Nucleotide and Amino Acid Sequences of oriT-traM-traJ-traY-traA-traL Regions and Mobilization of Virulence Plasmids of Salmonella enterica Serovars Enteritidis, Gallinarum-Pullorum, and Typhimurium

Abstract

The virulence plasmid of Salmonella enterica serovar Gallinarum-Pullorum (pSPV) but not those of Salmonella enterica serovars Enteritidis (pSEV) and Typhimurium (pSTV) can be readily mobilized by an F or F-like conjugative plasmid. To investigate the reason for the difference, the oriT-traM-traJ-traY-traA-traL regions of the three salmonella virulence plasmids (pSVs) were cloned and their nucleotide and deduced amino acid sequences were examined. The cloned fragments were generally mobilized more readily than the corresponding full-length pSVs, but the recombinant plasmid containing the oriT of pSPV was, as expected, more readily mobilized, with up to 100-fold higher frequency than the recombinant plasmids containing the oriT of the other two pSVs. The nucleotide sequences of the oriT-traM-traJ-traY-traA-traL region of pSEV and pSTV were almost identical (only 4 bp differences), but differed from that of pSPV. Major nucleotide sequence variations were found in traJ, traY, and the Tra protein binding sites sby and sbm. sby of pSPV showed higher similarity than that of pSEV or pSTV to that of the F plasmid. The reverse was true for sbm: similarity was higher with pSEV and pSTV than with pSPV. In the deduced amino acid sequences of the five Tra proteins, major differences were found in TraY: pSEV's TraY was 75 amino acids, pSTV's was 106 amino acids, and pSPV's was 133 amino acids; and there were duplicate consensus βαα fragments in the TraY of pSPV and F plasmid, whereas there was only a single βαα fragment in that of pSEV and pSTV.

Seven Salmonella enterica serovars, Abortusovis, Choleraesuis, Dublin, Enteritidis, Gallinarum-Pullorum, Sendai, and Typhimurium, are known to harbor a serovar specific salmonella virulence plasmid (pSV) that contains the spv operon, which plays a role in the virulence of these serovars (3, 8). All except the Gallinarum-Pullorum virulence plasmid (pSPV) are incompatible with the Typhimurium virulence plasmid (pSTV) (17). These pSVs are all nonconjugative; however, pSPV can be readily mobilized by an F or F-like plasmid. In contrast, the pSVs of serovars Choleraesuis (pSCV), Dublin (pSDV), Enteritidis (pSEV), and Typhimurium (pSTV) are rarely or not mobilized by these conjugative plasmids (19).

DNA-DNA hybridization indicates that pSCV and pSDV are not mobilized, probably because of the absence of oriT (19). In contrast, pSPV contains oriT, as do pSEV and pSTV; yet, as mentioned above, the last two are rarely mobilized (19). To clarify this difference, the nucleotide sequences of the oriT region in pSPV, pSEV, and pSTV were examined and compared with each other as well as with that of F and F-like conjugative plasmids.

The oriT region of F plasmid contains binding sites for four proteins: IHF, the integration host factor (23); TraI, the relaxase/helicase (15); TraY, an accessory protein for efficient nicking in vivo (16); and TraM, a cytoplasmic protein associated with the inner membrane (1, 6). All four proteins are required for mobilization of the plasmid for transfer. IHF binds to two sites in the F oriT region and induces bending to fulfill the three-dimensional structure requirement at oriT for cleavage at nic (23). TraI contains an ATP-dependent helicase activity in the large carboxyl-terminal domain of the molecule (10), binds to the sbi site, and cleaves a single strand of the oriT DNA at a site called nic (2, 11). TraY, encoded by the first gene in the traYI operon, binds to two sites, sbyA and sbyC, in the F oriT (12, 14), but to only one site in the oriT of R100 (10), and the binding of TraY involves β-sheet residues (13).

In F plasmid, IHF, TraI, and TraY are all needed for an efficient nick formation (16), and the three proteins are assembled in a specific order to form the oriT region complex: the binding of TraI follows IHF and TraY binding (9). The fourth protein, TraM, binds to three sites, sbmA, sbmB, and sbmC, in F oriT (6). The sbmC site is associated with transfer, while the other two, sbmA and sbmB, are involved in the autoregulation of traM transcription (20). Salazar et al. (21) earlier suggested that the nucleotide sequences of the binding sites sbi, sby, and sbm might have coevolved independently of those of their own proteins, TraI, TraY, and TraM.

