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R N Reusch

Publications and source records attributed to R N Reusch.

35 records · Page 2Linked to original sources
PubMed1995

Genetic competence in Escherichia coli requires poly-beta-hydroxybutyrate/calcium polyphosphate membrane complexes and certain divalent cations.

In earlier studies of genetic competence in Escherichia coli induced with calcium-containing buffers, a strong correlation was found between transformation efficiency and the formation of poly-beta-hydroxybutyrate/calcium polyphosphate (PHB/Ca2+/PPi) complexes in the plasma membranes. In this study, we replaced Ca2+ with one of a number of other cations--monovalent, divalent, and trivalent--and found significant numbers of transformants (transformation efficiency, > 10(5)/micrograms of pBR322 DNA) only when the cells had high levels of PHB/Ca2+/PPi and the medium contained at least one of the divalent cations Ca2+, Mn2+, Sr2+, or Mg2+. Cells with high levels of the complexes were not competent when the medium did not contain these cations, but the cations were also ineffectual when the cells had few complexes. Surprisingly, Mn, Sr, and Mg were not incorporated into the complexes in place of Ca. These results indicate that PHB/Ca2+/PPi complexes and the above-mentioned divalent cations each have essential but disparate roles in genetic competence. Moreover, the strong selectivity of PHB/PPi for Ca2+ suggests the binding sites in the complexes are ionophoretic.

Calcium↗
PubMed1995

Low molecular weight complexed poly(3-hydroxybutyrate): a dynamic and versatile molecule in vivo.

It is increasingly clear that poly(3-hydroxybutyrate) (PHB) is not just an inert storage polymer, confined to certain bacteria, but a ubiquitous, interactive, solvating biopolymer involved in important physiological functions. Low molecular weight PHB, complexed to other macromolecules (c-PHB), is widely distributed in biological cells, being found in representative organisms of nearly all phyla. Complexation modifies the physical and chemical properties of c-PHB, allowing it to pervade aqueous as well as hydrophobic regions of the cell, and as a result c-PHB can be found in cytoplasm and intracellular fluids as well as in membranes and lipoproteins. The lipidic homopolymer associates with other macromolecules primarily via its ester carbonyl oxygens, which can act as hydrogen-bond acceptors or as ligands for coordinate bonds to cations. The spacing of the electron-donating groups along the flexible backbone allows for multiple bonding interactions, and forms the basis for the ability of c-PHB to bind to proteins, or to form ion-conducting complexes with salts. The singular ability of c-PHB to dissolve salts and facilitate their transfer across hydrophobic barriers defines a potential physiological niche for c-PHB in cell metabolism.

Hydroxybutyrates↗
PubMed1994

Isolation and 1H-NMR spectroscopic identification of poly(3-hydroxybutanoate) from prokaryotic and eukaryotic organisms. Determination of the absolute configuration (R) of the monomeric unit 3-hydroxybutanoic acid from Escherichia coli and spinach.

Trace amounts of poly[(R)-3-hydroxybutanoate] were isolated from competent Escherichia coli, spinach, bovine serum albumin, beef heart mitochondria, and aortal tissues, all sources in which it is not accumulated as storage material. Its identity was in all cases proved by 1H-NMR spectroscopy. In some runs, the poly[(R)-3-hydroxybutanoate] isolated from competent E. coli also contained some 3-hydroxyvalerate, an observation confirmed by 1H-NMR spectroscopy and gas chromatography. The absolute configuration of the polymers isolated from E. coli and spinach was shown to be (all-R) by gas chromatography on chiral columns.

Animals↗
PubMed1993

Methoxylated fatty acids reported in Rhizobium isolates arise from chemical alterations of common fatty acids upon acid-catalyzed transesterification procedures.

We obtained from a phospholipid extract of wild-type Rhizobium leguminosarum bv. trifolii ANU843 methoxylated fatty acids that had been previously reported as constitutive unusual Rhizobium fatty acids. The use of deuterated reagents and subsequent gas-liquid chromatography-mass spectrometry analyses showed that these methoxylated fatty acid derivatives are the products of chemical alterations of common cyclopropane-containing and unsaturated fatty acids occurring during various acid-catalyzed transesterification treatments aimed at producing the methyl ester derivatives. Similar results were obtained from a phospholipid extract of Escherichia coli K-12. In contrast, these chemical alterations were not induced by an alkaline methanolysis method of transesterification. If an acidic treatment is needed to release the fatty acids from the source molecule, the finding of unusual methoxylated fatty acids should be carefully confirmed with deuterated reagents.

