A procedure for the preparative isolation of Escherichia coli cell wall, membrane, and deoxyribonucleic acid (DNA)-envelope complex fragments has been developed. The envelope fragments were produced by controlled mechanical cell breakage and isolated by density gradient centrifugation and subsequent preparative free-flow electrophoresis. The DNA-envelope complex fragments were shown to contain biochemical markers of both the cell wall and the membrane and by electron microscopy to be cell envelope fragments containing wall/membrane adhesion zones.

The different Escherichia coli envelope fractions (cell wall, cytoplasmic membrane, and DNA-envelope complex fragments) were isolated by free-flow electrophoresis and analyzed by sodium dodecylsulfate-acrylamide gel electrophoresis. The DNA-envelope complex fragments possess a specific protein (mol wt 80,000-90,000). Upon treatment with trypsin, this protein disappears and the complex breaks down, thus releasing DNA, cell wall, and cytoplasmic membrane. Disaggregation of the complex can also be achieved by high salt concentrations. Lysozyme treatment dissolves the murein layer within the complex but does not disaggregate the complex. From these and other results on the stability of the DNA-envelope complex, conclusions can be drawn about the possible linkage within the described envelope particles.

Phospholipids containing photolysable carbene precursors ($l-trifluoro-a-diazopropionoxy and m-diazirinophenoxy groups) in w-positions of sn-2 fatty acyl chains were prepared. Photolysis of their vesicles produced crosslinked products in 40-60% yields. Crosslinking was mostly intermolecular and occurred by carbene insertion into the C-H bonds of a second fatty acyl chain. Crosslinking products were characterized by (i) Photolysis of Vesicles. The sample was introduced into a quartz vessel with a jacket through which circulated aqueous potassium hydrogen phthalate solution (0.5 or 2.0%, wt/vol). The vessel was placed in the center of a Rayonet photochemical reactor equipped with 16 symmetrically placed RPR 3000 A or RPR 3500 A lamps. The temperature of the circulating solution was controlled by a thermostat. RPR 3000 A lamps emitted 15% of the total radiation at 254 nm; RPR 3500 A lamps emitted no radiation below 290 nm. The radiation at 254 nm emitted by RPR 3000 A lamps was filtered out by 0.05% phthalate solution; the 2% solution filtered out radiation essentially completely uip to 315 nm. Progress of photolysis was followed either by monitoring the disappearance of the characteristic infrared frequency of azido (2100 cm-l) and diazo (2140 cm-l) groups or the UV absorption of diazirine and ca,/-unsaturated carbonyl groups after extraction (8) of the reaction mixture aliquots.Separation of Photolysis Products. After photolysis, the reaction mixtures were extracted (8), the organic phase was evaporated, and the residue, as a concentrated solution in chloroform/methanol 1:1 (vol/vol), was applied to a Sephadex LI-20 column (2.5 X 100 cm). Elution was performed with the same solvent at a rate of about 60 ml/hr. Fractions were monitored by their phosphorus content or radioactivity.Digestion with Phospholipase A2. This was performed as described by Chakrabarti and Khorana (1).Digestion with Phospholipase C. The digestions were performed and the resulting 1,2-diacylglycerides were isolated as described by Ottolenghi (9).Abbreviations: TLC(', thin-laver chromatography; 1[4ClPam2PtdCho, 1,2-dil( 4Cjpalmitoyl-sn-glycero(3)phosphocholine (di[14C]palmitoyl phosphatidylcholine); [I4CJOle2PtdCho, 1,2-di114CQoleoyl-sn-glycero(3)phosphocholine (dilr14C oleoyl phosphatidylcholine); GCMS, gas chromatography/mass spectrometry.

Infection of the temperature-sensitive E. eli mutant HfrH 165/70 (dnaB) with the filamentous ..1-Stranded DNA phage M13 is abortive at the restrictive temperature. Upon infection at 410, singlestranded phage DNA penetrates the cell and is converted in a rifampicin-sensitive step to the double-stranded replicative form (RF). The parental RF attaches to the cell membrane, but subsequent replication of the RF is blocked. It is concluded that in M13 infection semiconservative RF replication of a double strand to a double strand, in contrast to single-stranded DNA synthesis, depends specifically on the dnaB function.In strains of E. coli containing a temperature-sensitive mutation in the dnaB region, infection by the single-stranded DNA phage M13 is inhibited at the restrictive temperature (1). However, synthesis of phage single-stranded (ss) DNA and liberation of mature phage particles continues after cells that are actively producing phage are shifted to the higher temperature (1)t. This result implies that there is an earlier step in the replication process of M13 that depends on the dnaB function. Since a precise localization of the block in M13 replication may contribute information both to the replicative process of single-stranded DNA phages and to the biochemical lesion in the dnaB mutant, abortive M13 infection at the restrictive temperature was studied in more detail. At the restrictive temperature, M13 ss DNA penetrates the cell and is readily converted to RF. In contrast, neither the parental nor progeny RF could replicate at the high temperature. The data indicate that the block in DNA replication in the dnaB mutant involves the inability to perform semiconservative replication of double-stranded RF DNA. For determination of membrane-bound DNA, cells were lysed by lysozyme treatment in the presence of 15% (w/w) sucrose at 00 for 30 min, followed by 3-4 cycles of freezethawing. The lysate was then analyzed by centrifugation in a 20-40% sucrose gradient over a shelf of 60% sucrose (3).Preparation of 82P-labeled M13 phage and 3H-labeled ss M13 DNA and radioactivity assays have been described (4). RESULTS Abortive infection at 410To detect abortive infection under the conditions used, E. coli HfrH 165/70 (dnaB) cells were infected with M13 both at the permissive (340) and restrictive (410)

The various stages of M 13 DNA replication were investigated in an Escherichia coli dnaE mutant thermosensitive in DNA polymerase III after a shift to the restrictive temperature (45°C). The following results were obtained. (1) DNA polymerase III is not needed for the conversion of the infecting single‐stranded phage DNA into the parental replicative form. (2) Semi‐conservative replicative form replication is completely blocked in the absence of a functional DNA polymerase III. (3) Synthesis of phage‐specific DNA continues after a temperature shift late in the infection. However, due to an inherent thermosensitivity of M 13 single‐stranded circular DNA production no free single strands are detected.