FacZ is a GpsB-interacting protein that prevents aberrant division-site placement in Staphylococcus aureus

Staphylococcus aureus is a Gram-positive pathogen responsible for antibiotic-resistant infections. To identify vulnerabilities in cell envelope biogenesis that may overcome resistance, we enriched for S. aureus transposon mutants with defects in cell surface integrity or cell division by sorting for cells that stain with propidium iodide or have increased light-scattering properties, respectively. Transposon sequencing of the sorted populations identified more than 20 previously uncharacterized factors impacting these processes. Cells inactivated for one of these proteins, factor preventing extra Z-rings (FacZ, SAOUHSC_01855), showed aberrant membrane invaginations and multiple FtsZ cytokinetic rings. These phenotypes were suppressed in mutants lacking the conserved cell-division protein GpsB, which forms an interaction hub bridging envelope biogenesis factors with the cytokinetic ring in S. aureus. FacZ was found to interact directly with GpsB in vitro and in vivo. We therefore propose that FacZ is an envelope biogenesis factor that antagonizes GpsB function to prevent aberrant division events in S. aureus.

Table summarizes the relevant metrics from Tn-Seq analysis of the initial transposon library, END and CSD enrichments, and the ungated control sort.The numbers 2 and 3 refer to the sorting round (e.g., CSD 2 has been subjected to two rounds of sorting increased light scattering).The total number of unique TA sites and the percentage of total TA sites in the genome with transposon insertions are given, as well as median and mean number of insertions per gene in the library following enrichments.The number of genes in each library hit at least once, twice, or five times is shown at right, along with the percent of annotated genes bearing at least that many hits.Also, the number of genes meeting or exceeding these thresholds is compared to the number of mutants in the NTML ordered transposon library 2 , which is commonly used in S. aureus research.

Table S2. Tn-seq data for the END and CSD enrichments.
Tn-seq results for the second and third rounds of sorting of the CSD and END enrichments.
Relative enrichments compare the results of a given round of either CSD or END sorting to the ungated control.P-values are from Mann-Whitney U test.Genes are sorted by SAOUHSC locus number.

Table S3. Relative enrichment of transposon insertions in each gene following END and CSD enrichments versus the ungated control.
Tn-seq data showing the relative enrichments of the END and CSD sorts compared to an ungated control.Genes are sorted by SAOUHSC locus number.Table S4.PCA-adjusted Tn-Seq results.
Following 2D-PCA rotation of the CSD and END sorted data, each gene's enrichment was assigned a new (X,Y) location, with X representing a genes relative enrichment along PC2, and Y representing the relative enrichment along PC1.These data were used to plot the PCA panel in Fig. S1.Genes are sorted by SAOUHSC locus number.

Table S5. PCA-adjusted hit list.
The top 50 hits from the PCA analysis based on maximum relative enrichment along PC1."Δ" indicates the change in PC1 or PC2 relative enrichment between rounds of sorting."Mean fold increase" and "% increasing in enrichment" are metrics to assess whether hits identified by PCA in sort 2 increase in PC1 enrichment from additional rounds of CSD and END sorting.In general, hits identified by PC1 enrichment enrich further from additional sorts, where those from PC2 do not, consistent with PC1-enrichment serving as a proxy for mutual CSD and END enrichment.
Genes are displayed by decreasing relative enrichment along PC1.

Table S6. ΔfacZ synthetic lethal Tn-seq hits
Tn-seq analysis of transposon libraries constructed in WT S. aureus [aTB015] versus one constructed in a ΔfacZ [aTB259] strain.P-values are from Mann-Whitney U test.Genes are sorted by increasing ∆facZ:WT relative enrichment ratio.Loci were identified with gene names and brief descriptions from AureoWiki.Genes previously described as having a role in cell wall biosynthesis, morphogenesis, cell division, and cell separation are highlighted in yellow.

Table S8 : S. aureus strains used in this study 84 S. aureus strain Background Relevant genotype Source Construction notes
All S. aureus strains used in the course of this study are noted in this table.See Methods section for a description of how strains ∆facZ::specR ∆attB(f11)::Orf5, pTM381 This study ⏀85 lysate of aTB243 was used to transduce ∆facZ::specR into aTB016 85 were made.

Table S9 : B. subtilis strains used in this study
All plasmids produced in the course of this study were made by isothermal assembly (ITA) or restriction digest and ligation using pairs of restriction enzymes, as noted in this table.Plasmids were isolated from and are stored in either DH5α or BL21(DE3) E. coli strains, and are available upon request.
All B. subtilis strains used in the course of this study are noted in this table.See Methods section for a description of how strains were made.
All oligos used in the course of this study are noted in this table.