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A putative mobile genetic element carrying a novel type IIF restriction-modification system (PluTI).

Khan F, Furuta Y, Kawai M, Kaminska KH, Ishikawa K, Bujnicki JM, Kobayashi I - Nucleic Acids Res. (2010)

Bottom Line: Subsequently, the protein, named R.PluTI, was expressed in vivo, purified and found to be a novel type IIF restriction enzyme that recognizes 5'-GGCGC/C-3' (/ indicates position of cleavage).These results suggested that they constitute a restriction-modification system, present on the putative mobile element.Our approach thus allowed detection of a previously uncharacterized family of DNA-interacting proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, Japan.

ABSTRACT
Genome comparison and genome context analysis were used to find a putative mobile element in the genome of Photorhabdus luminescens, an entomopathogenic bacterium. The element is composed of 16-bp direct repeats in the terminal regions, which are identical to a part of insertion sequences (ISs), a DNA methyltransferase gene homolog, two genes of unknown functions and an open reading frame (ORF) (plu0599) encoding a protein with no detectable sequence similarity to any known protein. The ORF (plu0599) product showed DNA endonuclease activity, when expressed in a cell-free expression system. Subsequently, the protein, named R.PluTI, was expressed in vivo, purified and found to be a novel type IIF restriction enzyme that recognizes 5'-GGCGC/C-3' (/ indicates position of cleavage). R.PluTI cleaves a two-site supercoiled substrate at both the sites faster than a one-site supercoiled substrate. The modification enzyme homolog encoded by plu0600, named M.PluTI, was expressed in Escherichia coli and shown to protect DNA from R.PluTI cleavage in vitro, and to suppress the lethal effects of R.PluTI expression in vivo. These results suggested that they constitute a restriction-modification system, present on the putative mobile element. Our approach thus allowed detection of a previously uncharacterized family of DNA-interacting proteins.

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Genome contexts of plu0597 and plu0598 homologs (shaded boxes) in eubacterial genomes. Repeat sequences are drawn as triangles. The accession numbers of the sources are: (a) Refseq NC_010645; (b) Refseq NC_010524; (c) Refseq NC_010409; (d) Genbank AJ851089; (e) Refseq NC_006816; (f) Refseq NC_009837.
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Figure 6: Genome contexts of plu0597 and plu0598 homologs (shaded boxes) in eubacterial genomes. Repeat sequences are drawn as triangles. The accession numbers of the sources are: (a) Refseq NC_010645; (b) Refseq NC_010524; (c) Refseq NC_010409; (d) Genbank AJ851089; (e) Refseq NC_006816; (f) Refseq NC_009837.

Mentions: We also found several homologs of plu0597 and plu0598 genes (e-value: 1e-110 to 2e-93 for plu0597 and 1e-141 to 1e-118 for plu0598) adjacent to each other on putative mobile elements including integrated plasmids, transposon-like structures or composite transposon-like structures, suggesting that these two genes might be involved in genome mobility (Figure 6). A homologous pair (Figure 6a) was found in a region annotated as a plasmid integrated into the 3′-terminus of a tRNA gene resulting in 40-bp long flanking direct repeats. Another homologous pair (Figure 6b) was flanked by 21-bp inverted repeats of 19/21 bp identity next to a transposase homolog. This structure is similar to those of transposons such as Tn3 in which a transposase and a resolvase catalyze site-specific recombination for cointegrated resolution (51). In three other cases (Figure 6c–e), the homologous pair is next to an IS1 copy. The relative position and orientation of the homologous pair and IS1 are the same in these three clusters, suggesting that they form a mobile unit or a composite transposon. In these three cases (Figure 6c–e), the homologous pair is also adjacent to the VagCD system, a toxin–antitoxin system (52). In another case (Figure 6f), the homologous pair lies next to a vagD homolog, but its linkage to an IS1 copy is interrupted by insertion of ISEc12.Figure 6.


