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Genomic alterations of <jats:italic><jats:styled-content style=\"fixed-case\">S<\/jats:styled-content>.\u2009pneumoniae<\/jats:italic> <jats:styled-content style=\"fixed-case\">R<\/jats:styled-content>6 introduced during four successive transformations with <jats:styled-content style=\"fixed-case\">DNA<\/jats:styled-content> of the high\u2010level penicillin\u2010resistant <jats:italic><jats:styled-content style=\"fixed-case\">S<\/jats:styled-content>treptococcus mitis<\/jats:italic> <jats:styled-content style=\"fixed-case\">B<\/jats:styled-content>6 with beta\u2010lactam selection have now been determined and the contribution of genes to high resistance levels was analysed genetically. Essential for high level resistance to penicillins of the transformant <jats:styled-content style=\"fixed-case\">CCCB<\/jats:styled-content> was the combination of <jats:styled-content style=\"fixed-case\"><jats:italic>murM<\/jats:italic><jats:sub>B<\/jats:sub><\/jats:styled-content><jats:sub>6<\/jats:sub> and the 3\u2032 region of <jats:italic>pbp2b<\/jats:italic><jats:styled-content style=\"fixed-case\"><jats:sub>B<\/jats:sub><\/jats:styled-content><jats:sub>6<\/jats:sub>. Sequences of both genes were detected in clinical isolates of <jats:italic><jats:styled-content style=\"fixed-case\">S<\/jats:styled-content>.\u2009pneumoniae<\/jats:italic>, confirming the participation of <jats:italic><jats:styled-content style=\"fixed-case\">S<\/jats:styled-content>.\u2009mitis<\/jats:italic> in the global gene pool of beta\u2010lactam resistance determinants. The <jats:italic><jats:styled-content style=\"fixed-case\">S<\/jats:styled-content>.\u2009mitis<\/jats:italic> <jats:styled-content style=\"fixed-case\">PBP1b<\/jats:styled-content> gene which contains an authentic stop codon within the transpeptidase domain is now shown to contribute only marginal to resistance, but it is possible that the presence of its transglycosylase domain is important in the context of cognate <jats:styled-content style=\"fixed-case\">PBPs<\/jats:styled-content>. The genome sequence of <jats:styled-content style=\"fixed-case\">CCCB<\/jats:styled-content> revealed 36 recombination events, including deletion and acquisition of genes and repeat elements. 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