We report here a confirmation of efficient mobilization of pSPV in contrast to the inefficient mobilization of pSEV and pSTV and determination and comparison of the nucleotide sequences and the deduced amino acid sequences of the oriT regions of the three pSVs. We found that major variations resided in the nucleotide sequence of sby, sbm, and their corresponding gene traY, as well as in the sequence of TraY, but not in that of traM.

MATERIALS AND METHODS

Bacterial strains, plasmids, media, and chemicals.

Bacterial strains used were various plasmid-containing derivatives of plasmidless strains OU5048 (Typhimurium), OU7351 (Pullorum), and OU7044 (Enteritidis); these derivatives are described in the appropriate places in the text. Escherichia coli K-12 HB101 (F⁻ pro leu thi recA13 Δ[mcrC-mrr] lac rpsL), DH10B (F⁻ mcrA Δ[mrr-hsdRMS-mcrBC] lac recA1 rpsL), and DH5α (F⁻ phoA recA hsdR17 thi gyrA) were used as the recipients in conjugation or transformation. The F′ lac⁺ plasmid, used as the mobilization agent, was derived from E. coli K-12 NH4111 lac gal arg metA28 tsx thi λ⁻ Sm^s harboring the F′ lac⁺ plasmid (pOU507) (19). Plasmids used are listed in Table 1.

TABLE 1.

Plasmids

Plasmid	Size (kb)	Descriptiona
pOU206	98.5	Tra−, wild-type pSTV with Tn5 insertion at rsk, Kmr
pOU127	42	Tra−, recombinant pBR322 carring 37.1-kb HindIII fragment derived from pSTV, insertion from samA to RepFIIA, Apr
pOU1505	5.1	Tra−, recombinant pUC18 carrying a 2.4-kb EcoRI fragment derived from pSTV, insert spanning from gene 32 to traM, Apr
pOU1204	65.8	Tra−, wild-type pSEV with Tn5 insertion at spvC, Kmr
pOU1504	10.8	Tra−, recombinant pUC18 carrying 8.1-kb PstI fragment derived from pSEV, insert spanning from samA to traE, Apr
pOU1401	91.6	Tra−, wild-type pSPV with Tn5 insertion at an unknown site outside of the oriT region, Kmr
pOU1506	13.3	Tra−, recombinant pUC18 carrying a 10.6-kb PstI fragment derived from pSPV, insert spanning from an unknown locus to traE, Apr
pOU1507	5.4	Tra−, recombinant pUC18 carrying a 2.7-kb HpaI fragment derived from pOU1506, insert spanning from gene 19 to traJ, Apr
pUC18	2.7	Tra− Apr
pOU507	>200	Tra+, F′ lac+

^aKm^r, resistant to kanamycin; Ap^r, resistant to ampicillin.

Bacteria were routinely grown in Penassay broth without shaking or on Luria-Bertani (LB) agar medium at 37°C. MacConkey agar with or without drugs was used to detect the lac gene: red color indicated Lac⁺. When needed, antibiotics used were ampicillin (50 μg/ml), kanamycin (25 μg/ml), and streptomycin (100 μg/ml).

DNA preparation and DNA-DNA hybridization.

Plasmid DNA was extracted and purified by the CsCl gradient method described elsewhere (18). DNA-DNA hybridization was performed according to the method of Southern (22) and that described by the material (Zeta-probe membrane) supplier (Bio-Rad). The probe for oriT was prepared by PCR amplification of the oriT of pOU507 in F′ NH4111 and purified by a PCR purification kit (Promega). The probe was labeled with [³²P]dCTP (specific activity, 3,000 Ci/mmol; Amersham Pharmacia) by the random primer labeling method (Gibco-BRL). After hybridization, the sample was exposed to X-ray film with an intensifying screen.

Cloning of DNA fragment containing oriT.

