Study of envelope proteins in E. coli cet and recA mutants by SDS acrylamide gel electrophoresis

Disc-electrophoresis of E. coli envelope proteins on SDS acrylamide gels reproducibly revealed up to 50 distinct polypeptide bands. Corresponding molecular weights ranged from 105,000 to 20,000 daltons or less. Major bands corresponded to molecular weights of 73,000, 48,000, 36,000 and 30,000 with the latter constituting up to 20% of the total envelope protein depending upon the method of isolation. Minimum levels of detection using stained gels equaled 0.25 μg protein or 1% of total sample analyzed; for a polypeptide of molecular weight 40,000 daltons this was calculated to be equivalent to 1,200 molecules per cell envelope. In envelopes from a cetB^? mutant strain (refractory to colicin E2), an additional band, constituting up to 5% of the total envelope protein was present. The molecular weight of this protein, which was maximally present in wild type envelopes in only trace amounts, is 44,000 daltons, indicating a cellular concentration of approximately 6 × 10³ molecules per envelope. This new band was not affected by heating envelope preparations to 100° prior to electrophoresis, but was largely eliminated by washing isolated envelopes in low ionic strength buffer, or by pre-incubating cells with trypsin prior to preparation of envelopes. Treatment of isolated envelopes with Triton X-100, which preferentially releases inner membrane proteins from the envelope (18), resulted in the extraction of a preponderance of the high molecular weight polypeptides, including the 44,000 dalton protein from envelopes of the mutant. The major polypeptides of the envelope and the low molecular weight components were not extracted by Triton X-100. The properties of the 44,000 dalton protein indicated that it is relatively loosely associated with the surface envelope and may be exposed on the external surface of the cytoplasmic membrane. Possible explanations for the appearance of this protein in mutant strains and its relationship to the inability of these to respond, specifically to surface bound colicin E2, will be discussed. Extensive analysis of envelopes from recA^? mutants was also carried out and revealed an unusual amount of variation in polypeptide profiles obtained from different preparations. However, no consistent quantitative or qualitative difference between recA and rec⁺ strains was obtained. In recA, cetB double mutants, the increased level of the 44,000 dalton polypeptide was identical to that found in the rec⁺, cetB mutant.