Table 1 RM systems of pneumococcal strain D39.

RM system name	Type	ORFs ^*	Specificity ^†	TRD	Modified base	Number of sites ^*	Predicted no. of sites ^*	Difference (%)	Strand specificity (%) ^‡
SpnD39ORF1631P (DpnI)	Type II	SPD_1630-1	5′-GATC-3′ 3′-CTAG-5′	—	—	7,164	7,324	−2	—
SpnD39I (SpnD39ORF1260P)	Type II	SPD_1259-60	5′-TCTAGA-3′ 3′-AGATCT-5′	—	^m6A	644	438	47^§	—
SpnD39II^\|\| (SpnD39ORF1079AP)	Type II	SPD_1079-80	5′-TCGAG-3′ 3′-AGCTG-5′	—	^m6A	1,509	1454	4	—
SpnD39IIIA (SpnD39ORF454P)	Type I	SPD_0450-5	5′-CRAAN₈CTG-3′ 3′-GYTTN₈GAC-5′	1.1, 2.1	^m6A	720	438	64^§	66^§
SpnD39IIIB	Type I	SPD_0450-5	5′-CRAAN₉TTC-3′ 3′-GYTTN₉AAG-5′	1.1, 2.2	^m6A	1,029	665	55^§	64^§
SpnD39IIIC	Type I	SPD_0450-5	5′-CACN₈TTC-3′ 3′-GTGN₈AAG-5′	1.2, 2.2	^m6A	641	876	−27^§	66^§
SpnD39IIID	Type I	SPD_0450-5	5′-CACN₇CTG-3′ 3′-GTGN₇GAC-5′	1.2, 2.1	^m6A	428	577	−26^§	67^§
SpnD39IIIE	Type I	SPD_0450-5	5′-CRAAN₈CTT-3′ 3′-GYTTN₈GAA-5′	1.1, 2.3	^m6A	1,028	665	55^§	63^§
SpnD39IIIFP	Type I	SPD_0450-5	5′-CACN₇CTT-3′ 3′-GTGN₇GAA-5′	1.2, 2.3	^m6A	796	876	−9^§	64^§
SpnD39ORF782P	Type I	SPD_0782-4^¶	not active; see below	—	—	—	—	—	—
SpnD39McrBCP	Type IV	SPD_1108-9	not known	—	—	—	—	—

ORF, open reading frame; RM, restriction-modification system; TRD, target recognition domain.
Using strains WCH16 and WCH43 (ref. 14), we identified the target sites of the two possible variants of this Type I RM system to be 5′-G(^m6A)YN₆TATC-3′ and 3′-CTRN₆(^m6A)TAG-5′ for S.SpnWCH16IVA (GenBank nucleotide database accession code KM030255) and 5′-TG(^m6A)N₇TATC-3′ and 3′-ACTN₇(^m6A)TAG-5′ for S. SpnWCH43IVB (GenBank code KM030256).
^*Open reading frame (ORF) numbering and calculations are done for strain D39 (GenBank nucleotide database accession code CP000410.1).
^†Methylated adenine in bold underlined.
^‡Percentage of sites mapping to the lagging strand of the genome (3′-5′ strand from start to 1,047,526 and 5′-3′ from 1,047,527 to end).
^§Differences were analysed by χ² analysis and found to be significant for all values (P<0.001).
^||The target site TCG(^m6A)G could be associated to Spn39II due to absence of both this locus and TCG(^m6A)G methylation in strain WCH43 (ref. 14).
^¶The gene encoding S.SpnD39ORF782P is truncated in D39 and no methylation was detected.

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