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.     

Surface exclusion genes traS and traT of the F sex factor of Escherichia coli K-12. Determination of the nucleotide sequence and promoter and terminator activities

The DNA encoding the surface exclusion genes traS and traT of the F sex factor of Escherichia coli K-12 has been sequenced and the biological activity of the various terminators and promoters determined. The data show that traS encodes a 16,861 Mr protein with no apparent signal sequence, as expected for its cytoplasmic membrane location. The protein is extremely hydrophobic. traS has its own promoter and a weak terminator region follows the gene. After the traS termination loop there is a small intergenic region before the traT promoter. The traT gene encodes a 25,932 Mr precursor for the 23,709 Mr mature protein. The amino-terminal signal peptide is 21 amino acid residues, consistent with it being an outer membrane lipoprotein. A very strong termination loop follows the gene and adjacent to this a further loop can be predicted from the sequence. These secondary structures would be expected to enhance the stability of the mRNA in the presence of 3' specific ribonucleases accounting for the apparent long half-life of the messenger. The amino acid sequence of the mature product of traT of F differs from that of R100 by only a single amino acid substitution (Gly for Ala at position 119), whereas that of pED208 (Folac) differs at 40 positions. traT lies in a region of heteroduplex homology between F and R100, and the nucleotide sequence confirms this and demonstrates that this homology breaks down immediately preceding and following the coding region. Sequence analysis shows that this is also so for pED208. Thus the entire traS of F, R100 and pED208 are very different at the DNA level. An open reading frame, preceded by a typical promoter sequence and a weak and poorly located Shine-Dalgarno sequence, follows traT and corresponds to the start of traD. Alone, this promoter appears to be inactive.     

traJ independence in expression of traT on F.

Studies of F plasmids in the same cell as a transfer-repressed IncFII R plasmid showed a 100 to 1,000-fold decrease in transfer and a 75-fold decrease in surface exclusion, but no detectable change was shown in the amount of TraTp synthesized. Moreover, a mutation in traJ on F which caused a 10(4)-fold reduction in transfer caused only a 3.6-fold decrease in TraTp. These two findings suggest that a significant amount of traT expression on F is independent of traJ. Furthermore, we showed, using immunoprecipitation of TraTp, that normal amounts of this protein could be present in the cell without producing normal levels of surface exclusion.     

Characterization of the oriT region of the IncFV plasmid pED208

DNA sequence analysis of a 2.2kb EcoRI-HindIII fragment from pED208, the derepressed form of the IncFV plasmid Folac, revealed sequences highly homologous to the oriT region, traM, and traJ genes of other IncF plasmids. The TraM protein was purified and immunoblots of fractionated cells containing pED208 or Folac showed that TraM was predominantly in the cytoplasm. Using DNA retardation assays and the DNase I footprinting technique, the TraM protein was found to bind to three large motifs in the oriT region: (I) an inverted repeat, (II) two direct repeats, and (III) the traM promoter region. These three footprint regions contained a Hinfl-like sequence (GANTC) that appeared 16 times, spaced 11-12 bp (or multiples thereof) apart, suggesting that TraM protein binds in a complex manner over this entire region.     

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.     

Nucleotide sequence of the tra YALE region from IncFV plasmid pED208

The pED208 plasmid is a 90-kilobase conjugative plasmid which is the derepressed form of Fo lac plasmid (IncFV). A 3.3-kilobase HindIII-PstI fragment from the pED208 plasmid was cloned and sequenced and was found to contain four open reading frames which were highly homologous to the traA, traL, traE, and traY gene products of the F plasmid. The pED208 traA propilin protein was 119 amino acids in length, consisting of a leader sequence of 55 amino acids and a mature pilin subunit of 64 residues. The leader sequence contained a hydrophobic region followed by a classic signal peptidase cleavage site (Ala-Ser-Ala-55). F and pED208 pilin proteins shared 27 conserved residues and had similar predicted secondary structures. The pED208 traA and traL genes were separated by a single base pair, and no ribosome binding site preceded the traL gene. The pED208 traY gene contained an IS2 insertion element in orientation II 180 nucleotides (60 residues) upstream of the traY stop codon. This insertion of IS2 resulted in a predicted fusion peptide of 69 residues for traY which may provide the observed traY activity. Since IS2 is absent in the wild-type plasmid, Fo lac, derepression and concomitant multipiliation may be due to the insertion of IS2 providing constitutive expression of the pED208 tra operon.     