A putative mobile genetic element carrying a novel type IIF restriction-modification system (PluTI).

Khan F, Furuta Y, Kawai M, Kaminska KH, Ishikawa K, Bujnicki JM, Kobayashi I - Nucleic Acids Res. (2010)

Genome contexts of plu0597 and plu0598 homologs (shaded boxes) in eubacterial genomes. Repeat sequences are drawn as triangles. The accession numbers of the sources are: (a) Refseq NC_010645; (b) Refseq NC_010524; (c) Refseq NC_010409; (d) Genbank AJ851089; (e) Refseq NC_006816; (f) Refseq NC_009837.
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Related In: Results  -  Collection

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Figure 6: Genome contexts of plu0597 and plu0598 homologs (shaded boxes) in eubacterial genomes. Repeat sequences are drawn as triangles. The accession numbers of the sources are: (a) Refseq NC_010645; (b) Refseq NC_010524; (c) Refseq NC_010409; (d) Genbank AJ851089; (e) Refseq NC_006816; (f) Refseq NC_009837.
Mentions: We also found several homologs of plu0597 and plu0598 genes (e-value: 1e-110 to 2e-93 for plu0597 and 1e-141 to 1e-118 for plu0598) adjacent to each other on putative mobile elements including integrated plasmids, transposon-like structures or composite transposon-like structures, suggesting that these two genes might be involved in genome mobility (Figure 6). A homologous pair (Figure 6a) was found in a region annotated as a plasmid integrated into the 3′-terminus of a tRNA gene resulting in 40-bp long flanking direct repeats. Another homologous pair (Figure 6b) was flanked by 21-bp inverted repeats of 19/21 bp identity next to a transposase homolog. This structure is similar to those of transposons such as Tn3 in which a transposase and a resolvase catalyze site-specific recombination for cointegrated resolution (51). In three other cases (Figure 6c–e), the homologous pair is next to an IS1 copy. The relative position and orientation of the homologous pair and IS1 are the same in these three clusters, suggesting that they form a mobile unit or a composite transposon. In these three cases (Figure 6c–e), the homologous pair is also adjacent to the VagCD system, a toxin–antitoxin system (52). In another case (Figure 6f), the homologous pair lies next to a vagD homolog, but its linkage to an IS1 copy is interrupted by insertion of ISEc12.Figure 6.

Bottom Line: Subsequently, the protein, named R.PluTI, was expressed in vivo, purified and found to be a novel type IIF restriction enzyme that recognizes 5'-GGCGC/C-3' (/ indicates position of cleavage).These results suggested that they constitute a restriction-modification system, present on the putative mobile element.Our approach thus allowed detection of a previously uncharacterized family of DNA-interacting proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, Japan.

ABSTRACT
Genome comparison and genome context analysis were used to find a putative mobile element in the genome of Photorhabdus luminescens, an entomopathogenic bacterium. The element is composed of 16-bp direct repeats in the terminal regions, which are identical to a part of insertion sequences (ISs), a DNA methyltransferase gene homolog, two genes of unknown functions and an open reading frame (ORF) (plu0599) encoding a protein with no detectable sequence similarity to any known protein. The ORF (plu0599) product showed DNA endonuclease activity, when expressed in a cell-free expression system. Subsequently, the protein, named R.PluTI, was expressed in vivo, purified and found to be a novel type IIF restriction enzyme that recognizes 5'-GGCGC/C-3' (/ indicates position of cleavage). R.PluTI cleaves a two-site supercoiled substrate at both the sites faster than a one-site supercoiled substrate. The modification enzyme homolog encoded by plu0600, named M.PluTI, was expressed in Escherichia coli and shown to protect DNA from R.PluTI cleavage in vitro, and to suppress the lethal effects of R.PluTI expression in vivo. These results suggested that they constitute a restriction-modification system, present on the putative mobile element. Our approach thus allowed detection of a previously uncharacterized family of DNA-interacting proteins.

Show MeSH
Related in: MedlinePlus