Limits...
Genome-wide DNA methylation analysis of Haloferax volcanii H26 and identification of DNA methyltransferase related PD-(D/E)XK nuclease family protein HVO_A0006.

Ouellette M, Jackson L, Chimileski S, Papke RT - Front Microbiol (2015)

Bottom Line: Further bioinformatic analysis of the HVO_A0006 gene demonstrated that the gene is rare among the Halobacteria.It is surrounded by two transposition genes suggesting that HVO_A0006 is a fragment of a Type IIG RM gene, which has likely been acquired through gene transfer, and affects restriction-modification activity by interacting with another RM system component(s).Here, we present the first genome-wide characterization of DNA methylation in an archaeal species and examine the function of a DNA methyltransferase related gene HVO_A0006.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of Connecticut Storrs, CT, USA.

ABSTRACT
Restriction-modification (RM) systems have evolved to protect the cell from invading DNAs and are composed of two enzymes: a DNA methyltransferase and a restriction endonuclease. Although RM systems are present in both archaeal and bacterial genomes, DNA methylation in archaea has not been well defined. In order to characterize the function of RM systems in archaeal species, we have made use of the model haloarchaeon Haloferax volcanii. A genomic DNA methylation analysis of H. volcanii strain H26 was performed using PacBio single molecule real-time (SMRT) sequencing. This analysis was also performed on a strain of H. volcanii in which an annotated DNA methyltransferase gene HVO_A0006 was deleted from the genome. Sequence analysis of H26 revealed two motifs which are modified in the genome: C(m4)TAG and GCA(m6)BN6VTGC. Analysis of the ΔHVO_A0006 strain indicated that it exhibited reduced adenine methylation compared to the parental strain and altered the detected adenine motif. However, protein domain architecture analysis and amino acid alignments revealed that HVO_A0006 is homologous only to the N-terminal endonuclease region of Type IIG RM proteins and contains a PD-(D/E)XK nuclease motif, suggesting that HVO_A0006 is a PD-(D/E)XK nuclease family protein. Further bioinformatic analysis of the HVO_A0006 gene demonstrated that the gene is rare among the Halobacteria. It is surrounded by two transposition genes suggesting that HVO_A0006 is a fragment of a Type IIG RM gene, which has likely been acquired through gene transfer, and affects restriction-modification activity by interacting with another RM system component(s). Here, we present the first genome-wide characterization of DNA methylation in an archaeal species and examine the function of a DNA methyltransferase related gene HVO_A0006.

No MeSH data available.


Related in: MedlinePlus

PCR confirmation of HVO_A0006 deletion strain. The template DNA amplified was from H. volcanii DS2 (Lane 1), pΔHVO_A0006 (lane 2; as a positive control), unsuccessful pop-outs (lanes 3, 4, and 10) and successful pop-out colonies (lanes 5–9). A Mid-Range DNA ladder (Fisher Scientific) is shown in Lane 11.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4389544&req=5

Figure 1: PCR confirmation of HVO_A0006 deletion strain. The template DNA amplified was from H. volcanii DS2 (Lane 1), pΔHVO_A0006 (lane 2; as a positive control), unsuccessful pop-outs (lanes 3, 4, and 10) and successful pop-out colonies (lanes 5–9). A Mid-Range DNA ladder (Fisher Scientific) is shown in Lane 11.

Mentions: The annotated adenine methyltransferase gene HVO_A0006 (accession number ADE01899) was selected for deletion in H. volcanii strain H53. The gene deletion strategy used in this study was modified from the methodology previously developed (Blaby et al., 2010) and uses the In-Fusion HD Cloning Kit (Clontech). The primer sequences used in this study are listed in Table 2. H. volcanii strain H53 was then transformed with pΔHVO_A0006 using the PEG-mediated transformation of haloarchaea protocol from Cline et al. (1989), Bitan-Banin et al. (2003), Allers et al. (2004), and Blaby et al. (2010). The transformed cells were then plated on Hv-CA agar media without uracil and incubated for 5–7 days at 42°C. Colony PCR was performed using forward and reverse M13 primers to screen for pop-ins. The positive colonies from the pop-in screen were plated on Hv-CA plates with 50 μg mL−1 5-FOA and 50 μg mL−1 uracil to obtain pop-outs. Final pop-outs were confirmed through PCR (see primers in Table 2) as visualized through gel electrophoresis (Figure 1).