Analysis and characterization of the IncFV plasmid pED208 transfer region

pED208 is a transfer-derepressed mutant of the IncFV plasmid, F(0)lac, which has an IS2 element inserted in its traY gene, resulting in constitutive overexpression of its transfer (tra) region. The pED208 transfer region, which encodes proteins responsible for pilus synthesis and conjugative plasmid transfer, was sequenced and found to be very similar to the F tra region in terms of its organization although most pED208 tra proteins share only about 45% amino acid identity. All the essential genes for F transfer had homologs within the pED208 transfer region with the exception of traQ, which encodes the chaperone for stable F-pilin expression. F(0)lac appears to have a fertility inhibition system different than the FinOP system of other F-like plasmids, and its transfer efficiency was increased in the presence of F or R100, suggesting that it could be mobilized by these plasmids. The F-like transfer systems specified by F, R100, and F(0)lac were highly specific for their cognate origins of transfer (oriT) as measured by their abilities to mobilize chimeric oriT-containing plasmids.     

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.     

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.     

Cloning, mutation, and location of the F origin of conjugal transfer.

pED806 , a pBR322 derivative carrying the origin of transfer ( oriT ) of F, was rapidly lost from cells carrying an F tra+ plasmid. Instability was increased in a RecA- host, and depended in particular upon the Ftra YZ genes that produce the nick at oriT at which transfer is initiated. Instability was also correlated with the orientation of the oriT fragment in the vector plasmid. Mutants of pED806 selected as being stable in the presence of Flac proved to carry cis-dominant oriT mutations. The oriT site was subcloned from pED806 on a HaeII fragment including a HaeII-Bg/II segment of F DNA approximately 385 base pair (bp) long into the 2.25 kilobase (kb) vector plasmid pED825 , giving pED822 . pED822 was fully proficient for oriT function, and recircularised in recipient cells by a recA- and tra-independent oriT -specific ligation/recombination event. ' Phasmids ' constructed by cloning pED806 or an oriT - mutant into a lambda vector were used to confirm that the nick site in lambda oriT phages grown in the presence of Flac tra+ is indeed at oriT . The nick site in a further lambda oriT phage (ED lambda 102) was then located 140 +/- 20 bp from the Bg/II site forming one terminus of the F fragment cloned in pED806 and pED822 .     

Characterization and sequence analysis of pilin from F-like plasmids.

Conjugative pili are expressed by derepressed plasmids and initiate cell-to-cell contact during bacterial conjugation. They are also the site of attachment for pilus-specific phages (f1, f2, and QB). In this study, the number of pili per cell and their ability to retract in the presence of cyanide was estimated for 13 derepressed plasmids. Selected pilus types were further characterized for reactivity with anti-F and anti-ColB2 pilus antisera as well as two F pilus-specific monoclonal antibodies, one of which is specific for a sequence common to most F-like pilin types (JEL92) and one which is specific for the amino terminus of F pilin (JEL93). The pilin genes from eight of these plasmids were cloned and sequenced, and the results were compared with information on F, ColB2, and pED208 pilin. Six pilus groups were defined: I, was F-like [F, pED202(R386), ColV2-K94, and ColVBtrp]; IIA was ColB2-like in sequence but had a lowered sensitivity to f1 phage due to its decreased ability for pilus retraction [pED236(ColB2) and pED203(ColB4)]; IIB was ColB2-like but retained f1 sensitivity [pED200(R124) and pED207(R538-1)]; III contained R1-19, which had a ColB2-like amino terminus but had an additional lysine residue at its carboxy terminus which may affect its phage sensitivity pattern and its antigenicity; IV was R100-1-like [R100-1 and presumably pED241(R136) and pED204(R6)] which had a unique amino-terminal sequence combined with a carboxy terminus similar to that of F. pED208(Folac) formed group V, which was multipiliated and exhibited poor pilus retraction although it retained full sensitivity to f1 phage. The pED208 pilin gene could not be cloned at this time since it shared no homology with the pilin gene of the F plasmid.     

