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Query: UMLS:C0038187 (starvation)
 24,951 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The oprO gene of Pseudomonas aeruginosa codes for a polyphosphate-specific porin and terminates 458 bp upstream of the start codon for the phosphate-specific porin OprP. OprO was found to be expressed only under phosphate-starvation conditions in both wild-type and oprP::Tn501 mutant P. aeruginosa strains. However, unlike the rest of the genes of the Pho regulon, including oprP, expression of oprO required cells to be in the stationary growth phase in addition to phosphate starvation. Wild-type P. aeruginosa cells were grown in fermentor culture under these conditions and fractionated by selective solubilization in octylpolyoxyethylene detergent solution. Solubilized OprO was separated from OprP by application to a Mono Q FPLC column and elution with a salt gradient and shown to be functionally identical to cloned OprO produced in Escherichia coli. DNA sequencing of oprO showed the gene product to be highly homologous to OprP, with 76% identity and 16% conserved substitutions. Most genes of the Pho regulon possess a modified -35 region called the Pho box. Two such elements, separated by 4 bp were found in oprO. DNA sequencing also revealed a second Pho box in the oprP gene with the same spacing.
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PMID:Polyphosphate-selective porin OprO of Pseudomonas aeruginosa: expression, purification and sequence. 140 71

Escherichia coli transports inorganic phosphate (Pi) by the low-affinity transport system, Pit. When the level of the external Pi is lower than 20 microM, another transport system, Pst, is induced with a Kt of 0.25 microM. An outer-membrane porin, PhoE, with a Km of about 1 microM is also induced. The outer membrane allows the intake of organic phosphates which are degraded to Pi by phosphatases in the periplasm. The Pi-binding protein will capture the free Pi produced in the periplasm and direct it to the transmembrane channel of the cytoplasmic membrane. The channel consists of two proteins, PstA and PstC, which have six and five transmembrane helices, respectively. On the cytoplasmic side of the membrane the channel is linked to the PstB protein, which carries a nucleotide (probably ATP)-binding site. PstB probably provides the energy required by the channel to free Pi. The Pst system has two functions in E. coli: (i) the transport of Pi, and (ii) the negative regulation of the phosphate regulon (a complex of 20 proteins mostly related to organic phosphate transport). It is remarkable that these two functions are not related, since the repressibility of the regulon depends on the integral structure of Pst (PiBP + PstA + PstC + PstB) and not on the Pi transported. Another gene of the pst operon, phoU, produces a protein involved in the negative regulation of the Pho regulon, but the mechanism of this function has not been explained. Thus the regulatory function of the Pst system remains obscure. Its basal level, present when Pi is abundant, is sufficient to repress the Pho regulon but the negative regulatory function is lost upon Pi starvation.
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PMID:Molecular aspects of phosphate transport in Escherichia coli. 170 Feb 57

The composition and antibiotic permeability barrier of the outer membrane of Serratia marcescens were assessed in cells grown in vivo and in vitro. Intraperitoneal diffusion chambers implanted in rats were used for the in vivo cultivation of bacteria. Outer membranes isolated from log-phase bacterial cells recovered from these chambers were compared with membranes isolated from cells grown in vitro. Analysis revealed that the suspected 41-kilodalton porin and the OmpA protein were recovered on sodium dodecyl sulfate-polyacrylamide gels in equal quantities. Several high-molecular-weight proteins, thought to be iron starvation induced, appeared in the diffusion chamber-grown cells. The outer membrane permeability barriers to cephaloridine were similar in in vivo- and in vitro-grown cells based on permeability coefficient calculations. The permeability coefficient of cephaloridine in S. marcescens cells (30.3 x 10(-5) to 38.9 x 10(-5) cm s-1) was greater than that obtained for an Escherichia coli strain expressing only porin OmpC but smaller than those obtained for the E. coli wild type and a strain expressing only porin OmpF. Functional characterization of the suspected porin was performed by using the planar lipid bilayer technology. The sodium dodecyl sulfate-0.4 M NaCl-soluble porin from both in vitro- and in vivo-grown cells showed an average single-channel conductance in 1 M KCl of 1.6. A partial amino acid sequence (19 residues) was obtained for the S. marcescens porin. The sequence showed a very high homology to the E. coli OmpC porin. These data identified the S. marcescens outer membrane 41-kilodalton protein as a porin by both functional and amino acid analyses. Also, the methodology used allowed for efficient growth and recovery of diffusion chamber-grown bacterial cells and permitted identification of specific in vivo-induced changes in bacterial cell membrane composition.
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PMID:Outer membrane and porin characteristics of Serratia marcescens grown in vitro and in rat intraperitoneal diffusion chambers. 215 67

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.
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PMID: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. 241 13

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.
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PMID:Phosphate-selective porins from the outer membranes of fluorescent Pseudomonas sp. 243 33

