The transport of group 2 capsular polysaccharides across the periplasmic space in Escherichia coli. Roles for the KpsE and KpsD proteins.

The cell surface expression of group 2 capsular polysaccharides involves the translocation of the polysaccharide from its site of synthesis on the inner face of the cytoplasmic membrane onto the cell surface. The transport process is independent of the repeat structure of the polysaccharide, and translocation across the periplasm requires the cytoplasmic membrane-anchored protein KpsE and the periplasmic protein KpsD. In this paper we establish the topology of the KpsE protein and demonstrate that the C terminus interacts with the periplasmic face of the cytoplasmic membrane. By chemical cross-linking we show that KpsE is likely to exist as a dimer and that dimerization is independent of the other Kps proteins or the synthesis of capsular polysaccharide. No interaction between KpsD and KpsE could be demonstrated by chemical cross-linking, although in the presence of both KpsE and Lpp, KpsD could be cross-linked to a 7-kDa protein of unknown identity. In addition, we demonstrate that KpsD is present not only within the periplasm but is also in both the cytoplasmic and outer membrane fractions and that the correct membrane association of KpsD was dependent on KpsE, Lpp, and the secreted polysaccharide molecule. Both KpsD and KpsE showed increased proteinase K sensitivity in the different mutant backgrounds, reflecting conformational changes in the KpsD and KpsE proteins as a result of the disruption of the transport process. Collectively the data suggest that the trans-periplasmic export involves KpsD acting as the link between the cytoplasmic membrane transporter and the outer membrane with KpsE acting to facilitate this transport process.

A functional analysis of the Streptococcus pneumoniae genes involved in the synthesis of type 1 and type 3 capsular polysaccharides.

Type 3 pneumococci produce a capsule composed of cellobiuronic acid units connected in a beta (1-->3) linkage. Cellobiuronic acid is a disaccharide consisting of D-glucuronic acid (GlcA) beta (1-->4) linked to D-glucose (Glc). The genes implicated in the biosynthesis of the type 3 capsule have been cloned, expressed, and biochemically characterized. The three type 3-specific genes--designated as cap3ABC--are transcribed together. However, the two complete open reading frames located upstream of cap3A are not transcribed and, consequently, are not required for capsule formation. The promoter of the cap3 operon was localized by primer extension analysis. The products of cap3A, cap3B, and cap3C were biochemically characterized as a UDP-Glc dehydrogenase, the type 3 polysaccharide synthase, and a Glc-1-P uridyltransferase, respectively. The Cap3B synthase was expressed in Escherichia coli, and pneumococcal type 3 polysaccharide was synthesized in this heterologous system. When a recombinant plasmid (pLSE3B) containing cap3B was introduced by transformation into encapsulated pneumococci of types 1, 2, 5, or 8, the lincomycin-resistant transformants displayed a binary type of capsule, this is, they showed a type 3 capsule in addition to that of the recipient type. Unencapsulated (S2) laboratory strains of S. pneumoniae also synthesized a type 3 capsule when transformed with pLSE3B. On the other hand, we have cloned and sequenced seven type 1-specific genes (designated as cap1A-G), and their functions have been preliminarily assigned based on sequence similarities.

Type 3-specific synthase of Streptococcus pneumoniae (Cap3B) directs type 3 polysaccharide biosynthesis in Escherichia coli and in pneumococcal strains of different serotypes.

The cap3B gene, which is involved in the formation of the capsule of Streptococcus pneumoniae type 3, encodes a 49-kD protein that has been identified as a polysaccharide synthase. Escherichia coli cells harboring the recombinant plasmid pTBP3 (cap3B) produced pneumococcal type 3 polysaccharide, as demonstrated by immunological tests. Biochemical and cell fractionation analyses revealed that this polysaccharide had a high molecular mass and was localized in substantial amounts in the periplasmic space of E. coli. Unencapsulated (S-2), laboratory pneumococcal strains synthesized type 3 polysaccharide by transformation with plasmid pLSE3B harboring cap3B. In addition, encapsulated pneumococci of types 1, 2, 5, or 8 transformed with pLSE3B can direct the synthesis of pneumococcal type 3 polysaccharide, leading to the formation of strains that display binary type of capsule.

