H Protein of Bacteriophage
            <i>16-3</i>
            and RkpM Protein of
            <i>Sinorhizobium meliloti</i>
            41 Are Involved in Phage Adsorption

Putnoky, Péter; Deák, Veronika; Békási, Krisztina; Pálvölgyi, Adrienn; Maász, Anita; Palágyi, Zsuzsanna; Hoffmann, Gyula; Kerepesi, Ildikó

doi:10.1128/jb.186.6.1591-1597.2004

Cited by 8 publications

(17 citation statements)

References 29 publications

(51 reference statements)

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“…Phage strains 16-3⌬NC (9) and 16-3cti3 (24) were used as back-ground strains for the isolation of host range and insertional mutants, respectively. Host range mutant h109 was isolated earlier on strain GH4180 (32). New host range mutants described in this study were isolated on different bacterial mutants by the same procedure: h182 was isolated on strain AT313 (AK631 rkpZ::Tn5) (20), while h842 and h843 were isolated on strain PP4073 (25).…”

Section: Methodsmentioning

confidence: 99%

“…In order to elucidate the molecular mechanism of phage 16-3 and S. meliloti 41 recognition, bacterial mutants carrying an altered phage receptor and host range phage mutants able to overcome the adsorption block have been characterized previously (32). It was shown that the RkpM protein, together with other yet uncharacterized elements, is a component of the phage receptor.…”

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“…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.…”

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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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Section: Methodsmentioning

confidence: 99%

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

et al. 2010

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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).…”

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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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H Protein of Bacteriophage 16-3 and RkpM Protein of Sinorhizobium meliloti 41 Are Involved in Phage Adsorption

Cited by 8 publications

References 29 publications

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

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

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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