Sequence and conservation of genes at the distal end of the transfer region on plasmids F and R6-5

The nucleotide sequence of the region downstream of transfer gene traI, including fertility inhibition gene finO, on the conjugative plasmids F and R6-5, has been determined. Analysis of the F sequence revealed two open reading frames (ORF's), ORF248 and ORF186; ORF186 (finO) is interrupted by the insertion of IS3. The R6-5 sequence also contained ORF248 and an intact ORF186, although an additional ORF (ORF286) was located between the two genes. ORF248, which we have designated traX, and ORF186 (finO) are highly conserved on both plasmids. The organisation of these genes indicates that traI and traX on F, and traI, traX and ORF286 on R6-5 are co-transcribed from their respective promoters upstream of traI. Sequences homologous to traX were detected on a range of conjugative F-like plasmids, whereas sequences homologous to ORF286 were only found on plasmids R6-5, R100 and R1. The conservation of traX sequences suggests a functional importance for that gene and/or its product.     

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.     

Conjugal transfer of cloning vectors derived from ColE1.

The transfer properties of five cloning vectors derived from ColE1 were studied. Two of the vectors (pSF2124 and pGM706) behaved like wild type ColE1 in that they could be transferred efficiently in the presence of the conjugative plasmid F. The mobilization of the remaining three vectors (pMB9, PBR313 and pBR322) by F was barely detectable. The transfer defect in pBR313 and pBR322 could be complemented by ColK when R64drd11, but not F, was used as the conjugative plasmid. The transferred plasmids could be recovered unchanged from recipients. Conjugal transfer is a potentially useful technique for screening hybrid plasmids in low-risk cloning experiments involving poorly transformable strains.     

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.     

finO sequences on conjugally repressed and derepressed F-like plasmids

DNA-DNA hybridization studies have demonstrated the physical relatedness of the fertility inhibition gene, finO, among both FinO+ and FinO- F-like conjugative plasmids, viz. ColV2-K94, R100-1,R1drd19,R1,R6-5, UCR123, R386, p307, R453, R773, and pIP162-1. Furthermore, the data indicate that finO sequences on the FinO- plasmid ColV2-K94 map downstream of the transfer region, within 93.6-95.3 ColV2-K94.     

Partial suppression of the phenotype of Escherichia coli K-12 dnaG mutants by some I-like conjugative plasmids.

Three I-like conjugative plasmids, ColIdrd1, R144drd3, and R64drd11, which are derepressed for functions involved in conjugation, were found to suppress at least partially the phenotype of temperature-sensitive dnaG mutants of Escherichia coli K-12, as judged from the kinetics of deoxyribonucleic acid synthesis at elevated temperature in newly formed and established plasmid-containing strains. In contrast, the corresponding wild-type plasmids and three F-like derepressed conjugative plasmids, F101, R100drd1, and R1drd16, all failed to suppress. Suppression is presumably caused by a different plasmid-determined function from that which promotes survival of ultraviolet-irradiated bacteria, because both the wild-type I-like plasmids and their drd mutants protected irradiated bacteria. One possible interpretation of these results is that the product of a gene carried by certain I-like plasmids can substitute for the bacterial dnaG gene product during ongoing deoxyribonucleic acid replication.     

Complete nucleotide sequences of 84.5- and 3.2-kb plasmids in the multi-antibiotic resistant Salmonella enterica serovar Typhimurium U302 strain G8430

The multi-antibiotic resistant (MR) Salmonella enterica serovar Typhimurium phage type U302 strain G8430 exhibits the penta-resistant ACSSuT-phenotype (ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline), and is also resistant to carbenicillin, erythromycin, kanamycin, and gentamicin. Two plasmids, 3.2- and 84.5-kb in size, carrying antibiotic resistance genes were isolated from this strain, and the nucleotide sequences were determined and analyzed. The 3.2-kb plasmid, pU302S, belongs to the ColE1 family and carries the aph(3')-I gene (Kan(R)). The 84.5-kb plasmid, pU302L, is an F-like plasmid and contains 14 complete IS elements and multiple resistance genes including aac3, aph(3')-I, sulII, tetA/R, strA/B, bla(TEM-1), mph, and the mer operon. Sequence analyses of pU302L revealed extensive homology to various plasmids or transposons, including F, R100, pHCM1, pO157, and pCTX-M3 plasmids and TnSF1 transposon, in regions involved in plasmid replication/maintenance functions and/or in antibiotic resistance gene clusters. Though similar to the conjugative plasmids F and R100 in the plasmid replication regions, pU302L does not contain oriT and the tra genes necessary for conjugal transfer. This mosaic pattern of sequence similarities suggests that pU302L acquired the resistance genes from a variety of enteric bacteria and underscores the importance of a further understanding of horizontal gene transfer among the enteric bacteria.     

