A traC mutant that retains sensitivity to f1 bacteriophage but lacks F pili.

An F lac pro mutant which was temperature sensitive for infection by the filamentous bacteriophage f1 but resistant to the F-specific icosahedral RNA phage f2 was isolated. Cells carrying the F' mutation failed to elaborate F pili at all temperatures. Mutant cells were able to pair with recipient cells during bacterial conjugation, but transfer of conjugal DNA occurred at a greatly reduced frequency. Complementation analyses showed the F' mutation to be in the traC gene. When a plasmid carrying traC was introduced into hosts harboring the F' mutation, phage sensitivity, the ability to elaborate F pili, and conjugation efficiency were restored. The mutation was named traC1044. The F lac pro traC1044 mutant appears to be unique among traC mutants in retaining host sensitivity to the filamentous phage f1 in the absence of expression of extended F pili. Phage f1 attachment sites appeared to be present at the cell surface in traC1044 mutants. The reduced accessibility of these sites may account for the reduced efficiency of phage f1 infection of traC1044 hosts, although the possibility that a defect was present in the receptor site itself was not eliminated. Membranes of hosts carrying the F' mutation contained a full complement of mature F-pilin subunits, so the product of traC is presumably required for pilus assembly but not for pilin processing. This, together with the deficiency in conjugal DNA transfer, suggests that traC may be part of a membrane-spanning tra protein complex responsible for pilus assembly and disassembly and conjugal DNA transmission.     

Nucleotide sequence and characterization of the traABCD region of IncI1 plasmid R64.

A 3.6-kb BglII-SmaI segment of the transfer region of IncI1 plasmid R64drd-11 was sequenced and characterized. Analysis of the DNA sequence indicated the presence of four genes, traA, traB, traC, and traD, in this region. The expression of the traB, traC, and traD genes was examined by maxicell experiments and that of the traA gene was examined by constructing the traA-lacZ fusion gene. The introduction of frameshift mutations into the four genes indicated that the traB and traC genes are essential for conjugal transfer in liquid medium and on a solid surface. Both were also required for the formation of the thin pilus, which is the receptor for phages I alpha and PR64FS. Upstream of the traA gene, a promoter sequence for sigma 70 of E. coli RNA polymerase was identified by S1 nuclease mapping and primer extension experiments.     

The tra region of the nopaline-type Ti plasmid is a chimera with elements related to the transfer systems of RSF1010, RP4, and F.

The Ti plasmids of Agrobacterium tumefaciens encode two transfer systems. One mediates the translocation of the T-DNA from the bacterium to a plant cell, while the other is responsible for the conjugal transfer of the entire Ti plasmid from one bacterium to another. The determinants responsible for conjugal transfer map to two regions, tra and trb, of the nopaline-type Ti plasmid pTiC58. By using transposon mutagenesis with Tn3HoHo1, we localized the tra determinants to an 8.5-kb region that also contains the oriT region. Fusions to lacZ formed by transposon insertions indicated that this region is expressed as two divergently transcribed units. We determined the complete nucleotide sequence of an 8,755-bp region of the Ti plasmid encompassing the transposon insertions defining tra. The region contains six identifiable genes organized as two units divergently transcribable from a 258-bp inter-genic region that contains the oriT site. One unit encodes traA, traF, and traB, while the second encodes traC, traD, and traG. Reporter insertions located downstream of both sets of genes did not affect conjugation but were expressed, suggesting that the two units encode additional genes that are not involved in transfer under the conditions tested. Proteins of the predicted sizes were expressible from traA, traC, traD, and traG. The products of several Ti plasmid tra genes are related to those of other conjugation systems. The 127-kDa protein expressed from traA contains domains related to MobA of RSF1O1O and to the helicase domain of TraI of plasmid F. The translation product of traF is related to TraF of RP4, and that of traG is related to TraG of RP4 and to VirD4 of the Ti plasmid T-DNA transfer system. Genetic analysis indicated that at least traG and traF are essential for conjugal transfer, while sequence analysis predicts that traA also encodes an essential function. traB, while not essential, is required for maximum frequency of transfer. Patterns of sequence relatedness indicate that the oriT and the predicted cognate site-specific endonuclease encoded by traA share lineage with those of the transfer systems of RSF1010 and plasmid F, while genes of the Ti plasmid encoding other essential tra functions share common ancestry with genes of the RP4 conjugation system.     

