Outer membrane of Escherichia coli: properties of the F sex factor traT protein which is involved in surface exclusion.

The traT protein (TraTp) of the F sex factor is the product of one of the two genes involved in surface exclusion. Several detergents were examined under different conditions in order to determine their ability to solubilize TraTp from membrane vesicles. These experiments showed that TraTp behaved similar to a number of peptidoglycan-associated outer membrane proteins and that it existed in multimeric aggregates within the membrane. However, unlike other major outer membrane proteins, the amount of TraTp incorporated into the membrane was not affected by lipopolysaccharide-deficient mutants, even when mutants totally lacking the neutral sugars in their lipopolysaccharide backbone were used. TraTp wqs also examined by two-dimensional gel electrophoresis, where it ran as a discrete spot with a very basic isoelectric point. By coupling cyanogen bromide-activated dextran onto whole cells and by labeling whole cells with 125I (via lactoperoxidase), it was shown that TraTp was exposed on the cell surface. TraTp in a membrane environment was also insensitive to proteolytic attack by trypsin.     

Mutants that overproduce TraTp, a plasmid-specified major outer membrane protein of Escherichia coli

Summary The isolation of a series of plasmid mutant derivatives that overproduce the traT outer membrane protein, TraTp, is described. Some of the mutants directed the synthesis of 10-fold more TraTp (200,000 copies/cell) than did the parental plasmid (20,000 copies/cell). The proteins specified by all mutant plasmids except one were correctly inserted into the outer membrane and exposed on the cell surface. The TraTp that was not correctly inserted did not mediate the expected levels of surface exclusion and serum resistance, suggesting that surface localization is a requirement of TraTp function. The overproduction of TraTp was deleterious to bacterial growth, particularly that of minicell mutants of E. coli K-12.     

Nucleotide sequence of the surface exclusion genes traS and traT from the IncF0 lac plasmid pED208.

pED208 is a 90-kilobase conjugative plasmid belonging to the incompatibility group IncF0 lac. The surface exclusion system from this plasmid was cloned and sequenced, and two genes demonstrated exclusion ability. traS encoded a 186-amino-acid hydrophobic protein which, when transcribed from a vector promoter, caused exclusion of pED208. The product of traT (TraTp) was a 245-residue protein which was highly expressed independently of a vector promoter in Escherichia coli minicells. The TraTp from pED208 was homologous with traT products from the IncF plasmids R-100 and F (80% homology), but recombinants containing the pED208 surface exclusion system excluded F poorly.     

Purification and characterization of pro-TraTp, the signal sequence-containing precursor of a secreted protein encoded by the F sex factor.

The structural gene for the F sex factor outer membrane surface exclusion protein ( traT ) was cloned onto a high-copy-number plasmid where it is expressed from the phage lambda promoter pL. Conditional control over expression was provided by a temperature-sensitive lambda cI repressor. Induction of pL produced large quantities of the traT gene product ( TraTp ) and, in rich growth media, even larger amounts of a higher-molecular-weight form of TraTp . This polypeptide was purified and characterized as a pro- TraTp precursor, which contains at its amino terminus a typical signal-like sequence, which is not present in the mature form of TraTp as isolated from the outer membrane of F-containing cells. Accumulation of pro- TraTp seemed not to result from the jamming of export sites, as in another system for obtaining precursors of secreted proteins, but rather from overwhelming kinetically the ability of the cell to process exported proteins. Although pro- TraTp appeared to be successfully translocated to the outer membrane, it was defective in forming the oligomeric structure required for surface exclusion function. The procedure used is not a general method but can be applied to certain other secreted proteins.     