Genome-wide DNA methylation analysis of Haloferax volcanii H26 and identification of DNA methyltransferase related PD-(D/E)XK nuclease family protein HVO_A0006.

Ouellette M, Jackson L, Chimileski S, Papke RT - Front Microbiol (2015)

PCR confirmation of HVO_A0006 deletion strain. The template DNA amplified was from H. volcanii DS2 (Lane 1), pΔHVO_A0006 (lane 2; as a positive control), unsuccessful pop-outs (lanes 3, 4, and 10) and successful pop-out colonies (lanes 5–9). A Mid-Range DNA ladder (Fisher Scientific) is shown in Lane 11.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4389544&req=5

Figure 1: PCR confirmation of HVO_A0006 deletion strain. The template DNA amplified was from H. volcanii DS2 (Lane 1), pΔHVO_A0006 (lane 2; as a positive control), unsuccessful pop-outs (lanes 3, 4, and 10) and successful pop-out colonies (lanes 5–9). A Mid-Range DNA ladder (Fisher Scientific) is shown in Lane 11.
Mentions: The annotated adenine methyltransferase gene HVO_A0006 (accession number ADE01899) was selected for deletion in H. volcanii strain H53. The gene deletion strategy used in this study was modified from the methodology previously developed (Blaby et al., 2010) and uses the In-Fusion HD Cloning Kit (Clontech). The primer sequences used in this study are listed in Table 2. H. volcanii strain H53 was then transformed with pΔHVO_A0006 using the PEG-mediated transformation of haloarchaea protocol from Cline et al. (1989), Bitan-Banin et al. (2003), Allers et al. (2004), and Blaby et al. (2010). The transformed cells were then plated on Hv-CA agar media without uracil and incubated for 5–7 days at 42°C. Colony PCR was performed using forward and reverse M13 primers to screen for pop-ins. The positive colonies from the pop-in screen were plated on Hv-CA plates with 50 μg mL−1 5-FOA and 50 μg mL−1 uracil to obtain pop-outs. Final pop-outs were confirmed through PCR (see primers in Table 2) as visualized through gel electrophoresis (Figure 1).

Bottom Line: Further bioinformatic analysis of the HVO_A0006 gene demonstrated that the gene is rare among the Halobacteria.It is surrounded by two transposition genes suggesting that HVO_A0006 is a fragment of a Type IIG RM gene, which has likely been acquired through gene transfer, and affects restriction-modification activity by interacting with another RM system component(s).Here, we present the first genome-wide characterization of DNA methylation in an archaeal species and examine the function of a DNA methyltransferase related gene HVO_A0006.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of Connecticut Storrs, CT, USA.

ABSTRACT
Restriction-modification (RM) systems have evolved to protect the cell from invading DNAs and are composed of two enzymes: a DNA methyltransferase and a restriction endonuclease. Although RM systems are present in both archaeal and bacterial genomes, DNA methylation in archaea has not been well defined. In order to characterize the function of RM systems in archaeal species, we have made use of the model haloarchaeon Haloferax volcanii. A genomic DNA methylation analysis of H. volcanii strain H26 was performed using PacBio single molecule real-time (SMRT) sequencing. This analysis was also performed on a strain of H. volcanii in which an annotated DNA methyltransferase gene HVO_A0006 was deleted from the genome. Sequence analysis of H26 revealed two motifs which are modified in the genome: C(m4)TAG and GCA(m6)BN6VTGC. Analysis of the ΔHVO_A0006 strain indicated that it exhibited reduced adenine methylation compared to the parental strain and altered the detected adenine motif. However, protein domain architecture analysis and amino acid alignments revealed that HVO_A0006 is homologous only to the N-terminal endonuclease region of Type IIG RM proteins and contains a PD-(D/E)XK nuclease motif, suggesting that HVO_A0006 is a PD-(D/E)XK nuclease family protein. Further bioinformatic analysis of the HVO_A0006 gene demonstrated that the gene is rare among the Halobacteria. It is surrounded by two transposition genes suggesting that HVO_A0006 is a fragment of a Type IIG RM gene, which has likely been acquired through gene transfer, and affects restriction-modification activity by interacting with another RM system component(s). Here, we present the first genome-wide characterization of DNA methylation in an archaeal species and examine the function of a DNA methyltransferase related gene HVO_A0006.

No MeSH data available.


Related in: MedlinePlus