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-l, 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-i 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.Conjugative transposons promote bacterial conjugation, during which they excise from a donor and then insert into a new target in the recipient genome (recent reviews include references 6, 21, 22, and 29). All such transposons share regions of DNA homology and carry the tetM gene, a determinant of tetracyline resistance which is expressed in bacteria of many different genera (6). The promiscuity of conjugative transposons is an important clinical problem, since they are transferred among unrelated gram-positive bacteria and serve as vectors for the spread of antibiotic resistance. Although these elements do not promote conjugation between Escherichia coli strains (7, 11), they do transpose in this organism. These elements integrate into many different sites in the genome of their hosts, and multiple transposons are often found in transconjugants (10). Furthermore, the presence of a conjugative transposon in the recipient strain in mating does not affect the frequency of integration of a second such element (8, 16). These properties make conjugative transposons valuable for mutagenesis, especially in gram-positive bacteria.The best-studied conjugative transposons are the 16.4-kb Tn916 (9) and TnI545, a 25.3-kb transposon. In addition to tetM, the latter also carries antibiotic resistance determinants for erythromycin and kanamycin (3). The terminal 250 bp of these two elements are essentially identical, and they have similar restriction maps.

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.

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 regon was fused to the lacZ gene. ,-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 ,B-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.Soon after the initiation of sporulation, Bacillus subtilis synthesizes an intracellular serine protease (ISP-1) (3,16). The isp gene has been cloned, and studies have shown that ISP-1 is not essential for normal sporulation (2, 10). The exact role of this enzyme in sporulation has not been established, and little is known about the regulation of ISP-1 expression and control of protease activity. It has been suggested that ISP-1 activity might be regulated by interaction with a proteinaceous inhibitor, calcium availability, or activation by membrane-bound proteases (12,13,19). A recent report presented immunological evidence that ISP-1 was initially synthesized as an enzymatically inactive protein that was subsequently converted to an active protease (3).Several mutations affecting the production of extracellular enzymes in B. subtilis have been described. Mutations in the sacU and sacQ genes are pleiotropic in nature, affecting the prpduction of many extracellular enzymes and some sporulation-related events (11,22). Other mutations (catA, hpr, ScoC), although from independently isolated mutants, may be mutations of the same gene. These mutations affect production of the extracellular proteases and catabolite repression of sporulation eyents (5,8,9). The only common feature of the target genes of these hyperproduction mutations seems to be their participation in adapting to starvation conditions requiring the utilization of a complex carbon or nitrogen source. Since one of the postulated roles of ISP-1 is to provide amino acids by degrading proteins during sporulation, it wa...

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