Genetic organization, sequence and transcriptional analysis of the genes implicated in the synthesis of pneumococcal types 3 and 1 capsules

GARCíA E; ARRECUBIETA C; MOLLERACH M; LóPEZ R

Leicester, Inglaterra

Workshop; Third European Meeting on the Molecular Biology of the Pneumococcus; 1995

European Team on the Molecular Biology of the Pneumococcus

The nucleotide (nt) sequence of a 9704-bp EcoR1 fragment of Streptococcus pneumoniae type 3 has been determined and found to contain one partial and five complete open reading frames (ORFs). One the these ORFs corresponds to the cap3A gene coding for the UDP-glucose (UDPGlc) dehydrogenase which is directly responsible for the transformation of some unencapsulated serotype 3 mutants to the encapsulated phenotype. Two ORFs downstream of this gene (cap3B and cap3C) encode proteins with molecular masses of 49-kDa and 34-kDa. Analysis of the predicted aminoacid (aa) sequences of Cap3B and of Cap3C shows homology to polysaccharide synthases and UDPGIc pyrophosphorylases, respectively. Furthermore, genetic complementation analysis showed that cap3C restored the galU defect of an Escherichia coli mutant. Northern blots have shown that cap3A, cap3B and cap3C constitute a single transcriptional unit and primer extension analysis has revealed that the transcriptional start site is preceded by a nt sequence identical to the ó70 consensus promoter sequence of E. coli. The DNA sequence upstream of this cluster also has a high degree of similarity with genes postulated to be essential for capsular production in several Gram-positive bacteria. However, Northern blot analysis and insertion-duplication mutagenesis indicated that genes located in this region are not necessary for type 3 capsule production in the pneumococcal strain 406. On the other hand, we have also identified a chromosomal region of S. pneumoniae serotype 1 apparently involved in capsule polysaccharide synthesis. The organization of this region is being currently studied.