Research Article | Molecular Biology and Physiology

Reading between the Lines: Utilizing RNA-Seq Data for Global Analysis of sRNAs in Staphylococcus aureus

Hailee M. Sorensen, Rebecca A. Keogh, Marcus A. Wittekind, Andrew R. Caillet, Richard E. Wiemels, Elizabeth A. Laner, Ronan K. Carroll
Paul D. Fey, Editor
DOI: 10.1128/mSphere.00439-20

Article Figures & Data

Figures

  • FIG 1
    FIG 1

    Overall design of global sRNA expression study. Numbers in parentheses represent numbers of studies, data sets, or analyses as indicated.

  • FIG 2
    FIG 2

    Analysis of sRNA stability. All sRNA expression values were normalized using the expression values for hup, which is included as a comparison (blue line). (A) Highly stable sRNAs. Thirty four sRNAs were deemed highly stable based on an overall stability profile similar to that of hup. The highly stable sRNAs were either more stable than hup or their stability was 2-fold less than that of hup. FMN, flavin mononucleotide; SAM, S-adenosylmethionine. (B) Highly unstable sRNAs. A total of 23 sRNAs were deemed highly unstable, with stability 100-fold less than that of hup.

  • FIG 3
    FIG 3

    Northern blot analysis of selected stable sRNAs. (A) RNAIII. (B) Teg41. Order of lanes: L (RNA ladder), 0 (0 min sample—prior to rifampin addition), 2.5 (2.5 min after rifampin addition), 5 (5 min after rifampin addition), 10 (10 min after rifampin addition). The T10/T0 values for RNAIII and Teg41 were 1.39 and 0.89, respectively.

  • FIG 4
    FIG 4

    Analysis of sRNA ribosome protected fragments. (A) RPF/expression ratios of 303 sRNAs from S. aureus. A total of 65.68% of sRNAs had an RPF/expression ratio of >3. (B) RPF/expression ratios of 2,649 CDS genes from S. aureus.

  • FIG 5
    FIG 5

    Ribosome profiling data for the RNAIII locus. Visualization of reads mapped to the RNAIII locus is provided. RPF alignment, read alignment of duplicate ribosome protected fragment data sets; RNAseq alignment, read alignment of duplicate RNA-Seq transcriptomic data sets. Mapping was generated using the CLC Genomics Workbench software package.

  • FIG 6
    FIG 6

    Western immunoblot detection of tsr-encoded potential small peptides. (A) Western blot analysis, performed using an anti-his antibody, to detect His-tagged, tsr-encoded peptides/proteins. Protein samples from the cell envelope, cytoplasm, and secreted fraction were analyzed. (B) Table of predicted sizes of potential tsr-encoded small peptides. (C) Predicted membrane topology of peptides encoded by tsr21C, tsr22, and trs37. All three peptides are predicted to contain transmembrane regions. Membrane topology was predicted using SACS MEMSAT2 Transmembrane Prediction (18).

  • FIG 7
    FIG 7

    Quantitative analysis of clumping phenotype observed in tsr37 overexpressor strain. A clumping assay was performed using S. aureus strain AH1263 overexpressing tsr37 (pMK4 tsr37-his), and results were compared to those seen with an empty vector control (pMK4 empty vector). The proportion of clumping of the empty vector control after 2 h was 7.9% compared to 79.2% for the tsr37-overexpressing strain.

  • FIG 8
    FIG 8

    Analysis of sRNA expression in vivo. (A) A total of 93 sRNAs were upregulated >2-fold in the CF lung, whereas 60 sRNAs were upregulated >2-fold in the mouse vagina. A total of 29 sRNAs were upregulated in both data sets. (B) Table of 29 sRNAs upregulated in both in vivo data sets. Fold increase in expression in the vagina is indicated for each time point and compared to CDM/SCFM data. Also included are stability values (taken from Data Set S3), RPF/expression ratios (taken from Data Set S4), and the number of pairwise analyses where a >2-fold change in expression was observed (taken from Data Set S2).

Tables

  • TABLE 1

    22 studies used for global sRNA expression analysisa

    TABLE 1

    • ↵a DHBP, 2,4-dihydroxybenzophenone; ID, identifier; NA, not applicable; MRSA, methicillin-resistant Staphylococcus aureus; nor, nitric oxide reductase gene; nos, nitric oxide synthase gene; ssRNA, single-stranded RNA; WT, wild type.

  • TABLE 2

    64 pairwise analysesa

    TABLE 2

    • ↵a KO, knockout; nor, nitric oxide reductase gene; nos, nitric oxide synthase gene; DEA NONOate, nitric oxide donor diethylamine NONOate; ES, early stationary; LE, late exponential; EE, early exponential; fad, fatty acid desaturase gene; mocR, mocR regulator gene; cstR, CsoR-like sulfurtransferase repressor gene; PBT2, zinc ionophore; walK, sensor kinase of WalKR two-component system gene; DHBP, 2,4-dihydroxybenzophenone; aTc, anhydrotetracycline; floxuridine, chemotherapy drug (liver cancer); streptozotocin, chemotherapy drug (pancreatic cancer); fosfomycin, cell wall synthesis inhibiting antibiotic; linoleic acid, polyunsaturated omega-6 fatty acid; MT02, DNA replication inhibiting antibiotic; spermine NONOate, nitric oxide donor; TTO, tea tree oil; MRSA16791, methicillin-resistant chicken isolate; ATCC 29213, methicillin-sensitive strain; rsp, transcriptional regulator gene; BUSA2288, human nasal isolate; saeS, histidine kinase sensor of SaePQRS system gene.

    • ↵b Values represent numbers of sRNAs with expression increased or decreased >3-fold (total = 303).

  • TABLE 3

    sRNAs displaying altered expression in >20 of the 64 pairwise analyses

    TABLE 3
  • TABLE 4

    Conditions under which Teg41 expression is altered

    TABLE 4

    • ↵a Expression values are expressed in RPKM. Expression value 1 and expression value 2 represent the expression values of Teg41 under the two conditions tested. For details of each specific condition, see Supplemental Data Set S2.

  • TABLE 5

    Strains and plasmids

    TABLE 5

    • ↵a CMr, chloramphenicol resistance.

Supplemental Material

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KEYWORDS

RNA stability
RNA-seq
Staphylococcus aureus
genome annotation
regulatory RNA
sRNA
small peptides

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