The lytA gene and the DNA region located downstream of this gene are not involved in the formation of the type 3 capsular polysaccharide of Streptococcus pneumoniae.

Streptococcus pneumoniae strain M31, an unencapsulated, serotype 2 (S2(-)) mutant having a deletion of at least 5.5 kb containing the gene lytA that encodes the main pneumococcal autolysin, was transformed to the encapsulated serotype 3 (S3(+)) with DNA from the clinical pneumococcal strain 406. Hybridization analysis revealed that the S3(+) transformants also have the deletion demonstrating that lytA and the DNA region located downstream of this gene are not involved in the encapsulation of S. pneumoniae.

Demonstration of UDP-glucose dehydrogenase activity in cell extracts of Escherichia coli expressing the pneumococcal cap3A gene required for the synthesis of type 3 capsular polysaccharide.

The gene cluster of Streptococcus pneumoniae coding for the type 3 capsular polysaccharide contains four genes (cap3ABCD). A DNA fragment containing the cap3A gene was amplified by PCR and cloned under the control of a T7 RNA polymerase-dependent promoter. Overexpression of this gene in Escherichia coli resulted both in a 47-kDa protein in the cytoplasm of isopropyl-beta-D-thiogalactopyranoside-induced bacteria and in high levels of UDP-glucose dehydrogenase activity. These data demonstrate, in a direct experimental way, that cap3A encodes the UDP-glucose dehydrogenase of pneumococcus type 3.

Sequence and transcriptional analysis of a DNA region involved in the production of capsular polysaccharide in Streptococcus pneumoniae type 3.

The nucleotide (nt) sequence of a 9704-bp EcoRI fragment of Streptococcus pneumoniae (Sp) type-3 DNA has been determined and found to contain one partial and five complete open reading frames (ORFs). One of these ORFs corresponds to the cap3 A gene coding for the UDP-glucose (UDPGlc) dehydrogenase which is directly responsible for the transformation of some unencapsulated serotype-3 Sp mutants to the encapsulated phenotype [Arrecubieta et al., J. Bacteriol. 176 (1994) 6375-6383]. The two ORFs downstream from this gene (cap3B and cap3C) encode proteins with molecular masses of 49 and 34 kDa. Analysis of the deduced amino acid (aa) sequences of Cap3B and Cap3C shows homology to polysaccharide synthases and UDPG1c 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 transcription start point is preceded by a nt sequence identical to the sigma 70 consensus promoter sequence of E. coli. The sequence upstream from this cluster also has a high degree of similarity with genes postulated to be essential for capsular production in several Gram+ 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 Sp strain 406.

Molecular characterization of cap3A, a gene from the operon required for the synthesis of the capsule of Streptococcus pneumoniae type 3: sequencing of mutations responsible for the unencapsulated phenotype and localization of the capsular cluster on the pneumococcal chromosome.

The complete nucleotide sequence of the cap3A gene of Streptococcus pneumoniae, which is directly responsible for the transformation of some unencapsulated, serotype 3 mutants to the encapsulated phenotype, has been determined. This gene encodes a protein of 394 amino acids with a predicted M(r) of 44,646. Twelve independent cap3A mutations have been mapped by genetic transformation, and three of them have been sequenced. Sequence comparisons revealed that cap3A was very similar (74.4%) to the hasB gene of Streptococcus pyogenes, which encodes a UDP-glucose dehydrogenase (UDP-GlcDH) that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid, the donor substances in the pneumococcal type 3 capsular polysaccharide. Furthermore, a PCR-generated cap3A+ gene restored encapsulation in our cap3A mutants as well as in a mutant previously characterized as deficient in UDP-GlcDH (R. Austrian, H. P. Bernheimer, E.E.B. Smith, and G.T. Mills, J. Exp. Med. 110:585-602, 1959). These results support the conclusion that cap3A codes for UDP-GlcDH. We have also identified a region upstream of cap3A that should contain common genes necessary for the production of capsule of any type. Pulsed-field gel electrophoresis and Southern blotting showed that the capsular genes specific for serotype 3 are located near the genes encoding PBP 2X and PBP 1A in the S. pneumoniae chromosome, whereas copies of the common genes (or part of them) appear to be present in different locations in the genome.