To the editor:

The study by Nakamura and coworkers1 offers insight into the computational analysis of horizontal gene transfer. Their results seem to be convincingly supported by phylogenetic validation of the supplied examples of calculated horizontal gene transfer events. An outstanding example, validating their method, concerns the presence of the gene encoding an rRNA adenine N-6-methyltransferase, NMB0066, in the genome of Neisseria meningitidis MC58 (ref. 2). According to the authors' results, NMB0066 originates from plasmids naturally occurring in Staphylococcus aureus, such as pE5. In fact, this gene, being an erythromycin resistance cassette (ermC), was horizontally acquired, because it was deliberately introduced in the N. meningitidis MC58 genome by genetic modification using plasmid pIP10 (ref. 3) to reduce virulence. In the pIP10 construct, the gene encoding the polysialic acid capsule biosynthesis protein SiaD (NMB0067) is inactivated by insertion of cloning vector sequences and the ermC gene originally derived from plasmid pIM13, a naturally occurring plasmid found in Bacillus subtilis4. Remnants of cloning vector sequences flanking NMB0066 are noticeable in the genome sequence of N. meningitidis MC58. The sequences of NMB0066 and ermC of pIM13 are identical, whereas that of ermC of pE5 contains one nonsynonymous mutation and an insertion of 107 nucleotides upstream of the open reading frame. This means that, although NMB0066 is clearly horizontally acquired by N. meningitidis MC58, its origin remains at best obscure. In addition, it is implausible that the surrounding genes, NMB0065 through NMB0070, were acquired in one event from the same donor as ermC, opposing the authors' suggestion that they were transferred simultaneously with NMB0066. In conclusion, although the algorithm by Nakamura and coworkers correctly identified the acquisition of NMB0066 by N. meningitidis, their suggestion that S. aureus was the donor organism is improbable. Moreover, their interpretation concerning the simultaneous acquisition of NMB0066 and its surrounding genes is inappropriate.