A <i>proline tRNA(CGG)</i> gene encompassing the attachment site of temperate phage <i>16‐3</i> is functional and convertible to suppressor tRNA

Blaha, Béla; Semsey, Szabolcs; Ferenczi, Szilamér; Csiszovszki, Zsolt; Papp, Péter; Orosz, László

doi:10.1111/j.1365-2958.2004.04300.x

Cited by 4 publications

(3 citation statements)

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“…(2) Seven ORFs in phage RsoM1USA annotated as hypothetical/other proteins have no corresponding ORFs in phage RSA1. (3) Myoviruses are known to integrate into bacterial genomes mediated by a portion of bacterial tRNA, an att site, as in the cases of phages RSA1 and RSY 1 of R. solanacearum (Fujiwara et al, 2008; Askora et al, 2017), phage 16-3 of Rhizobium meliloti (Blaha et al, 2004), and phage vB_RleM_PPF1 on Rhizobium leguminosarum (Halmillawewa et al, 2016). We confirmed experimentally that phage RsoM1USA uses the 3′ 45-base portion of the threonine tRNA (TGT) gene as its att site (Figure 5), which is different from RSA1 which uses the 3′ 45-base of the arginine tRNA (CCG) gene (Fujiwara et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

Molecular and Biological Characterization of Ralstonia Phage RsoM1USA, a New Species of P2virus, Isolated in the United States

2019

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The first Ralstonia -infecting bacteriophage from soil of the United States, designated RsoM1USA, was isolated from a tomato field in Florida. Electron microscopy revealed that phage RsoM1USA is member of the genus P2virus in the family Myoviridae with an icosahedral head of about 66 nm in diameter, a contractile tail of about 152 nm in length, and a long “neck.” Phage RsoM1USA infected 12 of the 30 tested R. solanacearum species complex strains collected worldwide in each of the three Ralstonia species: R. solanacearum , R. pseudosolanacearum , and R. syzygii . The phage completed its infection cycle 180 min post infection with a burst size of about 56 particles per cell. Phage RsoM1USA has a genome of 39,309 nucleotides containing 58 open reading frames (ORFs) and is closely related to Ralstonia phage RSA1 of the species Ralstonia virus RSA1 . The genomic organization of phage RsoM1USA is also similar to that of phage RSA1, but their integrases share no sequence homology. In addition, we determined that the integration of phage RsoM1USA into its susceptible R. solanacearum strain K60 is mediated by the 3′ 45-base portion of the threonine tRNA (TGT), not arginine tRNA (CCG) as reported for phage RSA1, confirming that the two phages use different mechanism for integration. Our proteomic analysis of the purified virions supported the annotation of the main structural proteins. Infection of a susceptible R. solanacearum strain RUN302 by phage RsoM1USA resulted in significantly reduced growth of the infected bacterium in vitro , but not virulence in tomato plants, as compared to its uninfected RUN302 strain. Due to its differences from phage RSA1, phage RsoM1USA should be considered the type member of a new species with a proposed species name of Ralstonia virus RsoM1USA.

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Section: Discussionmentioning

confidence: 99%

Molecular and Biological Characterization of Ralstonia Phage RsoM1USA, a New Species of P2virus, Isolated in the United States

2019

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“…Genes, proteins, and chromosomal sites for several functions of the temperate phage 16-3 of Rhizobium meliloti 41 have been studied in detail (1,2,5,8,9,11,19,22,24,25). The most thoroughly analyzed region of the 16-3 genome has been the immC regulatory region which encodes a lambda type CI repressor (16-3 C repressor protein), and the cognate c cistron is flanked by two operator regions, O L and O R (3,4,6,(16)(17)(18)20).…”

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Repressor of Phage 16 - 3 with Altered Binding Specificity Indicates Spatial Differences in Repressor-Operator Complexes

2006

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The C repressor protein of phage 16-3, which is required for establishing and maintaining lysogeny, recognizes structurally different operators which differ by 2 bp in the length of the spacer between the conserved palindromic sequences. A "rotationally flexible protein homodimers" model has been proposed in order to explain the conformational adaptivity of the 16-3 repressor. In this paper, we report on the isolation of a repressor mutant with altered binding specificity which was used to identify a residue-base pair contact and to monitor the spatial relationship of the recognition helix of C repressor to the contacting major groove of DNA within the two kinds of repressor-operator complexes. Our results indicate spatial differences at the interface which may reflect different docking arrangements in recognition of the structurally different operators by the 16-3 repressor.The regulation of gene expression occurs mostly at the level of transcription through sequence-specific DNA-protein interactions. In several cases, even a single change in the binding sequences results in loss of function due to loss or weakened binding of the protein. Alternatively, examples are also known of when the DNA binding protein has relaxed binding specificity, that is, when several changes at given positions still allow proper binding (12,23). Only a few naturally existing systems are known where the protein has the ability to bind specifically to sequences with different lengths. The Escherichia coli cyclic AMP receptor protein (CRP) recognizes 16-and 18-bp-long binding sites, in which 6-and 8-bp central spacers, respectively, are bracketed by the recognition sequences. According to Adhya's "geometric homeostasis" method of resolution in CRP-DNA complexes, a conformational shift from B-to Aform DNA over one helical turn covering the longer spacer allows sequence-specific binding of CRP (13). The CytR repressor recognizes binding sites consisting of two octamer repeats, in direct or inverted orientation, separated by 2 bp. However, in the presence of cyclic AMP-CRP, CytR instead recognizes inverted repeats separated by 10 to 13 bp or direct repeats separated by 1 bp. It was shown that the bases for recognizing the structurally different sites were conformational changes within the CytR protein induced by protein-protein interactions between CRP and CytR (14, 21). The CI repressor of E. coli phage 186 was found to recognize two distinct DNA sequences, termed A-type and B-type sites. The A-type binding sites were different in length since half-sites were separated by either 4-bp or 5-bp spacers (26). The binding of 16-3 C repressor to its operators is another example for recognition of structurally different DNA sites by the same protein. In this system, the possibility that either conformational shifts from B-to A-form DNA within the binding sites or interactions of the repressor with another protein is involved in formation of 16-3 repressor-operator complexes was ruled out (20).Genes, proteins, and chromosomal sites for several f...

