Terminal restlessness--its management and the role of midazolam.
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Humans↗
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K Poole
Publications and source records attributed to K Poole.
Interaction of aminoglycosides with the outer membranes and purified lipopolysaccharide and OmpF porin of Escherichia coli.
The mechanism of uptake of aminoglycosides across the outer membrane of Escherichia coli was reevaluated. Porin-deficient mutants showed no alteration in gentamicin or kanamycin susceptibility. Furthermore, the influence of kanamycin on intrinsic tryptophan fluorescence of porin OmpF (Y. Kobayashi, and T. Nakae, Eur. J. Biochem. 151:231-236, 1985) was shown to be strongly influenced by protein concentration and EDTA. This led to the hypothesis that aminoglycoside-mediated increases and decreases in intrinsic tryptophan fluorescence were due to aggregation-disaggregation of OmpF mediated by interaction at a divalent cation binding site on OmpF. Gentamicin, kanamycin, and polymyxin B increased E. coli outer membrane permeability to the hydrophobic fluorescent compound 1-N-phenyl-naphthylamine (NPN) and the peptidoglycan-degrading enzyme lysozyme. Addition of Mg2+ blocked these permeabilizing activities. Furthermore, gentamicin and polymyxin B bound to Mg(2+)-binding sites on E. coli lipopolysaccharide, as determined in dansyl polymyxin displacement experiments. A polymyxin-resistant, lipopolysaccharide-altered pmr mutant of E. coli had a fourfold-lower MIC of gentamicin and kanamycin and was more poorly permeabilized to 1-N-phenylnaphthylamine than was its parent strain. These data were consistent with uptake of aminoglycosides across the E. coli outer membrane by the self-promoted uptake mechanism.
Anti-Bacterial Agents↗
Pyochelin-mediated iron transport in Pseudomonas aeruginosa: involvement of a high-molecular-mass outer membrane protein.
An iron-regulated outer membrane protein of 75,000 daltons was strongly expressed following iron limitation of strains of Pseudomonas aeruginosa which fail to produce pyoverdine. A mutant nonderepressible for this protein (K372) was deficient in pyochelin-mediated iron transport at 150 nM FeCl3, consistent with a role for the 75-kDa protein in ferripyochelin transport. Moreover, ferripyochelin specifically protected the 75-kDa protein against trypsin digestion, supporting an interaction between ferripyochelin and the 75-kDa protein. Previous reports implicated a 14,000-dalton outer membrane protein as the receptor for ferripyochelin (P.A. Sokol and D.E. Woods, Infect. Immun. 40:665-669, 1983) and demonstrated that a mutant (FBP-28) expressing a defective 14-kDa outer membrane protein did not exhibit pyochelin-mediated iron transport (P.A. Sokol, J. Bacteriol. 169:3365-3368, 1987). Nonetheless, we were able to demonstrate (i) that FBP-28 was inducible for the 75-kDa protein under iron-limiting conditions and (ii) that concomitant with the induction of this protein in FBP-28, pyochelin-mediated iron uptake at 150 nM FeCl3 was observed. Interestingly, strain K372 did transport ferripyochelin at higher (750 nM) FeCl3 concentrations, suggesting that a second pyochelin-mediated iron transport system, perhaps involving the 14-kDa outer membrane protein identified previously, operates in P. aeruginosa.
Bacterial Outer Membrane Proteins↗
Pyoverdine-mediated iron transport in Pseudomonas aeruginosa: involvement of a high-molecular-mass outer membrane protein.
Reduced expression of an iron-regulated outer membrane protein (IROMP) of approximate molecular mass 90,000 was observed in Pseudomonas aeruginosa concomitant with a loss of pyoverdine production in a wild type strain grown at 43 degrees C and in a mutant deficient in pyoverdine production. Consistent with an implied role in pyoverdine-mediated iron transport a mutant lacking the 90 kDa protein transported barely detectable levels of ferri-pyoverdine. Interestingly, the mutant still exhibited pyoverdine-dependent growth in an iron-deficient medium containing the synthetic iron chelator ethylene diamine-di(omicron-hydroxyphenol acetic acid) (EDDHA) suggesting that a second uptake system for ferri-pyoverdine may exist in P. aeruginosa.
