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Comparative Ser/Thr/Tyr phosphoproteomics between two mycobacterial species: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG.

Nakedi KC, Nel AJ, Garnett S, Blackburn JM, Soares NC - Front Microbiol (2015)

Bottom Line: Moreover, our study identified a number of conserved Ser/Thr phosphorylated sites and conserved Tyr phosphorylated sites across different mycobacterial species.In particular, M. bovis BCG exponential cells exhibited a much more complex and sophisticated protein phosphorylation network regulating important cellular cycle events such as cell wall biosynthesis, elongation, cell division including immediately response to stress.The differences in the two phosphoproteomes are discussed in light of different mycobacterial growth rates.

View Article: PubMed Central - PubMed

Affiliation: Blackburn Lab, Applied Proteomics and Chemical Biology Group, Division of Medical Biochemistry, Institute of Infectious Disease and Molecular Medicine, University of Cape Town Cape Town, South Africa.

ABSTRACT
Ser/Thr/Tyr protein phosphorylation plays a critical role in regulating mycobacterial growth and development. Understanding the mechanistic link between protein phosphorylation signaling network and mycobacterial growth rate requires a global view of the phosphorylation events taking place at a given time under defined conditions. In the present study we employed a phosphopeptide enrichment and high throughput mass spectrometry-based strategy to investigate and qualitatively compare the phosphoproteome of two mycobacterial model organisms: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG. Cells were harvested during exponential phase and our analysis detected a total of 185 phospho-sites in M. smegmatis, of which 106 were confidently localized [localization probability (LP) = 0.75; PEP = 0.01]. By contrast, in M. bovis BCG the phosphoproteome comprised 442 phospho-sites, of which 289 were confidently localized. The percentage distribution of Ser/Thr/Tyr phosphorylation was 39.47, 57.02, and 3.51% for M. smegmatis and 35, 61.6, and 3.1% for M. bovis BCG. Moreover, our study identified a number of conserved Ser/Thr phosphorylated sites and conserved Tyr phosphorylated sites across different mycobacterial species. Overall a qualitative comparison of the fast and slow growing mycobacteria suggests that the phosphoproteome of M. smegmatis is a simpler version of that of M. bovis BCG. In particular, M. bovis BCG exponential cells exhibited a much more complex and sophisticated protein phosphorylation network regulating important cellular cycle events such as cell wall biosynthesis, elongation, cell division including immediately response to stress. The differences in the two phosphoproteomes are discussed in light of different mycobacterial growth rates.

No MeSH data available.


Related in: MedlinePlus

Phosphorylation of M. bovis BCG Cell division FtsQ (Thr24) and probable conserved protein membrane mmpS3 (Tyr70). (A) Identification of phosphorylated residue by mass spectrometry. Fragmentation spectra for modified peptide bearing the phosphorylated Thr24. (B) Fragmentation spectra for modified peptide bearing the phosphorylated Tyr70.
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Figure 1: Phosphorylation of M. bovis BCG Cell division FtsQ (Thr24) and probable conserved protein membrane mmpS3 (Tyr70). (A) Identification of phosphorylated residue by mass spectrometry. Fragmentation spectra for modified peptide bearing the phosphorylated Thr24. (B) Fragmentation spectra for modified peptide bearing the phosphorylated Tyr70.

