of phenol-extracted lipopolysaccharide derived fromP. aeruginosa ATCC 9027 and a lipopolysaccharide “core”-defective strain indicated that changes in this region of the molecule were responsible for gentamicin-biding differences. Lysozyme and magnesium ion were both capable of disrupting this interaction. The nature of the interaction appeared to be related to the amount of organic phosphate associated with the lipopolysaccharide and may play a role in the barrier function of the cell envelope.     

Respiration and cyanogenesis inPseudomonas aeruginosa

The respiratory ability of batch cultures ofPseudomonas aeruginosa strain 9-D2 peaks during midlog phase at 3.8 nmol O₂/min/10⁸ cells. This ability declines in late log phase, just prior to the time the culture begins to produce cyanide. The respiration of this organism is particularly sensitive to cyanide inhibition during midlog-phase growth, but is extremely resistant to this compound in stationary phase. These inhibition patterns are biphasic for each of these situations and indicate several respiratory responses to HCN. Addition of cyanide to midlog-phase cells resulted in the production of a stationary-phase type of cyanide respiration pattern in 2 h. A non-cyanideproducing mutant of this organism produced significantly less of the cyanide-resistant respiration components.     

Variability in Pseudomonas aeruginosa Lipopolysaccharide Expression during Crude Oil Degradation

Bacterial utilization of crude oil components, such as the n-alkanes, requires complex cell surface adaptation to allow adherence to oil. To better understand microbial cell surface adaptation to growth on crude oil, the cell surface characteristics of two Pseudomonas aeruginosa strains, U1 and U3, both isolated from the same crude oil-degrading microbial community enriched on Bonny Light crude oil (BLC), were compared. Analysis of growth rates demonstrated an increased lag time for U1 cells compared to U3 cells. Amendment with EDTA inhibited U1 and U3 growth and degradation of the n-alkane component of BLC, suggesting a link between cell surface structure and crude oil degradation. U1 cells demonstrated a smooth-to-rough colony morphology transition when grown on BLC, while U3 cells exhibited rough colony morphology at the outset. Combining high-resolution atomic force microscopy of the cell surface and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracted lipopolysaccharides (LPS), we demonstrate that isolates grown on BLC have reduced O-antigen expression compared with that of glucose-grown cells. The loss of O-antigen resulted in shorter LPS molecules, increased cell surface hydrophobicity, and increased n-alkane degradation.     

Transport of inorganic phosphate inPseudomonas aeruginosa

Inorganic phosphate transport by wild-typePseudomonas aeruginosa cells grown in a phosphate-limited medium involves a biphasic process. The uptake obeys Michaelis-Menten kinetics with respective apparentK _m values of 1.1 M and 10 M for the high- and low-affinity systems. These systems may be also differentiated by their sensitivity to osmotic shock, by their specificity towards phosphite, pyrophosphate, arsenate, and some phosphonates and also by their energy requirements. The two phosphate transport systems fromP. aeruginosa are compared with the two major systems (Pst and Pit) characterized inEscherichia coli.     

Regulation of urea uptake inPseudomonas aeruginosa

The energy-dependent urea permease was studied in two strains ofPseudomonas aeruginosa, measuring the uptake (transport and metabolism) of¹⁴C-urea. In both strains urea uptakein vivo and urease activityin vitro differed significantly with respect to kinetic parameters, temperature and pH dependence and response to metabolic inhibitors. Ammonium strongly interfered both with the expression of the urea uptake system and its activity. The inhibition of the uptake activity by ammonium was partially relieved by hydraziniumsulfate, which prevented the translocation of ammonium into the cell, and in a methylammonium/ammonium transport-defective mutant of strain DSM 50071. Furthermore, methionine-sulfoximine, which prevented the intracellular glutamine formation from ammoniumvia inhibition of glutamine synthetase, relieved the inhibition of urea uptake by ammonium. These findings suggested that urea uptake activity inP. aeruginosa is regulated by intracellular glutamine.Abbreviations CCCP carbonylcyanide-m-chlorphenylhydrazone - DCCD dicyclohexylcarbodiimide - GS glutamine synthetase - MSX methionine-sulfoximine     

