Sodium-dependent transport of L-leucine in membrane vesicles prepared from Pseudomonas aeruginosa.

Membrane vesicles were prepared by osmotic lysis of spheroplasts of Pseudomonas aeruginosa strain P14, and the active transport of amino acids was studied. D-Glucose, gluconate, and L-malate supported active transport of various L-amino acids. The respiration-dependent leucine transport was markedly stimulated by Na+. Moreover, without any respiratory substrate, leucine was also transported transiently by the addition of Na+ alone. This transient uptake of leucine was not inhibited either by carbonyl cyanide p-trifluoromethyoxyphenylhydrazone or by valinomycin, but was completely abolished by gramicidin D. Increase in the concentration of Na+ of the medium resulted in a decrease of the Km for L-leucine transport, whereas the Vmax was not significnatly affected. Active transport of leucine was inhibited competitively by isoleucine or by valine, whose transport was also stimulated by Na+. On the other hand, Na+ was not required for the uptake of other L-amino acids tested, but rather was inhibitory for some of them. These results show (i) that a common transport system for branched-chain amino acids exists in membrane vesicles, (ii) that the system requires Na+ for its activity, and (iii) that an Na+ gradient can drive the system.     

Characterization of phospholipids in membrane vesicles derived from Pseudomonas aeruginosa

Many Gram-negative bacteria release membrane vesicles (MVs), but their phospholipid properties are poorly understood. Phosphatidylglycerol was present at high levels in MVs derived from Pseudomonas aeruginosa, but not in the cellular outer membrane. The ratio of stearic acid in MVs was high compared to that in the cellular outer membrane. These findings suggest that membrane rigidity is associated with MV biogenesis.     

Virulence factors are released from Pseudomonas aeruginosa in association with membrane vesicles during normal growth and exposure to gentamicin: a novel mechanism of enzyme secretion.

Pseudomonas aeruginosa blebs-off membrane vesicles (MVs) into culture medium during normal growth. Release of these vesicles increased approximately threefold after exposure of the organism to four times the MIC of gentamicin. Natural and gentamicin-induced membrane vesicles (n-MVs and g-MVs and g-MVs, respectively) were isolated by filtration and differential centrifugation, and several of their biological activities were characterized. Electron microscopy of both n-MVs and g-MVs revealed that they were spherical bilayer MVs with a diameter of 50 to 150 nm. Immunoelectron microscopy and Western blot (immunoblot) analysis of the vesicles demonstrated the presence of B-band lipopolysaccharide (LPS), with a slightly higher proportion of B-band LPS in g-MVs than in n-MVs. A-band LPS was occasionally detected in g-MVs but not in n-MVs. In addition to LPS, several enzymes, such as phospholipase C, protease, hemolysin, and alkaline phosphatase, which are known to contribute to the pathogenicity of Pseudomonas infections were found to be present in both vesicle types. Both types of vesicles contained DNA, with a significantly higher content in g-MVs. These vesicles could thus play an important role in genetic transformation and disease by serving as a transport vehicle for DNA and virulence factors and are presumably involved in septic shock.     

htpR-like gene controls cell division and proteolysis in Pseudomonas aeruginosa

As shown in hybridization experiments, the genome of Pseudomonas aeruginosa cells contains a htpR-like gene which controls the expression of heat shock genes in cells of Escherichia coli. By means of specially constructed plasmids, the synthesis of htpR antisense RNA has been found to disturb cell division and proteolytic processes in P. aeruginosa, suggesting the functional relationship of htpR genes in E. coli and Pseudomonas bacteria.     

Cell division in Pseudomonas aeruginosa: participation of alkaline phosphatase.

Pseudomonas aeruginosa grows at an apparent reduced rate at 46 C as compared with the rate at 37 C, when growth is measured as an increase in absorbance. Cells at 46 C are long, plasmolyzed, nonmotile filaments. The filaments contain phase-dark material that may be chromosomal in nature. When the 46 C culture is shifted to 37 C, the filaments fragment at polar ends after flagella form, and the final number of cells is equal to the number of chromosomal "packets" observed within the filament. The outer envelope of the filament appears to be structurally complete as determined by biochemical, thin section, and freeze-etch examination. When filaments are treated with lysozyme, they form large spheroplasts, suggesting that the outer wall and the cytoplasmic membrane are continuous within the filament. Filaments produce little or no periplasm-located alkaline phosphatase (APase), but activity appears immediately after a shift to 37 C. Cells grown at 37 C and shifted to 46 C remain as single, nonmotile, rods or doublets, and the APase formed at 37 C remains stable at 46 C. The addition of APase or inorganic phosphate is partially or completely effective as an inducer of filament fragmentation at 46 C. The results suggest that periplasm-located APase is an important enzyme in the final stages of cell division when P. aeruginosa is cultured on inorganic phosphate-limiting media.     