The PstI-digested DNA fragments of pSEV and pSPV and EcoRI-digested DNA fragments of pSTV were electrophoresed in a 0.8% low-melting-point agarose gel with TAE buffer (40 mM Tris acetate, 2 mM EDTA, pH 5.8). The DNA fragment containing oriT was first identified by DNA-DNA hybridization with the oriT probe and then cut from the gel to elute it. The DNA fragment was then ligated to dephosphorylated PstI- and EcoRI-digested pUC18 in 5 U of T4 DNA ligase (Promega). The ligation product was transferred into E. coli DH10B by electroporation at 2.5 kV, 200 Ω, and 25 μF.

E. coli competent cells were prepared as follows. Fifty milliliters of bacteria in broth (optical density at 600 nm = 0.6 to 1.0) was centrifuged at 3,000 × g for 10 min, and the pellet was washed once with 25 ml of 1 mM HEPES (pH 7.2) and resuspended in 10 ml of 10% glycerol. The step was repeated, and the pellet was finally resuspended in 2 to 3 ml of 10% glycerol and used in electroporation. The plasmid, confirmed to carry oriT, was extracted, purified from DH10B, and transferred into appropriate target strains such as plasmidless Salmonella strains. The presence of oriT was also confirmed by the mobilization test: all fragments showing positive DNA-DNA hybridization to the oriT probe were mobilizable by an F plasmid.

Conjugation.

In conjugation, the donor and recipient strains were grown to the late exponential phase (4 × 10⁸ bacteria/ml) and mixed 1:1. All donors were F′ strains; i.e., all harbored the F′ lac⁺ plasmid pOU507 regardless of the presence or absence of any other plasmid. The mixture was incubated without shaking at 37°C for 18 h and plated on MacConkey plates containing an appropriate drug (kanamycin, streptomycin, or ampicillin) for transconjugant selection. The efficiency of mating was calculated by dividing the number of transconjugants by the input number of the minority parent.

DNA sequencing.

The nucleotide sequences of oriT and its flanking region were first determined with M13 universal forward and reverse primers. These newly determined sequences were used to design new primers to obtain overlapping reading in both strands. Sequencing was conducted with an autosequencer, ABI Prism 3100. The search for homologous sequences was done in the GenBank database using the FastA software through the Internet.

RESULTS

Mobilization of recombinant plasmids that contained oriT.

The oriT regions of each of the three virulence plasmids pSEV, pSPV, and pSTV were cloned into pUC18. The mobilizability of these recombinant plasmids with various sizes containing oriT and their respective full-length plasmids, shown in Table 2, was tested. The strains harboring the full-length plasmid and recombinant plasmids were converted into F′ strains by transferring F′ lac⁺ (pOU507) into them, and the resultant transconjugant F plasmids served as the donors in mobilization experiments. The recipient was E. coli HB101 in all matings. The vector pUC18 was not mobilized for transfer, whether or not it was in an F′ Salmonella or F′ E. coli (data not shown).

TABLE 2.

Frequency of mobilization of Salmonella plasmids in conjugation^a

F′ donor	pSV size (kb)	Frequency of transconjugant formation
		Expt 1	Expt 2	Expt 1	Expt 2
Pullorum plasmids
Sp F′ lac+/pOU1401	91.7	0.51 × 10−3	1.09 × 10−3
Ec F′ lac+/pOU1401	91.7			2.45 × 10−4	3.13 × 10−4
Sp F′ lac+/pOU1506	14.1	0.90 × 10−2	1.36 × 10−2
Ec F′ lac+/pOU1506	14.1			3.15 × 10−3	3.74 × 10−3
Sp F′ lac+/pOU1507	5.4	5.67 × 10−1	8.9 × 10−1
Ec F′ lac+/pOU1507	5.4			4.12 × 10−2	6.03 × 10−2
Enteritidis plasmids
Se F′ lac+/pOU1204	65.8	9.21 × 10−5	8.92 × 10−5
Ec F′ lac+/pOU1204	65.8			7.32 × 10−5	8.91 × 10−5
Se F′ lac+/pOU1504	10.8	1.40 × 10−2	1.61 × 10−2
Ec F′ lac+/pOU1504	10.8			4.23 × 10−3	6.12 × 10−3
Typhimurium plasmids
St F′ lac+/pOU206	98.5	1.33 × 10−4	5.96 × 10−4
Ec F′ lac+/pOU206	98.5			1.03 × 10−5	3.02 × 10−5
St F′ lac+/pOU1505	5.1	4.01 × 10−3	5.4 × 10−3
Ec F′ lac+/pOU1505	5.1			1.80 × 10−4	3.16 × 10−4

