DNA target recognition domains in the Type I restriction and modification systems of Staphylococcus aureus

Abstract Staphylococcus aureus displays a clonal population structure in which horizontal gene transfer between different lineages is extremely rare. This is due, in part, to the presence of a Type I DNA restriction–modification (RM) system given the generic name of Sau1, which maintains different patterns of methylation on specific target sequences on the genomes of different lineages. We have determined the target sequences recognized by the Sau1 Type I RM systems present in a wide range of the most prevalent S. aureus lineages and assigned the sequences recognized to particular target recognition domains within the RM enzymes. We used a range of biochemical assays on purified enzymes and single molecule real-time sequencing on genomic DNA to determine these target sequences and their patterns of methylation. Knowledge of the main target sequences for Sau1 will facilitate the synthesis of new vectors for transformation of the most prevalent lineages of this ‘untransformable’ bacterium.


S.SauNU
Site determined to be ACC-6-TTRG or ACC-6-YTRG. Note that the underlined site was determined by SMRT and is accepted since if Y is a cytosine, then it can't be methylated. 1-soluble cell extract 2-Nickel column flow through 3-Nickel column wash 4-Nickel column eluate 5-eluate after conc. and PD10 desalting 6-final protein after concentration 7-CC398-1 purified protein marker DNA cleavage assay.

S.SauXE
TCTA-6-RTGA The degeneracy in the target determined by SMRT sequencing can be resolved by reference to targets from other systems. 1-marker 2-soluble cell extract 3-Nickel column flow through 4-Nickel column wash 5-Nickel column eluate 6-eluate after conc. step and PD10 desalting 7-final concentrated protein 8-CC398-1 purified protein marker DNA cleavage assay showed cutting of all plasmids so the ATPase assay was used given that we knew the individual TRD specificities. 1-marker 2-soluble cell extract 3-Nickel column flow through 4-Nickel column wash 1 5-Nickel column wash 2 6-Nickel column eluate 7-eluate after PD10 desalting 8-final protein after concentration 9-NQ purified protein marker The DNA cleavage assay showed cutting of all plasmids so the ATPase assay was used since we knew the TRD specificities.

S.SauAc* CCAY-6-RTC
The Ac* TRD combination is found in CC97-1. The MTase was not purified but instead used to methylate the genome of E. coli ER2796 for SMRT analysis. The target is CCAYNNNNNNRTC. There are a few minor amino acid differences in the S.SauAc* between members of CC97. 1-marker 2-soluble cell extract 3-Nickel column flow through 4-Nickel column wash 1 5-Nickel column wash 2 6-Nickel column eluate 7-eluate after conc. and PD10 desalting 8-final protein after concentration 9-CC75-1 purified protein marker

CC97
Although purified, this enzyme cut all plasmids in the DNA cleavage assay so the ATPase assay was used as we knew the specificities of the TRDs.  SMRT results for S. aureus strains LGA251 and NCTC13435 LGA251 NCTC13435 SUPPLEMENTARY INFORMATION FOR TABLES 5 AND 6.

GGA-6-RTGA Sub species 21262, a member of ST49
CLUSTAL O(1.2.1) multiple sequence alignment TRD R and TRD f* against EHO91218, the second HsdS in this strain.