Induction of cell division: role of cell membrane sites

Density dependent inhibition of cell replication is released in stationary cultures of BALB/c-3T3 fibroblasts by the potent tumor promoter for mouse skin, phorbol myristate acetate (PMA). The saturation density of these cultures, which is dependent on the serum concentration of the medium, is increased by PMA. Cell division, which can be induced by PMA or serum in stationary monolayers of BALB/c-3T3 cells, is not blocked by several inhibitors of proteolytic enzymes or the plant lectins, concanavalin A or wheat germ agglutinin. Induction of cell replication by PMA or serum does not appear to be dependent on proteolytic activity, and the membrane sites associated with this induction appear to be distinct from the agglutinin-binding sites.     

Kinetics of Pseudomonas aeruginosa adhesion to 304 and 316-L stainless steel: role of cell surface hydrophobicity.

Fifteen different isolates of Pseudomonas aeruginosa were used to study the kinetics of adhesion to 304 and 316-L stainless steel. Stainless steel plates were incubated with approximately 1.5 X 10(7) CFU/ml in 0.01 M phosphate-buffered saline (pH 7.4). After the plates were rinsed with the buffer, the number of adhering bacteria was determined by a bioluminescence assay. Measurable adhesion, even to the electropolished surfaces, occurred within 30 s. Bacterial cell surface hydrophobicity, as determined by the bacterial adherence to hydrocarbons test and the contact angle measurement test, was the major parameter influencing the adhesion rate constant for the first 30 min of adhesion. A parabolic relationship between the CAM values and the logarithm of the adhesion rate constants (In k) was established. No correlation between either the salt aggregation or the improved salt aggregation values and the bacterial adhesion rate constants could be found. Since there was no significant correlation between the bacterial electrophoretic mobilities and the In k values, the bacterial cell surface charge seemed of minor importance in the process of adhesion of P. aeruginosa to 304 and 316-L stainless steel.     

Effect of growth conditions on cell surface hydrophobicity of multiresistant Pseudomonas aeruginosa strains

Bacterial cell surface hydrophobicity (CSH) may promote colonization. The aim of this study was evaluation of the influence of growth conditions and sensitivity to selected antibiotics on hydrophobic properties of multiresistant P. aeruginosa strains by means of salt aggregation test (SAT) and bacterial adhesion to hydrocarbons (BATH). 30 multiresistant P. aeruginosa strains were included in this study. The variables were: microbiological media type (trypticase-soy agar, trypticase-soy agar with 5% sheep blood and trypticase-soy broth), growth temperature (22, 30 and 37 degrees C) and growth time (24 and 48 h). Most of the investigated strains presented hydrophilic properties in both methods. Cultivation in trypticase-soy broth caused statistically relevant decrease of CSH. Growth temperature did not influence CSH. 48 hours of incubation caused statistically relevant drop of the CSH when compared with 24 h incubation. The sensitivity to selected antibiotics did not vary investigated strains, except form cefepime sensitive and intermediate sensitive strains.     

Pyocin R1 inhibits active transport in Pseudomonas aeruginosa and depolarizes membrane potential.

Pyocin R1, a bacteriocin of Pseudomonas aeruginosa, inhibited active transport of proline in the presence of high concentrations of malate and magnesium salt. Pyocin R1 did not affect the respiration of sensitive cells nor induce cell lysis, but it caused a decrease in the intracellular ATP level. In addition, a passive inflow of [14C]thiocyanate anion, a probe of membrane potential, was induced by pyocin R1, showing a depolarization of the cytoplasmic membrane. It is considered that membrane depolarization is a primary action of pyocin R1.     