Genetic and physical characteristics of an enterotoxin plasmid.

We are engaged in the genetic and physical characterization of an enterotoxin (Ent) plasmid, Ent P307, which contains genes for the production of a hear-labile and a heat-stable enterotoxin. We are using an Escherichia coli K-12 strain, 711 (P307), constructed by S. Falkow, which contains no other plasmids besides Ent P307. Our genetic studies have shown that the plasmid is incompatible with the sex factor F, both in the integrated (Hfr) and the autonomous (F-prime) state. Ent P307 can thus be assigned to incompatibility group FI. An R factor, R386, which belongs to the same incompatibility group, was also found to be incompatibile with Ent P307, whereas five other R factors belonging to different incompatibility groups were compatible with Ent P307. In the presence of Ent P307, conjugal transfer and sensitivity to a male-specific phage of a derepressed F-like R factor, R1drd19, were repressed. Ent P307 is, thus, finO+. Presumably, it also causes repression of its own transfer genes since conjugal transfer of Ent P307 could not be demonstrated. Unlike F, it does not restrict the growth of female-specific phage phiII. From physical studies on extracted deoxyribonucleic acid, the molecular weight of Ent P307 was determined to be 54 X 10(6). By electron microscope heteroduplex analysis, the plasmid was found to be homologous with F in four regions, encompassing about half of its length. One long region and two short ones contain genes for conjugal transfer; the other short region carries genes for replication and incompatibility.     

Promoter-distal region of the tra operon of F-like sex factor R100 in Escherichia coli K-12.

The distal region of the tra (transfer) operon of F-like plasmid R100 was investigated, using small plasmids derived from R100, primarily the plasmid pSM6. The transposon Tn5 (which confers kanamycin resistance) was inserted at different positions into pSM6, and the transposition derivatives were tested for ability to complement defined tra mutants of the F sex factor. Thus, the tra genes traH, G, T, and D were localized on the plasmid R100. A restriction map of pSM6 was constructed, and the locations of the insertions were mapped, using restriction endonuclease digestion of the plasmid DNA and exploiting the fact that several restriction sites are localized in the inverted repeat regions of the transposon. The gene products of the genes traG, S, T, and D were identified by radioactive labeling of proteins synthesized in minicells carrying the various insertion plasmids followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of another transfer gene, traI, was inferred from these data. Another protein, the r2-A protein, was also identified, and its gene was mapped. On the basis of the data, a best-fit physical map of this region of the tra operon of R100 was constructed. The results confirmed that the general order and size of the distal transfer genes is as in the F sex factor, but showed that differences exist with respect to all of the gene products. The significance of these differences are discussed in the light of the genetic and physical homology (Manning et al., J. Bacteriol. 150:76-88) of the transfer regions.     

Complementation analysis of derepressed mutants of F-like plasmids of Escherichia coli

Complementation analysis of a number of conjugative transfer functions was performed in derepressed (drd) mutants of E. coli F-like plasmids. The major part of double plasmid complexes investigated has revealed the formation of complementation transfer inhibitor of Fin V-type, or less frequently--the formation of Fin U-type inhibitor. An additional complementation analysis of drd plasmids defective at Fin V region genes has demonstrated at least three genes (denoted A, B, C) in the structure of this region.     

Physical Properties and Mechanism of Transfer of R Factors in Escherichia coli

The physical properties of F-like and I-like R factors have been compared with those of the wild-type F factor in Escherichia coli K-12 unmated cells and after transfer to recipient cells by conjugation. The F-like R factor R538-1drd was found to have a molecular weight of 49 x 10(6), whereas the molecular weight of the I-like R factor R64drd11 was 76 x 10(6). The wild-type F factor, F1, had a molecular weight of 62 x 10(6). When conjugation experiments are performed by using donor strains carrying these derepressed F-like or I-like R factors, the transferred deoxyribonucleic acid can be isolated as a covalently closed circle from the recipient cells. This circular deoxyribonucleic acid was characterized by making use of the observation that the complementary strands of these R factors can be separated in a CsCl-poly (U, G) equilibrium gradient. The results of the strand-separation experiments show that only one of the complementary strands of the R factor is transferred from the donor to the recipient. With both the F-like and I-like R factors, this strand is the heavier strand in CsCl-poly (U, G). These results indicate that even though F-like and I-like R factors differ greatly in many properties (phage specificity, size, compatability, etc.), they are transferred by a similar mechanism.     

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.     

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.     

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.     

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.     

Molecular cloning of restriction fragments and construction of a physical and genetic map of the Escherichia coli plasmid R538-1.