Acids↗
PubMed1992

Transport of poly-beta-hydroxybutyrate in human plasma.

Poly-beta-hydroxybutyrate (PHB) is an amphiphilic lipid that has been found to be a ubiquitous component of the cellular membranes of bacteria, plants and animals. The distribution of PHB in human plasma was investigated using chemical and immunological methods. PHB concentrations proved highly variable; in a random group of 24 blood donors, total plasma PHB ranged from 0.60 to 18.2 mg/l, with a mean of 3.5 mg/l. In plasma separated by density gradient ultracentrifugation, lipoproteins carried 20-30% of total plasma PHB; 6-14% in the very low density lipoproteins (VLDL), 8-16% in the low density lipoproteins (LDL), and less than 3% in the high density lipoproteins (HDL). The majority of plasma PHB (70-80%) was found in protein fractions of density greater than 1.22 g/ml. Western blot analysis of the high density fractions with anti-PHB F(ab')2 identified albumin as the major PHB-binding protein. The affinity of albumin for PHB was confirmed by in vitro studies which demonstrated transfer of 14C-PHB from chloroform into aqueous solutions of human and bovine serum albumins. PHB was less tightly bound to LDL than to other plasma components; the polymer could be isolated from LDL by extraction with chloroform, or by digestion with alkaline hypochlorite, but it could not similarly be recovered from VLDL or albumin. PHB in the LDL correlated positively with total plasma cholesterol and LDL cholesterol, and negatively with HDL cholesterol. The wide concentration range of PHB in plasma, its presence in VLDL and LDL and absence in HDL, coupled with its physical properties, suggest it may have important physiological effects.

Binding Sites↗
PubMed1992

Biological complexes of poly-beta-hydroxybutyrate.

Short-chain complexed poly-beta-hydroxybutyrate, 130-170 monomer units, is a ubiquitous constituent of cells, wherein it is usually associated with other macromolecules by multiple coordinate bonds, or by hydrogen bonding and hydrophobic interactions. This conserved PHB has been isolated from the plasma membranes of bacteria, from a variety of plant tissues, and from the plasma membranes, mitochondria, and microsomes of animal cells. In bacterial membranes, PHB has been found complexed to the calcium salts of inorganic polyphosphates, and to single-stranded DNAs. The ability of PHB to solvate salts, consisting of cations having high solvation energies and large delocalized anions, is in accordance with its molecular characteristics, that of a flexible linear molecule possessing a large number of electron-donating ester oxygens suitably spaced to replace the hydration shell of cations. In turn, PHB may be rendered soluble in aqueous media by complexation to water-soluble proteins, such as serum lipoproteins and albumin. Such solvates are highly resistant to hydrolytic enzymes. These findings suggest that the physiological roles of this unique biopolymer may include the solvation of salts of polymeric anions to facilitate their movement through hydrophobic barriers, and the protection of cellular polymers from enzymatic degradation.

Animals↗
PubMed1990

Influence of alkali metal cations on the transport of calcium by phosphatidate in multi-phase systems.

The effects of alkali metal cations on the rates at which Ca2+ and phosphatidic acid were cotransported from aqueous to hydrocarbon medium were examined. The alkali metal cations remained in the aqueous phase yet specifically influenced the transport of Ca2+ into the hydrocarbon solvent. For the physiological cations, Na+ and K+, there were critical concentration ranges in which small changes in concentration effected sharp changes in transport rates. The maximal rate observed with Na+ was an order of magnitude greater than that with K+; however, unlike Na+, K+ promoted low levels of transport below the critical concentration range. Li+ effected only low levels of transport even at high concentrations, whereas Rb+ and Cs+ induced transport at rates proportional to their concentrations. These results are discussed in terms of a classical ionophore model for the complex composed of a neutral phosphatidic acid dimer bridged by Ca2+.

Biological Transport, Active↗
PubMed1989

Poly-beta-hydroxybutyrate/calcium polyphosphate complexes in eukaryotic membranes.