Structural basis of cooperative DNA recognition by the plasmid conjugation factor, TraM

The conjugative transfer of F-like plasmids such as F, R1, R100 and pED208, between bacterial cells requires TraM, a plasmid-encoded DNA-binding protein. TraM tetramers bridge the origin of transfer (oriT) to a key component of the conjugative pore, the coupling protein TraD. Here we show that TraM recognizes a high-affinity DNA-binding site, sbmA, as a cooperative dimer of tetramers. The crystal structure of the TraM-sbmA complex from the plasmid pED208 shows that binding cooperativity is mediated by DNA kinking and unwinding, without any direct contact between tetramers. Sequence-specific DNA recognition is carried out by TraM's N-terminal ribbon-helix-helix (RHH) domains, which bind DNA in a staggered arrangement. We demonstrate that both DNA-binding specificity, as well as selective interactions between TraM and the C-terminal tail of its cognate TraD mediate conjugation specificity within the F-like family of plasmids. The ability of TraM to cooperatively bind DNA without interaction between tetramers leaves the C-terminal TraM tetramerization domains free to make multiple interactions with TraD, driving recruitment of the plasmid to the conjugative pore.     

Expression of F plasmid traT: independence of traY----Z promoter and traJ control

We have shown, using an F-derived Tra+ cosmid in conjunction with the infected-cell translational system and a time-course study, that one of the surface exclusion genes, traT, can be expressed independently of the promoter of the traY----Z operon, PYZ, and in the absence of a normal quantity of traJ gene product. Studies with deleted derivatives of the cosmid pRS2405 confirmed this independence and also indicated that expression of traD can be independent of PYZ. We propose that the expression of traT by these deleted plasmids is directed from a traJ-independent promoter, PT, located adjacent to traT.     

Nucleotide sequence of the traD region in the Escherichia coli F sex factor

The complete nucleotide sequence has been determined of a 3635-bp region, extending from the HpaI site in traT, at F coordinate 90.3 kb, to beyond the end of traD, of the F sex factor plasmid of Escherichia coli K-12. This region contains the C-terminal coding part of traT and the entire traD gene. An open reading frame (ORF) of 2148 bp within the sequence confirms that traD encodes an 81.4-kDa cytoplasmic membrane protein. The TraD protein has several regions with an unusually high pI (greater than 10), suggesting that they may correspond to the DNA-binding domains. Several other ORFs were detected within the region including the gene (ORF1) for a 26.3-kDa protein and ORF2, probably corresponding to traI, which continues to the end of the sequence. An ORF for an 8.5-kDa protein preceded by an excellent promoter and ribosome-binding site is present in the region following traD but on the opposite strand. This promoter is thought to correspond to the major RNA polymerase binding site in this region, implying that traI does not have its own promoter. The lack of a typical terminator following traD and ORF1 and the translational coupling provided by overlapping stop and start codons is consistent with this conclusion.     

Biochemical and immunological characterization of an R plasmid-encoded protein with properties resembling those of major cellular outer membrane proteins.

