M Klose

The outer membrane protein OmpA of Escherichia coli K-12 serves as a receptor for a number of T-even-like phages. We have isolated a series of ompA mutants which are resistant to such phages but which still produce the OmpA protein. None of the mutants was able to either irreversibly or reversibly bind the phage with which they had been selected. Also, the OmpA protein is required for the action of colicins K and L and for the stabilization of mating aggregates in conjugation. Conjugal proficiency was unaltered in all cases. Various degrees of colicin resistance was found; however, the resistance pattern did not correlate with the phage resistance pattern. DNA sequence analyses revealed that, in the mutants, the 325-residue OmpA protein had suffered the following alterations: Gly-65----Asp, Gly-65----Arg, Glu-68----Gly, Glu-68----Lys (two isolates), Gly-70----Asp (four isolates), Gly-70----Val, Ala-Asp-Thr-Lys-107----Ala-Lys (caused by a 6-base-pair deletion), Val-110----Asp, and Gly-154----Ser. These mutants exhibited a complex pattern of resistance-sensitivity to 14 different OmpA-specific phages, suggesting that they recognize different areas of the protein. In addition to the three clusters of mutational alterations around residues 68, 110, and 154, a site around residue 25 has been predicted to be involved in conjugation and in binding of a phage and a bacteriocin (R. Freudl, and S. T. Cole, Eur. J. Biochem, 134:497-502, 1983; G. Braun and S. T. Cole, Mol. Gen. Genet, in press). These four areas are regularly spaced, being about 40 residues apart from each other. A model is suggested in which the OmpA polypeptide repeatedly traverses the outer membrane in cross-beta structure, exposing the four areas to the outside.

A series of overlapping deletions has been constructed in the ompA gene which encodes the 325-residue Escherichia coli outer membrane protein OmpA. Immunoelectron microscopy showed that the OmpA fragments were either located in the periplasmic space or were associated with the outer membrane. Apparently an area between residues 154 and 180 is required for this association; all proteins missing this area were found to be periplasmic. The nature of this association remained unknown; no membrane-protected tryptic fragments could be identified for any of these polypeptides. Hybrid genes were constructed encoding parts of the periplasmic maltose binding protein and an area of the ompA gene coding for residues 154-274. The corresponding proteins were not localized to the outer membrane but remained attached to the outer face of the plasma membrane, possibly because the normal mechanism of release from this membrane was impaired. In the OmpA protein the conspicuous sequence Ala180-Pro-Ala-Pro-Ala-Pro-Ala-Pro187 exists. Frameshift mutants were constructed to eliminate this sequence. There was no effect on the incorporation of the mutant proteins into the outer membrane. Thus, this "hinge" region is not involved in sorting. A proposal suggesting the existence of a sorting signal common to several outer membrane proteins (Benson, S. A., Bremer, E., and Silhavy, T. J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3830-3834) was subsequently rejected (Bosch, D., Leunissen, J., Verbakel, J., de Jong, M., van Erp, H., and Tommassen, J. (1986) J. Mol. Biol. 189, 449-455; Freudl, R., Schwarz, H., Klose, M., Movva, N. R., and Henning, U. (1985) EMBO J. 4, 3593-3598). Although it is not known whether or not the outer membrane association observed represents a step in the normal sorting mechanism, it is concluded that it remains an open question whether or not a sorting signal, as proposed originally, exists in outer membrane proteins.

SecA is the precursor protein binding subunit of the bacterial precursor protein translocase, which consists of the SecY/E protein as integral membrane domain. SecA is an ATPase, and couples the hydrolysis of ATP to the release of bound precursor proteins to allow their proton-motive-force-driven translocation across the cytoplasmic membrane. A putative ATP-binding motif can be predicted from the amino acid sequence of SecA with homology to the consensus Walker A-type motif. The role of this domain is not known. A lysine residue at position 106 at the end of the glycine-rich loop in the A motif of the Bacillus subtilis SecA was replaced by an asparagine through site-directed mutagenesis (K106N SecA). A similar replacement was introduced at an adjacent lysine residue at position 101 (K101N SecA). Wild-type and mutant SecA proteins were expressed to a high level and purified to homogeneity. The catalytic efficacy (kcat/km) of the K106N SecA for lipid-stimulated ATP hydrolysis was only 1% of that of the wild-type and K101N SecA. K106N SecA retained the ability to bind ATP, but its ATPase activity was not stimulated by precursor proteins. Mutant and wild-type SecA bind with similar affinity to Escherichia coli inner membrane vesicles and insert into a phospholipid monolayer. In contrast to the wild type, membrane insertion of the K106N SecA was not prevented by ATP. K106N SecA blocks the ATP and proton-motive-force-dependent chase of a translocation intermediate to fully translocated proOmpA. It is concluded that the GKT motif in the amino-terminal domain of SecA is part of the catalytic ATP-binding site. This site may be involved in the ATP-driven protein recycling function of SecA which allows the release of SecA from its association with precursor proteins, and the phospholipid bilayer.

The SecA protein is a major component of the cellular machinery that mediates the translocation of proteins across the Escherichia coli plasma membrane. The secA gene from Bacillus subtilis was cloned and expressed in E. coli under the control of the lac or trc promoter. The temperature-sensitive growth and secretion defects of various E. coli secA mutants were complemented by the B. subtilis SecA protein, provided the protein was expressed at moderate levels. Under overproduction conditions, no complementation was observed. One of the main features of the SecA protein is the translocation ATPase activity which, together with the protonmotive force, drives the movement of proteins across the plasma membrane. A putative ATP-binding motif can be identified in the SecA protein resembling the consensus Walker A type motif. Replacement of a lysine residue at position 106, which corresponds to an invariable amino acid residue, in the consensus motif by asparagine (K106N) resulted in the loss of the ability of the B. subtilis SecA protein to complement the growth and secretion defects of E. coli secA mutants. In addition, the presence of the K106N SecA protein interfered with protein translocation, most likely at an ATP-requiring step. We conclude that lysine 106 is part of the catalytic ATP-binding site of the B. subtilis SecA protein, which is required for protein translocation in vivo.