Localization of TraC, a protein involved in assembly of the F conjugative pilus.

TraC is one of the proteins encoded by the F transfer region of the F conjugative plasmid which is required for the assembly of F pilin into the mature F pilus structure. Overproduction of this protein from the plasmid pKAS2, which carries only traC, resulted in the formation of inclusion bodies from which soluble TraC was purified. When small amounts of TraC were produced from pKAS2, the protein was localized to the cytoplasm by using anti-TraC antibodies. Similar analysis of a set of TraC-alkaline phosphatase fusion proteins localized all of these fusion proteins to the cytoplasm. However, when TraC was expressed from the F plasmid, much of it appeared associated with the bacterial membrane fraction. Under these conditions, TraC does not appear to be part of the tip of the F pilus, as neither anti-TraC antibodies nor purified TraC had any effect on the infection of F-containing bacteria by the filamentous bacteriophage f1. These data suggest that TraC is normally associated with the membrane through interactions with other proteins specified by the tra region. This interaction may be via the carboxyl-terminal region of the TraC protein, as a mutant TraC protein containing an Arg-Cys substitution at amino acid 811 exhibits an interaction with the membrane weaker than that of the wild-type protein in the presence of the other Tra proteins.     

Characterization of transfer regions within the HII incompatibility group plasmid pHH1508a.

A circular map of the 208-kilobase IncHII plasmid pHH1508a was constructed by using the restriction endonucleases XbaI, XhoI, and NotI. Tn5 insertion mutagenesis showed that transfer genes were distributed widely over the plasmid map. Analysis of the deletion mutant pDT1178 indicated that genes required for conjugative pilus production are located in a 96.0-kilobase region of the plasmid.     

Characterization of conjugative plasmid EDP208.

EDP208 is a conjugative plasmid belonging to incompatibility group IncF0 lac, A restriction endonuclease map of this plasmid was constructed using five restriction enzymes: BamHI, HindIII, PvuI, SstI, and XhoI. On the basis of these mapping studies, the plasmid was found to be 90 kilobases in length. Clones were constructed from four large HindIII fragments of plasmid EDP208. One fragment, HindIII-20.5, was found to contain the lac genes and the origin of vegetative replication (oriV). Another fragment, HindIII-27.5, was found to contain all of the genes necessary for sex pilus formation, but it was nontransmissible. However, when used to complement a plasmid carrying an adjacent fragment, HindIII-23, the transfer of the latter occurred, suggesting that HindIII-23 contains the origin of transfer (oriT). The further localization of genes concerned with pilus biosynthesis was achieved by transposon mutagenesis. Six EDP208::Tn1 and thirty-seven EDP208::Tn5 mutants were isolated on the basis of their resistance to f1, a filamentous phage which adheres to intact pilus tips. The positions of the inserted transposons were determined on the restriction map and a 16.5-kilobase region was found to be required for pilus synthesis.     

General organization of the conjugal transfer genes of the IncW plasmid R388 and interactions between R388 and IncN and IncP plasmids.

The complete conjugal transfer gene region of the IncW plasmid R388 has been cloned in multicopy vector plasmids and mapped to a contiguous 14.9-kilobase segment by insertion mutagenesis. The fertility of the cloned region could still be inhibited by a coresident IncP plasmid. The transfer region has been dissected into two regions, one involved in pilus synthesis and assembly (PILW), and the other involved in conjugal DNA metabolism (MOBW). They have been separately cloned. PILW also contains the genes involved in entry exclusion. MOBW contains oriT and the gene products required for efficient mobilization by PILW. MOBW plasmids could also be mobilized efficiently by PILN, the specific pilus of the IncN plasmid pCU1, but not by PILP, the specific pilus of the IncP plasmid RP1.     

Characterization, localization, and sequence of F transfer region products: the pilus assembly gene product TraW and a new product, TrbI.

