Genome sequence of Avery's virulent serotype 2 strain D39 of Streptococcus pneumoniae and comparison with that of unencapsulated laboratory strain R6

Abstract

Streptococcus pneumoniae (pneumococcus) is a leading human respiratory pathogen that causes a variety of serious mucosal and invasive diseases. D39 is an historically important serotype 2 strain that was used in experiments by Avery and coworkers to demonstrate that DNA is the genetic material. Although isolated nearly a century ago, D39 remains extremely virulent in murine infection models and is perhaps the strain used most frequently in current studies of pneumococcal pathogenesis. To date, the complete genome sequences have been reported for only two S. pneumoniae strains: TIGR4, a recent serotype 4 clinical isolate, and laboratory strain R6, an avirulent, unencapsulated derivative of strain D39. We report here the genome sequences and new annotation of two different isolates of strain D39 and the corrected sequence of strain R6. Comparisons of these three related sequences allowed deduction of the likely sequence of the D39 progenitor and mutations that arose in each isolate. Despite its numerous repeated sequences and IS elements, the serotype 2 genome has remained remarkably stable during cultivation, and one of the D39 isolates contains only five relatively minor mutations compared to the deduced D39 progenitor. In contrast, laboratory strain R6 contains 71 single-base-pair changes, six deletions, and four insertions and has lost the cryptic pDP1 plasmid compared to the D39 progenitor strain. Many of these mutations are in or affect the expression of genes that play important roles in regulation, metabolism, and virulence. The nature of the mutations that arose spontaneously in these three strains, the relative global transcription patterns determined by microarray analyses, and the implications of the D39 genome sequences to studies of pneumococcal physiology and pathogenesis are presented and discussed.

FIG. 1.

Pedigree of strains D39 (NCTC), D39 (Lilly), and R6 whose genome sequences are compared in the present study. (The figure was modified from reference .) See the introduction and reference for details.

FIG. 2.

Primer-walk strategy used to sequence the genomes of strains D39 (NCTC) and D39 (Lilly). Overlapping PCR amplicons were synthesized and sequenced by using primers based on the published sequence of strain R6 (48). See the text for details. The figure is not drawn to scale.

FIG. 3.

PCR analysis of the repeated region in the spd0080 gene of D39 (spr0075 of R6). Primers III-F-036 and III-R-042 flanking the multiple 456-bp repeats of the SSURE domain (21) were used in PCRs to amplify the region as described in Materials and Methods. Amplification products from strains R6, D39 (Lilly), and D39 (NCTC) are shown next to a 1-kb molecular mass marker ladder (left lane) and the expected sizes of amplicons containing one to eight repeat units. The predominant species indicated repeats of eight or six SSURE units in this gene in R6 or D39, respectively. The faint ladder of bands differing by one repeat unit are likely a PCR amplification artifact and were not observed in Southern blots (see text). The extent to which this region was sequenced is indicated at the bottom of the figure. As was done previously (48), we inserted a placeholder sequence containing the five repeats reported previously for R6 into this region of the D39 genome sequence (see the text).

FIG. 4.

Microarray analysis of relative transcript amounts in strains D39 (NCTC) and R6 grown exponentially in BHI. Microarray analyses were performed as described in Materials and Methods. A representative log-scale scatter plot of relative transcript amounts is shown, and fold changes and Bayesian P values for transcripts changing at least 1.9 are listed in Table S6 in the supplemental material. Genes comprising the capsule locus are circled. Symbols indicate coregulated gene clusters as follows: stars, competence-induced genes (cibBC [allolysis], single-strand binding protein [ssb], and cglABCD comYD [peptioglycan-spanning structural proteins]); circles (spd0501 to spd0503; spr0504 to spr0506), genes coregulated with licT (spd0501; spr0504); diamonds (spd0559 to spd0562; spr0562 to spr0565), genes coregulated with ptsN (spd0559; spr0562); squares (spd1958 to spd1961; spr1937 to spr1940), genes coregulated with a BglG-family transcriptional regulator (spd1961; spr1940); triangles (spd1029 to spd1034; spr1052 to spr1057), genes coregulated with an HAD superfamily hydrolase (spd1034; spr1057). IC, mutation in upstream intercistronic region; Pt, point mutation within gene coding sequence. See the text for additional details.

FIG. 5.

Biophotonic imaging of ICR male mice (ca. 25 to 30 g) infected with D39 (NCTC) luxABCDE or D39 (Lilly) luxABCDE. Each mouse was infected intratracheally (IT) with 50 μl of buffered saline containing ∼2.5 × 10⁶ CFU of each strain (see Materials and Methods) (96). Bioluminescence was monitored and recorded at various times in separate animals (87, 88). Typical images from 1-min exposures are shown 19 h (A) and 85 h (B) after infection. The mouse in the lower right corner has cleared the D39 (Lilly) infection.

FIG. 6.

Microarray analysis of relative transcript amounts in strains D39 (NCTC) and D39 Lilly) grown exponentially in BHI. Microarray analyses were performed as described in Materials and Methods. A representative log-scale scatter plot of relative transcript amounts is shown, and the fold changes and Bayesian P values for transcripts changing at least 1.8-fold are listed in Table S7 in the supplemental material. Symbols are as follows: squares (spd1664 to spd1670), genes partially or completely deleted in D39 (Lilly); circles (spd0404 to spd0408), ilvBNC gene cluster likely regulated to compensate for deletion of amiCDEF; stars, ciaRH two-component signal transduction system; triangles, genes regulated in a “cia-on” mutant (74).