Overproduction, purification and characterization of the F traT protein

Summary A lambda transducing phage (ED110) which carries the sex factor F surface exclusion genes, traS and traT, was characterized by both genetic and physiochemical techniques. The transducing segment consists of 5.2 kilobases of F tra DNA, and carries the carboxy-terminal onehalf of the upstream traG gene, as well as traS, traT, and the adjacent downstream gene traD. These tra proteins could be identified in infected UV-irradiated cells, and the major part of their synthesis was found to occur from the phage's late promoter p_R under Q control.Lysogens for ED110 were induced and found to greatly overproduce the traT gene product (TraTp), an outer membrane protein normally found in about 20,000 copies per cell, to levels which exceeded the major outer membrane proteins. This led to the development of a simple purification procedure for TraTp, the most important step of which was the construction of an appropriate ompB derivative to eliminate the major outer membrane porin proteins, which have several physical properties in common with TraTp.Purified TraTp was added to mixtures of donor and recipient cells and found to inhibit mating. The specificity of this assay was demonstrated by using an R100-1 donor, which responds to a heterologous surface exclusion system, and by using an altered TraTp containing a missense amino acid substitution.A mechanism by which TraTp mediates surface exclusion is proposed.     

Mapping Loci for Surface Exclusion and Incompatibility on the F Factor of Escherichia coli K-12

A series of Hfr deletion strains carrying deletions extending different distances into the integrated F factor have been used to map loci for surface exclusion (traS) and for incompatibility (inc) on the Escherichia coli K-12 sex factor F. traS mapped between traG and traD. It forms a part of the large operon, including all the known transfer genes except traJ, and is co-controlled with these. The product of traS is not required for formation of the F pilus. inc mapped between the phi(R) (11) locus and the origin of transfer; it is therefore one of the earliest loci transferred during conjugation.     

Five control systems preventing transfer of Escherichia coli K-12 sex factor F.

The transfer inhibition systems of 28 Fin+ plasmids have been characterized, using Flac mutants insensitive to inhibition by R100 or R62. All F-like plasmids (except R455) and one N group plasmid determined systems analogous to that of R100; this is designated the FinOP system. None of these plasmids could supply a FinP component of the transfer inhibitor able to replace that of F itself. In addition to the FinOP and R62 transfer inhibition systems described previously, new systems were encoded by the F-like plasmid R455, the I-like plasmid JR66a, and the group X plasmid R485. Besides inhibiting F transfer, JR66a also inhibited F pilus formation and surface exclusion, whereas R485 inhibited only pilus formation and R455 inhibited neither. All three R factors inhibited transfer of J-independent Flac elements, indicating that they act directly on one or more genes (or products) of the transfer operon, rather than directly via traJ. The tral products and transfer origin sequences of two Fin+ F-like plasmids, ColB2 and R124, appear to have similar specificities to those of F itself.     

Inhibition of Flac Transfer by the Fin+ I-Like Plasmid R62

Flac mutants have been isolated in Escherichia coli K-12 which carry dominant mutations resulting in insensitivity to transfer inhibition by the Fin(+) I-like plasmid R62. These mutants were still sensitive to transfer inhibition by the fin(+) F-like plasmid R100 and, conversely, FlactraO(-) and traP(-) mutants, which are insensitive to R100 inhibition, were still sensitive to R62. The sites of action of the two inhibition systems are therefore different. Furthermore, inhibition by R62, unlike R100, did not require an F-specified product. Like R100, R62 prevented transfer, pilus formation, and surface exclusion and, therefore, probably inhibits expression of the transfer operon traA through traI. However, R62 was different from R100 in inhibiting transfer of J-independent mutants, indicating that its effect on the transfer operon is probably direct rather than via traJ. This is consistent with the different sites of action of the two inhibition systems. None of the Flac mutants overproduced pili in the absence of R62, although one mutant differing from those described above showed increased levels of transfer and surface exclusion.     

Transfer inhibition of RP4 by F factor

When RP4 and F factors were brought together into one E. coli cell, the F factor reduced 500-1000-fold the frequency of transfer of RP4. However, F had almost no effect on the surface exclusion and pilus formation by RP4. In contrast, RP4 did not affect the transfer of F. Using in vitro recombinant DNA techniques, a gene of F responsible for the above-mentioned transfer inhibition of RP4 was located within the BamHI fragment (40.4-42.8 kb) of the mini-F sequence on F. From the result of product analysis using minicells, the responsible gene in the BamHI fragment was inferred to encode the 33 K protein.