The traW gene of the Escherichia coli K-12 sex factor, F, encodes one of the numerous proteins required for conjugative transfer of this plasmid. We have found that the nucleotide sequence of traW encodes a 210-amino-acid, 23,610-Da polypeptide with a characteristic amino-terminal signal peptide sequence; in DNA from the F lac traW546 amber mutant, the traW open reading frame is interrupted at codon 141. Studies of traW expression in maxicells in the presence and absence of ethanol demonstrate that the traW product does undergo signal sequence processing. Cell fractionation experiments additionally demonstrated that mature TraW is a periplasmic protein. Electron microscopy also showed that F lac traW546 hosts do not express F pili, confirming that TraW is required for F-pilus assembly. Our nucleotide sequence also revealed the existence of an additional gene, trbI, located between traC and traW. The trbI gene encodes a 128-amino-acid polypeptide which could be identified as a 14-kDa protein product. Fractionation experiments demonstrated that TrbI is an intrinsic inner-membrane protein. Hosts carrying the pOX38-trbI::kan insertion mutant plasmids that we constructed remained quite transfer proficient but exhibited increased resistance to F-pilus-specific phages. Mutant plasmids pOX38-trbI472 and pOX38-trbI473 expressed very long F pili, suggestive of a pilus retraction deficiency. Expression of an excess of TrbI in hosts carrying a wild-type pOX38 plasmid also caused F-pilus-specific phage resistance. The possibility that TrbI influences the kinetics of pilus outgrowth and/or retraction is discussed.     

The conjugal transfer system of Agrobacterium tumefaciens octopine-type Ti plasmids is closely related to the transfer system of an IncP plasmid and distantly related to Ti plasmid vir genes.

We have determined the DNA sequences of two unlinked regions of octopine-type Ti plasmids that contain genes required for conjugal transfer. Both regions previously were shown to contain sequences that hybridize with tra genes of the nopaline-type Ti plasmid pTiC58. One gene cluster (designated tra) contains a functional oriT site and is probably required for conjugal DNA processing, while the other gene cluster (designated trb) probably directs the synthesis of a conjugal pilus and mating pore. Most predicted Tra and Trb proteins show relatively strong sequence similarity (30 to 50% identity) to the Tra and Trb proteins of the broad-host-range IncP plasmid RP4 and show significantly weaker sequence similarity to Vir proteins found elsewhere on the Ti plasmid. An exception is found in the Ti plasmid TraA protein, which is predicted to be a bifunctional nickase-helicase that has no counterpart in IncP plasmids or among Vir proteins but has homologs in at least six other self-transmissible and mobilizable plasmids. We conclude that this Ti plasmid tra system evolved by acquiring genes from two or three different sources. A similar analysis of the Ti plasmid vir region indicates that it also evolved by appropriating genes from at least two conjugal transfer systems. The widely studied plasmid pTiA6NC previously was found to be nonconjugal and to have a 12.65-kb deletion of DNA relative to other octopine-type Ti plasmids. We show that this deletion removes the promoter-distal gene of the trb region and probably accounts for the inability of this plasmid to conjugate.     

Location of F plasmid transfer operon genes traC and traW and identification of the traW product.

As part of an analysis of the conjugative transfer genes associated with the expression of F pili by plasmid F, we have investigated the physical location of the traC and traW genes. We found that plasmid clones carrying a 2.95-kilobase EcoRI-EcoRV F transfer operon fragment were able to complement transfer of F lac traC mutants and expressed an approximately 92,000-dalton product that comigrates with TraC. We also found that traW-complementing activity was expressed from plasmids carrying a 900-base-pair SmaI-HincII fragment. The traW product was identified as an approximately 23,000-dalton protein. The two different F DNA fragments that expressed traC and traW activities do not overlap. Our data indicate that the traC gene is located in a more-tra operon promoter-proximal position than suggested on earlier maps and that traW is distal to traC. These results resolve a long-standing question concerning the relationship of traW to traC. The clones we have constructed are expected to be useful in elucidating the role of proteins TraC and TraW in F-pilus assembly.     

Twelve pil genes are required for biogenesis of the R64 thin pilus

The IncI1 plasmid R64 produces two kinds of sex pili: a thin pilus and a thick pilus. The thin pilus, which belongs to the type IV family, is required only for liquid matings. Fourteen genes, pilI to -V, were found in the DNA region responsible for the biogenesis of the R64 thin pilus (S.-R. Kim and T. Komano, J. Bacteriol. 179:3594-3603, 1997). In this study, we introduced frameshift mutations into each of the 14 pil genes to test their requirement for R64 thin pilus biogenesis. From the analyses of extracellular secretion of thin pili and transfer frequency in liquid matings, we found that 12 genes, pilK to -V, are required for the formation of the thin pilus. Complementation experiments excluded the possible polar effects of each mutation on the expression of downstream genes. Two genes, traBC, were previously shown to be required for the expression of the pil genes. In addition, the rci gene is responsible for modulating the structure and function of the R64 thin pilus via the DNA rearrangement of the shufflon. Altogether, 15 genes, traBC, pilK through pilV, and rci, are essential for R64 thin pilus formation and function.     

