Lipopolysaccharide Variability inPseudomonas aeruginosa

The lipopolysaccharide (LPS) ofPseudomonas aeruginosa (ATCC 9027) exhibited gross changes in both the amount of LPS produced per cell and the composition of the LPS in response to changes in magnesium ion concentrations. Compositional variation in the LPS was detected under both batch and continuous culture conditions, and was particularly apparent as changes in the levels of heptose in the molecule. Compositional changes in the molecule were also reflected as functional alterations in the LPS. However, functionally, these alterations were counterbalanced by increased production of the lipopolysaccharide. We propose that LPS composition changed in accordance with environmental factors, and that cells responded to these changes by increasing or decreasing the amount of LPS produced.     

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.     

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.     

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.     

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     

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.     

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.     

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.     

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.     

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.     

Adaptive resistance to polymyxin in Pseudomonas aeruginosa due to an outer membrane impermeability mechanism

The isolated outer membrane from cells of a Pseudomonas aeruginosa strain exhibiting adaptive resistance to polymyxin was not affected by polymyxin treatment, as monitored by electron microscopy of negatively stained preparations. This was in sharp contrast with extensive disruption by polymyxin of the outer membranes of the parent polymyxin-sensitive strain and the resistant strain following reversion to greater polymyxin sensitivity. The isolated cytoplasmic membrane of the polymyxin-resistant strain, on the other hand, remained sensitive to the disruptive effects of polymyxin treatment. The permeability characteristics of the resistant strains appear to be altered, as indicated by differences in minimal inhibitory concentrations for a variety of antibiotics between the polymyxin-sensitive and polymyxin-resistant strains. No evidence was found for a polymyxin-inactivating enzyme in osmotic shock fluid from the polymyxin-resistant strain. No evidence for a cytoplasmic membrane repair mechanism was found in the polymyxin-resistant strain. These observations suggest that the mechanism of adaptive polymyxin resistance in this model system is the alteration of the outer membrane so that it excludes polymyxin from reaching the still sensitive cytoplasmic membrane.     

Pyoverdine-mediated iron transport Fate of iron and ligand inPseudomonas aeruginosa

Summary Incubated in the presence of [⁵⁵Fe]ferri[¹⁴C]pyoverdine, iron-poorPseudomonas aeruginosa accumulated more⁵⁵Fe than¹⁴C over a 60-min period. Distribution studies showed (a) more¹⁴C than⁵⁵Fe in the soluble fraction during the first 20 min, (b) approximately 60% of the⁵⁵Fe associated with the membranes at 60 min, and (c) approximately 85% of the¹⁴C in the soluble fraction at 60 min. Cells osmotically shocked after incubating with [⁵⁵Fe]ferri[¹⁴C]pyoverdine for 60 min released⁵⁵Fe but not¹⁴C, suggesting separation of metal and ligand in the periplasmic space. Whereas the mechanism of dissociation of iron and ligand is not known, the decrease in transport observed in the presence of dipyridyl suggests involvement of reduction in this process. Transport of iron was energized by the proton motive force instead of by intracellular levels of ATP. The hydrogen ion gradient was the major driving force of transport. Cyanide-poisoned cells accumulated more¹⁴C than⁵⁵Fe over 60 min. Here, iron accumulated in the soluble fraction instead of on the membranes.     

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.     

On the regulation of L-hydroxyproline dissimilatory pathway inPseudomonas aeruginosa PAO

The enzymes involved in the regulation of L-hydroxyproline degradation inPseudomonas aeruginosa PAO were investigated. L-hydroxyproline when present in the growth medium induces all the four enzymes in the pathway. Growth of the cells in L-proline also weakly induced the enzymes. The organism failed to utilize D-allo-hydroxyproline due to permeability factors. Mutants blocked in the oxidative pathway of L-hydroxyproline were isolated and enzymatically characterized. In all the mutants lacking any one of enzymes of the metabolic pathway, L-hydroxyproline is still active in inducing the remaining enzymes of the pathway suggesting that L-hydroxyproline has intrinsic inducer activity.