^aE. coli HB101 was used as the recipient. Sp, serovar Pullorum OU7351; Ec, E. coli DH5α; Se, serovar Enteritidis OU7044; St, serovar Typhimurium OU5048. Mobilization of pUC18, whether in a Salmonella or E. coli host as the donor strain, was nil. All donors harbored F′ lac⁺, which was used to mobilize the Salmonella plasmid. The F′ lac⁺ donor that contained the Salmonella plasmid (first column) was mixed with the recipient for mating as described in Materials and Methods, and the frequency of Salmonella plasmid transconjugant formation was measured. Matings were repeated three to five times. The mating frequency varied greatly from one experiment to another with the same donors because of subtle differences in growth stage, aeration, and donor-to-recipient ratio in each mating, but the trend was consistent. Therefore, only two representative mating experiments are shown here. In each experiment, the transfer frequency of the F′ lac⁺ plasmid was similar regardless of the presence or absence of Salmonella plasmids in the F′ donor, and therefore its frequency, as high as 100% in overnight matings, is not shown.

Table 2 also shows the results of matings with various strains. Consistent with the earlier observation (19), the F′ lac⁺ plasmid was rarely segregated out, and its transfer was unperturbed by the presence of Salmonella plasmids, full-length or cloned, indicating the stable nature of these F′ strains and the absence of finO. The mobilization frequency of a full-length virulence plasmid was consistent with the earlier observation regardless of the donor strain: the full-length pSPV was readily mobilized and transferred into the recipient whether the donor was serovar Gallinarum-Pullorum or E. coli; in contrast, regardless of the donor species, the natural salmonella or E. coli host, the transfer frequency of the full-length pSEV or pSTV was very low, generally about 100-fold lower than that of pSPV (Table 2).

In general, a similar trend could be seen in the mating with recombinant plasmids: the rate of transfer was much higher with the recombinant plasmid derived from pSPV than with that from pSEV or pSTV (Table 2). When the transfer frequency was compared between the full-length and the recombinant plasmids, on the other hand, in all cases, the mobilization frequency was generally higher with the shorter-length recombinant plasmid than the corresponding full-length plasmid. For pSPV, the mating frequency of the recombinant plasmid was 10- to 100-fold higher than that of the full-length plasmid (Table 2), and for pSEV or pSTV, it was also generally 100-fold higher with the recombinant plasmid (Table 2).

Nucleotide sequence and structure of the oriT regions.

Next, the nucleotide sequence and the structure of the oriT region in the cloned fragments listed in Table 2 were determined. The restriction map (not shown) and orientation in terms of the known genes in the area were determined. The region stretched from samA on the left through oriT to traE on the right (see reference 4 for the location on the plasmids). The entire nucleotide sequences of these fragments, except for a portion of the 10.6-kb fragment (about 6 kb, located on the left end and outside of the tra region) in pOU1506, were determined.

The sequence of 550 bp in the immediate vicinity of each oriT is shown in Fig. 1, where the oriT regions of the three are juxtaposed to the corresponding base pairs of the F plasmid oriT region: arbitrarily, −157 bp from the nick (nic, C-C) site is assigned as the first base pair (bp 1, A[GCAA…]). Note that the gene sequence (gene 19 on the left) shown is in the mirror image of that shown by Chu et al. (4); this is to conform to the conventional order of the tra region. Variations in sequences were seen scattered all over the region, with the most varied area concentrated in RBS2 (the ribosome-binding site for traM) region from about bp 415 to bp 470.

FIG. 1.

