Site-specific integration of bacteriophage VWB genome into Streptomyces venezuelae and construction of a VWB-based integrative vector

Mellaert, Lieve Van; Mei, Lijuan; Lammertyn, Elke; Schacht, S; Anné, Jozef

doi:10.1099/00221287-144-12-3351

Cited by 52 publications

(31 citation statements)

References 37 publications

(70 reference statements)

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“…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).…”

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confidence: 63%

“…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.…”

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confidence: 99%

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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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confidence: 63%

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confidence: 99%

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 pSOK804 plasmid vector, containing an integration function (an integrase gene and AttP) from the streptomycete temperate phage VWB (37) and part of the pSET152 vector (6), was constructed (Table 1). Plasmid pSOK804 was able to integrate site-specifically into one site in the genome of S. noursei (data not shown), at a frequency about 2 orders of magnitude higher than that for the pSET152 vector previously used for gene expression in S. noursei (41).…”

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confidence: 99%

In Vivo Analysis of the Regulatory Genes in the Nystatin Biosynthetic Gene Cluster of Streptomyces noursei ATCC 11455 Reveals Their Differential Control Over Antibiotic Biosynthesis

et al. 2004

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Six putative regulatory genes are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. Gene inactivation and complementation experiments revealed that nysRI, nysRII, nysRIII, and nysRIV are necessary for efficient nystatin production, whereas no significant roles could be demonstrated for the other two regulatory genes. To determine the in vivo targets for the NysR regulators, chromosomal integration vectors with the xylE reporter gene under the control of seven putative promoter regions upstream of the nystatin structural and regulatory genes were constructed. Expression analyses of the resulting vectors in the S. noursei wild-type strain and regulatory mutants revealed that the four regulators differentially affect certain promoters. According to these analyses, genes responsible for initiation of nystatin biosynthesis and antibiotic transport were the major targets for regulation. Data from cross-complementation experiments showed that nysR genes could in some cases substitute for each other, suggesting a functional hierarchy of the regulators and implying a cascade-like mechanism of regulation of nystatin biosynthesis.

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“…Adaptations to growth in the streptomycetes were identified, including the prob-the predicted prophage genome. In this case, the sequence was identical to the core sequence in the VWB attP site (20), and the adjacent integrase (WP_009066700) also has very high identity to the VWB integrase. This sequence was therefore designated attP for prophage SPBP78.1.…”

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confidence: 81%

Evolutionary Relationships among Actinophages and a Putative Adaptation for Growth in Streptomyces spp

et al. 2013

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eThe genome sequences of eight Streptomyces phages are presented, four of which were isolated for this study. Phages R4, TG1, Hau3, and SV1 were isolated previously and have been exploited as tools for understanding and genetically manipulating Streptomyces spp. We also extracted five apparently intact prophages from recent Streptomyces spp. genome projects and, together with six phage genomes in the database, we analyzed all 19 Streptomyces phage genomes with a view to understanding their relationships to each other and to other actinophages, particularly the mycobacteriophages. Fifteen of the Streptomyces phages group into four clusters of related genomes. Although the R4-like phages do not share nucleotide sequence similarity with other phages, they clearly have common ancestry with cluster A mycobacteriophages, sharing many protein homologues, common gene syntenies, and similar repressor-stoperator regulatory systems. The R4-like phage Hau3 and the prophage StrepC.1 (from Streptomyces sp. strain C) appear to have hijacked a unique adaptation of the streptomycetes, i.e., use of the rare UUA codon, to control translation of the essential phage protein, the terminase. The Streptomyces venezuelae generalized transducing phage SV1 was used to predict the presence of other generalized transducing phages for different Streptomyces species. Bacteriophages are the most abundant and diverse genetic entities on planet Earth. Despite this, it has been proposed that all double-stranded DNA (dsDNA) phages share the same gene pool and that there are clear examples where common ancestry is evident between orthologous gene pairs from very different phages (for example, the capsid-encoding genes from coliphage HK97 and Streptomyces phage C31) (1, 2). Understanding the mechanisms through which phages generate such vast diversity is still a challenge, but the drivers of selection and adaptation to growth in different bacterial hosts are clearly strong influences. We are interested in the adaptations of phages that infect the Actinobacteria, a class of high-GC, Gram-positive bacteria that have very diverse morphology, physiology, and growth properties.Actinobacteria, particularly those in the genus Streptomyces, have a remarkable capacity to synthesize a diverse array of secondary metabolites of all the major classes. Indeed, 70% of clinically useful antibiotics come from Streptomyces spp. Genetic tools derived from Streptomyces phages have been invaluable in investigations aimed at understanding the fundamental biology of these bacteria and in manipulating antibiotic pathways (3, 4). Phages were used initially as cloning vectors, but since the early 1990s, integrating plasmid vectors based on phage-encoded site-specific recombination systems have been widely used, enabling the stable insertion of gene constructs into a defined site (attB) in the chromosome (5, 6). Moreover, as streptomycetes are mycelial and undergo a developmental cycle, phages that infect this group of bacteria are likely to have undergone host-specific evolu...

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Site-specific integration of bacteriophage VWB genome into Streptomyces venezuelae and construction of a VWB-based integrative vector

Cited by 52 publications

References 37 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

In Vivo Analysis of the Regulatory Genes in the Nystatin Biosynthetic Gene Cluster of Streptomyces noursei ATCC 11455 Reveals Their Differential Control Over Antibiotic Biosynthesis

Evolutionary Relationships among Actinophages and a Putative Adaptation for Growth in Streptomyces spp

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