The OmpA and OmpF proteins are major outer membrane proteins ofEscherichia coli K12. Their precursors, the pro‐OmpA and pro‐OmpF proteins, have been detected in vivo in pulse‐labelling experiments carried out with [35S]methionine at 25°C. When the pulse was at 37°C, however, no precursors were detected. The pulse‐labelled precursors were processed rapidly and quantitatively into mature protein at 25°C. The apparent half‐life of the pro‐OmpF protein was estimated to be 30 s, and the pro‐OmpA protein may be processed even faster. In short pulses (10 s) the precursors of both proteins were the predominant labelled species, indicating that at 25°C processing does not start until chain elongation of the precursor is almost, if not entirely, complete. When French press lysates of cells pulse‐labelled for 10 s were subjected to sucrose gradient centrifugation to separate the inner and outer membranes, both precursors comigrated with the inner membrane.

A mutant of the R plasmid Rl which synthesizes a f,-lactamase with altered kinetic characteristics was isolated. The level of penicillin resistance specified by this plasmid was correctly predicted from the properties of the wild-type Rl according to a simple theoretical model published by Zimmermann and Rosselet (Antimicrob. Agents Chemother. 12:368-372, 1977). The model also accounts for the high level of penicillin resistance specified by the R plasmid RP1.A theoretical model which attempts quantitatively to account for the level of penicillin resistance determined by periplasmic /-lactamases in gram-negative bacteria has been described (22

We describe here the isolation of a mutant derivative of the drug resistance factor R1 (Meynell and Datta, 1966) that carries a nonsense mutation in a gene determining resistance to penicillins. We have used this mutant R1 to isolate derivatives of Escherichia coli and Klebsiella pneumoniae that contain nonsense suppressors (Sup- strains) by screening penicillin-resistant revertants of strains containing the mutant R factor for the presence of such suppressors. This obviates the need to have known nonsense mutations in chromosomal genes. Theoretically, suppressor-containing derivatives of any bacterial species that can maintain and express R1 can be constructed.

The rate of synthesis of the OmpA and OmpF proteins, two of the major outer membrane proteins of E.sckerichia coli K12, was determined. At 25 "C both proteins were translated at 6.5 amino acids/s, and the OmpF protein was translated at 15 amino acids/s at 37 "C. The former rate corresponded to a synthesis time of just over SO s for both proteins, which is significantly faster than their reported rates of assembly into the outer membrane at 25 "C. The kinetics of processing of the pro-OmpF protein were also investigated in detail, and the pro-OmpF half-life estimated to be 3 -5 s at 25 "C. However a fraction of the precursor was processed more slowly, which may explain the discrepancy between these data and our earlier published estimate of 30 s. Pro-OnipA protein was processed with similar kinetics. These results demonstrate that the rate-limiting step in the assembly of both proteins into the outer membrane is post-translational and follows the processing step.The outer membrane proteins of gram-negative bacteria share a number of biosynthetic steps with two other classes of exported protein, the inner membrane and periplasmic proteins. These include: translation, export (i.e. transfer across the inner membrane) and processing (for proteins synthesized initially in precursor form). In addition, outer membrane proteins must be translocated to the outer membrane. Despite these similarities the rate of assembly of outer membrane proteins is two or three times slower than that of inner membrane proteins (though perhaps faster than that of periplasmic proteins) at 25-30 "C [I -41. Intuitively it would seem that the rate-limiting step in outer membrane protein assembly must be either translocation (the only step unique to outer membrane proteins) or processing (which should not affect the kinetics of inner membrane protein assembly). In an earlier paper [ S ] we reported that newly synthesized pro-OmpF protein had a relatively long half-life of 30 s, suggesting that the processing reaction might be ratelimiting for assembly. However, we also found that newly synthesized, processed molecules of both OmpF and OmpA proteins were present in the inner membrane after a 10-s pulse ; these results suggest that translocation may be rate-These results are at odds with those of Lin and Wu [2], who have suggested that the rate-limiting step in outer membrane protein assembly is translation of the niRNA. However, their evidence is indirect and not in accordance with the data of Boyd and Holland [6], who could detect no difference between the 'run-out' times (a crude measure of the translation rate) of the OmpF protein and a representative cytoplasmic protein in Escherichia coli Bjr.In view of these problems we decided to reinvestigate the kinetics of processing of the OmpF and OmpA proteins and to measure directly their rates of synthesis. Our results indicate that neither processing nor translation are slow enough to permit them to be considered rate-limiting. Since the only steps to follow processing are translocation and irre...

Thirty-eight mutants of Rl, an R plasmid specifying the type IIIa (TEM) ,Blactamase, were isolated; these mutants are partially or totally unable to synthesize the type IIIa f8-lactamase. The loss of fl-lactamase activity was associated with a reduction in the level of penicillin resistance conferred by the mutants upon their host strain. At least two of the mutants synthesized a ,B-lactamase with altered substrate specificity. These properties are compared with those of two f8-lactamase-deficient mutants of plasmid RP1. The results suggest that, for both R plasmids, penicillin resistance is entirely attributable to the presence of f,-lactamase activity. The properties of two Rl derivatives, pUB251 and pUB252, which have phenotypes similar to that of RP1, support this conclusion.