Bacterial Outer Membrane Proteins↗
Enterobactin-mediated iron transport in Pseudomonas aeruginosa.
A pyoverdine-deficient strain of Pseudomonas aeruginosa was unable to grow in an iron-deficient minimal medium in the presence of the nonmetabolizable iron chelator ethylene diamine-di(omega-hydroxyphenol acetic acid) (EDDHA), although addition of enterobactin to EDDHA-containing minimal media did restore growth of the pyoverdine-deficient P. aeruginosa. Consistent with the apparent ability of enterobactin to provide iron to P. aeruginosa, enterobactin-dependent 55Fe3+ uptake was observed in cells of P. aeruginosa previously grown in an iron-deficient medium containing enterobactin (or enterobactin-containing Escherichia coli culture supernatant). This uptake was energy dependent, was observable at low concentrations (60 nM) of FeCl3, and was absent in cells cultured without enterobactin. A novel protein with a molecular weight of approximately 80,000 was identified in the outer membranes of cells grown in iron-deficient minimal medium containing enterobactin, concomitant with the induction of enterobactin-dependent iron uptake. A Tn501 insertion mutant lacking this protein was isolated and shown to be deficient in enterobactin-mediated iron transport at 60 nM FeCl3, although it still exhibited enterobactin-dependent growth in iron-deficient medium containing EDDHA. It was subsequently observed that the mutant was, however, capable of enterobactin-mediated iron transport at much higher concentrations (600 nM) of FeCl3. Indeed, enterobactin-dependent iron uptake at this concentration of iron was observed in both the mutant and parent strains irrespective of whether they had been cultured in the presence of enterobactin. Apparently, at least two uptake systems for ferrienterobactin exist in P. aeruginosa: one of higher affinity which is specifically inducible by enterobactin under iron-limiting conditions and the second, of lower affinity, which is also inducible under iron-limiting conditions but is independent of enterobactin for induction.
Biological Transport↗
Iron regulation of Serratia marcescens hemolysin gene expression.
The hemolytic activity of Serratia marcescens was examined as a function of iron availability. Restriction of iron by the nonmetabolizable chelator 2,2'-dipyridyl or the iron-binding protein transferrin produced a marked increase in hemolytic activity. The hemolytic activity of S. marcescens is determined by two adjacent genes, 5'-shlB-shlA-3', where shlA encodes the hemolysin which requires the ShlB protein for activity. A gene fusion between the promoter-proximal portion of shlA and phoA, the Escherichia coli alkaline phosphatase gene, was subcloned into a medium-copy-number vector, and the recombinant plasmid was introduced into S. marcescens. The expression of shlA was measured as a function of alkaline phosphatase activity, which increased threefold under iron-restricted conditions. Removal of the 5' noncoding region upstream of shlB in the fusion vector resulted in a 10-fold decrease in alkaline phosphatase activity under iron-sufficient conditions, with no effect of iron limitation on this residual activity. This suggested that the site mediating iron regulation of shlA expression occurs upstream of shlB. Consistent with this, we observed iron-regulated synthesis of the ShlB protein in Western immunoblots of isolated outer membranes. The hemolysin determinant was subsequently expressed on a medium-copy-number vector in fur+/fur isogenic strains of E. coli K-12, where a 10-fold-higher activity was observed in the mutant strain compared with the wild type. A sequence exhibiting some homology to the Fur-binding consensus sequence was identified upstream of the shlB coding region, overlapping the -35 region of a putative promoter.
Alkaline Phosphatase↗
Influence of growth temperature and lipopolysaccharide on hemolytic activity of Serratia marcescens.
