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Characterizing the DNA Methyltransferases of Haloferax volcanii via Bioinformatics, Gene Deletion, and SMRT Sequencing

DNA methyltransferases (MTases), which catalyze the methylation of adenine and cytosine bases in DNA, can occur in bacteria and archaea alongside cognate restriction endonucleases (REases) in restriction-modification (RM) systems or independently as orphan MTases. Although DNA methylation and MTases...

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Autores principales: Ouellette, Matthew, Gogarten, J. Peter, Lajoie, Jessica, Makkay, Andrea M., Papke, R. Thane
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5867850/
https://www.ncbi.nlm.nih.gov/pubmed/29495512
http://dx.doi.org/10.3390/genes9030129
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author Ouellette, Matthew
Gogarten, J. Peter
Lajoie, Jessica
Makkay, Andrea M.
Papke, R. Thane
author_facet Ouellette, Matthew
Gogarten, J. Peter
Lajoie, Jessica
Makkay, Andrea M.
Papke, R. Thane
author_sort Ouellette, Matthew
collection PubMed
description DNA methyltransferases (MTases), which catalyze the methylation of adenine and cytosine bases in DNA, can occur in bacteria and archaea alongside cognate restriction endonucleases (REases) in restriction-modification (RM) systems or independently as orphan MTases. Although DNA methylation and MTases have been well-characterized in bacteria, research into archaeal MTases has been limited. A previous study examined the genomic DNA methylation patterns (methylome) of the halophilic archaeon Haloferax volcanii, a model archaeal system which can be easily manipulated in laboratory settings, via single-molecule real-time (SMRT) sequencing and deletion of a putative MTase gene (HVO_A0006). In this follow-up study, we deleted other putative MTase genes in H. volcanii and sequenced the methylomes of the resulting deletion mutants via SMRT sequencing to characterize the genes responsible for DNA methylation. The results indicate that deletion of putative RM genes HVO_0794, HVO_A0006, and HVO_A0237 in a single strain abolished methylation of the sole cytosine motif in the genome (C(m4)TAG). Amino acid alignments demonstrated that HVO_0794 shares homology with characterized cytosine CTAG MTases in other organisms, indicating that this MTase is responsible for C(m4)TAG methylation in H. volcanii. The CTAG motif has high density at only one of the origins of replication, and there is no relative increase in CTAG motif frequency in the genome of H. volcanii, indicating that CTAG methylation might not have effectively taken over the role of regulating DNA replication and mismatch repair in the organism as previously predicted. Deletion of the putative Type I RM operon rmeRMS (HVO_2269-2271) resulted in abolished methylation of the adenine motif in the genome (GCA(m6)BN(6)VTGC). Alignments of the MTase (HVO_2270) and site specificity subunit (HVO_2271) demonstrate homology with other characterized Type I MTases and site specificity subunits, indicating that the rmeRMS operon is responsible for adenine methylation in H. volcanii. Together with HVO_0794, these genes appear to be responsible for all detected methylation in H. volcanii, even though other putative MTases (HVO_C0040, HVO_A0079) share homology with characterized MTases in other organisms. We also report the construction of a multi-RM deletion mutant (ΔRM), with multiple RM genes deleted and with no methylation detected via SMRT sequencing, which we anticipate will be useful for future studies on DNA methylation in H. volcanii.
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spelling pubmed-58678502018-03-27 Characterizing the DNA Methyltransferases of Haloferax volcanii via Bioinformatics, Gene Deletion, and SMRT Sequencing Ouellette, Matthew Gogarten, J. Peter Lajoie, Jessica Makkay, Andrea M. Papke, R. Thane Genes (Basel) Article DNA methyltransferases (MTases), which catalyze the methylation of adenine and cytosine bases in DNA, can occur in bacteria and archaea alongside cognate restriction endonucleases (REases) in restriction-modification (RM) systems or independently as orphan MTases. Although DNA methylation and MTases have been well-characterized in bacteria, research into archaeal MTases has been limited. A previous study examined the genomic DNA methylation patterns (methylome) of the halophilic archaeon Haloferax volcanii, a model archaeal system which can be easily manipulated in laboratory settings, via single-molecule real-time (SMRT) sequencing and deletion of a putative MTase gene (HVO_A0006). In this follow-up study, we deleted other putative MTase genes in H. volcanii and sequenced the methylomes of the resulting deletion mutants via SMRT sequencing to characterize the genes responsible for DNA methylation. The results indicate that deletion of putative RM genes HVO_0794, HVO_A0006, and HVO_A0237 in a single strain abolished methylation of the sole cytosine motif in the genome (C(m4)TAG). Amino acid alignments demonstrated that HVO_0794 shares homology with characterized cytosine CTAG MTases in other organisms, indicating that this MTase is responsible for C(m4)TAG methylation in H. volcanii. The CTAG motif has high density at only one of the origins of replication, and there is no relative increase in CTAG motif frequency in the genome of H. volcanii, indicating that CTAG methylation might not have effectively taken over the role of regulating DNA replication and mismatch repair in the organism as previously predicted. Deletion of the putative Type I RM operon rmeRMS (HVO_2269-2271) resulted in abolished methylation of the adenine motif in the genome (GCA(m6)BN(6)VTGC). Alignments of the MTase (HVO_2270) and site specificity subunit (HVO_2271) demonstrate homology with other characterized Type I MTases and site specificity subunits, indicating that the rmeRMS operon is responsible for adenine methylation in H. volcanii. Together with HVO_0794, these genes appear to be responsible for all detected methylation in H. volcanii, even though other putative MTases (HVO_C0040, HVO_A0079) share homology with characterized MTases in other organisms. We also report the construction of a multi-RM deletion mutant (ΔRM), with multiple RM genes deleted and with no methylation detected via SMRT sequencing, which we anticipate will be useful for future studies on DNA methylation in H. volcanii. MDPI 2018-02-27 /pmc/articles/PMC5867850/ /pubmed/29495512 http://dx.doi.org/10.3390/genes9030129 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ouellette, Matthew
Gogarten, J. Peter
Lajoie, Jessica
Makkay, Andrea M.
Papke, R. Thane
Characterizing the DNA Methyltransferases of Haloferax volcanii via Bioinformatics, Gene Deletion, and SMRT Sequencing
title Characterizing the DNA Methyltransferases of Haloferax volcanii via Bioinformatics, Gene Deletion, and SMRT Sequencing
title_full Characterizing the DNA Methyltransferases of Haloferax volcanii via Bioinformatics, Gene Deletion, and SMRT Sequencing
title_fullStr Characterizing the DNA Methyltransferases of Haloferax volcanii via Bioinformatics, Gene Deletion, and SMRT Sequencing
title_full_unstemmed Characterizing the DNA Methyltransferases of Haloferax volcanii via Bioinformatics, Gene Deletion, and SMRT Sequencing
title_short Characterizing the DNA Methyltransferases of Haloferax volcanii via Bioinformatics, Gene Deletion, and SMRT Sequencing
title_sort characterizing the dna methyltransferases of haloferax volcanii via bioinformatics, gene deletion, and smrt sequencing
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5867850/
https://www.ncbi.nlm.nih.gov/pubmed/29495512
http://dx.doi.org/10.3390/genes9030129
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