Autolytic nature of iridescent lysis inPseudomonas aeruginosa

Attempts to demonstrate a filterable agent to be the cause of iridescent lysis inPseudomonas aeruginosa were uniformly negative. It was not possible to transmit the principle by needle transfer from iridescent plaques to non-iridescent cultures, and plaques produced byP. aeruginosa bacteriophages were never iridescent. Iridescent lysis and bacteriophage lysis were subjected to antibiotics, anti-metabolites, agar at different pH values, antisera to bacteriophage-lysed and to iridescent-lysed bacteria, different oxygen concentrations, and to different nutritional sources. Certain antibiotics, notably tetracycline, streptomycin and polymyxin inducedde novo or enhanced formation of metallic lysis in nutrient agar surface cultures ofP. aeruginosa. Bacteriophage was not induced. Antimetabolites of amino acids, carbohydrates and vitamins inhibited iridescence, or inhibited it at high concentrations and enhanced it at low concentrations. Bacteriophage action was unaffected. Metallic lysis was completely inhibited at pH 6.0; it was inhibited on media containing dye or bile salt and at lowered oxygen concentrations. Bacteriophage action was not affected under these conditions. Antisera to iridescent lysates and to bacteriophage lysates ofP. aeruginosa were tested. Phage antiserum strongly neutralized phage lysis but had no effect on iridescent lysis; antisera to iridescent lysates had no effect on either. No evidence for phage mediation of iridescent lysis was seen in any of the experiments. Iridescent lysis ofP. aeruginosa was demonstrated to be based on metabolic autolysis.     

R-bodies inPseudomonas aeruginosa strain 44T1

For the first time R-bodies are described in a new strain 44T1 ofPseudomonas aeruginosa. Its size was measured as being 0.22 to 0.37 m of width per 0.27 to 0.41 m of length and 5 to 9 spiral turns about 16 nm. These structures are similar to previously observed in bacteria and are related with physiological state of bacteria in minimal conditions of growth.     

Induction and selection of formaldehyde-based resistance inPseudomonas aeruginosa

Summary A formaldehyde resistant (R) phenotype ofPseudomonas aeruginosa was isolated from a formaldehydesensitive (S) parent by sequential treatment with 1,3,5-tris-(ethyl)hexahydro-s-triazine (ET). The resistance of the (R) strain to treatment with ET was approximately 3-fold higher than the parental (S) strain. Two modes of resistance to ET, and simultaneous resistance to formaldehyde, are demonstrated: (1) transient or induced resistance is expressed during shor-term exposure to ET, and this resistance is gradually lost during subsequent growth in the absence of ET, and (2) resistance that results from a stable phenotypic change in the (S) strain following sequential treatment with ET ((R) strain phenotype). The observed activities of three forms of the formaldehyde oxidizing enzyme, formaldehyde dehydrogenase, are strongly correlated with the relative response of the (S) and (R) strains to treatment with ET. The observed resistance of the (R) strain appears to be due to high levels of an NAD⁺-linked, glutathione-dependent form of formaldehyde dehydrogenase as well as a dye-linked formaldehyde dehydrogenase. The transient or induced response of the (R) strain involves an increase in activity of the dye-linked formaldehyde dehydrogenase. The induced response of the (S) strain and an ATCC strain ofP. aeruginosa, however, is correlated with the two forms of the NAD⁺-linked enzyme (glutathione-dependent (EC 1.2.1.1) and independent (EC 1.2.1.46)) with no contribution from the dye-linked enzyme.     

Studies on regulation of tetradecane oxidation inPseudomonas aeruginosa

Summary Using mainly the Warburg technique, the effect of intermediates of tetradecane metabolism on tetradecane oxidation inPseudomonas aeruginosa was investigated. Precultivation and simultaneous incubation of bacteria with acetate, succinate, fumarate, glycolate and malonate inhibit the oxidation of tetradecane as well as of all primary oxidation products including myristic acid in a different manner. C₂-units have a key function in the regulation of tetradecane oxidation. The inhibitory effect of tetradecane and its primary oxidation products on substrate oxidations may be unspecific.During the oxidation of tetradecane or its primary oxidation products O₂-consumption increases exponentionally but CO₂-production becomes stationary. The RQ-values obtained with myristic acid growing cells decrease to O.1, indicating that myristic acid is only partially oxidized via citric acid cycle.     

Relationship between oxygen and siderophore synthesis inPseudomonas aeruginosa

Pseudomonas aeruginosa strain PAO1, growing in low-iron medium, produces two siderophores, pyochelin and pyoverdin, in massive bursts as the culture shifts from logarithmic phase to stationary phase. Two medium components, oxygen and iron, prolonged the logarithmic phase when they were added to the medium. Oxygen and iron appeared to be in demand during this period because, as heme synthesis increased in response to the low oxygen concentration in the medium, a situation resulting from the high density of bacteria present in the medium during late log phase, the iron content of the bacteria decreased. These phenomena resulted in the production of massive amounts of siderophores late in the log phase to supply iron for the increased heme synthesis.     

Plasmid-determined resistance to arsenic and antimony inPseudomonas aeruginosa

Resistance to arsenic salts in aPseudomonas aeruginosa clinical isolate was shown to be determined by a 100 kb transferable plasmid. The resistance pattern included arsenate, arsenite, and antimonate ions. Arsenate and arsenite resistances were inducible by previous exposure of cultures to subinhibitory amounts of either of the two ions. Phosphate ions protectedP. aeruginosa cells from the toxic effects of arsenate but did not alter arsenite toxicity.     