A genetic and physical map of Escherichia coli plasmid R538-1 was constructed using restriction endonucleases and molecular cloning techniques. R538-1 DNA was cleaved into 12 fragments by endonuclease · R · EcoRI, 6 fragments by endonuclease R · HindIII, and 3 fragments by endonuclease R · BamHI. The order of these fragments was determined by standard restriction fragment mapping techniques. Endo · R · EcoRI, endo · R · HindIII, endo · R · BamHI, and endo · R · PstI fragments obtained from R538-1 and ColE1-derived plasmids (pMB9, ColE1Ap^r, and pBR322) were ligated in vitro and used to transform E. coli C600. Transformants were selected for antibiotic resistance markers carried by R538-1. Analysis of the R538-1 fragments contained in these hybrid plasmids permitted the construction of a genetic map of the R538-1 plasmid. The genetic map of this plasmid is very similar to that of plasmid R100.     

Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens

Transmission of ColE1/pMB1-derived plasmids, such as pBR322, from Escherichia coli donor strains was shown to be an efficient way to introduce these plasmids into Agrobacterium. This was accomplished by using E. coli carrying the helper plasmids pGJ28 and R64drd11 which provide the ColE1 mob functions and tra functions, respectively. For example, the broad host-range replication plasmid, pGV1150, a co-integrate plasmid between pBR322 and the W-type mini-Sa plasmid, pGV1106, was transmitted from E. coli to A. tumefaciens with a transfer frequency of 4.5 x 10(-3). As pBR322 clones containing pTiC58 fragments were unable to replicate in Agrobacterium, these clones were found in Agrobacterium only if the acceptor carried a Ti plasmid, thus allowing a co-integration of the pBR322 clones with the Ti plasmid by homology recombination. These observations were used to develop an efficient method for site-specific mutagenesis of the Ti plasmids. pTiC58 fragnents, cloned in pBR322, were mutagenized in vitro and transformed into E. coli. The mutant clones were transmitted from an E. coli donor strain containing pGJ28 and R64drd11 to an Agrobacterium containing a target Ti plasmid. Selecting for stable transfer of the mutant clone utilizing its antibiotic resistance marker(s) gave exconjugants that already contained a co-integrate plasmid between the mutant clone and the Ti plasmid. A second recombination can dissociate the co-integrate plasmid into the desired mutant Ti plasmid and a non-replicating plasmid formed by the vector plasmid pBR322 and the target Ti fragment. These second recombinants lose the second plasmid and they are identified by screening for the appropriate marker combination.     

Analysis of the traLEKBP sequence and the TraP protein from three F-like plasmids: F, R100-1 and ColB2.

The sequence of a region of the F plasmid containing the traLEKBP genes involved in plasmid transfer was compared to the equivalent regions of two IncFII plasmids, R100-1 and ColB2. The traLEK gene products of all three plasmids were virtually identical, with the most changes occurring in TraE. The TraB genes were also nearly identical except for an 11-codon extension at the 3' end of the R100-1 traB gene. The TraP protein of R100-l differed from those of F and ColB2 at its N terminus, while the ColB2 TraP protein contained a change of sequence in a predicted loop which was shown to be exposed in the periplasmic space by TnphoA mutagenesis. The effect of the altered TraP sequences was determined by complementing a traP mutant with clones expressing the traKBP genes of F, R100-1, and ColB2. The traP mutation in pOX38 (pOX38-traP474), a derivative of F, was found to have little effect on pilus production, pilus retraction, and filamentous phage growth and only a moderate effect on transfer. The transfer ability of pOX38-traP474 was shown to be affected by mutations in the rfa (lipopolysaccharide) locus and in ompA in the recipient cell in a manner similar to that for the wild-type pOX38-Km plasmid itself and could be complemented with the traP analogs from R100-1 and ColB2 to give an F-like phenotype. Thus, the TraP protein appears to play a minor role in conjugation and may interact with TraB, which varies in sequence along with TraP, in order to stabilize the proposed transmembrane complex formed by the tra operon products.     

The role of lipopolysaccharide structure in the recipient cell during plasmid-mediated bacterial conjugation

The role of the lipopolysaccharide (LPS) structure in the recipient cell during bacterial conjugation was studied using a series of well-defined LPS mutations in Salmonella minnesota. The plasmids Flac (IncFI) and R1drd19 (IncFII) transferred at a high frequency to the smooth S218 parent strain and the rough LPS mutants. However, R64drd1 1 (IncI alpha) transferred poorly to the LPS mutants compared with transfer to the smooth LPS parent strain. The decrease in R64drd1 1 transfer frequency correlated with the extent of the defect in LPS structure, suggesting that intact LPS on the recipient is a major requirement for R64drd1 1 mating. Transfer of the P-group plasmid, RK2, was not affected by changes in LPS structure. These results show that plasmids use different cell surface structures during conjugation, and that LPS is particularly important for R64drd1 1 transfer.