PubMed Health⌕ Search

Biomedical subjects

A P Pugsley

Publications and source records attributed to A P Pugsley.

16 recordsLinked to original sources
PubMed1992

Translocation of a folded protein across the outer membrane in Escherichia coli.

A mutation in the Escherichia coli dsbA gene (coding for a periplasmic disulfide oxidoreductase) reduces the rate of disulfide bond formation in the enzyme pullulanase and also reduces the rate at which the enzyme is secreted to the cell surface, as measured by protease accessibility. The enzyme did not become protease accessible when disulfide bond formation was completely prevented in the mutant strain by carboxymethylation. These results indicate that a disulfide bond may be required for, and certainly does not impede, the translocation of pullulanase across the outer membrane. Since it is unlikely that a disulfide bond could be formed and then reduced again in the periplasm, these results would appear to strengthen the argument that pullulanase polypeptides fold into or close to their final conformation before they are transported across the outer membrane. It is suggested that this might be a feature common to all proteins that are secreted by other Gram-negative bacteria by a pullulanase-like pathway.

Bacterial Proteins↗
PubMed1992

PulO, a component of the pullulanase secretion pathway of Klebsiella oxytoca, correctly and efficiently processes gonococcal type IV prepilin in Escherichia coli.

The PulO protein required for extracellular secretion of pullulanase by Klebsiella oxytoca is known to be highly homologous to two type IV prepilin peptidases, namely XcpA(PilD) (Pseudomonas aeruginosa) and TcpJ (Vibrio cholerae). The predicted prepilin peptidase activity of PulO was confirmed by showing that it could correctly process the product of the cloned pilE.1 type IV pilin structural gene from Neisseria gonorrhoeae in Escherichia coli. The P. aeruginosa prepilin peptidase and another putative prepilin peptidase, ComC from Bacillus subtilis, also processed prePilE. Subcellular fractionation showed that the pilE gene product that had been processed by PulO remained associated with the cytoplasmic membrane, as did the unprocessed precursor. PulO was also shown to process three of the four prePilE-PhoA hybrids tested. Southern hybridization experiments suggest that a pulO homologue is present in the N. gonorrhoeae chromosome.

Bacterial Outer Membrane Proteins↗
PubMed1979

Factors affecting the electrophoretic mobility of the major outer membrane proteins of Escherichia coli in polyacrylamide gels.

The outer membrane proteins of Escherichia coli can be resolved by polyacrylamide gel electrophoresis in the presence of anionic detergents. Factors such as the choice of detergent and buffer system and the presence of urea in the separation gel are all shown to affect the charge and/or the configuration of the detergent-protein complexes and will affect the relative migration of these complexes to different extents. The procedures described in this paper may be of use in the determination of the relatedness of the proteins from the same or different strains. In addition, detailed examinations of the effects of these different parameters and the effect of changes in acrylamide concentrations may be useful in the detection of unusual characteristics which may indicate the presence of posttranslational modification.

Cell Membrane↗
PubMed1979

Chemical heterogeneity of major outer membrane pore proteins of Escherichia coli.

Peptide mapping and isoelectric focusing were used to compare the major outer membrane pore proteins from various strains of Escherichia coli K-12, including strains carrying mutations in the nmpA, nmpB, and nmpC genes which result in the production of new membrane proteins. Proteins 1a, 1b, and 2 and the NmpA proteins each gave unique peptide and isoelectric focusing profiles, indicating that these are different polypeptides. The NmpA protein and the NmpB protein appeared to be identical by these criteria. The NmpC protein and protein 2 were nearly identical, although one different peptide was observed in comparing the proteolytic peptide maps of these proteins and there were slight differences in their isoelectric focusing profiles. Antiserum against protein 2 showed partial cross-reactivity with the NmpC protein. These results indicate that the various pore proteins of E. coli K-12 fall into four different classes.

Bacterial Proteins↗
PubMed1979

Colicin receptors and the mechanisms of colicin uptake.

This review deals in detail with the nature, synthesis, physiologic functions, and the regulation of colicin receptors, which represent components of transportsystems, as well as with the two mechanisms of the colicin uptake within the groups A and B of colicins.

Bacterial Proteins↗
PubMed1978

Outer membrane proteins of Escherichia coli. VII. Evidence that bacteriophage-directed protein 2 functions as a pore.

Protein 1, a major protein of the outer membrane of Escherichia coli, has been shown to be the pore allowing the passage of small hydrophilic solutes across the outer membrane. In E. coli K-12 protein 1 consists of two subspecies, 1a and 1b, whereas in E. coli B it consists of a single species which has an electrophoretic mobility similar to that of 1a. K-12 strains mutant at the ompB locus lack both proteins 1a and 1b and exhibit multiple transport defects, resistance to toxic metal ions, and tolerance to a number of colicins. Mutation at the tolF locus results in the loss of 1a, in less severe transport defects, and more limited colicin tolerance. Mutation at the par locus causes the loss of protein 1b, but no transport defects or colicin tolerance. Lysogeny of E. coli by phage PA-2 results in the production of a new major protein, protein 2. Lysogeny of K-12 ompB mutants resulted in dramatic reversal of the transport defects and restoration of the sensitivity to colicins E2 and E3 but not to other colicins. This was shown to be due to the production of protein 2, since lysogeny by phage mutants lacking the ability to elicit protein 2 production did not show this effect. Thus, protein 2 can function as an effective pore. ompB mutations in E. coli B also resulted in loss of protein 1 and similar multiple transport defects, but these were only partially reversed by phage lysogeny and the resulting production of protein 2. When the ompB region from E. coli B was moved by transduction into an E. coli K-12 background, only small amounts of proteins 1a and 1b were found in the outer membrane. These results indicate that genes governing the synthesis of outer membrane proteins may not function interchangeably between K-12 and B strains, indicating differences in regulation or biosynthesis of these proteins between these strains.