Mentions: In this study we considered all phosphorylation-events that were detected in any of the biological replicates, and only confidently localized phospho-sites (p-sites) (Figure 1), were considered for qualitative comparative analysis and further discussion. In the two M. bovis BCG replicates, we detected a total of 442 p-sites of which 289 were confidently localized (Localization probability (LP) = 0.75; PEP = 0.01) and 169/289 had a LP = 0.99 (Supplementary Table S1A and Figure S2c). We identified 88,822 MS/MS spectra corresponding to 7784 non-redundant peptide sequences (Supplementary Figure S2d) and 1765 protein groups (402 were identified by a single peptide) (Supplementary Table S1A). The estimated false discovery rate (FDR) was 0.32 at the peptide level, 0.30 at the modification level, and 1.03 at the protein level. Our initial analysis of two biological replicates of M. smegmatis revealed considerable differences in the number of identified p-sites between the two Mycobacterial species, 77 p-sites for M. smegmatis (Supplementary Figure S2e) compared to 289 for M. bovis BCG. To verify that these differences observed were biological, we further analyzed three additional biological replicates for M. smegmatis (Supplementary Table S1B). Phosphoproteomic analysis of five biological replicates of M. smegmatis protein extracts resulted in identification of 180, 396 MS/MS spectra, corresponding to 16, 185 non-redundant peptide sequences (Supplementary Figure S2f) and 2, 462 protein groups (464 were identified by a single peptide) (Supplementary Table S1B). The estimated false discovery rate (FDR) was 0.22 at the peptide level, 0.21 at the modification level, and 0.98 at the protein group level. We detected a total of 185 phospho-sites in M. smegmatis, of which 106 were confidently localized (LP = 0.75; PEP = 0.01) and 64/106 had a LP = 0.99 (Supplementary Table S1B). In detail, for M. bovis BCG, we detected 289 p-sites on 203 phoshoproteins: 35.3% on serine (pSer), 61.6% on threonine (pThr) and 3.1% tyrosine (pTyr). For M. smegmatis we detected 106 p-sites on 76 phosphoproteins: 39.47% on serine (pSer), 57.02% on threonine (pThr) and 3.51% on tyrosine (pTyr). Both phosphoproteomes were biased toward Thr compared with Ser (57–61%; 41–35%), which agrees with previous reports on M. tuberculosis H37Rv (Prisic et al., 2010). Importantly, the percentage of Tyr phosphorylation in M. bovis BCG was closer to that reported in M. tuberculosis (Kusebauch et al., 2014). Although it had previously been suggested that Tyr phosphorylation was non-existent within mycobacterial species, it was recently confirmed that Tyr phosphorylation does in fact occur on a number of diverse M. tuberculosis proteins (Kusebauch et al., 2014). Here, we have confidently identified nine Tyr p-sites in eight proteins in M. bovis BCG (Table 1, Figure 1B and Supplementary Table S1A) and four in M. smegmatis, supporting earlier suggestions that phosphorylation on Tyr residues occur in different mycobacterial species (Kusebauch et al., 2014).


Comparative Ser/Thr/Tyr phosphoproteomics between two mycobacterial species: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG.

Nakedi KC, Nel AJ, Garnett S, Blackburn JM, Soares NC - Front Microbiol (2015)

Phosphorylation of M. bovis BCG Cell division FtsQ (Thr24) and probable conserved protein membrane mmpS3 (Tyr70). (A) Identification of phosphorylated residue by mass spectrometry. Fragmentation spectra for modified peptide bearing the phosphorylated Thr24. (B) Fragmentation spectra for modified peptide bearing the phosphorylated Tyr70.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4389566&req=5

