A single gene (tts) located outside the cap locus directs the formation of Streptococcus pneumoniae type 37 capsular polysaccharide. Type 37 pneumococci are natural, genetically binary strains

Abstract

The molecular aspects of the type 37 pneumococcal capsular biosynthesis, a homopolysaccharide composed of sophorosyl units (beta-d-Glc-(1-->2)-beta-d-Glc) linked by beta-1,3 bonds, have been studied. Remarkably, the biosynthesis of the type 37 capsule is driven by a single gene (tts) located far apart from the cap locus responsible for capsular formation in all of the types characterized to date in Streptococcus pneumoniae. However, a cap37 locus virtually identical to the cap33f cluster has been found in type 37 strains, although some of its genes are inactivated by mutations. The tts gene has been sequenced and its transcription start point determined. Tts shows sequence motifs characteristic of cellulose synthases and other beta-glycosyltransferases. Insertion of the tts gene into the pneumococcal DNA causes a noticeable genome reorganization in such a way that genes normally separated by more than 350 kb in the chromosome are located together in clinical isolates of type 37. Encapsulated pneumococcal strains belonging to 10 different serotypes (or serogroups) transformed with tts synthesized type 37 polysaccharide, leading to the formation of strains that display the binary type of capsule. Type 37 pneumococcus constitutes the first case of a natural, genetically binary strain and represents a novel alternative to the mechanisms of intertype transformation.

Figure 1

Molecular characterization of the capsular gene locus of type 37 S. pneumoniae. (A) Long PCR amplification and restriction enzyme analysis of three different type 37 isolate DNAs, namely, 1235/89 (lane 1), 975/96 (lane 2), and 7077/39 (lane 3). (B) Genetic organization of the cap37 cluster of the 1235/89 strain. The gene organization of the cap33f locus (reference 11) is shown for comparison. Small arrows correspond to interrupted ORFs. Genes showing at least 95% identical nucleotides are indicated by identical shadings. The location of promoters (curved gray arrow) and of a putative transcription terminator (bold exclamation point) is indicated. (C) Type 37 laboratory transformants have not changed their recipient cap3 locus. PCR-amplified and restricted DNAs were prepared from the type 37 strain 1235/89 (lane 1), the S3⁻ M24 strain (lane 2), and two different S37⁺ transformants of M24, strains DN2 (lane 3) and DN5 (lane 4). The positions of some of the BstEII-digested λ DNA fragments used as size standards are indicated at the right of A and C.

Figure 2

Schematic representation of the procedure used for determination of the sequence of the ends of the PstI restriction fragment containing the gene(s) responsible for type 37 capsular polysaccharide biosynthesis. The type 37–specific DNA is indicated by a hatched bar. The open rectangles depict the polylinker region of pUCE191. Open and solid triangles represent the direct and reverse M13/pUC oligonucleotide primers, respectively. Ln^R, Ln-resistant; P, PstI; X, any restriction site not present in pUCE191.

Figure 3

Genetic organization of the type 37–specific polysaccharide synthase gene (tts) and its surrounding regions. (A) Localization of tts and flanking genes in type 37 clinical strains. The thin hatched arrow corresponds to the interrupted gene encoding the IS1167 transposase. The small divergent arrows show the location of a 105-bp repeat element characteristic of pneumococcus. The black star indicates the position of a 128-bp sequence where integration of the tts gene occurred. The location of ttsp is indicated (black curved arrow). Several pertinent restriction sites and oligonucleotide primers (triangles) are depicted. B and C show the gene structure of several contigs of the S. pneumoniae chromosome as deduced from a preliminary sequence (see text). Whenever possible, genes are identified by the designation of their most similar homologue. The nucleotide sequence of the indicated DNA fragment that has been recently reported 54 is available from EMBL/GenBank/DDBJ under accession number AF068901. The PstI sites flanking the tts gene and relevant to this study are circled.

Figure 4

Agarose gel electrophoresis demonstrating the insertion–duplication mutagenesis of the tts gene. Strain DN21 contains the ermC gene (open arrow) inserted into the DN2 tts gene (solid arrow). The wild-type and mutant genes were PCR amplified using oligonucleotides D90 and D91 and digested with MunI (M) or EcoRI (E).

Figure 5

Primer-extension mapping of the transcription initiation site for the tts gene. Total RNA was extracted from a culture of M31 harboring pDLP36. The final products were loaded on a 6% polyacrylamide 7 M urea sequencing gel, in parallel with a sequencing reaction using the same oligonucleotide primer (OL62) and pDLP36. The major extended product is indicated by an arrow, and the −10 consensus sequence of ttsp is also shown. Note that the indicated sequence corresponds to the coding strand.

Figure 6

Computer-generated alignment (PILEUP) of selected regions of the Tts synthase (SPNE_Tts) and several cellulose synthases and other glucosyltransferases. Stars indicate the conserved aspartic acid residues, and solid triangles indicate the QXXRW motif reported to be critical for UDP-Glc binding and/or catalysis 34. Residues in black boxes indicate aa residues identical in at least 7 out of the 13 proteins aligned. Conserved aa substitutions are shown in gray. The accession numbers of the selected proteins are also indicated: U58283 (Gossypium hirsutum CelA1); U58284 (G. hirsutum CelA2); D48636 (Oryza sativa CelA); U15857 (A. xylinum AcsAII): P19449 (A. xylinum BcsA); L38609 (A. tumefaciens CelA); AE000738 (Aquifex aeolicus BcsA); AL031004 (Arabidopsis thaliana ATF28M20); AF047687 (Bradyrhizobium japonicum NdvB); AJ000993 (L. lactis OrfD); D90912 (Synechocystis subspecies S11377); and U42580 (Paramecium bursaria Chlorella virus A473L).

Figure 7

(A) Partial physical and genetic maps of the S. pneumoniae strain M24 DNA. The locations of most of the restriction fragments and the genetic markers are taken from Gasc et al. 37. The genes gpmA and pyrDA that flank the tts gene in type 37 clinical isolates of S. pneumoniae have been localized in this work and are shown in bold. The psaA gene, located downstream of gpmA, has also been mapped here and is shown in bold. (B) PFGE of the DNAs obtained from strains 1235/89 (lane 1), M24 (lane 2), DN2 (lane 3), and DN5 (lane 4) digested with ApaI, SacII, or SmaI. Solid triangles indicate DNA fragments characteristic of DN2 DNA.

Figure 7

(A) Partial physical and genetic maps of the S. pneumoniae strain M24 DNA. The locations of most of the restriction fragments and the genetic markers are taken from Gasc et al. 37. The genes gpmA and pyrDA that flank the tts gene in type 37 clinical isolates of S. pneumoniae have been localized in this work and are shown in bold. The psaA gene, located downstream of gpmA, has also been mapped here and is shown in bold. (B) PFGE of the DNAs obtained from strains 1235/89 (lane 1), M24 (lane 2), DN2 (lane 3), and DN5 (lane 4) digested with ApaI, SacII, or SmaI. Solid triangles indicate DNA fragments characteristic of DN2 DNA.