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Biomedical subjects

J Konisky

Publications and source records attributed to J Konisky.

At least 55 records · Page 3Linked to original sources
PubMed1983

Complementation of argG and hisA mutations of Escherichia coli by DNA cloned from the archaebacterium Methanococcus voltae.

DNA derived from the methanogenic archaebacterium Methanococcus voltae was digested with PstI restriction endonuclease and cloned into the PstI site of pBR322. The recombinant plasmids generated were used to transform a multiply auxotrophic strain of Escherichia coli with selection for tetracycline resistance. Plasmids complementing the argG(pAW1) or hisA(pAW2) mutations were isolated and characterized. Nick-translated pAW1 and pAW2 hybridized to the predicted M. voltae PstI fragments but not to digested E. coli DNA. A novel 55,000-dalton protein was synthesized in UV-irradiated cells by pAW1, whereas pAW2 synthesized a novel 26,000-dalton protein. Derivatives of pAW1 carrying insertion elements no longer complemented the argG mutation and failed to produce the 55,000-dalton protein. When an AccI fragment was deleted from pAW2, complementation of hisA did not occur and no 26,000-dalton protein was synthesized. The effect of orientation of the cloned DNA within the vector on complementation and polypeptide synthesis was examined.

Arginine↗
PubMed1981

Mode of action of colicin ib: formation of ion-permeable membrane channels.

Addition of purified colicin Ib to whole Escherichia coli cells or cytoplasmic membrane vesicles inhibits their subsequent ability to generate a membrane potential. In addition, this colicin is shown to bring about a voltage-dependent increase in the conductance of an artificial planar bilayer membrane prepared from soybean phospholipids. This results from the formation of ion-permeable channels. These data provide strong evidence that the depolarization of Escherichia coli cells by this colicin results from an Ib-induced increase in membrane permeability to ions.

Cell Membrane↗
PubMed1981

Activation of plasminogen to plasmin by a protease associated with the outer membrane of Escherichia coli.

Preparations of outer membrane of two strains of Escherichia coli contain a protease that can activate the serum zymogen plasminogen to the active protease plasmin. The amount of plasmin formed is proportional to the membrane concentration. The kinetics of plasminogen activation are linear and obey the Michaelis--Menten rate equation. The Km(app) for the activation of dog plasminogen by E. coli outer membrane preparations is similar to the Km(app) for the activation of dog plasminogen by human urokinase. The E. coli enzyme is active in a membrane-associated form, as opposed to a secreted or soluble form, and is most likely a serine protease because it is inhibited by diisopropyl fluorophosphate. It is also inhibited from activating plasminogen by p-nitrophenyl-p-guanidinobenzoate and aprotinin. Analysis of the activation of plasminogen by the E. coli enzyme by NaDodSO4/polyacrylamide gel electrophoresis showed that the cleavage of plasminogen to plasmin was as specific as that exhibited in the activation of plasminogen to plasmin by urokinase. Possible in vivo roles for this plasminogen activator in E. coli outer membranes are discussed.

Cell Membrane↗
PubMed1981

Increased binding of a hydrophobic, photolabile probe to Escherichia coli inversely correlates to membrane potential but not adenosine 5'-triphosphate levels.

We describe conditions for a quantitative determination of azidopyrene binding to Escherichia coli cells. In addition, we define conditions whereby irradiation of azidopyrene in the presence of cells leads to irreversible association of probe with cells. This is presumably due to the light-dependent generation of reactive nitrenes and subsequent incorporation of nitrenopyrene moieties into cellular components. These methods allowed us to determine that the amount of azidopyrene bound to cells was inversely correlated with the magnitude of the cellular membrane potential, but was not correlated with high or low adenosine 5-triphosphate levels per se. Cells bound more azidopyrene if the delta psi was low. Cell-bound azidopyrene was found to be entirely associated with the inner and outer membrane. We suggest that the decreased association of hydrophobic probes upon energization of whole cells reflects a rapid transition in structural properties of the cell envelope.

Adenosine Triphosphate↗
PubMed1981

Cleavage of colicin Ia by the Escherichia coli K-12 outer membrane is not mediated by the colicin Ia receptor.

Colicin Ia can be cleaved by isolated outer membranes prepared from sensitive and resistant (lacking the colicin Ia receptor) strains of Escherichia coli. Both active and heat-denatured colicin Ia are extensively fragmented. Such proteolysis does not occur when colicin Ia is added to whole sensitive or resistant cells. These results demonstrate that cleavage of colicin Ia is not mediated by its outer membrane receptor.

Cell Membrane↗
PubMed1981

Cloning of immunity and structural genes for colicin V.

