The expression of fumarate reductase in Escherichia coli has been amplified over 30-fold by utilizing a recombinant plasmid, pFR63, carrying the fumarate reductase operon. More than 50% of the innermembrane protein could be accounted for by the enzyme, whereas the total amount of protein associated with the inembrane fraction doubled. The membrane accommodated this excess fumarate reductase without reducing the levels of other membrane-associated enzymes. At the same time, the amount of membrane lipid increased such that the lipid/protein ratio remained constant, indicating that the total amount of membrane had doubled. Small alterations in fatty acid composition as well as a large increase in cardiolipin were detected in the fumarate reductase-enriched memnbranes. The excess membrane was localized in novel tubular structures which were observed in thin-section and negatively stained electron-microscopic preparations. The tubules only appeared after the cytoplasmic membrane became highly enriched in fumarate reductase. They branched from the cytoplasmic membrane and were comnposed of an aggregate of fumarate reductase and lipid. * Corresponding author. amplification of fumarate reductase does not reduce the levels of other membrane-bound activities. MATERIALS AND METHODS Strains and plasmids. E. coli HB101 is F_ hsdR hsdM pro leu gal lac thi recA rpsL. Plasmids pBR322 and pFRD63 have previously been described (15). Preparation of everted envelopes, inner membranes, and

Ethidium fluorescence assays were adapted for the rapid and sensitive detection of precA; in addition, fluorescence measurements on binding precA to linear, OC and CCC PM2 DNAs have enabled the stoichiometry of precA binding as well as the precA-induced unwinding angle of DNA to be determined. The stoichiometry of binding was independently confirmed by sedimentation analysis to be one precA molecule per 3 bp. The unwinding angle was also independently confirmed by measurements of fluorescence changes induced by the binding of precA to CCC DNA which was relaxed by topoisomerase to give a precA-induced unwinding angle of 51 degrees. Electron microscopy of OC DNA molecules which bound nonsaturating amounts of precA revealed that the length increase in DNA due to precA was approximately 55%. Finally, examination of negatively stained precA complexes with a variety of linear DNAs showed that the minor groove is the primary site of interaction for this protein.

No abstract

SUMMARYFull-length measles virus RNA molecules isolated from purified virions or nucleocapsids and examined by electron microscopy were 5.12(+0.12) ~tm in length, corresponding to a molecular weight of 5.2(+0-1) x 106. Purified virions examined by negative staining in the electron microscope exhibited a pleomorphic range of particle sizes varying in diameter between 300 nm and 1000 nm. Purified nucleocapsids had dimensions of 21 nm (diameter) x 1254(_+7) nm (length) and a central core of diameter about 5 nm. Full-length nucleocapsids were composed of 204(+3) protein discs. The pitch of the nucleocapsid helix was calculated to be 6.1 nm and the helix angle, ,, to be 8 ° 16'. Approximate volume calculations indicate that each enveloped virus particle contains multiple nucleocapsids.

The arsenate poisoning of R17 phage eclipse in Escherichia coli cultures grown in glycerol-containing medium has been found to be mediated by a dramatic loss in cell-associated F pili. Poisoning was very rapid and was nearly complete within 3 min at 37 C. The loss of pili was reflected by a 90% reduction in the ability of these cells to attach ribonucleic acid phage and by a reduction in the pili-per-cell ratio from 1.36 to 0.04 as determined by electron microscopy. Neither the integrity of the pilus per se nor the attachment process was affected by arsenate, for cell-free pili treated with arsenate retained the ability to attach phage. The arsenate effect on F piliation could be reversed readily without any loss in cell viability. This restoration of pili occurred under conditions of inhibited protein synthesis, implying that pools of pili protein exist in cells. In contrast to this phenomenon, cells grown in glucose-containing media were mainly resistant to arsenate poisoning as determined by phage attachment and the number of pili per cell. These results implied that arsenate poisons the ability of cells to synthesize and maintain F pili under certain specific conditions. The possible mechanism of this poisoning is discussed.