Functional Expression of the PorAH Channel from Corynebacterium glutamicum in Cell-free Expression Systems

Rath, Parthasarathi; Demange, Pascal; Saurel, Olivier; Tropis, Marielle; Daffé, Mamadou; Dötsch, Volker; Ghazi, Alexandre; Bernhard, Frank; Milon, Alain

doi:10.1074/jbc.m111.276956

Cited by 30 publications

(36 citation statements)

References 38 publications

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“…Although the function and underlying mechanisms of O-mycoloylation remain elusive, the biological consequences of having long acyl moieties attached to proteins include enhancement of membrane binding as well as protein-protein interactions. For example, in vitro studies revealed that protein posttranslational modification (PTM) was essential for PorA/PorH pore-forming activity (8). Interestingly, the mycoloylated PorA, PorH, and ProtX proteins Significance Protein secretion is an essential determinant of bacterial physiology and virulence.…”

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confidence: 99%

Identification of specific posttranslational O -mycoloylations mediating protein targeting to the mycomembrane

Carel

Marcoux

Réat

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The outer membranes (OMs) of members of the Corynebacteriales bacterial order, also called mycomembranes, harbor mycolic acids and unusual outer membrane proteins (OMPs), including those with α-helical structure. The signals that allow precursors of such proteins to be targeted to the mycomembrane remain uncharacterized. We report here the molecular features responsible for OMP targeting to the mycomembrane of Corynebacterium glutamicum, a nonpathogenic member of the Corynebacteriales order. To better understand the mechanisms by which OMP precursors were sorted in C. glutamicum, we first investigated the partitioning of endogenous and recombinant PorA, PorH, PorB, and PorC between bacterial compartments and showed that they were both imported into the mycomembrane and secreted into the extracellular medium. A detailed investigation of cell extracts and purified proteins by top-down MS, NMR spectroscopy, and site-directed mutagenesis revealed specific and well-conserved posttranslational modifications (PTMs), including O-mycoloylation, pyroglutamylation, and N-formylation, for mycomembrane-associated and -secreted OMPs. PTM site sequence analysis from C. glutamicum OMP and other O-acylated proteins in bacteria and eukaryotes revealed specific patterns. Furthermore, we found that such modifications were essential for targeting to the mycomembrane and sufficient for OMP assembly into mycolic acid-containing lipid bilayers. Collectively, it seems that these PTMs have evolved in the Corynebacteriales order and beyond to guide membrane proteins toward a specific cell compartment.Corynebacteriales | O-acylation | sequence motif | top-down proteomics | NMR

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confidence: 99%

Identification of specific posttranslational O -mycoloylations mediating protein targeting to the mycomembrane

Carel

Marcoux

Réat

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Careful pH control in a fermenter was required since acidification of the growth medium led to a drastic reduction of PorH expression yield. The proteins extraction and purification was performed according to published procedures [5] and as described in the material and methods section, and was based on LDAO solubilisation of the cell walls. Since we had shown that cell-free expressed PorH can be solubilised and give nice HSQC NMR spectra in LDAO detergent micelles [5], we first tried to assign PorH Chis in this environment.…”

Section: Resultsmentioning

confidence: 99%

“…The proteins extraction and purification was performed according to published procedures [5] and as described in the material and methods section, and was based on LDAO solubilisation of the cell walls. Since we had shown that cell-free expressed PorH can be solubilised and give nice HSQC NMR spectra in LDAO detergent micelles [5], we first tried to assign PorH Chis in this environment. However detailed inspection of the spectra rapidly revealed strong spectral heterogeneity, which could arise from either a chemical heterogeneity (due to PTM on several residues) or from conformational heterogeneity, due to slow exchange between various conformers at the micelle interface.…”

Section: Resultsmentioning

confidence: 99%

“…In C. glutamicum, we recently showed that two Por, namely PorA and PorH, are O-acylated by a mycoloyl residue, a novel form of mycolate-containing substance, which represents the first O-linked acylation onto an hydroxylated amino-acid of bacterial polypeptides [4]. We further demonstrated that this modification is critical for the pore-forming activity of the PorA/PorH hetero-oligomer [5].…”

Section: Introductionmentioning

confidence: 99%

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NMR localization of the O‐mycoloylation on PorH, a channel forming peptide from Corynebacterium glutamicum

et al. 2013

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a b s t r a c tPorH and PorA are two small peptides that, in complex, form a voltage-dependent ion channel in the outer membrane of Corynebacterium glutamicum. Specific post-translational modifications on PorA and PorH are required for the formation of a functional ion channel. The assignment of PorH proton NMR chemical shifts in DMSO, allowed identifying unambiguously the exact position of the PorH Omycoloylation on Ser 56 side chain. This was further confirmed by site directed mutagenesis and mass spectrometry. Together with the previously published localization of PorA mycoloylation, this provides the complete primary structure characterization of this outer membrane porin.

