The genome of Streptococcus mitis B6--what is a commensal?

Abstract

Streptococcus mitis is the closest relative of the major human pathogen S. pneumoniae. The 2,15 Mb sequence of the Streptococcus mitis B6 chromosome, an unusually high-level beta-lactam resistant and multiple antibiotic resistant strain, has now been determined to encode 2100 genes. The accessory genome is estimated to represent over 40%, including 75 mostly novel transposases and IS, the prophage phiB6 and another seven phage related regions. Tetracycline resistance mediated by Tn5801, and an unusual and large gene cluster containing three aminoglycoside resistance determinants have not been described in other Streptococcus spp. Comparative genomic analyses including hybridization experiments on a S. mitis B6 specific microarray reveal that individual S. mitis strains are almost as distantly related to the B6 strain as S. pneumoniae. Both species share a core of over 900 genes. Most proteins described as pneumococcal virulence factors are present in S. mitis B6, but the three choline binding proteins PcpA, PspA and PspC, and three gene clusters containing the hyaluronidase gene, ply and lytA, and the capsular genes are absent in S. mitis B6 and other S. mitis as well and confirm their importance for the pathogenetic potential of S. pneumoniae. Despite the close relatedness between the two species, the S. mitis B6 genome reveals a striking X-alignment when compared with S. pneumoniae.

Figure 1. The S. mitis B6 genome.

A. Circular display of the S. mitis B6 chromosome. Black dots mark clusters larger than 15 kb which are absent in most or all S. pneumoniae and S. mitis; open circles indicate phages related islands. The outer two circles show open reading frames oriented in the forward and reverse direction, respectively. The third circle marks phage related elements including φB6 (pink; roman numbers) and gene clusters >15 kb of the accessory genome which are absent in most or all S. pneumoniae and S. mitis (blue; A: ntp cluster; B: unknown function; C: Tn5801; D: monX cluster; E: aminoglycoside resistance). The fourth circle shows IS (red) and the two group II introns (black), the fifth circle BOX elements (blue) and RUP (black). The sixth circle shows GC skew, purple indicating negative values; the sixth circle indicates the G+C content. B. Electron micrograph of φB6. Phage particles were purified from mitomycin C-induced S. mitis B6 cultures (0.2 µg/ml). The bar reprents 100 nm. C. Genome alignment of S. mitis B6 with S. pneumoniae R6. In the display using ACT, red areas mark regions of the same orientation in both species, blue indicates regions implicated in the X-alignment. Only regions >1 kb are shown.

Figure 2. The aminoglycoside resistance gene cluster.

The two genes smi_1689 and smi_1720 flanking the cluster are conserved in S. pneumoniae and S. mitis. Red: genes implicated in antibiotic resistance as indicated above the genes; black: IS and recombinases; hatched: S. mitis B6 specific hypothetical genes. Thick lines below mark regions with homology to genes in other species as indicated. The fat arrows left and right mark repeat sequences.

Figure 3. Alignment of the choline-binding modules with 40mer repeat sequences.

The S. pneumoniae R6 Spr583 (CBP11 homologue) is included.

Figure 4. Bacteriocin clusters in S. mitis B6.

A: the blp/pnc cluster; B: cluster II upstream comAB; C: components implicated in competence regulation. The gene designation of TIGR4 is given above, and S. mitis B6 gene numbers are indicated below. Grey areas indicate regions of >80% identity. Red: Response regulators and histidine kinases; dark blue: ABC peptide transporter; light blue: peptide pheromone; green: CAAX proteases; black: bacteriocins; grey: immunity proteins; striped: IS; white: hypothetical proteins. Black triangles mark the position of BOX elements.

Figure 5. Genomic comparison of S. mitis B6.

A. Genomic hybridization analysis of S. mitis strains using a S. mitis B6 specific microarray. Mobile elements and phage related gene clusters, and ambiguous signals were not considered. The percentage of positive hybridization signals is indicated on the left. The vertical fat black line indicates positive genes common to all S. mitis strains. The values for S. pneumoniae R6 are shown in red. The roman numbers specify the S. mitis groups as shown in (B) based on the MLST-derived tree . B. Genetic relationship of S. mitis. Geographic origin of the strains: D, Germany; GB: Great Britain; Hu: Hungary; SA: South Africa; Sp: Spain. C. Gene clusters of S. mitis B6 as detected by genomic hybridization of S. mitis on the B6-specific oligonucleotide microarray. The genes are arranged according to the annotated genome with the replication start on top. Low hybridization signals are indicated by black lines; genes that hybridized with DNA of all strains are not shown. Clusters that are not contained in the B6 strain are marked by boxes on the right, grey boxes indicate phage related gene clusters. * mark the presence of transposases/recombinases. Arrows indicate gene clusters >15 kb.

Figure 6. Genomic comparison of S. mitis and S. pneumoniae.

Genes represented on the oligonucleotide microarray (1760 features) excluding mobile elements and phage related gene clusters were used in this calculation. A. Comparison of 1760 S. mitis gene products with those annotated in six S. pneumoniae genomes. S. pneumoniae genomes: see text for details. Inner circle: deep red, percentage of features hybridizing with all ten S. mitis strains (S. mitis core); light red, with at least one S. mitis (S. mitis accessory genome); white: no hybridization with any S. mitis (B6 specific); outer circle: proteins present in six S. pneumoniae genomes according to in silico analysis of the annotated gene products, using 70% identity as cut off value and a 60% minimum coverage. Dark blue: genes present in all six S. pneumoniae genomes; light blue: genes present in at least one S. pneumoniae; white: absent in S. pneumoniae. Large numbers indicate the percentage of the 1760 genes represented on the microarray; the number of genes is given in small letters below. B. Homologues of the 109 S. mitis B6 genes not present in the six S. pneumoniae genomes listed in (A). Only >80% identity values were used, and only species with the best hit are listed. The number in brackets below the species name indicates genes exclusively found in this species. S. aga.: S. agalactiae; S. e.: S. equi; S. g.: S. gordonii; S. m.: S. mutans; S. p.: S. pneumoniae; S. py.: S. pyogenes; S. san.: S. sanguinis; S. therm: S. thermophilus; S. u.: Streptococcus uberis; C.d.: Clostridium difficile; R. g.: Ruminococcus gnavus; St.a.: Staphylococcus aureus.

Figure 7. Comparison of the ply/lytA island and flanking regions of S. pneumoniae R6 and the dinF region in S. mitis B6.

A: Black: conserved genes in S. mitis B6 and S. pneumoniae; hatched: S. pneumoniae ply and lytA; ovals: BOX elements; small rectangle: RUP elements; RL and RR designate the left and right direct repeats flanking the ply/lytA region (long rectangles). B: Sequences of the left (RL) and right (RR) direct repeat. Arrows above mark the inverted repeat within the direct repeat sequence, and matching nucleotides are underlined; non conserved nucleotides of the S. pneumoniae R6 sequences compared to S. mitis B6 are shown. The DinF gene is indicated below in small letters and as open arrow.