Excision of the Conjugative Transposon Tn916 in Lactococcus lactis

Abstract

In Lactococcus lactis excision of Tn916 is limited by the concentration of integrase and is increased by providing more excisionase. However, even with increased excision of Tn916 in L. lactis, no conjugative transfer is detectable. This suggests that L. lactis is deficient in a host factor(s) required for conjugative transposition.

Conjugative transposons mediate their own transfer from a donor DNA molecule in one bacterial cell to a target molecule in another cell. Excision of Tn916 from the donor molecule is required for conjugative transposition and results in a covalently closed circular transposon molecule that is the intermediate in conjugal transfer (2). A single DNA strand of the covalently closed circular transposon is transferred to the recipient cell, where the complementary strand is synthesized to recreate a double-stranded circular transposon, which inserts into a target site (15).

The host range of Tn916 includes more than 53 species of bacteria (4). Among these hosts is Lactococcus lactis, which is unique because Tn916 can enter and insert in L. lactis following conjugative transposition from another species but is not able to mediate its own transfer from L. lactis to another host (1). L. lactis can act as a donor of conjugative plasmids, so it does not have a general defect in conjugational transfer. The most likely explanation for the lack of conjugative transposition in L. lactis is that Tn916 is unable to excise from the donor DNA molecule and is therefore unable to undergo conjugative transposition.

Excision of Tn916 in gram-positive bacteria requires the expression of two transposon-encoded proteins, integrase and excisionase (Int and Xis, respectively). Int is a site-specific recombinase belonging to the λ-integrase family, which cleaves both ends of Tn916 (13, 16). The location and stoichiometry of Xis binding to Tn916 in vitro indicate that it may bend the DNA (14), perhaps to facilitate binding or cleavage by Int. To test whether the lack of Tn916 conjugative transposition from L. lactis donors is caused by the failure of the transposon to excise, we tried to increase excision by overexpressing Int and Xis.

The expression vector pEU327 (6), which includes the broad-host-range pSH71 origin of replication (5) and the Bacillus subtilis xylA promoter (P-xylA) (8), was used to overexpress Int and Xis in L. lactis. Int and Xis were expressed individually and together under control of P-xylA from pEU329 (Int), pEU357 (Xis), and pEU354 (Int and Xis) (11). As in Enterococcus faecalis, overexpression of these proteins from the plasmids was detected by using polyclonal antibodies and was constitutive (11; Western blot data not shown). Expression of Int and Xis from a single copy of Tn916 in L. lactis could not be detected by Western blotting (data not shown).

To assay excision of Tn916, the conjugative plasmid pEU333 (15) was mated into L. lactis FMCB1 (a fusidic acid-resistant derivative of strain MG1363 [1]) to give strain EFL1103. Plasmid pEU333 consists of pIP501 (7) with Tn916 inserted in the chloramphenicol acetyl transferase (cat) gene. Excision of Tn916 from pEU333 and repair of the donor molecule result in an intact cat gene, which makes the cells chloramphenicol resistant (Cm^r). Excision frequency was expressed in terms of number of Cm^r CFU/total CFU.

We overexpressed Int and/or Xis in EFL1103/pEU333 in order to determine if either protein was limiting for excision. Excision of Tn916 from pEU333 resulted in production of a low frequency of Cm^r colonies, and addition of the vector pEU327 did not affect the excision frequency (Table 1). Addition of the Int-encoding plasmid pEU329 to EFL1103/pEU333 resulted in a 30-fold increase in the excision frequency (Table 1). EFL1103/pEU333 containing the Xis-encoding plasmid pEU357 exhibited slightly greater excision frequency than the strain containing the vector exhibited (Table 1). Thus, in L. lactis, the amount of Xis appears to be almost adequate, while the concentration of Int appears to be too low for significant excision to occur. This result differs from the results obtained with B. subtilis and E. faecalis, in which overexpression of Int or Xis alone did not have a significant effect on excision of Tn916 (11). Additional Int and Xis, expressed from pEU354 in EFL1103/pEU333, resulted in a sevenfold increase in the excision frequency compared with the frequency obtained with Int alone (Table 1). This indicates that once the deficiency in the Int concentration is overcome by overexpression of Int, further increases in the excision frequency require overexpression of Xis as well as Int. In other gram-positive species, excision is increased only when both Int and Xis are expressed (11).

TABLE 1.

Effect of overexpression of Int and/or Xis on excision in L. lactis

Plasmid in EFL1103/pEU333	Protein encoded by plasmid	Excision frequency (Cmr CFU/total CFU)
None	NAa	4.5 × 10−8 ± 4.1 × 10−8b
pEU327	Vector	4.3 × 10−8 ± 5.2 × 10−8
pEU329	Int	1.3 × 10−6 ± 1.1 × 10−6
pEU357	Xis	1.3 × 10−7 ± 0.5 × 10−7
pEU354	Int and Xis	9.5 × 10−6 ± 3.6 × 10−6

^aNA, not applicable. 

