Mycobacterium tuberculosis SatS is a chaperone for the SecA2 protein export pathway
- University of North Carolina at Chapel Hill, United States
- Texas A&M University, United States
Figures
Figure 1 with 1 supplement
∆satS mutant suppresses SecA2 K129R phenotypes.
(A) Mueller-Hinton growth phenotypes and azide sensitivity of M. smegmatis ∆secA2 mutant expressing wild-type secA2 (∆secA2 +psecA2), an empty vector, or secA2 K129R (secA2 K129R), and two …
Figure 1—figure supplement 1
Characterization of the M. smegmatis ∆satS mutant.
(A) Liquid cultures of M. smegmatis mc2155 and ΔsatS were grown in 96-well plates with a starting cell count of 1 × 104 cells/well. After 24 hr, resazurin was added and relative fluorescence units …
Figure 2 with 4 supplements
SatS is required for the export of SapM.
(A) Equal protein from culture supernatants from M. tuberculosis H37Rv, ∆secA2, ∆satS and the complemented strain (∆satS +psatSMtb) were immunoblotted for SapM and Mpt32. (B) Phosphatase activity in …
Figure 2—figure supplement 1
satS and sapM are co-transcribed.
Visualization of the operon structure of sapM and satS and the location of the primers used. Primer set FR flanking the intergenic region between sapM and satS and a control primer set in the …
Figure 2—figure supplement 2
Characterization of the M.tuberculosis ∆satS mutant.
(A) The ΔsatS mutant of M. tuberculosis and the parental H37Rv strain were grown in liquid 7H9 medium with ADS supplementation. Growth was monitored by measuring optical density (OD600) over time. …
Figure 2—figure supplement 3
SatSMtb complementation and lysis controls.
(A) Equal protein from whole cell lysates of M. tuberculosis H37Rv, ∆secA2, ∆satS and the complemented strain (∆satS +psatS) were immunoblotted for SatS or SigA. (B) Equal protein from culture …
Figure 2—figure supplement 4
SatS does not affect sapM transcription or translation.
(A) RNA was isolated from M. tuberculosis H37Rv and ∆satS. sapM transcript levels were measured by quantitative RT-PCR and normalized to the level of sigA transcript. Data shown are for the mean of …
Figure 3
Mce proteins require SatS.
(A) Equalized cell wall fractions of M. tuberculosis H37Rv, ΔsecA2, ΔsatS and complemented (ΔsatS +psatSMtb) strains were analyzed by immunoblot using Mce1A, Mce1E, and 19 kDa antibodies to monitor …
Figure 4
SatS contributes to M. tuberculosis growth in macrophages.
Nonactivated BMDM were infected at an MOI of one with M. tuberculosis H37Rv + EV, ΔsecA2 + EV, ΔsatS + EV, or ΔsatS + psatS, and CFU burden was monitored over the course of a 4 day infection. The …
Figure 5 with 1 supplement
SatS and SapM interact.
(A) Lysate from M. smegmatis ∆secA2/∆satS expressing SapM-FLAG and either SatSMtb-HA or SatSMtb without a tag, ∆satS expressing ∆ss-SapM-FLAG and SatSMtb-HA, or mc2155 with two empty vectors (as …
Figure 5—figure supplement 1
Epitope tags do not disrupt SapM or SatS functions.
(A) Whole cell phosphatase activity assay of M. smegmatis strains expressing SapM-FLAG or an empty vector. The error bars represent standard deviation from the mean of three independent replicates. …
Figure 6
SatS functions prior to SecA2.
(A) Equal protein from culture supernatants (CFP) from M. smegmatis mc2155, ∆secA2, ∆satS, the ∆secA2/∆satS double mutant, and ∆secA2/∆satS expressing wild-type SatS (∆secA2/∆satS +psatSMsm) were …
Figure 7
SatS and SatSC prevent aggregation of SapM in vitro.
Denatured SapM-His was diluted 150 fold to a final concentration of 1 µM in 40 mM HEPES, 100 mM NaCl, pH 7.4. SapM-His aggregation was monitored by light scattered (350 nm) at 25°C in the presence …
Figure 8 with 1 supplement
SatS has a new fold and hydrophobic grooves that share similarity with the preprotein binding sites of the SecB chaperone.
(A) The overall secondary structure of SatSC. The hydrophilicity of SatSC is a colored gradient from cyan (hydrophilic) to maroon (hydrophobic). SatSC exposes ~2,900 Å2 of hydrophobic surface. The …
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Figure 8—source data 1
SatSC X-ray Structure Validation Details.
- https://doi.org/10.7554/eLife.40063.017
Figure 8—figure supplement 1
SatSC secondary structure and the predicted secondary structure of SatSN.
(A) SatS is composed of two domains of similar secondary structure joined by a potential linker of 60 amino acids with little predicted secondary structure. The disordered linker region was …
Figure 9
SatS has at least two separable roles in protein export.
(A) Equal protein from culture supernatants (CFP) from M. smegmatis mc2155, ∆secA2, ∆satS and the ∆satS mutant expressing either wild-type SatS (∆satS +psatSMsm) or SatS G134D (∆satS +psatSMsmG134D) …
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.40063.020
