Fig 1.
Pseudogenes, insertion sequences and mobile genetic elements in the Staphylococcus aureus subsp. anaerobius isolate MVF7.
(A) Circular map of the chromosome. Rings show, from outside to inside: annotated genes in the positive strand, those in the negative strand (with the 205 pseudogenes and 87 insertion sequences shown in red and blue, respectively), and the mobile genetic elements found across all isolates (gold: prophage ΦSaa1; cyan: pathogenicity island SaaPIMVF7). (B) Gene map of SaaPIMVF7. Genes in grey are pseudogenes and genes in orange are intact by comparison other SaPI relatives. int: integrase; pri: primase; scn: Staphylococcal complement inhibitor (SCIN); vwb: von Willebrand factor-binding protein (vWBP). The box shows the expression of the SaaPIMVF7-encoded vwb. The SaaPIMVF7 vwb gene was cloned into the expression vector pCN51 under the control of a cadmium-inducible promoter, transformed into a coagulase and vWbp-deficient derivative of strain RN4220 (RN4220 coa::tetM Δvwb) and the ability of SaaPIMVF7 vWbp to coagulate ruminant plasma was assessed. (C) Graphical summary of the main biological functions potentially disrupted by the presence of pseudogenes.
Fig 2.
Staphylococcus aureus subsp. anaerobius represents a single clade within the S. aureus phylogenetic tree.
Maximum Likelihood tree constructed from a SNP alignment of the studied S. aureus subsp. anaerobius sequences (clades in blue) and 787 sequences of different S. aureus subsp. aureus (in black). (A) Unrooted tree showing the divergence of 17 sequences of other Staphylococcus (S. schweitzeri and S. argenteus) whereas S. aureus subsp. aureus is embedded in the S. aureus diversity. (B) Subtree showing the position of the S. aureus subsp. anaerobius clade (collapsed in blue) with respect to the other S. aureus subsp. aureus clonal complexes (CC).
Fig 3.
S. aureus subsp. anaerobius evolved over the last 1000 years with a stable population size.
Bayesian maximum clade credibility tree of the Staphylococcus aureus subsp. anaerobius isolates. The x-axis is expressed in calendar years, branch colours denote sampling country. Purple bars at each node show its most recent common ancestor (MRCA) confidence interval. Numbers in nodes indicate the posterior probability. The estimated dates of the MRCAs of the main lineages are indicated. The isolate that was whole genome sequenced using PacBio technology (MVF7) is highlighted in orange. The bottom plot represents the changes in the effective population size over time, with the shadowed area representing the 95% credible interval, following the same timescale as the tree.
Fig 4.
S. aureus subsp. anaerobius has undergone 6 large intra-chromosomal rearrangements.
Map of the genomic translocations across the Staphylococcus aureus subsp. anaerobius isolate MVF7 chromosome. The green blocks represent the 6 large translocations detected in MVF7 when compared to the Staphylococcus aureus subsp. aureus RF122 strain (see also S3 Fig). The shaded areas indicate the original location of the translocated portions in the putative ancestral genome. The inner ring shows the GC skew (green for positive skew, purple for negative), which is unaffected by the translocations.
Fig 5.
Characterisation of the transcriptional terminators (TTs) present in the ISs.
(A) Schematic representation of IS-associated TTs. (B) Schematic representation of and functional assessment of the bi-directional TT1 localised in the 5’ region of the IS. The bi-directional TT1 is composed of two sub-TTs (represented by the blue and yellow triangles, respectively) that share the same sequence but are inverted repeats, thus generating a master bi-directional TT1. The unidirectional TT2 is represented by a pink triangle. The expression of the blaZ reporter in plasmid pCN42 is controlled by a cadmium-inducible promoter. The identified transcriptional terminators were cloned between the promoter and the blaZ reporter gene. β-lactamase expression was monitored either in uninduced (black bars) or Cd-induced (open bars) cultures 180 min after induction. Data are show the means of three independent biological replicates and error bars show the standard deviation from the mean. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s post-test. Multiplicity adjusted p-values are shown for uninduced to induced comparison and all terminator containing reporters showed significant differences when compared to their respective empty plasmid control. **** p<0.0001, ns not significant.
Fig 6.
Presence of IS influences the expression of downstream genes and genes product through multiple mechanisms.
(A-D) Reporter constructs for presence of the IS upstream and in sense orientation of the target gene were designed in plasmid pCN41 containing a β-lactamase reporter gene. The green triangle indicates the IS position relative to the target gene’s start. All reporters were constructed either by removing the IS gene from S. aureus subsp. anaerobius strain MVF84 or by introducing the IS into S. aureus subsp. aureus strain RF122 to confirm the IS role on gene expression variation. In (A) the IS removal results in a 207bp deletion to the promoter region not present in RF122. Reporter plasmids were introduced into RN4220 as described in Methods. Data show the mean of three biological replicates, error bars represent the mean’s standard deviation. One-Way ANOVA was performed followed by Tukey’s multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns not significant. (E&F) Western blot analysis of (E) mgrA and (F) sle1 expression constructs to address whether the IS impacts on their expression levels by interrupting expression of an antisense transcript. (E) mgrA encoding a 3xFLAG tag was cloned in antisense to a cadmium-inducible promoter into plasmid pCN51 to mimic the expression of an antisense transcript to mgrA. Constructs presented different combinations of presence/absence of IS and/or transcriptional terminator the IS transcript or expression from the plasmid-encoded cadmium-inducible promoter from the mgrA transcript. Expression of the cadmium-inducible promoter was induced with 5 μM CdCl2. (F) To assess the IS impact in the context of a natural antisense transcript, 3xFLAG-encoding expression constructs of sle1 including the gene downstream of sle1 in the ancestral genome were cloned into the plasmid pCN47. Samples were taken during different growth phases. Plasmids were introduced into RN4220 Δspa as described in Methods. Western blots shown are representative of at least two independent biological replicates. TT, transcriptional terminator.