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“…Genes, proteins, and chromosomal sites for several functions of the phage have been studied in more detail. These include (i) the main repressor protein, C, required for establishing and maintaining lysogeny, and the operator regions where the C protein binds (8,10,25); (ii) the components of the sitespecific recombination system (5,12,23,29,30); (iii) the immX regulatory region conferring immunity against homoimmune phages (7); and (iv) the recently identified h gene encoding the tail fiber protein (26).Identification of the cohesive ends of phage 16-3. The plasmids used during the course of the study are listed in Table 1.…”

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Identification of Cohesive Ends and Genes Encoding the Terminase of Phage16-3

et al. 2005

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Cohesive ends of 16-3, a temperate phage of Rhizobium meliloti 41, have been identified as 10-base-long, 3-protruding complementary G/C-rich sequences. terS and terL encode the two subunits of 16-3 terminase. Significant homologies were detected among the terminase subunits of phage 16-3 and other phages from various ecosystems.The terminase enzyme is part of a large nucleoprotein complex that packages viral DNA into the capsid (4, 16). It has been well studied in the case of phage , but quite a few other members have been identified in different phages and studied in detail (6). Studying the packaging reaction of new phages provides the opportunity to identify alternative mechanisms and can extend our understanding of the packaging process and the formation and functioning of nucleoprotein complexes in general. Studies of similar functions in diverse systems are also required to understand the evolution of complex biological machines.Phage 16-3 is a temperate phage of Rhizobium meliloti 41. The genetic and physical maps of the phage have been established previously (9,11,13,22) and summarized in reference 7. Genes, proteins, and chromosomal sites for several functions of the phage have been studied in more detail. These include (i) the main repressor protein, C, required for establishing and maintaining lysogeny, and the operator regions where the C protein binds (8,10,25); (ii) the components of the sitespecific recombination system (5,12,23,29,30); (iii) the immX regulatory region conferring immunity against homoimmune phages (7); and (iv) the recently identified h gene encoding the tail fiber protein (26).Identification of the cohesive ends of phage 16-3. The plasmids used during the course of the study are listed in Table 1. The 96BglII-14EcoRI fragment ( Fig. 1) (numbering was based on the map of restriction sites in reference 13) of phage 16-3 was subcloned from cosmid pDH31 (11), which contains the region where the covalently ligated ends of the phage chromosome are located. The resulting plasmid, pPAG165, was used to determine the nucleotide sequence of the entire fragment. Nucleotide sequence determination was performed by the dideoxy chain termination method (28) with the fmol DNA cycle sequencing system (Promega). Primer 1, 5Ј-CACGGCTTCGGCGGCGC TGTC-3Ј, and primer 2, 5Ј-GGCAAGAAGGTCGTGACCTA TG-3Ј, were designed close to the expected ends, and the isolated 92EcoRI-cos right and cos left -14EcoRI fragments from phage DNA were used as templates for a pair of sequencing reactions to distinguish between 5Ј and 3Ј overhangs (Fig. 2). A 10-bp sequence region existing in pPAG165 was absent from the sequences of both end-fragments, hence we concluded that the chromosome of phage 16-3 ended in 10-base-long, 3Ј-protruding, single-stranded, complementary sequences (5Ј-. . .CCG-GCGTCGG-3Ј and 3Ј-GGCCGCAGCC. . .-5Ј). The cos sequence has high G/C content and shows dyad symmetry. The symmetry can be recognized in patches within the duplex DNA flanking the single-stranded ends (Fig. 1).Identification of genes around...

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A proline tRNA(CGG) gene encompassing the attachment site of temperate phage 16‐3 is functional and convertible to suppressor tRNA

Cited by 4 publications

References 50 publications

Molecular and Biological Characterization of Ralstonia Phage RsoM1USA, a New Species of P2virus, Isolated in the United States

Molecular and Biological Characterization of Ralstonia Phage RsoM1USA, a New Species of P2virus, Isolated in the United States

Repressor of Phage 16 - 3 with Altered Binding Specificity Indicates Spatial Differences in Repressor-Operator Complexes

Identification of Cohesive Ends and Genes Encoding the Terminase of Phage16-3

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