Nucleotide sequences of the oriT regions of F plasmid and the three pSVs. Arbitrarily, −157 from the nick site (nic, C-C) of F plasmid is assigned as bp 1, to which all the other sequences are aligned. Colons indicate identical bp. Two boxes with an arrow above indicate the 8-bp inverted repeats. For the other site and gene (locus) designations, see the text and references 10 and 20. Short arrows facing each other within a pair of short vertical lines indicate the range of the site. Accession numbers: F plasmid sequence, NC_002483; sequences from oriT to traL, AF389529 (pSPV), AF389530 (pSTV), and AF389528 (pSEV).

In general, the sequence of pSPV showed a greater variation than those of the other two pSVs from that of F plasmid. The nucleotide sequences of genes 32 (not shown) and 19 (shown partially) of the three pSVs were almost identical, and they were also very similar to those of F plasmid. The length of the oriT from gene 19 (gene X) to traM was 416, 422, and 416 bp for pSEV, pSPV, and pSTV, respectively (Fig. 1). The oriT region of these three pSVs comprised binding sites: sbi (binding site for TraI), sby (binding site for TraY), sbm (binding site for TraM), and two integration host factor (IHF) binding sites, both of which were similar to those of F plasmid (20).

The oriT nucleotide sequences of pSEV and pSTV differed by only 4 bp but differed significantly from that of pSPV. The overall nucleotide sequence of the F oriT showed a higher homology (Table 3) to that of pSEV (65.7%) and pSTV (64.1%) than to that of pSPV (53.5%). Major differences were found in the protein binding regions; particularly the sby and sbm nucleotide sequences of these pSVs differed substantially from the corresponding sequences in F plasmid (Fig. 1 and Table 3). The putative sbi nucleotide sequence of each of the three pSVs, however, was identical to that of F plasmid (21). As to other loci, the nucleotide sequences of the two TraY binding sites, sbyC and sbyA, of pSPV were highly homologous to those of F plasmid (Table 3), whereas substantial variations could be discerned in that of pSEV and pSTV. TraM, on the other hand, binds to three sites, sbmA, -B, and -C, whose nucleotide sequences in pSEV and pSTV were similar to that of F plasmid, whereas that of pSPV was much less similar (Table 3). There is an 8-bp inverted repeat (GCAAAAAC) at the 3′ end of sbi in F plasmid (bp 168 to bp 185). The inverted repeat at the corresponding location in the three pSVs was GCAAAATT (Fig. 1).

TABLE 3.

Nucleotide (nt) sequence similarity of the oriT regions including binding sites for TraM and TraY between F plasmid and the pSVs

Plasmid	% Homology to F (no. of pSV nt/no. of F nt)
	oriT	sby		sbm
		sbyA	sbyC	sbmA	sbmB	sbmC
pSPV	53.5 (226/422)	72.2 (26/36)	76.2 (16/21)	34.3 (12/35)	33.3 (10/30)	35.9 (14/39)
pSTV	64.1 (267/416)	53.7 (19/36)	65.0 (13/20)	68.6 (24/35)	83.3 (25/30)	64.1 (25/39)
pSEV	65.7 (270/416)	55.5 (20/36)	65.0 (13/20)	68.6 (24/35)	83.3 (25/30)	64.1 (25/39)

Amino acid sequences of TraM, TraJ, TraY, TraA, and TraL of pSVs.

The gene order of the five known tra genes in the oriT region is traM-traJ-traY-traA-traL in F plasmid (7) and in the three pSVs (Table 4). However, the nucleotide sequences and consequently the sizes and amino acid sequences of the proteins encoded by these five tra genes in the three pSVs differed from those of F plasmid and some from each other among the three (Tables 3, 4, and 5). As shown in Table 4, the nucleotide coordinates and amino acids of each gene except those of traY were identical between pSEV and pSTV. The number of nucleotides of traA, traL, and traM of pSPV was quite different from that of the other two, yet the numbers of nucleotides and amino acids were identical. Substantial differences in the numbers of both nucleotides and amino acids in pSPV from the other two were observed in traJ and traY and the corresponding proteins (Table 4). There were 483 nucleotides coding for 161 amino acids in the traJ of pSPV, whereas in both pSEV and pSTV, there were 687 nucleotides encoding 228 amino acids.

TABLE 4.

