Conjugative transposition of Tn916: preferred targets and evidence for conjugative transfer of a single strand and for a double-stranded circular intermediate.

Transposition of conjugative transposons proceeds by excision and formation of a covalently closed circular intermediate that includes at its joint the six flanking bases from its previous host (coupling sequences). To elucidate the role of the coupling sequences in this process and to determine the sequence of targets used by Tn916, we studied its insertion into a plasmid following conjugation. The results differ from those previously observed when Tn916 was introduced by transformation. They suggest that only one specific strand of the transposon molecule is transferred during the conjugation event and that complementary strand synthesis produces a double-stranded transposon circle with no mismatches which serves as the reaction intermediate. Tn916 inserts preferentially at specific sites and the same targets are used when Tn916 comes from donors with different coupling sequences. An analysis of the sequences of preferred targets is presented.

Transfer of Tn916 between Lactococcus lactis subsp. lactis strains is nontranspositional: evidence for a chromosomal fertility function in strain MG1363.

Lactococcus lactis subsp. lactis MG1363 can act as a conjugative donor of chromosomal markers. This requires a chromosomally located fertility function that we designate the lactococcal fertility factor (Laff). Using inter- and intrastrain crosses, we identified other L. lactis strains (LMO230 and MMS373) that appear to lack Laff. The selectable marker in our crosses was Tcr, carried by Tn916, a transposon present on the chromosome. The transfer of Tcr was not due to Tn916-encoded conjugative functions, because (i) L. lactis cannot act as a donor in Tn916-promoted conjugation (F. Bringel, G. L. Van Alstine, and J. R. Scott, Mol. Microbiol. 5:2983-2993, 1992) and (ii) transfer occurred when the Tcr marker was present in a Tn916 derivative containing a mutation, tra-641, that prevents Tn916-directed conjugation in any host. In addition, we isolated a strain in which Tn916 appears to be linked to Laff; this strain should be useful for further analysis of this fertility factor. In this strain, Tn916 is on the same 600-kb SmaI fragment as Clu, a fertility factor previously shown to promote lactose plasmid transfer in L. lactis. Thus, it is possible that Clu and Laff are identical.

Conjugative transposition of Tn916: the transposon int gene is required only in the donor.

Conjugative transposition of transposon Tn916 has been shown to proceed by excision of the transposon in the donor strain and insertion of this element in the recipient. This process requires the product of the transposon int gene. We report here the surprising finding that the int gene is required only in the donor during conjugative transposition. We find that Tn916 int-1, whose int gene has been inactivated by an insertion mutation, transposes when a complementing wild-type int gene is present only in the donor during mating. When the int+ gene is present in a plasmid and is expressed from the spac promoter, conjugative transposition is very inefficient. However, when the Int+ function is supplied from a coresident distantly linked Tn916 tra-641 mutant, which is defective in a function required for conjugation, efficient conjugative transposition of Tn916 int-1 occurs. This suggests either that Int is not required for integration of Tn916 in gram-positive bacteria or that the protein is transferred from the donor to the transconjugant during the mating event. When the nonconjugative plasmid pAT145 was present in the donor, it was rarely cotransferred with Tn916. This suggests that complete fusion of mating cells is not common during conjugative transposition.

A host factor absent from Lactococcus lactis subspecies lactis MG1363 is required for conjugative transposition.

In matings between Lactococcus lactis strains, the conjugative transposons Tn916 and Tn919 are found in the chromosome of the transconjugants in the same place as in the chromosome of the donor, indicating that no transposition has occurred. In agreement with this, the frequency of L. lactis transconjugants from intraspecies matings is the same whether the donor contains the wild-type form of the transposon or the mutant Tn916-int1, which has an insertion in the transposon's integrase gene. However, in intergeneric crosses with Bacillus subtilis or Enterococcus faecalis donors, Tn916 and Tn919 transpose to different locations on the chromosome of the L. lactis transconjugants. Moreover, Tn916 and Tn919 could not be transferred by conjugation from L. lactis and B. subtilis, E. faecalis or Streptococcus pyogenes. This suggests that excision of these elements does not occur in L. lactis. When cloned into E. coli with adjacent chromosomal DNA from L. lactis, the conjugative transposons were able to excise, transpose and promote conjugation. Therefore, the inability of these elements to excise in L. lactis is not caused by a permanent structural alteration in the transposon. We conclude that L. lactis lacks a factor required for excision of conjugative transposons.

Control of intracellular serine protease expression in Bacillus subtilis.

Expression of the major intracellular serine protease (ISP-1) gene of Bacillus subtilis was studied by using a translational fusion plasmid in which the isp promoter region was fused to the lacZ gene. beta-Galactosidase activity, used to measure transcription from the isp promoter, was produced immediately after the end of exponential growth, whereas intracellular protease activity was not detected until 4 h later. These results are consistent with a previous suggestion that ISP-1 initially accumulates in the cell in an enzymatically inactive form. ISP-1 activity was detected in all of the sporulation-deficient strains examined, and the amount of protease activity always corresponded to the amount of beta-galactosidase activity. These results indicate that the activation of ISP-1 is not dependent on a sporulation-specific gene product. Expression of ISP-1 is regulated by a number of mutations known to affect the expression of extracellular enzymes. In sacU(h) and sacQ(h) mutants, the expression of ISP-1 was 10-fold higher than in the wild-type strain. In catA, hpr, and scoC strains, expression of ISP was stimulated two- to threefold, whereas in sacU mutants the expression of ISP-1 was reduced to less than 10% of the wild-type level. The temporal expression and activation of ISP-1 was not affected by any of these mutations. This is the first evidence that the expression of a native intracellular protein is affected by these hyperproduction mutations.