Characterization of the locus encoding the Streptococcus pneumoniae type 19F capsular polysaccharide biosynthetic pathway

Abstract

SummaryWe have previously reported the nucleotide sequence of the first six genes of the Streptococcus pneumoniae type 19F capsular polysaccharide biosynthesis locus (cps19f ). In this study we used plasmid insertion/rescue and inverse polymerase chain reaction (PCR) to clone an additional 10 kb downstream region containing the remainder of the cps19f locus, which was then subjected to sequence analysis. The cps19f locus is located in the S. pneumoniae chromosome between dexB and aliA, and consists of 15 open reading frames (ORFs), designated cps19fA to cps19fO, that appear to be arranged as a single transcriptional unit. Insertion-duplication mutants in seven out of the nine new ORFs have been constructed in a smooth type 19F strain, all of which resulted in a rough (nonencapsulated) phenotype, confirming that the operon is essential for capsule production. Comparison with sequence databases has allowed us to propose functions for 12 of the cps19f gene products, and a biosynthetic pathway for type 19F capsular polysaccharide. T7 expression studies confirmed that cps19fH, cps19fK, cps19fL, cps19fM and cps19fN directed the production of polypeptides of the expected size in Escherichia coli. The function of the cps19fK product was confirmed by its ability to complement a mutation in nfrC (rffE ) in E. coli, as judged by restoration of sensitivity to bacteriophage N4. Interestingly, the last four genes of the locus (cps19fL-O ) exhibit very strong homology (up to 70% amino acid identity) to a portion of the Shigella flexneri rfb gene cluster encoding biosynthesis of dTDP-rhamnose. When expressed in E. coli, cps19fL-O were capable of complementing a mutation deleting the respective Shigella flexneri homologues. Southern hybridization analysis indicated that cps19fA and cps19fB were the only cps genes found in all 16 S. pneumoniae serotypes/groups tested. The region from cps19fG to cps19fK was found only in members of serogroup 19, and, within this, cps19fI was unique to type 19F.

The complete cps gene cluster from Streptococcus thermophilus NCFB 2393 involved in the biosynthesis of a new exopolysaccharide

“…pneumoniae and Sal. enterica (Morona et al, 1997 ;Mun4 oz et al, 1997 ;Xiang et al, 1994). The GjC content of the NCFB 2393 cps cluster is 35n6 mol % ; this is close to the average GjC content reported for Str.…”

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

“…In contrast to the rmlD genes in other species like Shigella flexneri (Macpherson et al ., 1994) and Streptococcus pneumoniae serotype 19F (Morona et al ., 1997), the only GAS rmlD homologue gacA is not part of an rmlABCD rhamnose biosynthesis operon. Instead, gacA is located at the beginning of the recently identified GAC gene cluster and hence named gacA (van Sorge et al ., 2014).…”

In vitro expression of the first capsule gene of Streptococcus pneumoniae, cpsA, is associated with serotype-specific colonization prevalence and invasiveness

“…In this study the role of transcription of the capsule operon was investigated. Since the capsule operon appears to be arranged as a single transcriptional unit (Morona et al, 1997;Munoz et al, 1997), transcription of the first gene of the capsule operon, cpsA, was measured. This study was limited to in vitro cpsA expression with quantification of capsule polysaccharide for only two isolates.…”