1887

Abstract

Eukaryotic-like Ser/Thr protein kinases (STPKs) are present in many bacterial species, where they control various physiological and virulence processes by enabling microbial adaptation to specific environmental signals. PknJ is the only member of the 11 STPKs identified in that still awaits characterization. Here we report that PknJ is a functional kinase that forms dimers , and contains a single transmembrane domain. Using a high-density peptide-chip-based technology, multiple potential mycobacterial targets were identified for PknJ. We confirmed PknJ-dependent phosphorylation of four of these targets: PknJ itself, which autophosphorylates at Thr, Thr and Thr residues; the transcriptional regulator EmbR; the methyltransferase MmaA4/Hma involved in mycolic acid biosynthesis; and the dipeptidase PepE, whose encoding gene is located next to in the mycobacterial genome. Our results provide a number of candidate phospho-targets for PknJ and possibly other mycobacterial STPKs that could be studied to investigate the role of STPKs in physiology and virulence.

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2010-06-01
2019-08-19
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Plasmids and bacterial strains used in the study [ PDF] (64 kb) Oligonucleotide primers used in the study [ PDF] (14 kb) Whole dataset for the phospho-chip analysis [ Excel file] (168 kb) Growth kinetics of CDC1551 and the mutant strains in various culture media. Sauton's minimal medium was inoculated with CDC1551, or the ::Tn or ::Tn mutants, in the absence or presence (+ BSA) of BSA. Bacterial growth was measured by spectrophotometry (OD ) at various time points. The figure represents one representative experiment of four independent experiments. [ PDF] (496 kb) Lipid profile comparison between WT and ::Tn strains. Lipids were applied to TLC plates and developed with various migration solvents: petroleum ether/ether 95/5 (a) CH Cl /CH OH 85/12 (b) and CH Cl /CH OH/H O 65/25/4 (c). DIMA-B, phthiocerol dimycocerosate A or B; TDM, trehalose dimycolate; TMM, trehalose monomycolate; DAT, diacyltrehalose; PE, phosphatidylethanolamine; PIM , diacylated phosphatidyl- -inositol dimannoside; PI, phosphatidyl- -inositol; CL, cardiolipin; Ac PIM , tetraacylated phosphatidyl- -inositol dimannoside; PIM f, phosphatidyl- o-inositol hexamannoside family; WT, wild-type. [ PDF] (572 kb) Cytokine and chemokine secretion profile of THP1 macrophages infected with CDC1551 or the ::Tn mutant. Cells were infected with the two strains at an m.o.i. of 5 bacteria per cell. After 4 or 18 h, supernatants were collected and were analysed for cytokine secretion using the RayBio Human Cytokine Antibody Array 3 (RayBiotech). [ PDF] (633 kb) [ PDF] (8 kb)

PDF

Plasmids and bacterial strains used in the study [ PDF] (64 kb) Oligonucleotide primers used in the study [ PDF] (14 kb) Whole dataset for the phospho-chip analysis [ Excel file] (168 kb) Growth kinetics of CDC1551 and the mutant strains in various culture media. Sauton's minimal medium was inoculated with CDC1551, or the ::Tn or ::Tn mutants, in the absence or presence (+ BSA) of BSA. Bacterial growth was measured by spectrophotometry (OD ) at various time points. The figure represents one representative experiment of four independent experiments. [ PDF] (496 kb) Lipid profile comparison between WT and ::Tn strains. Lipids were applied to TLC plates and developed with various migration solvents: petroleum ether/ether 95/5 (a) CH Cl /CH OH 85/12 (b) and CH Cl /CH OH/H O 65/25/4 (c). DIMA-B, phthiocerol dimycocerosate A or B; TDM, trehalose dimycolate; TMM, trehalose monomycolate; DAT, diacyltrehalose; PE, phosphatidylethanolamine; PIM , diacylated phosphatidyl- -inositol dimannoside; PI, phosphatidyl- -inositol; CL, cardiolipin; Ac PIM , tetraacylated phosphatidyl- -inositol dimannoside; PIM f, phosphatidyl- o-inositol hexamannoside family; WT, wild-type. [ PDF] (572 kb) Cytokine and chemokine secretion profile of THP1 macrophages infected with CDC1551 or the ::Tn mutant. Cells were infected with the two strains at an m.o.i. of 5 bacteria per cell. After 4 or 18 h, supernatants were collected and were analysed for cytokine secretion using the RayBio Human Cytokine Antibody Array 3 (RayBiotech). [ PDF] (633 kb) [ PDF] (8 kb)

