Identification of site-specific recombination genes int and xis of the Rhizobium temperate phage 16-3

Abstract

Phage 16-3 is a temperate phage of Rhizobium meliloti 41 which integrates its genome with high efficiency into the host chromosome by site-specific recombination through DNA sequences of attB and attP. Here we report the identification of two phage-encoded genes required for recombinations at these sites: int (phage integration) and xis (prophage excision). We concluded that Int protein of phage 16-3 belongs to the integrase family of tyrosine recombinases. Despite similarities to the cognate systems of the lambdoid phages, the 16-3 int xis att system is not active in Escherichia coli, probably due to requirements for host factors that differ in Rhizobium meliloti and E. coli. The application of the 16-3 site-specific recombination system in biotechnology is discussed.

FIG. 1

Map of the pSEM91 expression vector constructed from the replicon of plasmid pCU1 (hatched bar), RP4 mob region (shaded bar), and kanamycin resistance gene (Km) (black arrow). The shaded triangle represents the tac promoter, while the open bar shows the location of transcription terminators. Restriction sites in bold letters are unique.

FIG. 2

Plasmid constructs and the locations of their 16-3 phage content. (A) The 16-3 genome is indicated at the top (the scale is given in kilobases). Bars represent the extent and topology of the 16-3-derived part of each cosmid or plasmid construct. The region present in pSEM35 is enlarged to show details. The shaded bar shows the region of the determined sequence (GenBank accession no. AJ131679; the scale is given in base pairs). Open bars indicate deletions. Black arrows indicate the gene of the phage repressor (c). Stippled boxes represent the attachment region of the phage (attP). Restriction sites with numbers in parentheses refer to physical map positions as shown in reference . The arrowhead marked p_LO_L indicates the promoter-operator unit to the left of the repressor gene. Binding of the repressor to the p_LO_L unit regulates not only the lytic-lysogenic decision but also influences the site-specific recombination process of phage 16-3. (B) ORFs of the potential candidates for encoding Int and Xis proteins (black arrows). Black bars indicate the regions carried by different plasmid constructs. Stippled boxes represent the attachment region of the phage (attP). Numbers in parentheses following the names of the plasmids indicate phage content by base positions (the scale is given in base pairs below the shaded bar).

FIG. 3

Site-specific integration of plasmids containing attP into the R. meliloti chromosome as detected by PCR (A) and by Southern blotting (B and C). (A) The appearance of a 186-bp-long PCR product indicating attL formation due to integration of pSEM167 (lane 1) and of pSEM6 in the presence of pSEM164 (lane 2) can be seen. The presence of pSEM6 alone (lane 3) or the pSEM91 expression vector (lane 4) in R. meliloti did not result in attL formation. attL is also not present in R. meliloti (lane 5). M indicates the AluI digest of pBluescript II KS used as a molecular size marker. (B) Southern hybridization with a ³²P-labelled attP fragment. The order of the samples is the same as in panel A except that a mixture of known attP-containing fragments of different sizes was used for molecular size markers. The presence of attL and attR carrying EcoRI restriction fragments identified the site-specific recombination event (lanes 1 and 2). The attP-carrying fragments of pSEM6 indicate extrachromosomal copies of the plasmid (lanes 2 and 3). With the construct pSEM167 (lane 1) the extrachromosomal copies of the plasmid were lost, probably due to interference with plasmid replication. (C) Southern hybridization with the ³²P-labelled attB fragment. The same filter as in panel B was used. attL and attR fragments were identified when site-specific recombination occurred (lanes 1 and 2), but only attB could be detected in the controls (lanes 3 to 5). Rm41, R. meliloti 41.

FIG. 4

Comparison of 16-3 Int protein with the conserved regions of tyrosine recombinases. The open bar indicates schematically the sequence of a protein, and shaded boxes represent the locations of homology regions. The conserved residues and the ranges of spacings between them (as per reference 13) are indicated below the bar. Seven integrases were chosen to show the strong similarity of the homology regions found among the selected integrases and 16-3 Int protein. Filled and shaded circles indicate identical and similar residues, respectively. Boldface residues indicate the R-H-R-Y tetrad. Numbers between the sequences indicate actual spacings. The database sources for accession numbers are SwissProt (those starting with “P”), GenBank, and EMBL. Ref., reference.

FIG. 5

(A) Schematic diagram of the assay used to identify the xis gene of phage 16-3. The presence of Xis protein expressed from the xis gene-containing derivative of pSEM91 results in the excision of pSEM168 from the R. meliloti 41 chromosome, regenerating the attB site from attL and attR, which can be identified by PCR with an attB-specific primer pair. A 23-kb fragment representing the integrated pSEM168 might have been amplified with the same primer pair, but the PCR conditions did not favor the accumulation of such a product. (B) Products of PCR amplification. Total DNA from R. meliloti 41 (Rm41) (lane 1), R. meliloti 41(pSEM168) (lane 2), R. meliloti 41(pSEM168) plus pSEM91 (lane 3), R. meliloti 41(pSEM168) plus pSEM163 (lane 4), and R. meliloti 41(pSEM168) plus pSEM161 (lane 5) were used for templates in attB-diagnostic PCR assays. M indicates the molecular size marker (AluI digest of pBluescript II KS).

FIG. 6

Detection of cointegrate formation between pSEM167 and pIP79. EcoRI digests of pSEM167 (lane 1), pIP79 (lane 2), and both plasmids derived from E. coli (lane 3) and R. meliloti 41 (Rm41) (lane 4) are presented. The λ PstI digest served as the molecular size marker (lane M). The appearance of a 1,122-bp EcoRI fragment in lane 4 instead of the 276-bp EcoRI fragment of pIP79 in lanes 2 and 3 indicates cointegrate formation.