The colicin V immunity and structural genes of plasmid pColV-B188 were cloned into the vectors pMB9, pBR322, and pMK16. Both genes are closely linked and can be isolated on a 900-base-pair deoxyribonucleic acid fragment. Insertion of the transposon Tn5 into this cloned sequence led to the construction of a mutant plasmid which conferred colicin V immunity, but not the ability to produce this colicin. Analysis of the products determined by these cloned genes in cells has led to the conclusion that the polypeptide involved in immunity has a molecular weight of about 6,500, whereas the colicin has a molecular weight of approximately 4,000.

Bacteriocin Plasmids↗
PubMed1981

Plasmid-determined immunity of Escherichia coli K-12 to colicin Ia Is mediated by a plasmid-encoded membrane protein.

The colicin Ia structural (cia) and immunity (iia) genes of plasmid pColIa-CA53 have been cloned into the cloning vector pBR322. These two genes are closely linked, and both of them can be isolated on a deoxyribonucleic acid fragment approximately 4,800 base pairs long. An analysis of the polypeptides synthesized in ultraviolet-irradiated cells containing these cloned genes led to the conclusion that the iia gene product is a polypeptide with a molecular weight of approximately 14,500. Insertion of transposon Tn5 into the iia gene led to a concomitant loss of the immune phenotype and the ability to produce this protein. Fractionation of ultraviolet-irradiated cells harboring a plasmid carrying the iia gene showed that the immunity protein is a component of the inner (cytoplasmic) membrane. Furthermore, the mechanism of immunity to colicin Ia appears to operate at the level of the cytoplasmic membrane. This conclusion is based on our finding that membrane vesicles prepared from colicin Ia-immune cells could be depolarized by colicins E1 and Ib but not by colicin Ia.

Bacterial Proteins↗
PubMed1980

tonB-independent ferrichrome-mediated iron transport in Escherichia coli spheroplasts.

Although a functional tonB gene product was required for ferrichrome-mediated iron transport in whole cells of Escherichia coli K-12, such transport did not require the tonB+ function in spheroplasts. We suggest that in spheroplasts ferrichrome has direct access to the cytoplasmic membrane and that this is reflected in tonB-independent accumulation of ferrichrome iron. Therefore, the tonB gene product does not function in the translocation of ferrichrome iron across the inner membrane.

Biological Transport, Active↗
PubMed1979

Effect of colicins Ia and E1 on ion permeability of liposomes.

Colicins Ia and E1 are shown to inhibit the formation and bring about the collapse of a potassium diffusion potential imposed across the membrane of liposomes prepared from soybean or Escherichia coli phospholipids. Such depolarization results from a colicin-induced increase in membrane ion permeability. Colicins E2 and E3 do not depolarize such membranes. In addition to the colicin Ia-induced rapid efflux of preloaded rubidium, sodium, phosphate, or choline from liposomes, a slower efflux of preloaded sucrose or glucose 6-phosphate occurs. However, treated liposomes do not leak inulin or dextran, demonstrating that the effects of E1 and Ia are not due to a general disruption of membrane structure. The fact that colicin-induced ion efflux is observed in the complete absence of a membrane potential shows that the action of these colicins on liposomes is not voltage dependent. These results provide strong evidence that the depolarization of E. coli cells by colicins Ia and E1 results from a colicin-induced increase in membrane permeability to ions. It is proposed that this is brought about by the direct interaction of the colicin molecules with the bacterial cytoplasmic membrane.

Cell Membrane Permeability↗
PubMed1979

Mode of action of colicins Ia, E1 and K.

Addition of colicins Ia, E1 or K to sensitive Escherichia coli leads to inhibition of macromolecular synthesis and an uncoupling of electron transport from active transport. Recent results indicate that these colicins affect energy metabolism by interacting directly with the cytoplasmic membrane. This results in a transmembrane flow of ions leading to membrane depolarization. It is proposed that the function of the outer membrane colicin receptor is to mediate access of the colicin molecule to the cytoplasmic membrane.

Biological Transport, Active↗
PubMed1978

In vitro depolarization of Escherichia coli membrane vesicles by colicin Ia.

