Purification and properties of the F sex factor TraD protein, an inner membrane conjugal transfer protein.

Using a traD overexpression plasmid, we purified the F sex factor TraD protein in milligram quantities. The purified protein has an apparent molecular weight of 82,000 and an amino acid composition rich in acidic residues. Using specific antibodies, TraD was localized to the inner membrane of F+ cells under conditions where it is produced in physiologically normal amounts. Furthermore, the protein was soluble only in the presence of detergents, but there is evidence that the carboxyl terminus is water-soluble. The purified protein shows pH-sensitive binding to DNA cellulose columns.

Biochemical characterization of Escherichia coli DNA helicase I.

The gene product of F tral is a bifunctional protein which nicks and unwinds the F plasmid during conjugal DNA transfer. Further biochemical characterization of the Tral protein reveals that it has a second, much lower, Km for ATP hydrolysis, in addition to that previously identified. Measurement of the single-stranded DNA-stimulated ATPase rate indicates that there is co-operative interaction between the enzyme monomers for maximal activity. Furthermore, 18O-exchange experiments indicate that Tral protein hydrolyses ATP with, at most, a low-level reversal of the hydrolytic step during each turnover.

Regulation and utilization of microsin promoter turn on in a chromosomal fusion.

Prior work has demonstrated that the microsin antibiotics are produced by enteric bacteria when the growth medium is depleted of nutrients. Because the control loci could have biotechnical potential, and general stress-response phenomena are of importance to understanding how bacteria survive in natural and bioreactor environments, we examined further the growth rate dependence of gene expression under the control of the microsin B17 promoter. This work entailed performing batch and chemostat growth experiments with a strain of E. coli K-12 containing a mcbA-lacZ gene fusion in the chromosome. Our results indicate that when a culture is presented with excess respiratory substrate, a well defined growth rate exists, below which a significant induction event occurs. However, cultures that are fermenting or highly glycolytic tend to express poorly. Additionally, the utility of the fusion strain was examined by performing fed-batch cultivation experiments. We found that sustained production in a fed-batch reactor can be accomplished by using a straightforward, exponential nutrient feeding profile.

Nucleotide sequence of the traI (helicase I) gene from the sex factor F.

A 6.9-kilobase region of the Escherichia coli F plasmid containing the 3' half of the traD gene and the entire traI gene (encodes the TraI protein, DNA helicase I and TraI, a polypeptide arising from an internal in-frame translational start in traI) has been sequenced. A previously unidentified open reading frame (tentatively trbH) lies between traD and traI.

Evidence that DNA helicase I and oriT site-specific nicking are both functions of the F TraI protein.

Site-specific and strand-specific nicking at the origin of transfer (oriT) of the F sex factor is the initial step in conjugal DNA metabolism. Then, DNA helicase I, the product of the traI gene, processively unwinds the plasmid from the nick site to generate the single strand of DNA that is transferred to the recipient. The nick at oriT is produced by the combined action of two Tra proteins, TraY and TraZ. The traZ gene was never precisely mapped, as no available point mutation uniquely affected TraZ-dependent oriT nicking. With several new mutations, we have demonstrated that TraZ activity is dependent upon traI DNA sequences. The simplest interpretation of this finding is that the F TraI protein is bifunctional, with DNA unwinding and site-specific DNA nicking activities.

Revised genetic map of the distal end of the F transfer operon: implications for DNA helicase I, nicking at oriT, and conjugal DNA transport.

The DNA transfer stage of conjugation requires the products of the F sex factor genes traMYDIZ and the cis-acting site oriT. Previous interpretation of genetic and protein analyses suggested that traD, traI, and traZ mapped as contiguous genes at the distal end of the transfer operon and saturated this portion of the F transfer region (which ends with an IS3 element). Using antibodies prepared against the purified TraD and TraI proteins, we analyzed the products encoded by a collection of chimeric plasmids constructed with various segments of traDIZ DNA. We found the traI gene to be located 1 kilobase to the right of the position suggested on previous maps. This creates an unsaturated space between traD and traI where unidentified tra genes may be located and leaves insufficient space between traI and IS3 for coding the 94-kilodalton protein previously thought to be the product of traZ. We found that the 94-kilodalton protein arose from a translational restart and corresponds to the carboxy terminus of traI; we named it TraI*. The precise physical location of the traZ gene and the identity of its product are unknown. The oriT nicking activity known as TraZ may stem from unassigned regions between traD and traI and between traI and IS3, but a more interesting possibility is that it is actually a function of traI. On our revised map, the position of a previously detected RNA polymerase-binding site corresponds to a site at the amino terminus of traI rather than a location 1 kilobase into the coding region of the gene. Furthermore, the physical and genetic comparison of the F traD and traI genes with those of the closely related F-like conjugative plasmids R1 and R100 is greatly simplified. The translational organization we found for traI, together with its identity as the structural gene for DNA helicase I, suggests a possible functional link to several other genes from which translational restart polypeptides are expressed. These include the primases of the conjugative plasmids ColI and R16, the primase-helicase of bacteriophage T7, and the cisA product (nickase) of phage phi X174.

DNA transfer occurs during a cell surface contact stage of F sex factor-mediated bacterial conjugation.