Poly-beta-hydroxybutyrate/calcium polyphosphate (PHB-CaPolyPi) complexes exist as labile quasi-crystalline structures in bacterial plasma membranes. The composition, structure, and distribution of the complex suggest it may play a role in the regulation of intracellular calcium and in calcium signaling. The importance of these functions led to this investigation of the occurrence of PHB-CaPolyPi complexes in eukaryotes. A variety of plant and animal systems were analyzed and all were found to contain PHB associated with CaPolyPi. The intracellular location of the complex in bovine liver was primarily the mitochondria and microsomes, with smaller amounts in the plasma membranes. Eukaryotic PHB had the same narrow range of chain lengths (120-200 subunits) as PHB in bacterial membranes, and was associated with PolyPi of somewhat greater length (170-220) than the bacterial counterpart (130-170).

Animals↗
PubMed1988

Putative structure and functions of a poly-beta-hydroxybutyrate/calcium polyphosphate channel in bacterial plasma membranes.

A poly-beta-hydroxybutyrate complex extracted from the plasma membranes of genetically competent Escherichia coli contained polyhydroxybutyrate:polyphosphate:calcium in molar ratios approximating 1:1:0.5. The chain length of the polyhydroxybutyrate was estimated as 120-200 subunits, and that of the polyphosphate was estimated as 130-170 subunits. The extracted complex, when incorporated into liposomes, exhibited a lipid phase transition in the same temperature range as that of the membrane complex in whole cells as well as the same properties of irreversibility, lability, and sensitivity to chelating buffers. Space-filling molecular models and molecular energy minimization methods (Charmm) were used to develop and evaluate a plausible structure for the complex. It is proposed that the polyhydroxybutyrate forms an exolipophilic-endopolarophilic helix around an inner framework helix of calcium polyphosphate. The calcium ions link the two polymers by forming ionic bonds with phosphoryl oxygens of the polyphosphate and ion-dipole bonds with the ester carbonyl oxygens of the polyhydroxybutyrate. This symmetrical structure forms a channel through the membrane and may play a role in the transport of calcium, phosphate, and DNA.

Calcium Phosphates↗
PubMed1987

Protein synthesis during encystment of Azotobacter vinelandii.

Proteins synthesized during the encystment of Azotobacter vinelandii were radiolabeled with [35S]methionine and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pulse labeling was used to demonstrate that early encystment-specific proteins were beginning to be synthesized at 2 h and reached peak levels about 12 h after initiation of encystment. One such protein was identified as a beta-ketoacyl acyl-carrier protein synthase. The concentration of early proteins began to decrease at 16 h, when intermediate proteins specific to the differentiation process began to be synthesized. The cessation of synthesis of intermediate proteins began at 20 h postinitiation, and the labeling pattern of proteins then remained constant throughout the remaining 4 days of encystment.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed1986

Poly-beta-hydroxybutyrate membrane structure and its relationship to genetic transformability in Escherichia coli.

The effects of competence-inducing treatments on the composition and organization of membrane lipids in Escherichia coli K-12, DH1, DH5, HB101, and RR1 were investigated for two widely used protocols in which transformability is developed at low temperatures in Ca2+ buffers. At stages during each procedure, the lipid compositions of the cells were determined, and the thermotropic lipid phase transitions were observed in whole cell culture by fluorescence assay with the hydrophobic probe N-phenyl-1-naphthylamine. Competence was evaluated by determining transformation efficiencies with plasmid pBR322 DNA. The competence-inducing procedures effected only slight changes in phospholipid compositions which did not correlate with transformability. However, the induction of competence was coincident with de novo synthesis and incorporation of poly-beta-hydroxybutyrate into the cytoplasmic membranes and with the appearance of a sharp lipid phase transition above physiological temperatures. Transformation efficiencies correlated with poly-beta-hydroxybutyrate concentrations and with the intensity of the new phase transition. Transformability, poly-beta-hydroxybutyrate synthesis and the new phase transition were not significantly affected by inhibition of protein synthesis with chloramphenicol or inhibition of respiration or ATP synthesis with azide, cyanide, arsenate, or 2,4-dinitrophenol; however, when poly-beta-hydroxybutyrate synthesis was inhibited with acetaldehyde, the new phase transition was not observed, and competence failed to develop. These studies suggest that genetic transformability in E. coli may be physiologically regulated.

Cell Membrane↗
PubMed1985

A mechanism for phosphoglyceride and Ca2+ transbilayer movement.