MRB, a major R222 plasmid-encoded protein previously described by us, is synthesized in large amounts in host Escherichia coli cells, where it is located principally in the outer membrane. Most of this protein is also bound to the peptidoglycan layer in a form which is trypsin resistant. Its monomeric molecular weight is about 29,000, but it is isolated from cell membranes in aggregate molecular weights of more than 100,000. These properties demonstrate a strong similarity between MRB and porins, major outer membrane proteins of host E. coli cells. They suggest that MRB may have an as-yet unidentified transport function, as do cellular outer membrane proteins with similar biochemical properties. By using antiserum specific for MRB, we demonstrated identity between MRB and the product of the traT gene, one of the surface exclusion proteins on the F plasmid. The synthesis of MRB was found to be constitutive, in contrast to other tra genes, which appear to be under more rigid regulation by the tra operon. These findings suggest that on R222 and other F-like R plasmids this protein has its own promoter.     

The outer membrane permeability mutation of the virulence-associated plasmid of Salmonella typhimurium is located in a traT-like gene

Abstract The SS-A mutation carried by the virulence-as-associated plasmid of Salmonella typhimurium results in increased outer membrane permeability to hydrophobic compounds. A 7.8-kilobase pair Bam HI- Sal I fragment containing the SS-A mutation was cloned from the virulence-associated plasmid into the cloning vector pACYC184. The cloned DNA segment hybridized with a radioactive probed prepared from the traT gene of R6-5. A similar DNA fragment, cloned from the wild-type virulence-associated plasmid, complemented the SS-A mutant phenotype. Both clones produced a protein that immunologically resembled the R6-5 TraT protein; however, the protein produced by the SS-A containing clone appeared truncated by approximately M _r 1000 indicating an alteration in the primary structure or processing of the protein. We conclude that the mutation producing the SS-A phenotype has occured in a traT -like gene of the Salmonella plasmid.     

Tn2301, a transposon construct carrying the entire transfer region of the F plasmid.

The largest R . BamHI fragment of the plasmid F, which carries the entire F conjugation system, has been cloned into the single R . BamHI site of the ampicillin (Ap) resistance transposon TN1. pDS1106 (ColE1 mob::Tn1) was the vector plasmid, and the resultant conjugative plasmid, pED830, was characterized both genetically and by restriction enzyme analysis. The transposon construct, denoted Tn2301, was transposable at frequencies similar to Tn1 to small nonconjugative plasmids or to the Escherichia coli host chromosome. In the former case, Apr conjugative plasmids were obtained, whereas in the latter case, Hfr strains resulted. Representative Hfr strains were characterized by quantitative and interrupted mating experiments. Extension of this technique for Hfvr formation should aid chromosome mapping both in E. coli and in other bacterial genera.     

Mechanistic Basis of Plasmid-Specific DNA Binding of the F Plasmid Regulatory Protein,TraM

The conjugative transfer of bacterial F plasmids relies on TraM, a plasmid-encoded protein that recognizes multiple DNA sites to recruit the plasmid to the conjugative pore. In spite of the high degree of amino acid sequence conservation between TraM proteins, many of these proteins have markedly different DNA binding specificities that ensure the selective recruitment of a plasmid to its cognate pore. Here we present the structure of F TraM RHH (ribbon–helix–helix) domain bound to its sbmA site. The structure indicates that a pair of TraM tetramers cooperatively binds an underwound sbmA site containing 12 base pairs per turn. The sbmA is composed of 4 copies of a 5-base-pair motif, each of which is recognized by an RHH domain. The structure reveals that a single conservative amino acid difference in the RHH β-ribbon between F and pED208 TraM changes its specificity for its cognate 5-base-pair sequence motif. Specificity is also dictated by the positioning of 2-base-pair spacer elements within sbmA; in F sbmA, the spacers are positioned between motifs 1 and 2 and between motifs 3 and 4, whereas in pED208 sbmA, there is a single spacer between motifs 2 and 3. We also demonstrate that a pair of F TraM tetramers can cooperatively bind its sbmC site with an affinity similar to that of sbmA in spite of a lack of sequence similarity between these DNA elements. These results provide a basis for the prediction of the DNA binding properties of the family of TraM proteins.