Site-Specific Integrative Elements of Rhizobiophage 16-3 Can Integrate into Proline tRNA (CGG) Genes in Different Bacterial Genera

ABSTRACT 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.

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New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes forMethanosarcinaspecies

Archaea ◽

10.1155/2008/534081 ◽

2008 ◽

Vol 2 (3) ◽

pp. 193-203 ◽

Cited By ~ 60

Author(s):

Adam M. Guss ◽

Michael Rother ◽

Jun Kai Zhang ◽

Gargi Kulkkarni ◽

William W. Metcalf

Keyword(s):

Chromosomal Integration ◽

Recombination Site ◽

Integrase Gene ◽

Site Specific ◽

Site Specific Recombination ◽

Highly Efficient ◽

Recombination System ◽

Regulated Gene Expression ◽

Hybrid Promoters ◽

Site Specific Recombination System

A highly efficient method for chromosomal integration of cloned DNA intoMethanosarcina spp.was developed utilizing the site-specific recombination system from theStreptomycesphage φC31. Host strains expressing the φC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressedM. barkeriPmcrBpromoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strains that express thetetRgene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains withtetR-regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.

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Identification of Site-Specific Recombination Genesint and xis of the Rhizobium Temperate Phage 16-3

Journal of Bacteriology ◽

10.1128/jb.181.14.4185-4192.1999 ◽

1999 ◽

Vol 181 (14) ◽

pp. 4185-4192 ◽

Cited By ~ 13

Author(s):

Szabolcs Semsey ◽

IstvAn Papp ◽

Zsuzsanna Buzas ◽

Andras Patthy ◽

Laszlo Orosz ◽

...

Keyword(s):

Dna Sequences ◽

High Efficiency ◽

Rhizobium Meliloti ◽

Temperate Phage ◽

Host Chromosome ◽

Site Specific ◽

Site Specific Recombination ◽

E Coli ◽

Tyrosine Recombinases ◽

Recombination System

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 phage16-3 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.

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