Genetic variation in the Staphylococcus aureus 8325 strain lineage revealed by whole-genome sequencing

Abstract

Staphylococcus aureus strains of the 8325 lineage, especially 8325-4 and derivatives lacking prophage, have been used extensively for decades of research. We report herein the results of our deep sequence analysis of strain 8325-4. Assignment of sequence variants compared with the reference strain 8325 (NRS77/PS47) required correction of errors in the 8325 reference genome, and reassessment of variation previously attributed to chemical mutagenesis of the restriction-defective RN4220. Using an extensive strain pedigree analysis, we discovered that 8325-4 contains 16 single nucleotide polymorphisms (SNP) arising prior to the construction of RN4220. We identified 5 indels in 8325-4 compared with 8325. Three indels correspond to expected Φ11, 12, 13 excisions, one indel is explained by a sequence assembly artifact, and the final indel (Δ63bp) in the spa-sarS intergenic region is common to only a sub-lineage of 8325-4 strains including SH1000. This deletion was found to significantly decrease (75%) steady state sarS but not spa transcript levels in post-exponential phase. The sub-lineage 8325-4 was also found to harbor 4 additional SNPs. We also found large sequence variation between 8325, 8325-4 and RN4220 in a cluster of repetitive hypothetical proteins (SA0282 homologs) near the Ess secretion cluster. The overall 8325-4 SNP set results in 17 alterations within coding sequences. Remarkably, we discovered that all tested strains of the 8325-4 lineage lack phenol soluble modulin α3 (PSMα3), a virulence determinant implicated in neutrophil chemotaxis, biofilm architecture and surface spreading. Collectively, our results clarify and define the 8325-4 pedigree and reveal clear evidence that mutations existing throughout all branches of this lineage, including the widely used RN6390 and SH1000 strains, could conceivably impact virulence regulation.

Figure 1. Alignment of the twelve hypothetical proteins encoded in the 8325-4 genome with homology to SA0282.

The sequences were aligned using ClustalX and the alignment was visualized using the TEXshade package [51]. The top panels shows identical (blue), similar (pink) or non-conserved residues (white). The bottom panel shows each sequence on a single line with vertical gray scale lines indicating identical (black), similar (gray), or non-conserved residues (white), and with an indication of a variable region with little sequence identity.

Figure 2. Distribution of SNPs in the S. aureus 8325 and 8325-4 strain lineages.

Thirteen of the SNPs including the 63 bp deletion detected in 8325-4seq and described in Table 2 were analyzed by PCR from gDNA and direct sequencing for their distribution using strains collected from various laboratories worldwide. Shown in the matrix are 6 strains of 8325 pedigree group prior to prophage removal (white box), 10 strains from the 8325-4 pedigree after prophage Φ11, 12, and 13 removal (dark gray box), and one intermediate strain, RN25/NRS133, which retains prophage Φ13 (light gray box). Each SNP is indicated (top) together with coordinates from the 8325 reference genome (bottom) deposited as Genbank NC_007795.1. Strain abbreviations used are as described in Table 3. WT: wild type, +T: insertion of T, ‘ -‘ denotes SNP not tested.

Figure 3. High resolution 8325 pedigree based on the SNP analysis depicted in Figure 2.

Strains are shown in white boxes, mutagenesis treatments are shown in red boxes, and SNPs are shown in colored boxes, color coded as in Figure 2, on the line indicating strain evolution from 8325 to 8325-4seq. The placement of the SNPs along the line indicates their entry point into the pedigree. Five digit numbers indicate SAOUHSC locus tags. IG: intergenic.

Figure 4. Organization of the phenol soluble modulin αoperon showing the location of mutations (A5P, and L7 frameshift) in PSMα3 detected in the 8325-4 lineage.

(A) Shown is the non-annotated intergenic genomic region of S. aureus NCTC8325 sequence between sequence tags SAOUHSC_00411 and SAOUHSC_00412 encoding the PSMα operon. The position of the two detected mutations are shown in red on the antisense strand starting with NCTC8325 coordinate 412741, and the start codon is boxed in blue. (B) wild type and mutated PSMα3 translation products.

Figure 5. Diagram of the spa-sarS region showing the location of the 63 bp deletion (top panel) detected in a sub-lineage of 8325-4 strains.

The sarS transcriptional terminator as described in [28] is shown as a filled circle. Sequence analysis shows that the deletion arose by precise excision of sequence flanked by 14 bp direct repeats (bottom panel).

Figure 6. Assessment of 63 bp deletion in the spa-sarS intergenic region on the flanking genes by qRT-PCR analysis.

RNA extracts were prepared following entry to stationary phase (Materials and Methods). RNA was obtained from the various strains indicated and experiments normalized to 16S RNA followed by comparison to NRS77 (8325) fixed as reference control. * Indicates significant difference determined by Student’s two tail t test using three independent sample determinations. Pairwise comparisons revealed a pronounced reduction (75%) in sarS mRNA in strains 8325-4 (sequenced in this study) and SH1000, both harboring the deletion, compared with NRS77 with wild type intergenic sequence (P< 0.0001, respectively). No significant changes in spa mRNA were noted for 8325-4 or SH1000. No significant changes were observed with RNA obtained during exponential phase growth of the same cultures. All strains used for analysis gave clear zones of hemolysis on sheep blood agar and were agrA sequence 7A [16].

Figure 7. Phenotypic characteristics of strains used in this study.

Panel A: hemolysis on sheep blood agar; panel B: secreted proteolytic activity on casein agar; panel C: pigmentation. The bottom panel indicates the strain (Table 3) together with the AgrA sequence type [16] where noted.