Conditions are reported under which membrane vesicles prepared from Escherichia coli K12 are depolarized by colicin Ia. Although incubation of membrane vesicles with active colicin Ia affects neither transport activity nor the ability of such vesicles to generate a deltapH or deltapsi, a single freeze-thaw cycle of such vesicles in the presence of colicin Ia leads to 1) retention of the colicin by the vesicles, 2) inactivation of transport activity, and 3) membrane depolarization, with a concomitant increase in the transmembrane deltapH. These effects are dependent upon the presence of active colicin Ia during the freeze-thaw cycle. These findings are consistent with our previous results showing that Ia-treated whole cells or membrane vesicles prepared from such cells are defective in their ability to generate a deltapsi, yet generate an increased deltapH (Tokuda, H., and Konisky, J. (1978) Proc. Natl. Acad. Sci. U. S. A., 75, 2579--2583). In addition to its effect on vesicles prepared from sensitive cells, we show that vesicles prepared from both colicin Ia-resistant and -tolerant cells are depolarized by colicin treatment with a concomitant increase in deltapH. It is concluded that the final target of colicin Ia is the cytoplasmic membrane. A model for the mechanism of colicin Ia action is presented in which colicin Ia binds to the specific colicin Ia outer membrane receptor and is subsequently translocated to the cytoplasmic membrane where its integration leads to the formation of ion channels.

Biological Transport↗
PubMed1978

Mode of action of colicin Ia: effect of colicin on the Escherichia coli proton electrochemical gradient.

By use of the technique of flow dialysis, the membrane potential (deltapsi) and pH gradient (deltapH) have been measured in colicin Ia-treated Escherichia coli K-12 cells and in membrane vesicles prepared from such cells. Although such cells and vesicles are able to generate a transmembrane deltapH at pH 5.5, they do not generate a transmembrane deltapsi. Glucose-6-phospate uptake by cells is shown to be stimulated at pH 5.5 and inhibited at pH 7.5 by colicin Ia treatment. On the other hand, proline uptake is demonstrated to be inhibited progressively at pH 5.5, 6.6, and 7.5 in colicin Ia-treated cells. These data provide strong evidence for a colicin Ia-induced membrane deplorization and indicate that the membrane becomes permeable to ion(s) other than protons after treatment with colicin Ia.

Acid-Base Equilibrium↗
PubMed1976

Membrane changes in Escherichia coli induced by colicin Ia and agents known to disrupt energy transduction.

The addition of colicin Ia to a suspension of intact Escherichia coli in the presence of the hydrophobic fluorescent probe N-phenyl-l-naphthylamine causes dramatic changes in the fluorescence of the probe. The fluorescence intensity increases several fold, the emission spectrum shifts to the blue, the fluorescence lifetime approximately doubles, and the polarization increases. These changes do not appear to result from an increase in membrane microviscosity, as has been previously postulated to be the case for the N-phenyl-l-naphthylamine fluorescence changes seen with colicin El-treated cells (Helgerson, S.L., Cramer, W.A., Harris, J.M., and Lytle, F.E. (1974), Biochemistry, 13, 3057); rather, they result from an increased binding of the dye to the cell envelope. A variety of agents have been used to demonstrate that a very similar fluorescence response results whenever the cells are "deenergized." These agents include electron transport inhibitors (malonate, amytal, cyanide) as well as the uncouplers CCCP and azide. In addition, depleting the cells of either endogenous substrates or oxygen results in the same fluorescence response. In these cases, the fluorescence response is reversed upon addition of an oxidizable substrate or oxygen. It is clear that there are significant changes in the Escherichia coli envelope as energy transduction processes are disrupted and restored. The changes reported by the fluorescent probe may prove useful indeciphering structure-function relationships in the Escherichia coli envelope.

Anaerobiosis↗
PubMed1976

Relationship between the transport of iron and the amount of specific colicin Ia membrane receptors in Escherichia coli.

Strains of Escherichia coli K-12 defective in their ability to utilize exogenously supplied iron due to genetic defects in the entF, tonB, fes, or fep gene exhibited elevated levels of the specific outer-membrane receptor for colicin Ia when compared with parental strains. Although entF, fes, and fep strains showed a higher degree of Ia sensitivity than did the parental strains, tonB strains were resistant to colicin action. The colicin insensitivity of tonB strains was not due to hyperproduction of enterochelin. Growth in medium containing 101.8 muM Fe2+ led to a lowering of receptor levels in all the above strains and resulted in decreased colicin Ia sensitivity in all strains except tonB, which was already at maximal resistance. Growth in citrate plus iron (1.8 muM) or in ferrichrome resulted in a substantial reduction in both receptor levels and Ia sensitivity in ent, fes, and fep strains but had no effect on receptor levels in tonB strains. Growth in citrate did not lead to an alteration in receptor levels in a mutant specifically defective in citrate-mediated iron transport. The presence of enterochelin during growth led to a reduction in the number of receptors in the parental and ent strains but not in tonB, fes, or fep strains. Thus, in all cases examined, there was an inverse relationship between the number of colicin receptors per cell and the ability of the strain to take up iron from the growth medium. This suggests that under conditions of iron limitation there is a derepression of colicin Ia receptor biosynthesis. These results may point to a role of the colicin I receptor in iron uptake.

Binding Sites↗