Nucleotide (nt) and amino acid (aa) sequences of tra loci in the oriT-traM-traJ-traY-traA-traL region of the three pSVs^a

Gene	pSEV			pSTV			pSPV
	Location (nt)	No. of bp	No. of aa	Location (nt)	No. of bp	No. of aa	Location (nt)	No. of bp	No. of aa
oriT	1-523			1-523			1-531
traM	524-904	381	126	524-904	381	126	532-912	381	126
traJ	1097-1783	687	228	1097-1783	687	228	1107-1589b	483	161
traYc	1877-2104	228	75	1784-2104	321	106	1890-2291	402	133
traA	2159-2521	363	120	2159-2521	363	120	2333-2695	363	120
traL	2536-2847	312	103	2536-2847	312	103	2710-3021	312	103

^aSee reference 4 for approximate locations in the plasmids.

^bThis segment contained two open reading frames, and the first one on the left codes for 161 amino acids, which is listed.

^cThe traY sequence of pSTV, the nucleotide numbering of which is used here, is identical to the sequence with accession number AJO11572 and also, except for a few bases, to the corresponding segment of the nucleotide sequence in the database of Washington University, St. Louis, Mo.

TABLE 5.

Comparison of amino acid (aa) sequences of TraM, TraJ, TraY, TraA, and TraL between F-related plasmids and pSVs^a

Plasmid	% Homology (no. of identical aa/total)
	TraM		TraJ		TraY		TraA		TraL
	R1-19	F	R1-19	F	R1-19	F	R1-19	F	R1-19	F
pSEV	75 (95/126)	81 (102/126)	75 (150/199)	73 (182/246)	91 (68/75)	26 (34/130)	90 (108/120)	90 (110/121)	90 (81/90)	91 (94/103)
pSPV	85 (107/126)	74 (94/126)	0b	64 (145/228)	49 (37/75)	87 (113/128)	89 (107/120)	90 (109/121)	91 (82/90)	92 (95/103)
pSTV	75 (95/126)	81 (102/126)	73 (147/199)	72 (79/246)	91 (68/75)	26 (34/130)	88 (106/120)	89 (108/121)	91 (82/90)	92 (95/103)

^aF plasmid is incompatability group IncFI, and plasmid R1-19 is incompatability group IncFII.

^bLittle homology could be discerned.

As to traY, they were all different in both nucleotides and amino acids: 228 nucleotides encoding 75 amino acids for pSEV, 321 nucleotides encoding 106 amino acids for pSTV, and 402 nucleotides encoding 133 amino acids for pSPV. When the amino acid sequence was examined, differences, some quite substantial, appeared, as shown in Table 5, where the differences compared with plasmids R1-19 and F are listed. The peptide length and amino acid sequence of TraA and TraL were not identical but were very similar to those of F plasmid and R1-19 as well as among the three (Table 5). Those of the other three Tra proteins, however, were substantially different, not only from those of F plasmid and R1-19 but also from each other.

In general, TraY of pSEV and pSTV showed more homology to that of R1-19 than to that of F plasmid, whereas that of pSPV was much closer to that of F plasmid. The nucleotide sequences of traY of pSEV and pSTV differed in only 2 bp, and furthermore, there was a point mutation at the second nucleotide of the codon TTA (Val), altering it to TAA, a stop codon, in pSEV, reducing the amino acid length by 31 amino acids. Thus, the amino acid lengths of TraY of pSEV and pSTV were 75 and 106, respectively (Table 4). The amino acid composition in the 75-amino-acid TraY of pSEV was identical to that in the corresponding segment of the TraY of pSTV. Both TraYs showed very high homology to that of R1-19, but quite low homology to the TraY of pSPV and very low homology to that of F plasmid (Table 5). Conversely, TraY of pSPV showed higher homology than those of the other two to that of F plasmid.

Additionally, the TraY of both F plasmid and pSPV contained a duplicate βαα segment, whereas that of pSEV and pSTV contained only one unit of the βαα segment (Fig. 2). As to the TraM and TraJ of pSEV and pSTV, they consisted of the same numbers and nearly identical sequences of amino acids. TraM of these two showed a higher homology to that of F plasmid than to that of R1-19; the TraM of pSPV, however, showed higher homology to that of R1-19 than to that of F plasmid. TraJ of the three pSVs all showed from 64 to 75% homology to TraJ of both R1-19 and F plasmid, except that the TraJ of pSPV showed little homology to that of R1-19 (Table 5).