Analysis of the sequence and gene products of the transfer region of the F sex factor.

Bacterial conjugation results in the transfer of DNA of either plasmid or chromosomal origin between microorganisms. Transfer begins at a defined point in the DNA sequence, usually called the origin of transfer (oriT). The capacity of conjugative DNA transfer is a property of self-transmissible plasmids and conjugative transposons, which will mobilize other plasmids and DNA sequences that include a compatible oriT locus. This review will concentrate on the genes required for bacterial conjugation that are encoded within the transfer region (or regions) of conjugative plasmids. One of the best-defined conjugation systems is that of the F plasmid, which has been the paradigm for conjugation systems since it was discovered nearly 50 years ago. The F transfer region (over 33 kb) contains about 40 genes, arranged contiguously. These are involved in the synthesis of pili, extracellular filaments which establish contact between donor and recipient cells; mating-pair stabilization; prevention of mating between similar donor cells in a process termed surface exclusions; DNA nicking and transfer during conjugation; and the regulation of expression of these functions. This review is a compendium of the products and other features found in the F transfer region as well as a discussion of their role in conjugation. While the genetics of F transfer have been described extensively, the mechanism of conjugation has proved elusive, in large part because of the low levels of expression of the pilus and the numerous envelope components essential for F plasmid transfer. The advent of molecular genetic techniques has, however, resulted in considerable recent progress. This summary of the known properties of the F transfer region is provided in the hope that it will form a useful basis for future comparison with other conjugation systems.     

Stabilization of pSW100 from Pantoea stewartii by the F conjugation system

Plasmid pSW100 is 1 of the 13 plasmids from Pantoea stewartii subsp. stewartii SW2 which has a replicon that resembles that of ColE1. This work uses a pSW100 derivative, pSW140K, to study how the pSW100 replicon is stably maintained in its hosts. Our results indicate that although pSW140K is stable in Escherichia coli HB101, the plasmid is rapidly lost in another E. coli strain, DH5alpha, indicating that the genetic background of an E. coli strain affects the stability of pSW140K. Mutagenesis of E. coli HB101 with EZ::TN revealed that mutations in traC, traF, traG, traN, and traV, which encode the components of the sex pilus assembly, reduce plasmid stability. Furthermore, this work identified that a 38-bp region located immediately upstream of the RNAII promoter is critical to the maintenance of plasmid stability in E. coli HB101. TraC binds to the region, and in addition, deleting the region destabilizes the plasmid. Furthermore, inserting this 38-bp fragment into a plasmid that contains the minimal replicon from pSW200 stabilizes the plasmid in E. coli HB101. Fluorescence in situ hybridization and immunofluorescence staining also revealed that derivatives of pSW100, pSW128A, and TraC are colocalized in cells, suggesting that pSW100 may use the sex pilus assembly as a partition apparatus to ensure the even distribution of the plasmid during cell division, which may thus maintain the plasmid's stability.     

Nucleotide sequence of the F plasmid gene, traC, and identification of its product

The traC gene of the F plasmid tra operon is required for the assembly of mature F-pilin subunits into extended F pili. The nucleotide sequence of traC was determined with a determined with a deduced coding region of 875 amino acids (aa) and 99066 Da. The traC1044 mutant allele, which allows filamentous phage infection in the absence of piliation, contains a C-to-T transition leading to an Arg----Cys substitution. Confirmation of the translational start came from the direct N-terminal aa sequencing of a TraC-alkaline phosphatase fusion protein.     

Gene organization and nucleotide sequence of the primase region of IncP plasmids RP4 and R751.