A large-scale purification scheme was developed for lipopolysaccharide-free protein P, the phosphate-starvation-inducible outer-membrane porin from Pseudomonas aeruginosa. This highly purified protein P was used to successfully form hexagonal crystals in the presence of n-octyl-beta-glucopyranoside. Amino-acid analysis indicated that protein P had a similar composition to other bacterial outer membrane proteins, containing a high percentage (50%) of hydrophilic residues. The amino-terminal sequence of this protein, although not homologous to either outer membrane protein, PhoE or OmpF, of Escherichia coli, was found to have an analogous protein-folding pattern. Protein P in the native trimer form was capable of maintaining a stable functional trimer after proteinase cleavage. This suggested the existence of a strongly associated tertiary and quaternary structure. Circular dichroism studies confirmed these results in that a large proportion of the protein structure was determined to be beta-sheet and resistant to acid pH and heating in 0.1% sodium dodecyl sulphate.
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PMID:Large-scale purification and biochemical characterization of crystallization-grade porin protein P from Pseudomonas aeruginosa. 245 38

Plasmids pPBP and pRS-XP containing the cloned genes for the Pseudomonas aeruginosa phosphate-starvation-inducible periplasmic phosphate-binding protein and outer membrane porin P (oprP), respectively, were introduced into various Escherichia coli Pho-regulon regulatory mutants. Using Western immunoblots and specific antisera, the production of both gene products was observed to be under the control of regulatory elements of the E. coli Pho regulon. Sequencing of the region upstream of the translational start site of the oprP gene revealed a 'Pho box' with strong homology to the E. coli consensus 'Pho box', the putative binding site of the PhoB activator. Since P. aeruginosa and E. coli belong to different families and have quite different GC contents, these data suggest strong evolutionary conservation of regulatory elements of the Pho regulon.
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PMID:Regulation of components of the Pseudomonas aeruginosa phosphate-starvation-inducible regulon in Escherichia coli. 245 46

Lipid bilayer experiments were performed with one OmpF-PhoE and several OmpC-PhoE hybrid porins of Escherichia coli K-12. All hybrid pores had approximately the same pore-forming activity, which indicated that the structure of the pores remained essentially unchanged by the genetic manipulation. This result was supported by single-channel experiments because all pores had similar single-channel conductances in potassium chloride. Measurements with other salts indicated a drastic change in the ionic selectivity when the fusion site in the ompC-phoE hybrid genes passed along the sequence of the porins from the N-terminal to the C-terminal end. Selectivity measurements using zero-current membrane potentials showed that the selectivity suddenly changed from anion to cation selectivity when a relatively short portion from the N-terminal end of PhoE was replaced by the corresponding part of OmpC. The replacement of increasing portions led to an increase in the cation selectivity until that of OmpC was reached. The change in the anion to cation selectivity is correlated with exchange of lysine-18 and serine-28 by aspartic acids. The anion selectivity of the phosphate starvation-inducible PhoE porin is closely related to the presence of several lysines spread along the primary sequence of the polypeptide chain.
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PMID:Molecular basis of porin selectivity: membrane experiments with OmpC-PhoE and OmpF-PhoE hybrid proteins of Escherichia coli K-12. 247 Apr 9

Site-directed mutagenesis was performed with the phosphate starvation-inducible outer membrane porin PhoE of Escherichia coli K-12 to study the molecular basis of its anion selectivity. Lysines 18, 29, 64, and 125 were replaced by glutamic acids, and the properties of the mutant porins were investigated in in vivo and in vitro experiments. Lipid bilayer experiments showed that all these mutations had no influence on the pore structure because PhoE and the mutants had the same single channel conductance in KCl solution. Selectivity measurements revealed that the mutations changed the ionic selectivity of PhoE, but the change was dependent on the location of the lysine. Replacement of Lys18 and Lys29 by glutamic acid had a relatively small influence. The effect of the Lys64 substitution was somewhat larger, and the effect of the replacement of Lys125 resulted in the most drastic change in selectivity and in the loss of the interaction of PhoE with polyphosphate, whereas the replacement of the other lysines had no effect on the polyphosphate interaction behavior. The results are consistent with the assumption that the charge spot in PhoE consists of only 1 lysine per monomer, located in position 125 of the primary sequence and probably close to the pore interior.
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PMID:One single lysine residue is responsible for the special interaction between polyphosphate and the outer membrane porin PhoE of Escherichia coli. 247 43

Pseudomonas aeruginosa PAO1 was grown in vivo in chambers implanted into the peritoneums of mice and rats. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracts of bacterial cells taken from the chambers and washed to remove loosely bound host proteins revealed the presence of the major outer membrane proteins D2, E, F, G, and H2. Western immunoblotting with specific antisera confirmed the presence of porin protein F and lipoprotein H2. However, there was no apparent induction of the phosphate starvation-inducible porin P or the divalent cation starvation-inducible protein H1. Small amounts of proteins with molecular weights similar to those of the iron-regulated outer membrane proteins were found in cells grown in vivo; however, their presence could not be confirmed immunologically. The presence of pili and flagella on the cells grown in vivo was demonstrated by electron microscopy and Western immunoblotting. A consistent alteration in the lipopolysaccharide banding pattern was observed after growth in vivo. Compared with cells of strain PAO1 grown in vitro, cells grown in vivo appeared to lack a series of high-molecular-weight O-antigen-containing lipopolysaccharide bands and gained a new series of lower-molecular-weight lipopolysaccharide bands. This alteration in the lipopolysaccharide after growth in vivo did not affect the O-antigen serotype or the resistance of the bacteria to serum.
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PMID:Surface characteristics of Pseudomonas aeruginosa grown in a chamber implant model in mice and rats. 249 57


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