Bacterial Proteins↗
PubMed1978

Identification of three genes controlling production of new outer membrane pore proteins in Escherichia coli K-12.

Escherichia coli K-12 strains carrying mutations in the ompB gene or double mutations in the tolF and par genes lack the major outer membrane proteins 1a and 1b. These strains are deficient in the transport of small hydrophylic compounds and are multiply colicin resistant. When revertants of these strains were sought, a number of extragenic pseudorevertants were obtained which produced new outer membrane proteins. These new proteins could be divided into three classes by differences in electrophoretic mobility on polyacrylamide gels, by differing specificities for transport of small molecules, and by the identification of three different genetic loci for genes controlling their production. These genetic loci are designated as nmpA (at approximately 82.5 min on the E. coli K-12 genetic map), nmpB (8.6 min), and nmpC (12 min). The new proteins produced in strains carrying nmpA, nmpB, or nmpC mutations did not cross-react with antiserum against a mixture of proteins 1a and 1b, or with antiserum against phage-directed protein 2. Production of the new membrane proteins restored sensitivity to some of the colicins.

Bacterial Proteins↗
PubMed1977

Comparison of colicins B-K260 and D-CA23: purification and characterization of the colicins and examination of colicin immunity in the producing strains.

Colicins B-K260 and D-CA23 were purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography and were compared with respect to a number of physical and chemical properties. Both colicins were shown to be proteins and were found to have similar molecular weights, isoelectric points and amino acid compositions. The two colicins also have substantial antigenic similarities but are distinguished by the presence of non-cross-reacting antigens and by differences in stability and in sensitivity to heat and reducing conditions. In addition, strains of Escherichia coli K-12 producing colicins B-K260 and D-CA23 are not cross-immune. The similarities noted between the two colicins are compatible with their use of a common cell surface receptor while having different modes of action.

Amino Acids↗
PubMed1977

Uptake of ferrienterochelin by Escherichia coli: energy dependent stage of uptake.

The uptake of the siderophore-iron complex ferrienterochelin was found to be strongly dependent upon an energized membrane state, as demonstrated by its sensitivity to dinitrophenol, azide, and cyanide. Ferrienterochelin uptake may also be dependent upon phosphate bond energy, as indicated by sensitivity to arsenate and iodoacetic acid. Although the adenosine triphosphatase does not appear to be involved in this energy coupling mechanism, ferrienterochelin uptake was shown to be less dependent upon phosphate bond energy than was glutamine uptake. Sensitivity of ferrienterochelin uptake to osmotic shock was shown to be due to the release of a ferrienterochelin binding compound located in the outer membrane of the cells and probably identical to the colicin B receptor protein.

Adenosine Triphosphatases↗
PubMed1976

Iron uptake in colicin B-resistant mutants of Escherichia coli K-12.

Four classes of colicin B-resistant mutants of Escherichia coli K-12 were examined for defects in iron uptake. All four mutant classes (cbt, exbC, exbB, and tonB) were defective in the uptake of ferri-ennterochelin. The tonB mutant was also defective in citrate-, ferrichrome-, and rhodoturulic acid-mediated iron uptake. The defects in iron transport were reflected in increased sensitivity to iron chelators and to chromium and aluminium salts, and in hypersecretion of enterochelin. One of the mutants (cbt) was apparently defective in outer membrane ferri-enterochelin receptor activity. aroE derivatives (unable to synthesize enterochelin) of the four mutant classes and the parent strain produced increased amounts of two outer membranes polypeptides when grown under iron stress. These polypeptides are implicated in ferri-enterochelin receptor activity.

2,2'-Dipyridyl↗
PubMed1976

Characterization of group B colicin-resistant mutants of Escherichia coli K-12: colicin resistance and the role of enterochelin.

Nine classes of group B colicin-resistant mutants were examined to study the role of enterochelin in colicin resistance. Four of the mutants studied (cbt, exbC, exbB, and tonB) hypersecreted enterochelin. Enterochelin hypersecretion was apparently responsible for resistance of the exbC mutant to colicins G and H and for resistance of the exbB mutant to colicins G, H, Ia, Ib, S1, and V. All four mutants scored as colicin B tolerant, even in the absence of enterochelin synthesis. The mutants produced substantially increased amounts of two high-molecular-weight outer membrane polypeptides when grown under limiting iron conditions. The presence of these polypeptides was correlated with increased colicin B-neutralizing activity in the outer membrane preparations.

2,2'-Dipyridyl↗