A genetic dissection of the LlaJI restriction cassette reveals insights on a novel bacteriophage resistance system

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dc.contributor.author O'Driscoll, Jonathan
dc.contributor.author Heiter, Daniel F.
dc.contributor.author Wilson, Geoffrey G.
dc.contributor.author Fitzgerald, Gerald F.
dc.contributor.author Roberts, Richard
dc.contributor.author van Sinderen, Douwe
dc.date.accessioned 2012-11-29T12:19:30Z
dc.date.available 2012-11-29T12:19:30Z
dc.date.issued 2006-04-28
dc.identifier.citation O'Driscoll J, Heiter DF, Wilson GG, Fitzgerald GF, Roberts R, van Sinderen D. A genetic dissection of the LlaJI restriction cassette reveals insights on a novel bacteriophage resistance system. BMC Microbiol 2006;6: 40. http://www.biomedcentral.com/1471-2180/6/40 en
dc.identifier.volume 6 en
dc.identifier.startpage 40 en
dc.identifier.issn 1471-2180
dc.identifier.uri http://hdl.handle.net/10468/825
dc.identifier.doi 10.1186/1471-2180-6-40
dc.description.abstract Background: Restriction/modification systems provide the dual function of protecting host DNA against restriction by methylation of appropriate bases within their recognition sequences, and restriction of foreign invading un-methylated DNA, such as promiscuous plasmids or infecting bacteriphage. The plasmid-encoded LlaJI restriction/modification system from Lactococcus lactis recognizes an asymmetric,complementary DNA sequence, consisting of 5'GACGC'3 in one strand and 5'GCGTC'3 in the other and provides a prodigious barrier to bacteriophage infection. LlaJI is comprised of four similarly oriented genes, encoding two 5mC-MTases (M1.LlaJI and M2.LlaJI) and two subunits responsible for restriction activity R1.LlaJI and R2.LlaJI). Here we employ a detailed genetic analysis of the LlaJI restriction determinants in an attempt to characterize mechanistic features of this unusual hetero-oligomeric endonuclease. Results: Detailed bioinformatics analysis confirmed the presence of a conserved GTP binding and hydrolysis domain within the C-terminal half of the R1.LlaJI amino acid sequence whilst the N-terminal half appeared to be entirely unique. This domain architecture was homologous with that of the "B" subunit of the GTP-dependent, methyl-specific McrBC endonuclease from E.coli K-12. R1.LlaJI did not appear to contain a catalytic centre, whereas this conserved motif; PD....D/EXK, was clearly identified within the amino acid sequence for R2.LlaJI. Both R1.LlaJI and R2.LlaJI were found to be absolutely required for detectable LlaJI activity in vivo. The LlaJI restriction subunits were purified and examined in vitro, which allowed the assignment of R1.LlaJI as the sole specificity determining subunit, whilst R2.LlaJI is believed to mediate DNA cleavage. Conclusion: The hetero-subunit structure of LlaJI, wherein one subunit mediates DNA binding whilst the other subunit is predicted to catalyze strand hydrolysis distinguishes LlaJI from previously characterized restriction-modification systems. Furthermore, this distinction is accentuated by the fact that whilst LlaJI behaves as a conventional Type IIA system in vivo, in that it restricts un-methylated DNA, it resembles the Type IV McrBC endonuclease, an enzyme specific for methylated DNA. A number of similar restriction determinants were identified in the database and it is likely LlaJI together with these homologous systems, comprise a new subtype of the Type II class incorporating features of Type II and Type IV systems. en
dc.description.sponsorship Science Foundation Ireland (02/IN1/B198); Science Foundation Ireland (SFI-CSET) en
dc.format.mimetype application/pdf en
dc.language.iso en en
dc.publisher BioMed Central en
dc.relation.uri http://www.biomedcentral.com/1471-2180/6/40
dc.rights © 2006 O'Driscoll et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), en
dc.rights.uri http://creativecommons.org/licenses/by/2.0 en
dc.subject LlaJI en
dc.subject Lactococcus lactis en
dc.subject Hetero-oligomeric en
dc.subject Endonuclease en
dc.subject Restriction cassette en
dc.subject Bacteriophage en
dc.title A genetic dissection of the LlaJI restriction cassette reveals insights on a novel bacteriophage resistance system en
dc.type Article (peer-reviewed) en
dc.internal.authorurl http://research.ucc.ie/profiles/D010/dvansinderen en
dc.internal.authorcontactother Douwe van Sinderen, Department of Microbiology, University College Cork, Western Road, Cork, Ireland. Email: d.vansinderen@ucc.ie en
dc.internal.availability Full text available en
dc.description.version Published Version en
dc.internal.rssid 43336820
dc.contributor.funder Science Foundation Ireland en
dc.description.status Peer reviewed en
dc.identifier.journaltitle BMC Microbiology en
dc.internal.copyrightchecked You are free: to Share — to copy, distribute and transmit the work to Remix — to adapt the work to make commercial use of the work Under the following conditions: Attribution — You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). http://creativecommons.org/licenses/by/2.0 en
dc.internal.IRISemailaddress d.vansinderen@ucc.ie en


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© 2006 O'Driscoll et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), Except where otherwise noted, this item's license is described as © 2006 O'Driscoll et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
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