On the site specific recombination of phage 16-3 of Rhizobium meliloti: identification of genetic elements and att recombinations

Olasz, Ferene; Dorgai, László; Papp, Péter; Hermesz, Edit; Kósa, E.; Orosz, László

doi:10.1007/bf00425673

Cited by 11 publications

(12 citation statements)

References 17 publications

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“…AJ131679) was investigated in detail. This part carries the c repressor gene essential for lysogenic development (8,9,11,15,33,34) and the site-specific recombination system (13,32,35,44) controlled by the c gene (48). The attP site and the genes responsible for the integration and excision functions (int and xis) have been identified (12,13,44).…”

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

et al. 2002

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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...

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

et al. 2002

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“…pCU999 was used as the source of the kanamycin resistance cassette (27). Cosmid clones pDH1 and pDH79 (23) and pDH114 (7) of the 16-3cti3 chromosome and pMW23, harboring the rkpZ gene of S. meliloti 41 (41), have been described elsewhere. In triparental matings, helper plasmid pRK2013 (12) was used for the transfer of pBBR1MCS-5 and pLAFR1 cosmid clones, and pCU101 (39) was used for the transfer of pPAG160 derivatives.…”

Section: Methodsmentioning

confidence: 99%

“…In order to localize the temperature-sensitive (ts) mutations in phages h109 and the h843, cosmid clones pDH1, pDH79, and pDH114 (7,23) and pBBR1MCS-5 derivatives harboring the EcoRI-C, EcoRI-D, EcoRI-L, and EcoRI-H fragments of the 16-3cti3 phage chromosome were used (Fig. 1).…”

Section: Methodsmentioning

confidence: 99%

Identification of Tail Genes in the Temperate Phage 16 - 3 of Sinorhizobium meliloti 41

et al. 2010

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Genes encoding the tail proteins of the temperate phage 16-3 of the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti 41 have been identified. First, a new host range gene, designated hII, was localized by using missense mutations. The corresponding protein was shown to be identical to the 85-kDa tail protein by determining its N-terminal sequence. Electron microscopic analysis showed that phage 16-3 possesses an icosahedral head and a long, noncontractile tail characteristic of the Siphoviridae. By using a lysogenic S. meliloti 41 strain, mutants with insertions in the putative tail region of the genome were constructed and virion morphology was examined after induction of the lytic cycle. Insertions in ORF017, ORF018a, ORF020, ORF021, the previously described h gene, and hII resulted in uninfectious head particles lacking tail structures, suggesting that the majority of the genes in this region are essential for tail formation. By using different bacterial mutants, it was also shown that not only the RkpM and RkpY proteins but also the RkpZ protein of the host takes part in the formation of the phage receptor. Results for the host range phage mutants and the receptor mutant bacteria suggest that the HII tail protein interacts with the capsular polysaccharide of the host and that the tail protein encoded by the original h gene recognizes a proteinaceous receptor.The Sinorhizobium meliloti-Medicago symbiosis is an important model for endosymbiotic nitrogen fixation. The genome sequence of S. meliloti (strain 1021) has been established (14), and the Medicago truncatula genome is under intensive investigation (3). Phage 16-3 is a temperate, double-stranded DNA phage of S. meliloti strain 41. It is by far the best-studied rhizobiophage and serves as a tool in analyses of rhizobium genetics, in the isolation of some symbiotic mutants, and in the construction of special vectors. Genetic determinants and molecular mechanisms of many aspects of the 16-3 life cycle, such as phage integration and excision (8,26,38), regulation of the lytic/lysogenic switch (5, 6, 9, 24, 28), immunity to superinfection (4), phage DNA packaging (15), and the role of gene h in the host range (32), have been examined in detail. Moreover, the complete 60-kb phage genome sequence (accession no. DQ500118) has been determined recently (P. P. Papp et al., unpublished results). However, little is known about the genes and structural elements involved in the interaction between the phage and its host, and furthermore, only one study of the 16-3 virion proteins has been reported (11).The initial interaction between a tailed phage and its bacterial host cell is mediated by the distal part of the phage tail, which specifically binds to the phage receptor located on the host surface. Earlier results demonstrated that phage 16-3 adsorption is connected to the strain-specific capsular polysaccharide of S. meliloti 41, the K R 5 antigen. So far, three bacterial gene clusters involved in K R 5 antigen production, including the rkp-1, rkp-2, and rk...

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“…The vectors carrying the attP site of the 16‐3 phage [10] can readily integrate into the Rm41 chromosome when the integrase protein of the phage is provided in trans from a helper plasmid pSEM102 (the construction will be published elsewhere). The helper plasmid pSEM102 was introduced in standard triparental matings to Rm41 bacteria containing the promoter probe constructions.…”

Section: Methodsmentioning

confidence: 99%

Integrative promoter cloning plasmid vectors forRhizobium meliloti

Élö

Semsey

Kereszt

et al. 1998

FEMS Microbiology Letters

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A set of integrative 'promoter probe' plasmids were constructed for both translational and transcriptional fusions. The vectors are based on the broad host range, low copy number plasmid pRK290 (IncPl) in which the attachment site of Rhizobium phage 16-3 and the lacZ gene of Escherichia coli were combined. The vectors integrate into the chromosome of Rhizobium meliloti, providing also the advantages of the single copy promoter probe cassettes. Thus they fulfil the prerequisite of the systems used for investigating gene regulation. The plasmids were applied for the study of the transcription regulation of the 16-3 phage. Their versatile use is also demonstrated.

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On the site specific recombination of phage 16-3 of Rhizobium meliloti: identification of genetic elements and att recombinations

Cited by 11 publications

References 17 publications

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

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

Identification of Tail Genes in the Temperate Phage 16 - 3 of Sinorhizobium meliloti 41

Integrative promoter cloning plasmid vectors forRhizobium meliloti

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