Export without proteolytic processing of inner and outer membrane proteins encoded by F sex factor tra cistrons in Escherichia coli minicells.

Abstract

Most tra proteins encoded by the Escherichia coli F sex factor are incorporated into the minicell envelope. We have now assigned the tra proteins to cytoplasm (TraIp and 2b), inner membrane (TraEp, TraMp, and TraSp), and outer membrane (6e, TraAp, TraBp, TraJp, TraKp; TraLp, and TraTp). Two proteins, TraDp and 6d, were associated with both inner and outer membranes. The proteins exported to the inner or outer membranes did not undergo proteolytic cleavage (processing) whereas P-lactamase was processed normally.Many DNA transfer (tra) cistrons encoded by the Escherichia coli F sex factor have been defined (1,2). The F DNA carrying these cistrons has been cloned to yield hybrid plasmids (3, 4). The tra proteins were detected by radioactive labeling of minicells into which the plasmid DNAs had segregated or by adding purified plasmid DNA to an in vitro protein-synthesizing system (5). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis allowed the assignment of the proteins to tra cistrons (5-10). Most of the proteins were incorporated into the envelope of the minicells (5). In contrast, only one protein, TraTp, was detectable in normal cell envelopes (6). (The other tra proteins are synthesized too slowly or degraded too quickly for detection in the presence of cell proteins.) TraTp is an outer membrane protein present in numerous copies per cell (6). It is in part responsible for converting cells that carry the F factor into poor recipients in conjugation (surface exclusion). The other tra proteins are responsible for other aspects of conjugation (1). To understand these aspects better, we decided to separate the inner and outer membranes of minicells that contained the radioactively labelled tra proteins.It already had been noted (6) that TraTp has the same apparent molecular weight whether solubilized from outer membranes or synthesized in vitro. This result suggests that TraTp is exported to the outer membrane without proteolytic processing, unlike other periplasmic and outer membrane proteins (11-16). It seemed important to test the other proteins in this regard and to exclude possible experimental artifacts. MATERIALS AND METHODSMost of the techniques used here have been described (5-7, 17).Minicells were purified from derivatives of strain DS410 (18) carrying chimeric plasmids. We have named the tra proteins according to the cistrons encoding them (e.g., TraTp for the traT gene product) or, when unknown, according to the EcoRI DNA fragment encoding them (fragment f6 for 6c, 6d, 6e, and f2 for 2b). 2b was formerly incorrectly referred to as E7-A (9).Purified f3-lactamase was a gift from R. P. Ambler and G. W.Ross.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 4837Purification of Minicells. Four 2-liter erlenmeyer flasks, each containing 500 ml of L broth (19), were inoculated with 0.1 ml from cells stored in 30% (vol/vol) glycerol/...

Nucleotide sequence of the tra YALE region from IncFV plasmid pED208

“…3 and was 99 residues in length (compared with 94 residues for the equivalent F product). Although this gene did not contain 1 (18). Translation of traL in these two systems is probably coupled to that of traA in that both genes utilize the traA ribosome-binding site (18).…”

VirE1 protein mediates export of the single-stranded DNA-binding protein VirE2 from Agrobacterium tumefaciens into plant cells

“…Production and export of T strands may occur concomitantly at the bacterial membrane, as does nicking and transfer of F plasmid DNA during conjugation (1,17,19,40,41,53). F plasmid encodes an inner membrane protein, TraD, which plays a direct role in DNA translocation (40,41) and also appears to anchor TraI, an oriT-specific endonuclease, to the inner membrane (17).…”

Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri

“…The method used to fractionate E. coli K-12 cells to identify proteins located in the soluble fractions (cytoplasm and periplasm) and insoluble fractions (inner membrane [IM] and outer membrane [OM]) have been described previously (1,34). Briefly, the cultures (10 to 50 ml) were grown to mid-exponential phase (optical density at 600 nm of 0.6), and the cells were treated with a buffer containing Tris, EDTA, sucrose, and lysozyme.…”

“…Samples were adjusted to represent material from equivalent numbers of cells. Samples were solubilized at 100ЊC in sample buffer (25) before being applied to SDS-12 or 15% polyacrylamide gels as described previously (1).…”