PDF

Plasmids and bacterial strains used in the study [ PDF] (64 kb) Oligonucleotide primers used in the study [ PDF] (14 kb) Whole dataset for the phospho-chip analysis [ Excel file] (168 kb) Growth kinetics of CDC1551 and the mutant strains in various culture media. Sauton's minimal medium was inoculated with CDC1551, or the ::Tn or ::Tn mutants, in the absence or presence (+ BSA) of BSA. Bacterial growth was measured by spectrophotometry (OD ) at various time points. The figure represents one representative experiment of four independent experiments. [ PDF] (496 kb) Lipid profile comparison between WT and ::Tn strains. Lipids were applied to TLC plates and developed with various migration solvents: petroleum ether/ether 95/5 (a) CH Cl /CH OH 85/12 (b) and CH Cl /CH OH/H O 65/25/4 (c). DIMA-B, phthiocerol dimycocerosate A or B; TDM, trehalose dimycolate; TMM, trehalose monomycolate; DAT, diacyltrehalose; PE, phosphatidylethanolamine; PIM , diacylated phosphatidyl- -inositol dimannoside; PI, phosphatidyl- -inositol; CL, cardiolipin; Ac PIM , tetraacylated phosphatidyl- -inositol dimannoside; PIM f, phosphatidyl- o-inositol hexamannoside family; WT, wild-type. [ PDF] (572 kb) Cytokine and chemokine secretion profile of THP1 macrophages infected with CDC1551 or the ::Tn mutant. Cells were infected with the two strains at an m.o.i. of 5 bacteria per cell. After 4 or 18 h, supernatants were collected and were analysed for cytokine secretion using the RayBio Human Cytokine Antibody Array 3 (RayBiotech). [ PDF] (633 kb) [ PDF] (8 kb)

EXCEL

Plasmids and bacterial strains used in the study [ PDF] (64 kb) Oligonucleotide primers used in the study [ PDF] (14 kb) Whole dataset for the phospho-chip analysis [ Excel file] (168 kb) Growth kinetics of CDC1551 and the mutant strains in various culture media. Sauton's minimal medium was inoculated with CDC1551, or the ::Tn or ::Tn mutants, in the absence or presence (+ BSA) of BSA. Bacterial growth was measured by spectrophotometry (OD ) at various time points. The figure represents one representative experiment of four independent experiments. [ PDF] (496 kb) Lipid profile comparison between WT and ::Tn strains. Lipids were applied to TLC plates and developed with various migration solvents: petroleum ether/ether 95/5 (a) CH Cl /CH OH 85/12 (b) and CH Cl /CH OH/H O 65/25/4 (c). DIMA-B, phthiocerol dimycocerosate A or B; TDM, trehalose dimycolate; TMM, trehalose monomycolate; DAT, diacyltrehalose; PE, phosphatidylethanolamine; PIM , diacylated phosphatidyl- -inositol dimannoside; PI, phosphatidyl- -inositol; CL, cardiolipin; Ac PIM , tetraacylated phosphatidyl- -inositol dimannoside; PIM f, phosphatidyl- o-inositol hexamannoside family; WT, wild-type. [ PDF] (572 kb) Cytokine and chemokine secretion profile of THP1 macrophages infected with CDC1551 or the ::Tn mutant. Cells were infected with the two strains at an m.o.i. of 5 bacteria per cell. After 4 or 18 h, supernatants were collected and were analysed for cytokine secretion using the RayBio Human Cytokine Antibody Array 3 (RayBiotech). [ PDF] (633 kb) [ PDF] (8 kb)