Replicating vesicles as models of primitive cell growth and division

Primitive cells, lacking the complex bio-machinery present in modern cells, would have had to rely on the self-organizing properties of their components and on interactions with their environment to achieve basic cellular functions such as growth and division. Many bilayer-membrane vesicles, depending on their composition and environment, can exhibit complex morphological changes such as growth, fusion, fission, budding, internal vesicle assembly and vesicle-surface interactions. The rich dynamic properties of these vesicles provide interesting models of how primitive cellular replication might have occurred in response to purely physical and chemical forces.     

A novel technique for the preparation of transport-active membrane vesicles from Pseudomonas aeruginosa: observations on gluconate transport

Membrane vesicles were prepared from glucose-grown $\text{Pseudomonas aeruginosa}$ by osmotic lysis of cells treated with LiCl and lysozyme. These vesicles accumulated gluconate by coupling active transport with electron flow via FAD-linked l-malate dehydrogenase or d-glucose dehydrogenase. Glucose was not transported as the free sugar; instead, it was first oxidized to gluconate which was then transported by the gluconate transport system. Evidence was presented that suggested that a component(s) of the glucose transport system was lost during vesicle preparation.     

Bacteriolytic effect of membrane vesicles from Pseudomonas aeruginosa on other bacteria including pathogens: conceptually new antibiotics.

Pseudomonas aeruginosa releases membrane vesicles (MVs) filled with periplasmic components during normal growth, and the quantity of these vesicles can be increased by brief exposure to gentamicin. Natural and gentamicin-induced membrane vesicles (n-MVs and g-MVs, respectively) are subtly different from one another, but both contain several important virulence factors, including hydrolytic enzyme factors (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995). Peptidoglycan hydrolases (autolysins) were detected in both MV types, especially a periplasmic 26-kDa autolysin whose expression has been related to growth phase (Z. Li, A. J. Clarke, and T. J. Beveridge, J. Bacteriol. 178:2479-2488, 1996). g-MVs possessed slightly higher autolysin activity and, at the same time, small quantities of gentamicin. Both MV types hydrolyzed isolated gram-positive and gram-negative murein sacculi and were also capable of hydrolyzing several glycyl peptides. Because the MVs were bilayered, they readily fused with the outer membrane of gram-negative bacteria. They also adhered to the cell wall of gram-positive bacteria. g-MVs were more effective in lysing other bacteria because, in addition to the autolysins, they also contained small amounts of gentamicin. The bactericidal activity was 2.5 times the MIC of gentamicin, which demonstrates the synergistic effect of the antibiotic with the autolysins. n-MVs were capable of killing cultures of P. aeruginosa with permeability resistance against gentamicin, indicating that the fusion of n-MV to the outer membrane liberated autolysins into the periplasm, where they degraded the peptidoglycan and lysed the cells. g-MVs had even greater killing power since they liberated both gentamicin and autolysins into these resistant cells. These findings may help develop a conceptually new group of antibiotics designed to be effective against hard-to-kill bacteria.     

The cell wall amidase AmiB is essential for Pseudomonas aeruginosa cell division,drug resistance and viability

The physiological function of cell wall amidases has been investigated in several proteobacterial species. In all cases, they have been implicated in the cleavage of cell wall material synthesized by the cytokinetic ring. Although typically non‐essential, this activity is critical for daughter cell separation and outer membrane invagination during division. In Escherichia coli, proteins with LytM domains also participate in cell separation by stimulating amidase activity. Here, we investigated the function of amidases and LytM proteins in the opportunistic pathogen Pseudomonas aeruginosa. In agreement with studies in other organisms, ^PaAmiB and three LytM proteins were found to play crucial roles in P. aeruginosa cell separation, envelope integrity and antibiotic resistance. Importantly, the phenotype of amidase‐defective P. aeruginosa cells also differed in informative ways from the E. coli paradigm; ^PaAmiB was found to be essential for viability and the successful completion of cell constriction. Our results thus reveal a key role for amidase activity in cytokinetic ring contraction. Furthermore, we show that the essential function of ^PaAmiB can be bypassed in mutants activated for a Cpx‐like envelope stress response, suggesting that this signaling system may elicit the repair of division machinery defects in addition to general envelope damage.     