Figure 1: Phosphorylation of M. bovis BCG Cell division FtsQ (Thr24) and probable conserved protein membrane mmpS3 (Tyr70). (A) Identification of phosphorylated residue by mass spectrometry. Fragmentation spectra for modified peptide bearing the phosphorylated Thr24. (B) Fragmentation spectra for modified peptide bearing the phosphorylated Tyr70.
Mentions: In this study we considered all phosphorylation-events that were detected in any of the biological replicates, and only confidently localized phospho-sites (p-sites) (Figure 1), were considered for qualitative comparative analysis and further discussion. In the two M. bovis BCG replicates, we detected a total of 442 p-sites of which 289 were confidently localized (Localization probability (LP) = 0.75; PEP = 0.01) and 169/289 had a LP = 0.99 (Supplementary Table S1A and Figure S2c). We identified 88,822 MS/MS spectra corresponding to 7784 non-redundant peptide sequences (Supplementary Figure S2d) and 1765 protein groups (402 were identified by a single peptide) (Supplementary Table S1A). The estimated false discovery rate (FDR) was 0.32 at the peptide level, 0.30 at the modification level, and 1.03 at the protein level. Our initial analysis of two biological replicates of M. smegmatis revealed considerable differences in the number of identified p-sites between the two Mycobacterial species, 77 p-sites for M. smegmatis (Supplementary Figure S2e) compared to 289 for M. bovis BCG. To verify that these differences observed were biological, we further analyzed three additional biological replicates for M. smegmatis (Supplementary Table S1B). Phosphoproteomic analysis of five biological replicates of M. smegmatis protein extracts resulted in identification of 180, 396 MS/MS spectra, corresponding to 16, 185 non-redundant peptide sequences (Supplementary Figure S2f) and 2, 462 protein groups (464 were identified by a single peptide) (Supplementary Table S1B). The estimated false discovery rate (FDR) was 0.22 at the peptide level, 0.21 at the modification level, and 0.98 at the protein group level. We detected a total of 185 phospho-sites in M. smegmatis, of which 106 were confidently localized (LP = 0.75; PEP = 0.01) and 64/106 had a LP = 0.99 (Supplementary Table S1B). In detail, for M. bovis BCG, we detected 289 p-sites on 203 phoshoproteins: 35.3% on serine (pSer), 61.6% on threonine (pThr) and 3.1% tyrosine (pTyr). For M. smegmatis we detected 106 p-sites on 76 phosphoproteins: 39.47% on serine (pSer), 57.02% on threonine (pThr) and 3.51% on tyrosine (pTyr). Both phosphoproteomes were biased toward Thr compared with Ser (57–61%; 41–35%), which agrees with previous reports on M. tuberculosis H37Rv (Prisic et al., 2010). Importantly, the percentage of Tyr phosphorylation in M. bovis BCG was closer to that reported in M. tuberculosis (Kusebauch et al., 2014). Although it had previously been suggested that Tyr phosphorylation was non-existent within mycobacterial species, it was recently confirmed that Tyr phosphorylation does in fact occur on a number of diverse M. tuberculosis proteins (Kusebauch et al., 2014). Here, we have confidently identified nine Tyr p-sites in eight proteins in M. bovis BCG (Table 1, Figure 1B and Supplementary Table S1A) and four in M. smegmatis, supporting earlier suggestions that phosphorylation on Tyr residues occur in different mycobacterial species (Kusebauch et al., 2014).

Bottom Line: Moreover, our study identified a number of conserved Ser/Thr phosphorylated sites and conserved Tyr phosphorylated sites across different mycobacterial species.In particular, M. bovis BCG exponential cells exhibited a much more complex and sophisticated protein phosphorylation network regulating important cellular cycle events such as cell wall biosynthesis, elongation, cell division including immediately response to stress.The differences in the two phosphoproteomes are discussed in light of different mycobacterial growth rates.

View Article: PubMed Central - PubMed

Affiliation: Blackburn Lab, Applied Proteomics and Chemical Biology Group, Division of Medical Biochemistry, Institute of Infectious Disease and Molecular Medicine, University of Cape Town Cape Town, South Africa.

ABSTRACT
Ser/Thr/Tyr protein phosphorylation plays a critical role in regulating mycobacterial growth and development. Understanding the mechanistic link between protein phosphorylation signaling network and mycobacterial growth rate requires a global view of the phosphorylation events taking place at a given time under defined conditions. In the present study we employed a phosphopeptide enrichment and high throughput mass spectrometry-based strategy to investigate and qualitatively compare the phosphoproteome of two mycobacterial model organisms: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG. Cells were harvested during exponential phase and our analysis detected a total of 185 phospho-sites in M. smegmatis, of which 106 were confidently localized [localization probability (LP) = 0.75; PEP = 0.01]. By contrast, in M. bovis BCG the phosphoproteome comprised 442 phospho-sites, of which 289 were confidently localized. The percentage distribution of Ser/Thr/Tyr phosphorylation was 39.47, 57.02, and 3.51% for M. smegmatis and 35, 61.6, and 3.1% for M. bovis BCG. Moreover, our study identified a number of conserved Ser/Thr phosphorylated sites and conserved Tyr phosphorylated sites across different mycobacterial species. Overall a qualitative comparison of the fast and slow growing mycobacteria suggests that the phosphoproteome of M. smegmatis is a simpler version of that of M. bovis BCG. In particular, M. bovis BCG exponential cells exhibited a much more complex and sophisticated protein phosphorylation network regulating important cellular cycle events such as cell wall biosynthesis, elongation, cell division including immediately response to stress. The differences in the two phosphoproteomes are discussed in light of different mycobacterial growth rates.

No MeSH data available.


Related in: MedlinePlus