PDF

Plasmids and bacterial strains used in the study [ PDF] (64 kb) Oligonucleotide primers used in the study [ PDF] (14 kb) Whole dataset for the phospho-chip analysis [ Excel file] (168 kb) Growth kinetics of CDC1551 and the mutant strains in various culture media. Sauton's minimal medium was inoculated with CDC1551, or the ::Tn or ::Tn mutants, in the absence or presence (+ BSA) of BSA. Bacterial growth was measured by spectrophotometry (OD ) at various time points. The figure represents one representative experiment of four independent experiments. [ PDF] (496 kb) Lipid profile comparison between WT and ::Tn strains. Lipids were applied to TLC plates and developed with various migration solvents: petroleum ether/ether 95/5 (a) CH Cl /CH OH 85/12 (b) and CH Cl /CH OH/H O 65/25/4 (c). DIMA-B, phthiocerol dimycocerosate A or B; TDM, trehalose dimycolate; TMM, trehalose monomycolate; DAT, diacyltrehalose; PE, phosphatidylethanolamine; PIM , diacylated phosphatidyl- -inositol dimannoside; PI, phosphatidyl- -inositol; CL, cardiolipin; Ac PIM , tetraacylated phosphatidyl- -inositol dimannoside; PIM f, phosphatidyl- o-inositol hexamannoside family; WT, wild-type. [ PDF] (572 kb) Cytokine and chemokine secretion profile of THP1 macrophages infected with CDC1551 or the ::Tn mutant. Cells were infected with the two strains at an m.o.i. of 5 bacteria per cell. After 4 or 18 h, supernatants were collected and were analysed for cytokine secretion using the RayBio Human Cytokine Antibody Array 3 (RayBiotech). [ PDF] (633 kb) [ PDF] (8 kb)

PDF

Plasmids and bacterial strains used in the study [ PDF] (64 kb) Oligonucleotide primers used in the study [ PDF] (14 kb) Whole dataset for the phospho-chip analysis [ Excel file] (168 kb) Growth kinetics of CDC1551 and the mutant strains in various culture media. Sauton's minimal medium was inoculated with CDC1551, or the ::Tn or ::Tn mutants, in the absence or presence (+ BSA) of BSA. Bacterial growth was measured by spectrophotometry (OD ) at various time points. The figure represents one representative experiment of four independent experiments. [ PDF] (496 kb) Lipid profile comparison between WT and ::Tn strains. Lipids were applied to TLC plates and developed with various migration solvents: petroleum ether/ether 95/5 (a) CH Cl /CH OH 85/12 (b) and CH Cl /CH OH/H O 65/25/4 (c). DIMA-B, phthiocerol dimycocerosate A or B; TDM, trehalose dimycolate; TMM, trehalose monomycolate; DAT, diacyltrehalose; PE, phosphatidylethanolamine; PIM , diacylated phosphatidyl- -inositol dimannoside; PI, phosphatidyl- -inositol; CL, cardiolipin; Ac PIM , tetraacylated phosphatidyl- -inositol dimannoside; PIM f, phosphatidyl- o-inositol hexamannoside family; WT, wild-type. [ PDF] (572 kb) Cytokine and chemokine secretion profile of THP1 macrophages infected with CDC1551 or the ::Tn mutant. Cells were infected with the two strains at an m.o.i. of 5 bacteria per cell. After 4 or 18 h, supernatants were collected and were analysed for cytokine secretion using the RayBio Human Cytokine Antibody Array 3 (RayBiotech). [ PDF] (633 kb) [ PDF] (8 kb)

PDF

Plasmids and bacterial strains used in the study [ PDF] (64 kb) Oligonucleotide primers used in the study [ PDF] (14 kb) Whole dataset for the phospho-chip analysis [ Excel file] (168 kb) Growth kinetics of CDC1551 and the mutant strains in various culture media. Sauton's minimal medium was inoculated with CDC1551, or the ::Tn or ::Tn mutants, in the absence or presence (+ BSA) of BSA. Bacterial growth was measured by spectrophotometry (OD ) at various time points. The figure represents one representative experiment of four independent experiments. [ PDF] (496 kb) Lipid profile comparison between WT and ::Tn strains. Lipids were applied to TLC plates and developed with various migration solvents: petroleum ether/ether 95/5 (a) CH Cl /CH OH 85/12 (b) and CH Cl /CH OH/H O 65/25/4 (c). DIMA-B, phthiocerol dimycocerosate A or B; TDM, trehalose dimycolate; TMM, trehalose monomycolate; DAT, diacyltrehalose; PE, phosphatidylethanolamine; PIM , diacylated phosphatidyl- -inositol dimannoside; PI, phosphatidyl- -inositol; CL, cardiolipin; Ac PIM , tetraacylated phosphatidyl- -inositol dimannoside; PIM f, phosphatidyl- o-inositol hexamannoside family; WT, wild-type. [ PDF] (572 kb) Cytokine and chemokine secretion profile of THP1 macrophages infected with CDC1551 or the ::Tn mutant. Cells were infected with the two strains at an m.o.i. of 5 bacteria per cell. After 4 or 18 h, supernatants were collected and were analysed for cytokine secretion using the RayBio Human Cytokine Antibody Array 3 (RayBiotech). [ PDF] (633 kb) [ PDF] (8 kb)

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