^bMean ± standard deviation. 

A total of 103 of the Cm^r excisants isolated when Int and Xis were provided in trans from pEU354 were screened for tetracycline resistance (Tc^r) encoded by Tn916, and all were found to be Tc^r. This indicates that either Tn916 had not excised from all copies of pEU333 or that it was inserted elsewhere in the cell. Five excisants were tested by using primers in the cat gene and the ends of Tn916 to determine if some copies of pEU333 in the Cm^r cells still contained an insertion in the cat gene. In two of the five excisants examined, amplification products specific for cat::Tn916 were observed (data not shown), indicating that some excisants retained a copy of cat::Tn916 while others were likely to have Tn916 inserted at a new site (since the cells were Cm^r and Tc^r). The presence of an inserted copy of Tn916 in most (possibly all) of the L. lactis excisants differs from the results obtained with E. faecalis and B. subtilis (11). In B. subtilis all of the excisants lost Tn916, and the cells became tetracycline sensitive when Int and Xis were overexpressed from pEU354, and in E. faecalis less than one-half of the excisants retained a copy of Tn916. Therefore, reinsertion of Tn916 in the same cell after excision is more frequent in L. lactis than in the other two gram-positive hosts tested.

To determine whether L. lactis could serve as a conjugative donor under conditions that allowed measurable Tn916 excision, conjugative transposition was assayed in a strain containing plasmids supplying Int and Int plus Xis. Strain EFL1103/pEU333 was not used in these assays because pEU333 is capable of mediating conjugal transfer. Instead, strain LL411 (FMCB1::Tn916), which contains a single copy of Tn916 inserted in the chromosome (as determined by Southern blotting [data not shown]), was used as the donor and E. faecalis OG1X (9) was used as the recipient. No conjugative transposition (<3.2 × 10⁻¹¹ transconjugants per donor) was detected from LL411 carrying either pEU329 (Int overexpressed) or pEU354 (Int plus Xis overexpressed). Identical results were obtained when an L. lactis recipient strain was used in the mating experiments (data not shown). Similarly, increased excision caused by Int and Xis overexpression did not affect the frequency of conjugative transposition from B. subtilis or E. faecalis donors (12), although unlike L. lactis, conjugative transposition was detectable in these hosts.

It seemed possible that the absence of conjugative transposition in L. lactis might have resulted from deficient expression of the conjugation functions of Tn916. To examine this possibility, we tested the ability of Tn916-containing L. lactis strains to mobilize a plasmid containing the Tn916 origin of transfer (oriT). Plasmid pAM5160 contains the Tn916 oriT, the cat gene, and the replication origin of pIP501 (7) and can be mobilized by Tn916-containing E. faecalis and B. subtilis strains (3, 10). Excision of Tn916 is required for mobilization of pAM5160 in B. subtilis (3), although increased excision of Tn916 does not increase the frequency of pAM5160 mobilization in B. subtilis or E. faecalis (12). We found that none of the L. lactis strains tested was able to mobilize pAM5160 into E. faecalis OG1X, even when Int and Xis overexpression increased the excision frequency in the donor strain. Both strain LL411/pEU327 (vector) and strain LL411/pEU354 (Int and Xis) gave <2.4 × 10⁻¹¹ Cm^r transconjugants per donor, indicating that the Tn916-encoded transfer functions required for mobilization are not expressed in L. lactis. This implies that in addition to the Tn916 excision defect and unlike other gram-positive hosts, L. lactis is deficient for a factor needed for conjugal transfer from the Tn916 oriT.

We showed that in L. lactis the supply of Int limits the excision frequency of Tn916. Since Int was overproduced from a foreign promoter on an introduced plasmid, this implies that a host factor may affect transcription of at least this Tn916 gene and possibly others. In addition, conjugative transposition is not detectable in L. lactis, even when the excision frequency is increased by an oversupply of Int and Xis. Instead, Tn916 transfer functions are deficient in this host, and excision can result in reinsertion of the transposon in the same cell. The proposed host factor (see above) potentially involved in transcription of int may also be involved in transcription of genes required for conjugal transfer of the excised transposon, which would explain the defects in both excision and conjugal transfer observed in L. lactis. Alternatively, L. lactis may lack a host factor(s) directly involved in excision and conjugal transfer. Perhaps a host factor is required for a productive cleavage reaction between Int and the DNA at low Int concentrations and another host factor is required for transfer of the transposon DNA through the cell membrane. The use of L. lactis to study conjugative transposition may help elucidate host-encoded factors required for this process, and since the host-specific factor that leads to a defect in expression of a Tn916 gene(s) may be more general, it may also increase our understanding of gene expression in L. lactis.