Article Figures & Data
- FIG 1
Characterization of the S. aureus transposon mutant library. (A) Tn-seq mapping of the 71,700 transposon insertions (blue track; because of density, individual hash marks appear nearly solid). Analysis of initial mutant population recovered on BHI agar. (B) Cumulative number of unique transposon (Tn) insertions over the length of the 2.8-Mb NCTC8325 genome, illustrating absence of hot spots/large gaps in transposon insertion. (C) The number of unique transposon insertions per ORF (left), the transposon density of each ORF (middle), and the ORF dval (right) are shown for nonessential (red) and essential (blue) genes.
- FIG 2
Gene essentiality under laboratory conditions. Agreement between Tn-seq results for 24-h growth in BHI and previous identification of essential genes tested under similar conditions (11).
- FIG 3
Antisense RNA validation of contribution to fitness. Exponential cultures carrying xylose-inducible antisense constructs were diluted into Luria Broth supplemented with 0.2% glucose containing 4% xylose, and optical density at 600 nm (OD600) was monitored. (A to I) Induced control strain with the pEPSA5 vector lacking insert (red squares) compared to induced strain carrying antisense construct (purple X’s). Antisense clones of fragments from genes fabI (A), SAOUHSC_00486 (B), SAOUHSC_00889 (C), SAOUHSC_01263 (D), SAOUHSC_01203 (E), SAOUHSC_01857 (F), SAOUHSC_02383 (G), SAOUHSC_00022 (H), and SAOUHSC_01239 (I). Error bars show the deviations from the means for at least 9 replicates in 3 separate experiments.
- FIG 4
Metabolic genes contributing to S. aureus survival and proliferation in an abscess model.
- FIG 5
Nucleotide biosynthesis pathways of importance in abscess at 48 h. Pyrimidine (A) and purine (B) biosynthesis pathways. Genes that are critical for survival in abscess at 48 h but not in rich medium outgrowth control are highlighted in yellow.
Supplemental Material
Text S1
Supplemental materials and methods. Download Text S1, PDF file, 0.1 MB.
Copyright © 2014 Valentino et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S1
Genes contributing to S. aureus fitness across various environments. Table S1, PDF file, 0.1 MB.
Copyright © 2014 Valentino et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S1
Experimental strategy to determine genes essential for fitness in competitive, infection-relevant contexts. The transposon mutant pool (a) was first cultured in nutrient broth (b) to generate a reproducible inoculum for subsequent experiments. To identify genes contributing to fitness in infection, bacteria were inoculated in duplicate ex vivo into aqueous fluid, vitreous fluid, and whole blood (d). Additionally, genes contributing to persistence and proliferation in an abscess in vivo (e) were identified. Each output was sequenced and compared to nutrient broth outgrowth control (c). Download Figure S1, PDF file, 0.1 MB.
Copyright © 2014 Valentino et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S2
Comparison of genes curated as essential after growth in BHI in this study compared to previous study of essentiality in S. aureus. Table S2, PDF file, 0.1 MB.
Copyright © 2014 Valentino et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S2
Reproducibility of biological replicates. Regression statistics for independent biological replicates. Transposon density is the number of transposon insertions per kilobase, calculated independently for each ORF. dval is the number of transposon insertions found in a given ORF versus the number expected based on the size of the ORF. Download Figure S2, PDF file, 0.1 MB.
Copyright © 2014 Valentino et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S3
Genes which are either 4-fold underrepresented or 4-fold overrepresented following growth in infection compared to BHI outgrowth control. Table S3, PDF file, 0.1 MB.
Copyright © 2014 Valentino et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S3
Analysis of mariner mutant population changes in murine abscess by Tn-seq. (A) Flow chart of experimental strategy. (B) Analyses performed (abscesses generated on hind flank illustrated in yellow). (C) Representative abscesses at 24 h (a) and 48 h (b) prior to excision. Download Figure S3, PDF file, 0.1 MB.
Copyright © 2014 Valentino et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S4
Primers used in this study. Table S4, PDF file, 0.1 MB.
Copyright © 2014 Valentino et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Additional Files
Supplementary Data
Supplementary Data
Files in this Data Supplement:
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- Figure sf2, PDF - Figure sf2, PDF
- Figure sf3, PDF - Figure sf3, PDF
- Table st1, PDF - Table st1, PDF
- Table st2, PDF - Table st2, PDF
- Table st3, PDF - Table st3, PDF
- Table st4, PDF - Table st4, PDF
- Text s1, PDF - Text s1, PDF