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“…Because of its ease of handling, C. glutamicum serves as a model organism for M. tuberculosis studies. Studies with C. glutamicum have contributed significantly to deciphering the mechanism of lipid synthesis (12)(13)(14) and the function of proteins involved in cell wall synthesis (15,16) of M. tuberculosis.…”

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Visualization of Imbalances in Sulfur Assimilation and Synthesis of Sulfur-Containing Amino Acids at the Single-Cell Level

Hoffmann

Grünberger

Lausberg

et al. 2013

Appl Environ Microbiol

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We describe genetically encoded sensors which transmit elevated cytosolic concentrations of O-acetyl serine (OAS) and O-acetyl homoserine (OAH)-intermediates of L-cysteine and L-methionine synthesis-into an optical output. The sensor pSenOAS3 elicits 7.5-fold-increased fluorescence in cultures of a Corynebacterium glutamicum strain that excrete L-cysteine. Determination of the cytosolic OAS concentration revealed an increase to 0.13 mM, whereas the concentration in the reference strain was below the detection limit, indicating that incorporation of assimilatory sulfur is limited in the strain studied. In another strain, overexpression of metX encoding homoserine acetyltransferase resulted in an 8-fold increase in culture fluorescence at a cytosolic OAH concentration of 0.76 mM. We also assayed for consequences of extracellular sulfur supply and observed a graded fluorescence increase at decreasing sulfur concentrations below 400 M. Overall, this demonstrates the usefulness of the sensors for monitoring intracellular sulfur availability. The sensors also enable monitoring at the single-cell level, and since related and close homologs of the transcription factor used in the constructed sensors are widespread among bacteria, this technology offers a new possibility of assaying in vivo for sulfur limitation and of doing this at the single-cell level. Sulfur is an essential element for any microorganism. It is required for the synthesis of the sulfur-containing amino acids (L-cysteine and L-methionine) together with that of the sulfur-containing coenzymes or prosthetic groups and other compounds. In the context of applied microbiology, the assimilation of sulfur is crucial in at least two settings. One is related to pathogenic microorganisms in which there is an indication that such organisms face sulfur limitation in their natural environment. This is probably the case for the facultative intracellular pathogen Mycobacterium tuberculosis in its preferred host, the macrophage, since under conditions of sulfur limitation, the mycobacterial cysDNC operon enabling sulfur assimilation is upregulated (1). In addition to L-cysteine and L-methionine synthesis, the sulfate assimilation pathway of M. tuberculosis produces a number of sulfur-containing metabolites with important contributions to pathogenesis and survival (2). In different clinical M. tuberculosis isolates, significant differences in the abundance of sulfur (and iron) assimilatory proteins highlight the relevance of sulfur availability for the bacilli (3). In the opportunistic pathogen Pseudomonas aeruginosa E601, which causes infections of cystic fibrosis in patients, 43 proteins, among them a sulfatase with mucin as the substrate which makes the sulfur of the glycopeptide available, are upregulated more than 10-fold under conditions of sulfur limitation (4).Sulfur availability is also of relevance for the large-scale synthesis of L-cysteine and L-methionine. The bacterium Corynebacterium glutamicum is a prime candidate for amino acid production, because proce...

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Functional Expression of the PorAH Channel from Corynebacterium glutamicum in Cell-free Expression Systems

Cited by 30 publications

References 38 publications

Identification of specific posttranslational O -mycoloylations mediating protein targeting to the mycomembrane

Identification of specific posttranslational O -mycoloylations mediating protein targeting to the mycomembrane

NMR localization of the O‐mycoloylation on PorH, a channel forming peptide from Corynebacterium glutamicum

Visualization of Imbalances in Sulfur Assimilation and Synthesis of Sulfur-Containing Amino Acids at the Single-Cell Level

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