Table 1.
Oligonucleotides used in this study.
Fig 1.
Bioorthogonal labeling of mycoloylated proteins: Principle.
When added to the growth medium, a fatty-acid alkyne probe (17-ODYA), can be used by the cell to synthesize alkyne-modified mycolic acids. Specifically, mycolic acids are generated in the cytoplasm, by the condensation of two activated fatty acids, a reaction mediated by the polyketide synthase Pks13 [43]. Mycolic acids are then converted to trehalose monomycolate and transported to the envelope, where they are probably used as mycolate donor by MytC to modify a specific subset of proteins. Proteins that have incorporated an alkyne group can then be identified by Cu(I) catalyzed Azide-alkyne cycloaddition (CuAAC). In the presence of Cu(I), the azido group of azido-biotin reacts with the alkyne group, forming a triazole ring and resulting in the covalent attachment of biotin to the protein, which allows further detection of the labeled protein by streptavidin-HRP. CM: cytoplasmic membrane; MM: mycomembrane, CuAAC; Cu(I)-catalyzed azide-alkyne cycloaddition.
Fig 2.
C. glutamicum strains 13032, ΔmytC::Km, Δpks13::Km or CgΔporHporA over-expressing PorAhis were grown in the absence (lanes 1–2, 5–6) or in the presence (lanes 3–4, 7–8) of 17-ODYA. After purification, PorAhis was subjected to CuAAC in the presence (lanes 2,4,6,8) or absence (lanes 1,3,5,7) of Az-Biot. Samples were splitted and ran on two 16% Tricine-SDS-Page gels. One was colored with Coomassie blue (CB) while the other was used to transfer proteins on a nitrocellulose membrane that was then probed with a Streptavidin-HRP conjugate (Strep-HRP).
Fig 3.
C. glutamicum strains 13032 over-expressing PorHhis (lanes 1–4) or PorHhis-S56A (lanes 5–8) were grown in the absence (lanes 1–2, 5–6) or in the presence (lanes 3–4, 7–8) of 17-ODYA. After purification, PorHhis was subjected to CuAAC in the presence (lanes 2,4,6,8) or absence (lanes 1,3,5,7) of Az-Biot. Samples were splitted and ran on two 16% Tricine-SDS-Page gels. One was colored with Coomassie blue (CB) while the other was used to transfer proteins on a nitrocellulose membrane that was then probed with a Streptavidin-HRP conjugate (Strep-HRP).
Fig 4.
Mass spectrometry analysis of solvent-extracted proteins from wild-type and Δcg2875::Km strains.
Positive linear MALDI-TOF mass spectrum of the hydrophobic proteins extracts prepared from C. glutamicum wild-type (13032)(A) and Δcg2875::Km (B).
Fig 5.
ProtX metabolic labeling and mycoloylation site determination.
(A) C. glutamicum strains 13032, ΔmytC::Km, or Δpks13::Km over-expressing ProtXhis were grown in the absence (lanes 1–2, 5–6) or in the presence (lanes 3–4, 7–8) of 17-ODYA. After purification, ProtXhis was subjected to CuAAC in the presence (lanes 2,4,6,8) or absence (lanes 1,3,5,7) of Az-Biot. Samples were splitted and ran on two 16% Tricine-SDS-Page gels. One was colored with Coomassie blue (CB) while the other was used to transfer proteins on a nitrocellulose membrane that was then probed with a Streptavidin-HRP conjugate (Strep-HRP). (B) ProtXhis and ProtXhis single serine mutants were over-expressed in 13032 and Δpks13::Km or ΔprotX::Km, respectively in the presence of 17-ODYA, purified and subjected to CuAAC in the presence (lanes 1,2,4,6,8,10,12,14) or absence (lanes 3,5,7,9,11,13) of Az-Biot. Samples were splitted and ran on two 16% Tricine-SDS-Page gels. One was colored with Coomassie blue (CB) while the other was used to transfer proteins on a nitrocellulose membrane that was then probed with a Streptavidin-HRP conjugate (Strep-HRP).
Fig 6.
Mass spectrometry analysis of solvent-extracted proteins from CgΔporHporA strain over-expressing ProtXhis or ProtXhis-S37A.
Positive linear MALDI-TOF spectra of the hydrophobic proteins extracts from CgΔporHporA (A), CgΔporHporA transformed with pXMJ19-ProtXhis and grown in the presence of 1mM IPTG (B) or CgΔporHporA transformed with pXMJ19-ProtXhis-S37A and grown in the presence of 1mM IPTG (C).
Fig 7.
PorB and PorC metabolic labeling.
C. glutamicum strains 13032 over-expressing PorBhis (lanes 1–4) or PorChis (lanes 5–8) were grown in the absence (lanes 1–2, 5–6) or in the presence (lanes 3–4, 7–8) of 17-ODYA. After purification, PorBhis and PorChis were subjected to CuAAC in the presence (lanes 2,4,6,8) or absence (lanes 1,3,5,7) of Az-Biot. Samples were splitted and ran on two 16% Tricine-SDS-Page gels. One was colored with Coomassie blue (CB) while the other was used to transfer proteins on a nitrocellulose membrane that was then probed with a Streptavidin-HRP conjugate (Strep-HRP).
Fig 8.
PorB and PorC expression and localization.
C. glutamicum strains 13032, ΔmytC::Km, or Δpks13::Km transformed with either pXMJ19-PorBhis (A) or pXMJ19-PorChis (B) were grown overnight in the absence or in the presence of IPTG. Total cell extract (C), cell wall fraction (CW) and supernatant (S) were separated on Tricine-SDS-Page or Tris-Glycine-SDS-Page gels and immunoblotted with anti-PorB (A) or anti-PorC (B) antisera. The same samples were also probed with anti-aconitase (Acn, cytoplasmic marker) and anti-cMytA (envelope and supernatant marker).
Fig 9.
Cell wall and secreted PorB and PorC metabolic labeling and NaOH treatment.
C. glutamicum strains 13032 or ΔmytC::Km over-expressing PorBhis (A, B) or PorChis (C) were grown in the absence (lanes 1–2, 5–6) or in the presence (lanes 3–4, 7–8) of 17-ODYA. After purification from either the envelope or the culture filtrate, the proteins were subjected to CuAAC in the presence (lanes 2,4,6,8) or absence (lanes 1,3,5,7) of Az-Biot. Samples were splitted and ran on two 16% Tricine-SDS-Page gels. One was colored with Coomassie blue (CB) while the other was used to transfer proteins on a nitrocellulose membrane that was then probed with a Streptavidin-HRP conjugate (Strep-HRP). (D) PorChis purified from either the envelope or the supernatant of 13032 was treated with NaOH 0,1M for the indicated time. The reaction was stopped by addition of an equimolar amount of acetic acid. Samples were then splitted and ran on a 16% Tricine-SDS-Page gel and stained with Coomassie brilliant blue.