The primase genes of RP4 are part of the primase operon located within the Tra1 region of this conjugative plasmid. The operon contains a total of seven transfer genes four of which (traA, B, C, D) are described here. Determination of the nucleotide sequence of the primase region confirmed the existence of an overlapping gene arrangement at the DNA primase locus (traC) with in-phase translational initiation signals. The traC gene encodes two acidic and hydrophilic polypeptide chains of 1061 (TraC1) and 746 (TraC2) amino acids corresponding to molecular masses of 116,721 and 81,647 Da. In contrast to RP4 the IncP beta plasmid R751 specifies four large primase gene products (192, 152, 135 and 83 kDa) crossreacting with anti-RP4 DNA primase serum. As shown by deletion analysis at least the 135 and 83 kDa polypeptides are two separate translational products that by analogy with the RP4 primases, arise from in-phase translational initiation sites. Even the smallest primase gene products TraC2 (RP4) and TraC4 (R751) exhibit primase activity. Nucleotide sequencing of the R751 primase region revealed the existence of three in-phase traC translational initiation signals leading to the expression of gene products with molecular masses of 158,950 Da, 134,476 Da, and 80,759 Da. The 192 kDa primase polypeptide is suggested to be a fusion protein resulting from an in frame translational readthrough of the traD UGA stopcodon. Distinct sequence similarities can be detected between the TraC proteins of RP4 and R751 gene products TraC3 and TraC4 and in addition between the TraD proteins of both plasmids. The R751 traC3 gene contains a stretch of 507 bp which is unrelated to RP4 traC or any other RP4 Tra1 gene.     

A Novel Plasmid Gene Involved in Bacteriophage PRD1 Infection and Conjugative Host-Range

PRD1 infects bacteria carrying IncN plasmids by binding to their conjugative pili. Mutations in a plasmid locuskikAclose to the pilus region result in PRD1 resistance and reduced conjugation proficiency toKlebsiellabut not toEscherichia coli.One of the two genes ofkikAis sufficient to restore both normal phenotypes. PRD1 binds to cells carrying the mutant plasmid but fails to inject its genome.     

Characterization of the traC determinant of the Enterococcus faecalis hemolysin-bacteriocin plasmid pAD1: binding of sex pheromone.

pAD1, a conjugative, 60-kb, hemolysin-bacteriocin plasmid in Enterococcus faecalis, encodes a mating response to a small peptide sex pheromone, cAD1, secreted by potential recipient bacteria. A gene, traC, encoding a 60.7-kDa protein with a typical amino terminal signal peptide, was identified within a region that appears to encode a product that binds to exogenous pheromone. A cloned segment of DNA containing traC resulted in specific binding of cells to synthetic cAD1. The putative traC product has strong similarity to a product of the E. faecalis plasmid pCF10 as well as oligopeptide binding proteins of Escherichia coli, Salmonella typhimurium, and Bacillus subtilis.     

DNA sequence analysis of point mutations in traA, the F pilin gene, reveal two domains involved in F-specific bacteriophage attachment

Summary Six missense point mutations in traA (WPFL43,44,45,46,47 and 51), the gene encoding F pilin in the transfer region of the F plasmid, have been characterized for their effect on the transfer ability, bacteriophage (R17, QB and fl) sensitivity and levels of piliation expressed by the plasmid. The sequence analysis of the first five of these mutations revealed two domains in the F pilin subunit exposed on the surface of the F pilus which mediate phage attachment. These two domains include residues 14–17 (approximately) and the last few residues at the carboxy-terminus of the pilin protein. One of these mutants had a pleiotropic affect on pilus function and was thought to have affected pilus assembly. The sixthe point mutant (WPFL51), previously thought to be in traA, was complemented by chimeric plasmids carrying the traG gene of the F transfer region, which may be involved in the acetylation of the pilin subunit. A traA nonsense mutant (JCFL1) carried an amber mutation near the amino-terminus which is well suppressed in SuI⁺ (supD) and SuIII⁺ (supF) strains. Neither the antigenicity of the pilin nor the efficiency of plating of F-specific bacteriophages were affected when this plasmid was harbored by either suppressor strain. A second amber mutant (JCFL25) which is not suppressible, carried its mutation in the codon for the single tryptophan in F pilin, suggesting that this residue is important in subunit interactions during pilus assembly. Two other point mutants (JCFL32 and 44) carried missense mutations in the leader sequence (positions 9 and 13) which affected the number of pili per cell presumably by altering the processing of propilin to pilin.