Nucleotide Sequence Analysis ofGenesEssential for Capsular Polysaccharide Biosynthesis in Streptococcus pneumoniae Type19F

Previous studies haveshownthatthecapsular polysaccharide synthesis (cps)locusofthetype19F Streptococcus pneumoniae strain SSZwas closely linked toa copy oftheinsertion sequence IS1202 (J.K. Morona, A.Guidolin, R.Morona, D.Hansman, andJ.C.Paton, J.Bacteriol. 176:4437-4443, 1994). Inthe present study, we usedplasmid insertion andrescueandinverse PCR toclone6,322bpofflanking DNA upstream ofIS1202. Sequence analysis indicated thatthis region contains sixcomplete open reading frames (ORFs)andone partial ORFthat arearranged as a single transcriptional unit. Chromosomal disruption of anyofthese ORFsina smooth-type 19Fstrain leads toa rough(unencapsulated) phenotype, indicating that this operonisessential forcapsule production. TheORFshavetherefore beendesignated cpsl9fA tocpsl9fG, wherecpsl9fA isthefirst geneofthetype19Fcpslocus. Furthermore, many ofthegene products fromthis incomplete operonexhibit strong similarities toproteins knowntobeinvolved intheproduction ofcapsular polysaccharide, exopolysaccharide, teichoic acid, enterobacterial common antigen, andlipopolysaccharide fromnumerous other bacterial species. Thishasallowed us toproposefunctions for many ofthetype19Fcps geneproducts. Southern hybridization studies reveal thatcpsl9fA andcpsl9fB areconserved among all12 pneumococcal serotypes tested, whereas genesdownstream ofcpsl9fB areconserved among some,butnotall, oftheserotypes tested. Streptococcus pneumoniae (the pneumococcus) isan impor- tanthumanpathogen, causing invasive diseases suchaspneu- monia, meningitis, andbacteremia. Morbidity andmortality frompneumococcal infections remainhigh, even inregions whereeffective antibiotic therapy isfreely available. Indevel- oping countries, inexcessof3million children undertheageof 5yearsdieeachyearfrompneumonia, andS.pneumoniae is thecommonestcausative agent. S.pneumoniae also causesless serious, buthighly prevalent infections suchasotitis mediaand sinusitis, whichhaveasignificant impact onhealth-care costs in developed countries. Pneumococci arefrequently isolated fromthenasopharynx ofhealthy people. Virtually allhumansare colonized by pneumococci atsome stage, andpneumococcal carriage rates arehigher inyoungchildren andwherepeople areliving in crowded conditions. Inaproportion ofcarriers (approximately 15%inone pediatric study carried outintheUnitedStates (16)), thepneumococcusinvades tissues andcausesdisease. Thisscenario ismore likely inpersonswhohaveonlyrecently becomecolonized (2). An important feature ofS.pneumoniae isitscapacity to produce apolysaccharide capsule, whichisstructurally distinct foreachofthe84knownserotypes oftheorganism. The polysaccharide capsule isconsidered tobethesinequanon of pneumococcal virulence (2). Thisisbasedon theobservation thatallfresh isolates frompatients withpneumococcal infec- tionare encapsulated, and spontaneous nonencapsulated (rough) derivatives ofsuchstrains are almostcompletely