The ionophoretic capabilities of phosphoglycerides (PL) have been examined by measuring their translocation via cations from aqueous dispersions into linear and cyclic hydrocarbons. The PL surveyed were phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylinositol (PI). Only PA displayed ionophoretic activity in single lipid dispersions with a cation selectivity order of Mn greater than Ca. PG, PE and PC, but not PI, had a synergistic affect of PA induced translocation. These PL, inactive individually or in any combination, became strong Ca2+ ionophores of variable activity in association with PA. A dimeric structure proposed for the ionophoretic species forms the basis of a mechanism for transbilayer movement of PA, PG, PE and PC which would establish an asymmetric distribution of these lipids in the two faces of the bilayer by equilibrium processes.

Calcium↗
PubMed1983

D-(-)-poly-beta-hydroxybutyrate in membranes of genetically competent bacteria.

D-(-)-Poly-beta-hydroxybutyrate is a constituent of the membranes and the cytoplasms of genetically competent Azotobacter vinelandii, Bacillus subtilis, and Haemophilus influenzae cells. Within each species the concentration of D-(-)-poly-beta-hydroxybutyrate in the membranes and cytoplasm correlates with transformability. Fluorescence analysis of the thermotropic lipid phase transitions in A. vinelandii and B. subtilis cells indicates that D-(-)-poly-beta-hydroxybutyrate forms an organized gel structure in the membranes which is very labile. The concentration of organized D-(-)-poly-beta-hydroxybutyrate in the membranes, which can be estimated from the intensity of its phase transition, can be used to assess the competence of a culture.

Azotobacter↗
PubMed1981

Lipid metabolism during encystment of Azotobacter vinelandii.

The formation of cysts by Azotobacter vinelandii involves the synthesis of lipids as major metabolic products. Cells which encyst at low levels in aging glucose cultures undergo the same pattern of lipid synthesis as cells which undergo reasonably synchronous encystment in beta-hydroxybutyrate or n-butanol. The accumulation of poly-beta-hydroxybutyrate (PHB) precedes the synthesis of 5-n-heneicosylresorcinol and 5-n-tricosylresorcinol (AR1), which is then followed in about 6 h by the synthesis of the 5-n-alkylresorcinol galactosides (AR2). In the mature cyst, PHB, AR1, and AR2 account for 8, 5.6, and 4.5%, respectively, of the dry weight. Phospholipid formation levels off 4 h postinduction, which coincides with the final cell division, but fatty acids synthesis continues at a very low level throughout encystment, suggesting some turnover of fatty acid. Distribution studies show that AR1 and AR2 are found in roughly equal amounts in the exine and central body of the cysts, with only trace amounts recovered from the intine. Studies of cysts labeled during encystment with [14C]beta-hydroxybutyrate or during vegetative growth with [14C]glucose suggest that the exine structure is synthesized during encystment, but that the intine is composed largely of vegetative cell components.

Azotobacter↗
PubMed1981

Isolation and characterization of several unique lipids from Azotobacter vinelandii cysts.

Unique cyclic compounds were found in the lipid fraction of Azotobacter vinelandii cysts. In addition to two major molecular species which had already been identified, 5-n-alkylresorcinol and its galactoside derivative, five other molecular species (two alkyl side chain homologs of each) were isolated, and their structures were established by infrared, ultraviolet, nuclear magnetic resonance, and mass spectroscopy. These 10 compounds were 6-n-heneicosylresorcylic acid methyl ester and 6-n-tricosylresorcylic acid methyl ester, 5-n-(2-hydroxy)heneicosylresorcinol and 5-n-(2-hydroxy-tricosylresorcinol, 5-n-heneicosyl-4-acetylresorcinol and 5-n-tricosyl-4-acetylresorcinol, 6-n-heneicosyl-4-hydroxypyran-2-one and 6-n-tricosyl-4-hydroxypyran-2-one, and 6-(2-oxotricosyl)-4-hydroxy-pyran-2-one and 6-(2-oxopentacosyl)-4-hydroxypyran-2-one.

Azotobacter↗
PubMedDate not supplied

Novel lipid components of the Azotobacter vinelandii cyst membrane.

Phospholipids are ubiquitous components of biological membranes. In the vegetative cells of Azotobacter vinelandii, a Gram-negative free-living aerobic soil bacterium, the membrane lipids are phospholipids with polar head group and fatty acyl compositions similar to those of Escherichia coli. We report here that when A. vinelandii differentiates to form metabolically dormant cysts, the phospholipids in the membranes are replaced by a family of 5-n-alkylresorcinols and 6-n-alkylpyrones. These novel amphiphilic lipids form a unique membrane matrix which may contribute to the physiology and desiccation resistance of the cyst.

Azotobacter↗