A labile membrane domain, characterized by a sharp phase transition above physiological temperatures, has been identified in Azotobacter vinelandii, Escherichia coli and Bacillus subtilis which are genetically competent. The phase transition is observed by fluorescence emission spectroscopy with amphiphilic and hydrophobic probes located in all regions of the bilayer. Its intensity correlates closely with transformability and also with the number and size of semi-regular pebbled protein-free regions in the plasma membranes of A. vinelandii and E. coli as observed by freeze-fracture electron microscopy. There is substantive evidence that R-(-)-poly hydroxybutyrate (PHB) is a constituent of the domain. Ca2+ is required for the stability of the domain, and its uptake by cells developing genetic transformability is coincident with polyphosphate synthesis and with the formation of the domain. It is proposed that the domain structure is a PHB helix spanning the membrane cross-bridged by Ca2 to an inner helix of polyphosphate anion which neutralizes and delocalizes the cation charge and orients the structure perpendicular to the plane of the membrane. It is postulated to function as a Ca2+ and phosphate transporter in exponential-phase cells and as an importer of single-stranded DNA in competent cells. The participation of PHB in the domain will be further evaluated by studies of the rotational relaxation times of probes in model systems, membranes and intact cells. Intracellular free Ca2- and membrane Ca2+ will be measured as a function of the domain with Fura-2 and chlorotetracycline fluorescence, respectively. The factors influencing the synthesis of polyphosphate, PHB and their precursors during the competence protocols will be investigated. Complex formation between PHB and the polyanions: polyphosphate native DNA and ss-DNA will be studied to determine stability constants, cation selectivity sequences and rates of association and disassociation. PHB-Ca2+-polyphosphate complexes isolated from competent E. coli DH1 will be examined by bright field-dark field imaging, energy dispersive x-ray analysis, electron energy loss spectroscopy and microdiffraction with FE-STEM, and by optical rotatory dispersion, circular dichroism, infra-red dichroism and nuclear magnetic resonance spectroscopy. The ionophoretic activity of the complex will be examined in liposomes. Finally, the functioning of the domain will be studied in membrane vesicles and intact cells by examining its relationship to Ca2+ transport, polyphosphate transport and ss-DNA transport.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033375-02
Application #
3283035
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1986-04-01
Project End
1990-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
2
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Michigan State University
Department
Type
Schools of Arts and Sciences
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
Reusch, Rosetta N (2015) Poly-(R)-3-hydroxybutyrates (PHB) are Atherogenic Components of Lipoprotein Lp(a). Med Hypotheses 85:1041-3
Reusch, Rosetta N (2012) Insights into the structure and assembly of Escherichia coli outer membrane protein A. FEBS J 279:894-909
Reusch, R N (1999) Streptomyces lividans potassium channel contains poly-(R)-3-hydroxybutyrate and inorganic polyphosphate. Biochemistry 38:15666-72
Huang, R; Reusch, R N (1996) Poly(3-hydroxybutyrate) is associated with specific proteins in the cytoplasm and membranes of Escherichia coli. J Biol Chem 271:22196-202
Huang, R; Reusch, R N (1995) Genetic competence in Escherichia coli requires poly-beta-hydroxybutyrate/calcium polyphosphate membrane complexes and certain divalent cations. J Bacteriol 177:486-90
Reusch, R N; Huang, R; Bramble, L L (1995) Poly-3-hydroxybutyrate/polyphosphate complexes form voltage-activated Ca2+ channels in the plasma membranes of Escherichia coli. Biophys J 69:754-66
Reusch, R N (1995) Low molecular weight complexed poly(3-hydroxybutyrate): a dynamic and versatile molecule in vivo. Can J Microbiol 41 Suppl 1:50-4
Castuma, C E; Huang, R; Kornberg, A et al. (1995) Inorganic polyphosphates in the acquisition of competence in Escherichia coli. J Biol Chem 270:12980-3
Seebach, D; Brunner, A; Burger, H M et al. (1994) 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. Eur J Biochem 224:317-28
Reusch, R N (1992) Biological complexes of poly-beta-hydroxybutyrate. FEMS Microbiol Rev 9:119-29

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