FIG. 2.

Aligned amino acid sequences of TraY of F plasmid, pSPV, pSEV, and pSTV. Italicized bold letters signify conserved sequences between consensus 1 and 2. Underlined letters denote DNA-binding domains (13). The accession number for R1-19 is M19710. There was only one unit of the βαα segment in pSEV and pSTV, but two are shown here for easy viewing.

DISCUSSION

The frequency of mobilization was affected by the size (length) of the plasmids: the shorter the length, the higher the frequency. This is as expected, since a shorter plasmid would be transferred in a shorter time with little physical interference such as strand breaks than a long one, and as a result, as in an Hfr mating, there would be a gradient of transfer as a function of the size. However, we have no direct evidence here that this is so. On the mode of mobilization, we have earlier shown no formation of cointegrates and also, in Hfr mating, obtained results that seemed to disfavor the model of mobilization by cointegrate formation (19). In the present mating experiments also, no cointegrates were found. Therefore, the mobilization was probably not achieved by cointegrate formation. Also, the shorter plasmids were all recombinant plasmids with pUC18 as the vector; however, the effect of the vector on the mobilization frequency was likely small, if any, since the mobilization of pUC18 itself was nil.

Irrespective of the size, full length or shorter, the plasmids that contained the oriT region derived from either pSEV or pSTV were mobilized with very low frequency compared to those derived from pSPV (Table 2), confirming the earlier observation (19). The nucleotide sequence and structure of the oriT regions indicated that the immediate vicinity of the nic point was nearly identical for all plasmids. Therefore, this region was not the reason for the discrepancy in mobilization frequency. In the 8-bp inverted repeat of the three pSVs, two Ts replaced the AC of the 8-bp repeat of F plasmid. Yet only pSPV was efficiently mobilized, and therefore, the alteration of the base pair in the repeat was probably not the cause of the variance in mobilization. However, among all plasmids, including conjugative F and F-like plasmids, substantial variations were observed in other areas, particularly in the traJ, sby, and sbm regions. The last two are where TraY and TraM bind, respectively (6, 12, 14).

The traJ gene of pSPV was much shorter in both nucleotides and corresponding amino acids than those of the other two pSVs. On the other hand, the sby region of pSPV was highly homologous, but not that of pSEV and pSTV, to that of F; conversely, the sbm region of pSEV and pSTV but not that of pSPV showed more homology to that of F plasmid. The speculation would therefore be that, in addition to traJ, the sby region is responsible for the high mobilization frequency of pSPV. The sbys are the binding sites for TraY, and thus, the speculation is supported by the nucleotide sequence of traY and the structure, size, and amino acid sequence of TraY of the three pSVs. Because of a base replacement that converted the mutation site into a terminal codon, the size of the putative TraY of pSEV was only 75 amino acids, in contrast to 106 for pSTV and 133 for pSPV, both of which contained no terminal codon formed by mutation, and therefore, the last two TraYs should be full length.

When the amino acid sequences of these putative TraYs was examined, the TraY of pSPV showed very much higher homology than the other two to the amino acid sequence of F plasmid TraY (Table 5). On the other hand, the amino acid sequence of pSPV's TraY showed only 49% homology to that of R1-19 (IncFII), compared to 91% for the other two. Furthermore, the TraY of pSEV and pSTV carried only one copy of the βαα segment, which is involved in the binding of TraY (13), in contrast to two in the TraY of F and pSPV. Mobilization of the three pSVs by R1-19, however, has not been tested. These observations indicate that the key event in discriminating the mobilization of pSPV from that of pSEV and pSTV by F plasmid appears to reside in TraJ and TraY and particularly in the binding of TraY to the binding site sby in the oriT region.

Since TraJ and TraY would be produced by the coexisting mobilizing conjugative plasmid, the variation in these binding sites is presumably responsible for the anomalous mobilization efficiency by affecting the affinity of the Tra proteins for the specific binding sites. We are in the process of extracting TraY from F and the three pSVs to test its binding power to the sby regions of these pSVs by gel mobility test.