Sequence Elements Upstream of the Core Promoter Are Necessary for Full Transcription of the Capsule Gene Operon in Streptococcus pneumoniae Strain D39

Abstract

e Streptococcus pneumoniae is a major bacterial pathogen in humans. Its polysaccharide capsule is a key virulence factor that promotes bacterial evasion of human phagocytic killing. While S. pneumoniae produces at least 94 antigenically different types of capsule, the genes for biosynthesis of almost all capsular types are arranged in the same locus. The transcription of the capsular polysaccharide (cps) locus is not well understood. This study determined the transcriptional features of the cps locus in the type 2 virulent strain D39. The initial analysis revealed that the cps genes are cotranscribed from a major transcription start site at the ؊25 nucleotide (G) upstream of cps2A, the first gene in the locus. Using unmarked chromosomal truncations and a luciferasebased transcriptional reporter, we showed that the full transcription of the cps genes not only depends on the core promoter immediately upstream of cps2A, but also requires additional elements upstream of the core promoter, particularly a 59-bp sequence immediately upstream of the core promoter. Unmarked deletions of these promoter elements in the D39 genome also led to significant reduction in CPS production and virulence in mice. Lastly, common cps gene (cps2ABCD) mutants did not show significant abnormality in cps transcription, although they produced significantly less CPS, indicating that the CpsABCD proteins are involved in the encapsulation of S. pneumoniae in a posttranscriptional manner. This study has yielded important information on the transcriptional characteristics of the cps locus in S. pneumoniae. Streptococcus pneumoniae (pneumococcus) is a major cause of bacterial infections in humans, including otitis media, pneumonia, bacteremia, and meningitis (1). As the outermost structure, the capsule is the major virulence factor of S. pneumoniae and protects the bacterium by interfering with host phagocytic killing (2). Virtually all of the clinical isolates are encapsulated, and mutations in the cps locus result in the loss of virulence (3). The capsule of S. pneumoniae is composed of capsular polysaccharides (CPSs). The CPSs are immunogenic and thus serve as the target antigens for the current pneumococcal vaccines (4). Due to host immune selection that targets the capsule, the CPSs have undergone extensive chemical and antigenic variation via genetic diversity in the CPS biosynthesis genes. At least 94 antigenically distinct capsular serotypes have been identified in S. pneumoniae (5, 6). While the CPSs of types 3 and 37 are produced as relatively simple repeats of polysaccharides by the synthase pathway (7, 8), all other capsule types are much more complex and are synthesized by WZY-dependent polymerization, a mechanism of capsule production that is widely found in other bacteria (9-11).Except for type 37, all of the CPS types in S. pneumoniae are determined by a set of capsule biosynthesis genes, which are historically designated cps or cap (12). Bentley et al. recently renamed the pneumococcal capsule genes on the basis of their overall...

Addiction of Hypertransformable Pneumococcal Isolates to Natural Transformation for In Vivo Fitness and Virulence

“…Plasmids pTH5088 (comX), pTH5089 (ssbB), and pTH5275 (spxB) were constructed in pIB184 by the same procedures as in pIB166. Plasmid pTH3936 carrying the promoterless luciferase gene was constructed in pIB166 in our previous study (47). pTH5273, an equivalent of pTH3936, was generated in pIB184 by subcloning lux from pTH3936.…”

“…Luciferase reporter constructs of the com genes were prepared in the Escherichia coli-Streptococcus shuttle plasmid pIB166 (conferring chloramphenicol resistance [Cm r ]) (46) as described previously (47). Briefly, the entire 5= intergenic regions of comX, ssbB, and spxB were amplified from genomic DNA of R6 using primer pairs Pr7234/Pr7236, Pr7090/ Pr7091, and Pr6764/Pr6765, respectively.…”

Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae

“…The cotranscription of the ptvR-C genes was assessed by intergenic RT-PCR as described previously (73). A cDNA library was prepared using the total RNA of S. pneumoniae D39 (described above) with an iScript cDNA synthesis kit (Bio-Rad, Beijing, China) according to the supplier's instructions and used to amplify the intergenic regions of the ptvR-C genes with the following primer pairs: ptvR-ptvA (Pr7879/Pr7880), ptvA-ptvB (Pr7881/Pr7882), and ptvB-ptvC (Pr7883/Pr7884).…”

The Protease Locus of Francisella tularensis LVS Is Required for Stress Tolerance and Infection in the Mammalian Host

“…Promoter mapping in the protease locus was performed using quantitative RT-PCR (qRT-PCR) with the total RNA extract of LVS and isogenic transposon mutants essentially as described previously (44,45). The tig (tig::Tn, ST1284) and clpP (clpP::Tn, ST1292) transposon insertion mutants were generated in our previous study (8).…”

“…In-frame deletion in the coding region of lon, clpP, or clpX was done in F. tularensis LVS by allelic replacement and sacB-mediated counterselection as described previously (47). The up-and downstream sequences of each target gene were amplified by PCR from LVS genomic DNA with primer pairs Pr1235/Pr1238 and Pr1236/Pr1237 for lon, Pr1062/Pr1065 and Pr1063/ Pr1064 for clpP, and Pr1058/Pr1061 and Pr1059/Pr1060 for clpX and linked by fusion PCR as described previously (45). Each fusion product was initially cloned into the XhoI (for clpP and clpX) or XhoI/NotI (for lon) sites of pBluescript II (SKϪ) (Stratagene, La Jolla, CA) and subcloned into the ApaI/BamHI (for clpP and clpX) or ApaI/NotI (for lon) sites of pMP590 (Table 1).…”