Donor bacteria containing JCFL39, a temperature-sensitive traD mutant of the F sex factor, were used at the nonpermissive temperature to accumulate stable mating pairs with recipient cells. At this stage in conjugation, extracellular F pili were removed by treatment with 0.01% sodium dodecyl sulfate. Upon then shifting to the permissive temperature for JCFL39, transfer of the F plasmid was observed. The mating pairs that were accumulated with JCFL39 at the nonpermissive temperature were readily observed by electron microscopy in wall-to-wall contact with the recipient bacteria. These results demonstrate that the traD product, which is known to be required in transferring DNA to a recipient bacterium, acts after the stage at which extracellular F pili are required. In addition, we concluded that DNA transfer takes place while donor and recipient cells are in surface contact and not necessarily through an extended F pilus as envisioned in some models of bacterial conjugation.

Escherichia coli DNA polymerase I. Construction of a polA plasmid for amplification and an improved purification scheme.

Previous attempts to clone the Escherichia coli polA+ gene onto a high copy number plasmid were unsuccessful. The apparent lethality of unregulated overproduction of DNA polymerase I can be eliminated by cutting at a BglII site 100 nucleotides upstream from the ATG start codon of the polA gene. This permitted the construction of plasmid pMP5 which contains both the coding sequence for DNA polymerase I and the lambda pL promoter for conditional control of polA gene expression. BglII cutting only damages but does not eliminate the polA promoter activity; the BglII site thus lies within the polA promoter region. Leakiness of the damaged polA promoter results in overproduction of DNA polymerase I even under conditions where pL is fully repressed. This overproduction is inhibitory of cell growth, as reflected in both growth rate and in the frequency of appearance of mutant plasmids which are nonproducers of DNA polymerase I. Transformation of plasmid pMP5 into E. coli N4830 yields strain ATL100 which under inducing conditions provides 138-fold amplification of DNA polymerase I. Optimization of growth and expression conditions are presented together with an optimized rapid polymerase purification scheme. In addition to providing a convenient source for preparation of DNA polymerase I, this work serves as the basis for a future detailed molecular genetic analysis of the polA gene product.

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.

The product of the F sex factor traT surface exclusion gene is a lipoprotein.

The product of the Escherichia coli sex factor F traT gene (TraTp), an outer membrane protein of Mr = 25,000, is covalently modified in vivo by the addition of glycerol and fatty acids. Consistent with this result, and as would be expected for a bacterial lipoprotein, the novel amino acid glycerylcysteine can be detected in purified TraTp. Being a secreted protein, TraTp is made from a signal sequence containing precursor, and glycerol and fatty acids can be detected in both the precursor and mature (processed) species of TraTp. The peptide antibiotic globomycin inhibits the cleavage of the pro-TraTp signal sequence, but not the glycerol and fatty acid modification. Diglyceride modification of the Cys residue at the site of signal sequence cleavage probably precedes and is a prerequisite for processing of the TraTp signal sequence. Thus, TraTp appears to be a typical E. coli lipoprotein, having a pathway for modification and processing that is similar to that of Braun's lipoprotein (the major outer membrane lipoprotein).

Overproduction in Escherichia coli K-12 and purification of the TraJ protein encoded by the conjugative plasmid F.

The TraJ protein encoded by the conjugative plasmid F has been designated an Escherichia coli K-12 envelope protein that participates in a mechanism of gene regulation. We have purified the TraJ protein, using an Flac::lambda traJ lysogen that overproduces the protein after heat induction of the prophage. Sufficient TraJ protein was synthesized within 40 min after induction to follow the purification by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Our purification exploited the observation that the TraJ protein remains insoluble after repeated Triton X-100/EDTA extractions of crude envelope fractions. The protein was then solubilized in sodium dodecyl sulfate at 60 degrees C and fractionated further by gel filtration and hydroxylapatite chromatography, both in the presence of sodium dodecyl sulfate. After hydroxylapatite chromatography, the protein was greater than 95% pure. The identity of the purified protein was confirmed by analysis of its NH2-terminal amino acid sequence, which was the same as that predicted from the partial nucleotide sequence of the traJ gene (Thompson, R., and Taylor, L. (1982) Mol. Gen. Genet. 188, 513-518). This analysis also indicated that the TraJ protein is localized in the cell without proteolytic modification of its NH2-terminus. We discuss the possible significance of these observations with respect to the cellular functions of the TraJ protein.

Overproduction, purification and characterization of the F traT protein.

A lambda transducing phage (ED lambda 110) 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 one-half 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 pR' under Q control. Lysogens for ED lambda 110 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.

Identification of a membrane protein associated with expression of the surface exclusion region of the F transfer operon.

Membrane preparations from radioactively labeled male and female strains of Escherichia coli K-12 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. An intensely labeled band corresponding to a protein of molecular weight of 24,000 was readily apparent in preparations from Hfr and F-prime strains but not in those from female strains. When preparations from a series of Hfr strains containing transfer operon deletions were examined, presence of the band was found to be associated with retention of the region of the F transfer operon between ilzA and traD. Thus, the band ("protein S") appears to be the product of an F tra operon activity corresponding to traS (the gene for surface or entry exclusion), or an unknown gene in its vicinity. As predicted, protein S was subject to Fin+ control; only a faint band was detectable if the repressed plasmid R100 was also present in the F lac strain. A 24,000-dalton protein was also found in membrane preparations from strains carrying the derepressed plasmids R100-1 and R1-19 but not in those from strains carrying the repressed plasmids R100 or R1. Thus, the appearance of protein S in the membrane may be a general phenomenon resulting from transfer operon expression of F-like plasmids.