Article Figures & Data
Figures
- Fig 1
Level of Ag43 is reduced in an L22(Δ82-84) mutant strain. (A) Illustration of Ag43 showing the signal peptide (SP; residues 1 to 52), the passenger domain (residues 53 to 552), and the β domain (residues 553 to 1039). The pro form of the protein contains covalently linked passenger and β domains and is observed prior to the secretion and proteolytic processing of the passenger domain. (B) Soluble proteins from MG1655 and MNY15 [MG1655 rplV(Δ82-84)] were labeled with the fluorescent dyes Cy3 and Cy5, respectively, and analyzed by 2D-DIGE. Images of a section of each gel run in a representative experiment and an overlay image are shown. The spot that corresponds to Ag43 is denoted by an arrow. (C) Quantitative DeCyder analysis of the spot indicated in panel B reveals a 2.7-fold reduction of Ag43.
- Fig 2
Reduced synthesis of SecA in L22 mutant strains leads to a dramatic loss of Ag43 transcripts. (A) MG1655 (WT) and MG1655 derivatives containing the indicated mutation were grown at 37°C, and the steady-state level of Ag43 was measured by Western blotting using an antiserum raised against the Ag43 β domain. The cells shown in lane 6 were transformed with plasmid pMF8 (encoding secM-secA). pro, proAg43; β, Ag43 β domain. (B) Autoaggregation of the strains shown in panel A in static cultures. (C) Quantitative RT-PCR analysis of the level of the Ag43 gene, secA, and tnaA transcripts in MNY15 relative to MG1655. (D) The steady-state level of SecA in MG1655 (WT) and MG1655 derivatives containing the indicated mutation was measured by Western blotting. The cells shown in the last lane were transformed with plasmid pMF8.
- Fig 3
L22(Δ82-84) mutation does not affect the steady-state level of model envelope proteins. (A) MG1655 (WT) and MG1655 derivatives containing the indicated mutation were grown at 37°C, and the steady-state levels of various proteins were measured by Western blotting. The cells shown in the last lane were transformed with plasmid pMF8 (encoding secM-secA). (B) The growth of MG1655 (WT) and MG1655 derivatives containing the indicated mutation is shown.
- Fig 4
secY mutation dramatically reduces the steady-state level of Ag43. MG1655 (WT) and MNY37 (MG1655 secY39cs) were grown at 41°C and diluted into fresh medium. At an OD600 of 0.2, cultures were divided in half, and one half was shifted to 22°C. Cells were harvested 2 h later. The steady-state levels of Ag43, OmpA, and RpoD were determined by Western blotting. pro, proAg43; β, Ag43 β domain.
- Fig 5
Second-site mutations that disrupt genes encoding envelope proteins suppress the effect of L22 mutation on Ag43 production. The steady-state levels of Ag43 and RpoD were determined by Western blotting in MG1655 (WT) and MG1655 containing the indicated mutation(s). pro, proAg43; β, Ag43 β domain.
- Fig 6
Secretion stress reduces the stability of Ag43 mRNA. (A) MNY40 (MG1655 Ag43::His6) transformed with plasmid pMY28 (encoding Ag43PD-HA) or pMY29 (encoding ΔSP-Ag43PD-HA) or the cloning vector (pTrc99a) were grown to an OD600 of 0.4. Cultures were divided in half, and IPTG was added to one half to induce the expression of the plasmid-borne gene. After 30 min, steady-state levels of HA- and His-tagged proteins and SecA were determined by Western blotting. Analysis of one-tenth as much protein on a separate Western blot revealed the presence of precursor and mature forms of Ag43PD-HA. (B) Rifampin was added to cultures following the 30-min IPTG induction, and quantitative RT-PCR was performed to determine the percentage of the Ag43 gene transcripts present at t0 that remained at the indicated time points. gapA mRNA served as an internal control. The averages from two independent experiments are shown.
Additional Files
Files in this Data Supplement:
- Supplemental file 1 -
Supplemental methods
Table S1, oligonucleotides
Table S2, proteins that are differentially expressed in an L22(Δ82-84) mutant strain
Fig. S1, comparative 2D-DIGE proteomic profiling of wild-type and L22 mutant strains
Fig. S2, overproduction of an inactive form of SecA does not restore Ag43 synthesis in an L22 mutant strain
Fig. S3, deletion of oxyR partially suppresses the effect of an L22 mutation on Ag43 synthesis
Fig. S4, level of a His-tagged version of Ag43 is reduced in an L22 mutant strain
PDF, 4.0M
- Supplemental file 1 -