PilO of Pseudomonas aeruginosa 1244: subcellular location and domain assignment

PilO of Pseudomonas aeruginosa 1244 catalyses the attachment of an O-antigen repeating unit to the beta-carbon of the pilin C-terminal residue, a serine. The present study was conducted to locate the regions of this enzyme important in catalysis and to establish the cellular location of the pilin glycosylation reaction. While PilO was not detectable in extracts of P. aeruginosa or Escherichia coli, even under conditions of overexpression, it was found that an intact MalE-PilO fusion protein was produced in significant amounts. This fusion complemented a P. aeruginosa 1244 mutant containing a pilO deletion and targeted to the cytoplasmic membrane of E. coli. Wzy and WaaL, enzymes that also utilize the O-antigen repeating unit as substrate, were found to share a sequence pattern with PilO even though these proteins have little overall sequence similarity. PilO constructs in which portions of this common sequence were deleted or altered by site-directed mutagenesis lacked pilin glycosylating activity. Deletions of segments downstream from the common region also prevented enzyme activity. Topology studies showed that the two PilO regions associated with enzyme activity were located in the periplasm. These results establish regions of this enzyme important for catalysis and present evidence that pilin glycosylation occurs in the periplasmic space of this organism.     

Escherichia coli intracellular pH, membrane potential, and cell growth.

We studied the changes in various cell functions during the shift to alkaline extracellular pH in wild-type Escherichia coli and in strain DZ3, a mutant defective in pH homeostasis. A rapid increase in membrane potential (delta psi) was detected in both the wild type and the mutant immediately upon the shift, when both cell types failed to control intracellular pH. Upon reestablishment of intracellular pH - extracellular pH and growth in the wild type, delta psi decreased to a new steady-state value. The electrochemical proton gradient (delta muH+) was similar in magnitude to that observed before the pH shift. In the mutant DZ3, delta psi remained elevated, and even though delta muH+ was higher than in the wild type, growth was impaired. Cessation of growth in the mutant is not a result of cell death. Hence, the mutant affords an interesting system to explore the intracellular-pH-sensitive steps that arrest growth without affecting viability. In addition to delta muH+, we measured respiration rates, protein synthesis, cell viability, induction of beta-galactosidase, DNA synthesis, and cell elongation upon failure of pH homeostasis. Cell division was the only function arrested after the shift in extracellular pH. The cells formed long chains with no increase in colony-forming capacity.     

Interaction between hyaluronate and the cell surface of Pseudomonas aeruginosa

Abstract Treatment of Pseudomonas aeruginosa cells with the non-metabolizable polysaccharide hyaluronate led to a strong increase in extracellular lipase activity. Alteration of the cell surface either by treatment with the chelator EDTA or by selecting for phage-resistant mutants significantly altered the bacterial response to hyaluronate. Binding of ¹⁴C-labeled hyaluronate to the bacteria was shown to depend on polysaccharide concentration and on cell number. Cell-free exolipase interacted with chemically cross-linked hyaluronate. The results suggested an interaction between hyaluronate and the cell surface of P. aeruginosa as a prerequisite for the polysaccharide to be effective.     

Conservation of surface epitopes in Pseudomonas aeruginosa outer membrane porin protein OprF

Abstract The outer membrane proteins of several prominent bacterial pathogens demonstrate substantial variation in their surface antigenic epitopes. To determine if this was also true for Pseudomonas aeruginosa outer membraine protein OprF, gene sequencing of a serotype 5 isolate was performed to permit comparison with the published serotype 12 oprF gene sequence. Only 16 nucleotide substitutions in the 1053 nucleotide coding region were observed; none of these changed the amino acid sequence. A panel of 10 monoclonal antibodies (mAbs) reacted with each of 46 P. aeruginosa strains representing all 17 serotype strains, 12 clinical isolates, 15 environmental isolates and 2 laboratory isolates. Between two and eight of these mAbs also reacted with proteins from representatives of the rRNA homology group I of the Pseudomonadaceae . Nine of the ten mAbs recognized surface antigenic epitopes as determined by indirect immunofluorescence techniques and their ability to opsonize P. aeuroginosa for phagocytosis. These epitopes were partially masked by lipopolysacharide side chains as revealed using a side chain-deficient mutant. It is concluded that OprF is a highly conserved protein with several conserved surface antigenic epitopes.