Cell envelope impermeability to daptomycin inPseudomonas aeruginosa andPasteurella multocida

The exclusively gram-positive antibacterial spectrum of the lipopeptide daptomycin (LY146032) suggests that the underlying basis for intrinsic resistance in gram-negative organisms involves envelope impermeability. This study was undertaken to determine whether the outer membranes ofPseudomonas aeruginosa andPasteurella multocida can be rendered permeable to daptomycin by experimental modifications that result in susceptibility of gram-negative bacteria to lipophilic molecules. Turbidimetric growth assays revealed sublethal concentrations of polymyxin B or ethylenediaminetetraacetate (EDTA) sensitized all strains examined to the hydrophobic antibiotic novobiocin. Neither permeabilizer renderedPs. aeruginosa or a hydrophilicPa. multocida variant susceptible to daptomycin; however, polymyxin B sensitized a hydrophobicPa. multocida variant, whereas EDTA did not. Cells cultured with sublethal concentrations of polymyxin B or EDTA retained negatively charged cell surfaces comparable to those of control cells. Growth ofPa. multocida strains in the presence of polymyxin B did not result in modification of cell envelope lipid composition. These findings indicate that the ability of the outer membrane to retard the diffusion of daptomycin does not require normally intact structure, thereby suggesting that the residual negative charge of the cell surface may preclude interaction with the acidic antibiotic owing to electrostatic repulsion.     

Alterations inPseudomonas aeruginosa exoproducts by sub-MICs of some antibiotics

Aminoglycoside antibiotics, most effective at the level of 1/4 of the MIC, suppressed all the tested activities ofP. aeruginosa, except cytotoxicity. Proteinase activity was decreased to 60% (gentamicin) and 63% (streptomycin), permeability was reduced to 61% (gentamicin) and 73% (streptomycin), phospholipase C to 13% (gentamicin) and 51% (streptomycin) of the control values. Subinhibitory concentrations ofβ-lactams inhibited only phospholipase C activity to 89% (ticarcillin) and 64% (cefotaxim) of the control values. These antibiotics did not suppress the cytotoxic activity and increased protease activity up to 155% (ticarcillin) and 192% (cefotaxim) as well as permeability up to 121% (ticarcillin) and 154% (cefotaxim) of the control values.     

Mechanism of nitrite inhibition of cellular respiration inPseudomonas aeruginosa

One of the principal mechanisms of nitrite inhibition of cellular respiration has been considered to be the interference with the action of iron-containing enzymes. In procaryotic systems, the effect of nitrite on cellular metabolism remains unclear. This study provides evidence which shows a direct inhibition by a low concentration of nitrite on a highly purified oxidase inPseudomonas aeruginosa. The inhibition pattern was observed and was consistent at cellular, electron-transport membranous, and enzymic (oxidase) levels. This implies that the mechanism of nitrite inhibition on bacterial respiration is due to a direct inhibition at the terminal site of oxygen reduction. The uncompetitive inhibition pattern shown by nitrite strongly suggested a mechanism quite different from those of classic cytochrome oxidase inhibitors such as cyanide, azide, and carbon monoxide.     

The relationship between growth phase and cyanogenesis inPseudomonas aeruginosa

In batch cultures ofPseudomonas aeruginosa, hydrogen cyanide is produced primarily during the transition between logarithmic and stationary phases. This transient response is due to the synthesis of the enzyme system of cyanogenesis during mid to late logorithmic and the inactivation of this system in early stationary phase. Although glycine, the metabolic precursor of cyanide, stimulates cyanogenesis, it is not necessary to incorporate this amino acid in the growth medium to produce elevated enzyme levels. Under conditions of iron limitation (1×10⁻⁶ M), phosphate limitation (0.1 mM), and excess phosphate (250 mM), the culture produces low levels of the cyanogenic enzyme system. Increasing the carbon and energy source,l-glutamate, prolongs cyanogenesis and postpones the inactivation of the cyanogenic enzyme system.     

Na+(Li+)/Branched-chain amino acid cotransport inPseudomonas aeruginosa

Summary A transport system for branched-chain amino acids (designated as LIV-II system) inPseudomonas aeruginosa requires Na⁺ for its operation. Coupling cation for this system was identified by measuring cation movement during substrate entry using cation-selective electrodes. Uptakes of Na⁺ and Li^– were induced by the imposition of an inwardly-directed concentration gradient of leucine, isoleucine, or valine. No uptake of H^– was found, however, under the same conditions. In addition, effects of Na⁺ and Li⁺ on the kinetic property of the system were examined. At chloride salt concentration of 2.5mm, values of apparentK _m andV _max for leucine uptake were larger in the presence of Na⁺ than Li⁺. These results indicate that the LIV-II transport system is a Na⁺(Li⁺)/substrate cotransport system, although effects of Na⁺ and Li⁺ on kinetics of the system are different.