Diversity of CRISPR-Cas type II-A systems in Streptococcus anginosus

Abstract

Streptococcus anginosus is a commensal Streptococcal species that is often associated with invasive bacterial infections. However, little is known about its molecular genetic background. Many Streptococcal species, including S. anginosus, harbor clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems. A CRISPR-Cas type II-A system as well as a type II-C system have been reported for this species. To characterize the CRISPR-Cas type II systems of S. anginosus in more detail, we conducted a phylogenetic analysis of Cas9 sequences from CRISPR-Cas type II systems with a special focus on streptococci and S. anginosus. In addition, a phylogenetic analysis of S. anginosus strains based on housekeeping genes included in MLST analysis, was performed. All analyzed Cas9 sequences of S. anginosus clustered with the Cas9 sequences of CRISPR type II-A systems, including the Cas9 sequences of S. anginosus strains reported to harbor a type II-C system. The Cas9 genes of the CRISPR-Cas type II-C systems of other bacterial species separated into a different cluster. Moreover, analyzing the CRISPR loci found in S. anginosus, two distinct csn2 genes could be detected, a short form showing high similarity to the canonical form of the csn2 gene present in S. pyogenes. The second CRISPR type II locus of S. anginosus contained a longer variant of csn2 with close similarities to a csn2 gene that has previously been described in Streptococcus thermophilus. Since CRISPR-Cas type II-C systems do not contain a csn2 gene, the S. anginosus strains reported to have a CRISPR-Cas type II-C system appear to carry a variation of CRISPR-Cas type II-A harboring a long variant of csn2.

Keywords: CRISPR-Cas; CSN2 gene; Streptococcus anginosus; classification; phylogenetic analysis.

Figure 1

Genetic organization of CRISPR-Cas type II systems. (A) Conventional composition of CRISPR-Cas type II-A and II-C systems. (B) Genetic locus of CRISPR-Cas type II systems in Streptococcus anginosus. Green and yellow symbols represent repeat and spacer sequences of the CRISPR-Cas array. Genes present in all type II CRISPR-Cas systems are depicted in shades of blue, Csn2 genes are shown in yellow for the canonical form and red for the long version. Lilac depicts genes, which are not part of the CRISPR-Cas locus. Arrows illustrate the location of primers used to screen for the presence and absence of CRISPR-Cas type II systems in S. anginosus isolates.

Figure 2

Circular minimal evolution tree constructed on the sequence of seven housekeeping genes illustrating the distribution of CRISPR-Cas type II systems in S. anginosus isolates. The units of the number of base substitutions per site are depicted. Subspecies [S. anginosus subsp. whileyi (A), S. anginosus subsp. anginosus (B)] and genomosubspecies [S. anginosus genomosubsp. AJ1 (C) S. anginosus genomosubsp. Vellorensis (D)] are highlighted. Circles represent presence and absence of CRISPR-Cas type II systems. Yellow: type II-A. Red: type II-C. Black: no type II system. Green rectangles highlight sequenced strains derived from GenBank.

Figure 3

Cas9 phylogeny. Minimal Evolution tree constructed on the sequences of selected type IIA and IIC cas9 nucleases published by Chylinski et al. (2014); Fonfara et al. (2014) by bootstrap analysis using MEGA X. Multiple alignment of Cas9 amino acid sequences was constructed using the MUSCLE algorithm with default settings. The units of the number of amino acid substitutions per site are depicted. Type II-A systems are represented by circles, whereas type II-C systems are indicated by triangles. Corresponding csn2 types stated by Chylinski et al. (2014) are highlighted. Orange: csn2 type I, green: csn2 type II, black: csn2 type III and red: csn2 type V.

Figure 4

Multiple sequence alignment of Csn2 subfamilies found in S. anginosus. (A) Canonical 220 aa variant (Csn2 type I). (B) 350 aa long Csn2 variant (Csn2 type II). For each Csn2 subfamily the multiple sequence alignment of selected protein sequences was constructed separately using the MUSCLE algorithm with default settings. Homologs of each Csn2 subfamily were identified using HHPRED. Retrieved Csn2 sequences of S. thermophilus LMG 18311 (PDB: 3ZTH) and S. agalactiae ATCC 13813 (PDB: 3QHQ) were included. The positions strongly conserved (> 95% consensus) are shown by reverse shading, whereas above 70% consensus is highlighted in grey.