Log-phase cells of Serratia marcescens cultured at 30 degrees C were approximately 10-fold more hemolytic than those grown at 37 degrees C. By using a cloned gene fusion of the promoter-proximal part of the hemolysin gene (shlA) to the Escherichia coli alkaline phosphatase gene (phoA), hemolysin gene expression as a function of alkaline phosphatase activity was measured at 30 and 37 degrees C. No difference in alkaline phosphatase activity was observed as a function of growth temperature, although more hemolysin was detectable immunologically in whole-cell extracts of cells grown at 30 degrees C. The influence of temperature was, however, growth phase dependent, because the hemolytic activities of cells cultured to early log phase at 30 and 37 degrees C were comparable. Given the outer membrane location of the hemolysin, lipopolysaccharide (LPS) was examined as a candidate for mediating the temperature effect on hemolytic activity. Silver staining of LPS in polyacrylamide gels revealed a shift towards shorter O-antigen molecules at 37 degrees C relative to 30 degrees C. Moreover, there was less binding of O-antigen-specific bacteriophage to S. marcescens with increasing growth temperature, a finding consistent with temperature-mediated changes in LPS structure. Smooth strains of S. marcescens were 20- to 30-fold more hemolytic than rough derivatives, a result confirming that changes in LPS structure can influence hemolytic activity. The alkaline phosphatase activity of rough strains harboring the shlA-phoA fusion was threefold lower than that of smooth strains harboring the fusion plasmids, a result consistent with a decrease in hemolysin gene expression in rough strains. The absence of a similar effect of temperature on gene expression may be related to less-marked changes in LPS structure as a function of temperature compared with a smooth-to-rough mutational change.
Alkaline Phosphatase↗
Molecular characterization of the hemolysin determinant of Serratia marcescens.
The nucleotide sequence of a 7.3-kilobase-pair fragment of DNA encoding a hemolytic activity from Serratia marcescens was determined. Two large open reading frames were identified, designated shlA (Serratia hemolysin) and shlB, capable of encoding polypeptides of 165, 056 and 61,897 molecular weight, respectively. Both reading frames were expressed in vivo. The shlB gene product was localized to the outer membrane of Escherichia coli cells harboring the S. marcescens hemolysin determinant. Consistent with this location, a signallike sequence was identified at the N terminus of the polypeptide predicted from the nucleotide sequence of the shlB gene. Hyperexpression of the shlB locus permitted the identification of two shlB-encoded polypeptides of 65,000 and 62,000 molecular weight, respectively. Determination of the N-terminal amino acid sequence of the purified 62,000-molecular-weight protein confirmed that it was the mature form of the ShlB protein initially synthesized as a precursor (65,000-molecular-weight protein). By using polyclonal antisera raised against the purified proteins, ShlA and ShlB were identified in the outer membrane of S. marcescens. The shlA gene product was shown to interact with erythrocyte membranes, confirming it as the hemolysin proper. Both hemolysis and the interaction of ShlA with erythrocyte membranes did, however, require the ShlB function. Progressive deletion of the C terminus of the ShlA protein gradually reduced hemolytic activity until 37% of the amino acids had been removed. Elimination of 54% of the amino acids produced a nonhemolytic protein which, however, was still capable of associating with erythrocyte membranes.
Amino Acid Sequence↗
Phosphate-selective porins from the outer membranes of fluorescent Pseudomonas sp.
Phosphate starvation induced oligomeric proteins from the outer membranes of Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas aureofaciens, and Pseudomonas chlororaphis were purified to homogeneity. The incorporation of the purified proteins into planar lipid bilayer membranes resulted in stepwise increases in membrane conductance. Single channel conductance experiments demonstrated that these proteins were all capable of forming small channels, similar to the Pseudomonas aeruginosa phospsate porin protein P, with average single channel conductances in 1 M KCl of between 233 and 252 pS. Single channel conductance measurements made in salts of varying cation or anion size indicated that the channels were uniformly anion selective. The measurement of single channel conductance as a function of KCl concentration revealed that all channels saturated at higher salt concentrations, consistent with the presence of an anion-binding site in the channel. Apparent Kd values for Cl- binding were calculated and shown to vary only twofold (180-297 mM) among all channels, including protein P channels. Phosphate competitively inhibited chloride conductance through these channels with apparent I50 values of between 0.59 and 2.5 mM phosphate at 40 mM Cl- and between 9.7 and 27 mM phosphate at 1 m Cl-. These data were consistent with the presence of a phosphate-binding site in the channels of these phosphate-regulated proteins. Furthermore, they indicated that these channels exhibit at least a 20- to 80-fold higher affinity for phosphate than for chloride.
