Mosaic genes and mosaic chromosomes: intra- and interspecies genomic variation of Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae remains a major causative agent of serious human diseases. The worldwide increase of antibiotic resistant strains revealed the importance of horizontal gene transfer in this pathogen, a scenario that results in the modulation of the species-specific gene pool. We investigated genomic variation in 20 S. pneumoniae isolates representing major antibiotic-resistant clones and 10 different capsular serotypes. Variation was scored as decreased hybridization signals visualized on a high-density oligonucleotide array representing 1,968 genes of the type 4 reference strain KNR.7/87. Up to 10% of the genes appeared altered between individual isolates and the reference strain; variability within clones was below 2.1%. Ten gene clusters covering 160 kb account for half of the variable genes. Most of them are associated with transposases and are assumed to be part of a flexible gene pool within the bacterial population; other variable loci include mosaic genes encoding antibiotic resistance determinants and gene clusters related to bacteriocin production. Genomic comparison between S. pneumoniae and commensal Streptococcus mitis and Streptococcus oralis strains indicates distinct antigenic profiles and suggests a smooth transition between these species, supporting the validity of the microarray system as an epidemiological and diagnostic tool.

FIG. 1

Genomic DNA hybridizations on ROEZ06s microarray. (A) Fluorescence image of ROEZ06s 24-μm array containing more than 250,000 oligonucleotide probes complementary to all H. influenzae (top) and S. pneumoniae open reading frames and a selection of intergenic sequences (bottom). The array was hybridized against 15-μg total genomic DNA from S. pneumoniae KNR.7/87. An enlargement of each area shows the low cross-hybridization in the upper part of the chip and the specific signals in the lower part with intense rows of PM probes alternating with MM rows. (B) Scatter plot showing the correlation for the intensities of all genes obtained in two independent labeling reactions and hybridizations of genomic DNA from S. pneumoniae KNR.7/87 (experiment 1 versus 2). The two solid lines flanking the diagonal indicate an intensity ratio of 2. (C) Correlation of intensities for strain KNR.7/87 and a member of the Spanish clone SA17. All genes varying in strain SA17 appear on the lower right part of the graph.

FIG. 2

Cluster image showing genes with different intensity ratios in pairwise comparisons between S. pneumoniae strains. The genes are vertically sorted by position according to the unfinished genome of KNR.7/87 (http://www.tigr.org). Each column represents a pairwise comparison of the strains indicated on top of each lane; the number at the bottom indicates the pecentage of genes varying. (A) Comparison of 19 S. pneumoniae strains to KNR.7/87. All genes varying by an intensity ratio smaller than −4 in at least one of the comparisons are listed and marked by black lines (470 genes in total). Bars on the right side indicate clusters of variable genes covering more than 9 kb; *, the presence of transposases. Open arrowheads indicate the position of the bacteriocin cluster (blp or pcn) (9, 30) (top) and a Enterococcus cytolysin-related gene cluster (bottom) (41). (B) Comparison of D39 and its derivative R6 and members of the Spanish and Hungarian clone groups. All genes varying by an intensity ratio of less than −4 or greater than 4 in at least one of the comparisons are marked.

FIG. 3

Detailed analysis of pbp2x genes. (a) Intensity ratios for pbp2x genes in pairwise comparisons of 19 S. pneumoniae strains to KNR.7/87. Intensity ratios are 0 in penicillin-sensitive strains. The values for pbp2x range from −4 to −37 for pbp2x in all penicillin-resistant strains. Mosaic structures of the pbp2x genes are shown above the strain designation, with white bars representing conserved sequences in sensitive S. pneumoniae, and black bars representing mosaic blocks. Sequence differences between the mosaic blocks are not distinguished. (b) Probe performance graph for pbp2x in three strains. The results obtained for each of the 25 oligonucleotides representing the pbp2x gene are shown for the penicillin-sensitive strain Fi2306 and two resistant-strains, 496 and Hu-11. Signal intensity for the PM probe is shown in black, while MM probe signals are shown in white. Probe pairs are sorted from 5′ (left) to 3′.

FIG. 4

DNA sequences of dihydrofolate reductase genes in S. pneumoniae, S. mitis, and S. oralis strains. Only sites where at least one sequence differed from that of S. pneumoniae R6 dhfr are shown. The codons are indicated vertically in the first three rows and numbered according to the published sequence (2). The numbers 1, 2, and 3 refer to the position in the respective codon.

FIG. 5

Global allelic variations in oral streptococci. All;1,968 pneumococcus genes present on the microarray are represented vertically according to their position on the unfinished genome of KNR.7/87 (http://www.tigr.org). Genes varying by an intensity ratio smaller than −4 in at least one of the comparisons were marked. For comparison, genes with low intensity of the major multiple-antibiotic-resistant clones (Hu-11, SA17, 670) as well as the ancestor of the laboratory strain R6 (D39) are also listed, and the major variable gene clusters as indicated in Fig. 2 are marked by arrows on the right side.

FIG. 6

Variation of pneumococcal virulence genes and choline binding proteins (A), early competence genes (B), peptidoglycan biosynthesis-related genes (C) in oral streptococci. The genes are listed on the left side; black boxes indicate intensity ratios smaller than −4. Genes with low intensity in both S. mitis and S. oralis, as well as those with low intensity in S. oralis only, are boxed.

FIG. 7

Genomic variation of S. mitis, S. oralis, and S. pneumoniae strains. The percentage of genes with low intensity compared to S. pneumoniae KNR.7/87 is indicated; the last bar represents the variation observed between the two multiple-antibiotic-resistant S. pneumoniae clones.