The integrase protein of the Rhizobium meliloti 41 phage 16-3 has been classified as a member of the Int family of tyrosine recombinases. The site-specific recombination system of the phage belongs to the group in which the target site of integration (attB) is within a tRNA gene. Since tRNA genes are conserved, we expected that the target sequence of the site-specific recombination system of the 16-3 phage could occur in other species and integration could take place if the required putative host factors were also provided by the targeted cells. Here we report that a plasmid (pSEM167) carrying the attP element and the integrase gene (int) of the phage can integrate into the chromosomes of R. meliloti 1021 and eight other species. In all cases integration occurred at so-far-unidentified, putative proline tRNA (CGG) genes, indicating the possibility of their common origin. Multiple alignment of the sequences suggested that the location of the att core was different from that expected previously. The minimal attB was identified as a 23-bp sequence corresponding to the anticodon arm of the tRNA.Site-specific integration is an efficient way to introduce new genetic information into the chromosome of the cell in a targeted fashion and ensure stable maintenance of the feature gained. Integrative vectors developed by utilizing site-specific integration elements from temperate bacteriophages (i.e., attP and the integrase gene combined) provide tools for this technology. Since the presence of the appropriate target sequence (attB) in the recipient chromosome and specific host factors are required for the integrative process, utilization of a particular integrative recombination system is frequently restricted to the host range of a given phage. A number of known eubacterial temperate phages, such as L5 (37), HP1 (24), 16-3 (35), RP3 (21), U (50), P4 (38), Mx8 (28), Sfi21 (7), 10MC (22), T12 (31), A2 (2), mv4 (16), Ms6 (19), and VWB (51), use tRNA genes as target sequences for integration into the host chromosomes. There are some plasmids with integrative functions, such as pSAM2 (30), pSE101 (5), pSE211 (6), and pSLP1 (52), whose target sites are also within tRNA genes. The SSV1 virus of the thermophilic archaeon Sulfolobus shibatae can also integrate into a tRNA (40, 41). Considering the conservative nature of tRNAs, one can expect that the target sites for a particular integrative system could occur in several bacterial species at various levels of relatedness. This view was confirmed by well-documented examples: the site-specific recombination systems of Ms6, mv4, VWB, pSAM2, pSE101, and pSLP1 were shown to function in closely related bacteria (3,5,19,39,51,52). Moreover A2 provides an example of integration in both gram-negative and gram-positive species (2).The temperate bacteriophage 16-3 of Rhizobium meliloti 41 has been studied thoroughly. In addition to genetic and physical characterization of its genome (10,12,14), the central region of the phage chromosome (GenBank accession no. AJ131679) was investigated in de...