Bacterial Outer Membrane Proteins↗
Isolation of a Tn501 insertion mutant lacking porin protein P of Pseudomonas aeruginosa.
In order to demonstrate a role for anion-specific protein P channels in phosphate transport in Pseudomonas aeruginosa PAO, we wished to isolate a transposon insertion mutant deficient in protein P. A number of transposon delivery systems were tested which yielded, for the most part, whole plasmid inserts. Plasmid pMT1000 (Tsuda et al. 1984), a temperature-sensitive R68 plasmid carrying the transposon Tn501, was successfully employed in the isolation of a Tn501 insertion mutant lacking protein P under normally inducing conditions. To identify the mutant deficient in protein P, a protein P-specific polyclonal antiserum was used. This mutant, strain H576, was deficient in high-affinity phosphate transport exhibiting a Km for uptake (3.60 +/- 0.64 microM) almost ten times greater than that of the wild type strain (Km = 0.39 microM). There was, however, no change in the Vmax for high-affinity phosphate transport as a result of the loss of protein P in this mutant. The protein P-deficiency of the mutant correlated with a growth defect in a phosphate-limited medium, resulting in an 18%-35% decrease in growth when compared with the wild type.
Bacterial Outer Membrane Proteins↗
Lipopolysaccharide-free Escherichia coli OmpF and Pseudomonas aeruginosa protein P porins are functionally active in lipid bilayer membranes.
Escherichia coli porin OmpF and Pseudomonas aeruginosa porin protein P were eluted from sodium dodecyl sulfate-polyacrylamide gels. The resultant porin preparations were found to be devoid of detectable lipopolysaccharide (LPS) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining for LPS, direct enzyme-linked immunosorbent assays with LPS-specific monoclonal antibodies, and 2-keto-3-deoxyoctulosonic acid assays. The average conductances, ionic selectivities and incorporation rates of the electroeluted porins were identical to those of their conventionally purified counterparts. These data suggest that LPS is not required per se for porin function.
Bacterial Outer Membrane Proteins↗
Phosphate-starvation-induced outer membrane proteins of members of the families Enterobacteriaceae and Pseudomonodaceae: demonstration of immunological cross-reactivity with an antiserum specific for porin protein P of Pseudomonas aeruginosa.
Bacteria from members of the families Enterobacteriaceae and Pseudomonadaceae were grown under phosphate-deficient (0.1 to 0.2 mM Pi) conditions and examined for the production of novel membrane proteins. Of the 17 strains examined, 12 expressed a phosphate-starvation-induced outer membrane protein which was heat modifiable in that after solubilization in sodium dodecyl sulfate at low temperature the protein ran on gels as a diffuse band of higher apparent molecular weight, presumably an oligomer form, which shifted to an apparent monomer form after solubilization at high temperature. These proteins fell into two classes based on their monomer molecular weights and the detergent conditions required to release the proteins from the peptidoglycan. The first class, expressed by species of the Pseudomonas fluorescens branch of the family Pseudomonadaceae, was similar to the phosphate-starvation-inducible, channel-forming protein P of Pseudomonas aeruginosa. The second class resembled the major enterobacterial porin proteins and the phosphate-regulated PhoE protein of Escherichia coli. Using a protein P-trimer-specific polyclonal antiserum, we were able to demonstrate cross-reactivity of the oligomeric forms of both classes of these proteins on Western blots. However, this antiserum did not react with the monomeric forms of any of these proteins, including protein P monomers. With a protein P-monomer-specific antiserum, no reactivity was seen with any of the phosphate-starvation-inducible membrane proteins (in either oligomeric or monomeric form), with the exception of protein P monomers. These results suggest the presence of conserved antigenic determinants only in the native, functional proteins.
Antigens, Bacterial↗
Things our instructors never told us.
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Job Satisfaction↗