Organization and characterization of the capsule biosynthesis locus of Streptococcus pneumoniae serotype 9V

Bacteriology Division, Department of Infectious Diseases and Immunology, Utrecht University, PO Box 80.165, 3508 TD Utrecht, The Netherlands 1 Genencor International B. V., 2300 AE Leiden, The Netherlands 2 National Institute of Public Health and the Environment, 3720 BA Bilthoven, The Netherlands 3 Author for correspondence: Jos P. M. van Putten. Tel: +31 30 2534888. Fax: +31 30 2540784. e-mail: j.vanputten{at}vet.uu.nl The capsular polysaccharide (CPS) synthesis locus of Streptococcus pneumoniae serotype 9V was amplified by long-range PCR and sequenced. The locus was 17368 bp in size and contained 15 ORFs. The genetic organization of the cluster shared many features with other S. pneumoniae capsule loci, including the presence of four putative regulatory genes at the 5' end. Comparative sequence analyses allowed putative functions to be assigned to each of the gene products. The ORFs appeared to encode, besides the four regulatory genes, five glycosyltransferases, two O -acetyltransferases, an N -acetylglucosamine 2-epimerase, a glucose 6-dehydrogenase, an oligosaccharide transporter protein and a polysaccharide repeating unit polymerase. These functions covered the steps proposed in the CPS biosynthesis of serotype 9V. TLC of carbohydrate intermediates formed after incubation of bacterial membrane preparations with 14 C-labelled precursors demonstrated that the fifth ORF ( cps9vE ) encoded a UDP-glucosyl-1-phosphate transferase. This function was confirmed with the help of a cps9vE mutant that carried a deletion of a guanine residue located adjacent to a stretch of adenines. The identification and characterization of the serotype 9V locus is a major step in unravelling the 9V capsule biosynthesis pathway and broadens the insight into the genetic diversity of the S. pneumoniae capsule loci. Keywords: capsular polysaccharide biosynthesis, glucosyltransferase Abbreviations: CPS, capsular polysaccharide The GenBank accession number for the sequence reported in this paper is AF402095 .