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

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.


Introduction
Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), a major health concern worldwide. The current incidence of tuberculosis disease in South Africa is more than 900 cases per 100,000 people per year. Moreover, the World Health Organisation have estimated that roughly one third of the world's population is latently infected with M. tuberculosis. The majority of latently infected individuals will remain asymptomatic throughout their lives, with the risk of developing active TB disease from a latent infection being ∼10% per lifetime; in HIV positive individuals though, this risk increases to 10% per year (WHO, 2014). This latent infected population is thus a substantial reservoir of potential new TB cases and is therefore a significant public health concern. The ability of mycobacteria to adapt to, and overcome, hostile environmental conditions in order to survive within host cells depends on a fine-tuned and orchestrated series of events (e.g., response to stress; metabolic remodeling; changes in growth rate) that will ultimately define the success of establishing and maintaining long term infection (McKinney et al., 2000;Marrero et al., 2010). M. tuberculosis infection is often described by two distinct phases: an active phase, in which the microorganism is thought to be growing at or close to its maximum rate; and latent infection, in which the bacilli are thought to persist in a viable but perhaps more dormant-like state with lower or non-existent growth rate. Current thinking suggests that there is most likely a continuum of states between latent TB infection (LTBI), subclinical TB and active TB disease, but to date no M. tuberculosis bacilli have been observed in LTBI individuals, so the exact physiological state of M. tuberculosis during a latent infection remains unknown.
Alongside the increasing number of new TB infections there is another matter of great concern, which is the emergence and spread of multi-and extensively drug resistant M. tuberculosis strains. Here unique growth related mechanisms of mycobacteria which facilitate adaptation to different adverse microenvironments are thought to play an important role in the mechanisms of drug tolerance and acquired resistance that are observed during infection (Corper and Cohn, 1933;Wayne and Hayes, 1996). For instance, a recent study showed that diverse growth-limiting stresses trigger a common signal transduction pathway in M. tuberculosis that induces triglyceride synthesis, which is associated with slowing down of growth and reduced antibiotic efficacy (Baek et al., 2011). Therefore, further investigation of the signaling pathways which regulate mycobacterial growth rate might reveal important information regarding the capacity of M. tuberculosis to adapt to its environment and in particular how this relates to drug tolerance and to the ability to establish infection.
In M. tuberculosis, Ser/Thr phosphorylation is essential for growth and environmental adaptation (as reviewed by Kieser and Rubin, 2014). For instance, the Ser/Thr protein kinases (STPKs) PknA and PknB are essential for growth of M. tuberculosis in culture, indicating that phosphorylation plays a pivotal role in the survival of this bacterium (Sassetti et al., 2003;Kang et al., 2005;Fernandez et al., 2006;Molle and Kremer, 2010;Kusebauch et al., 2014). These STPKs are encoded by an operon which regulates genes involved in cell shape determination, cell wall synthesis, and cell division (Deol et al., 2005;Kang et al., 2005;Kusebauch et al., 2014). In addition to PknA and PknB, another group of STPKs comprised of PknG, PknL, and PknF appear to be involved in different aspects of M. tuberculosis growth regulation (Cowley et al., 2004;Deol et al., 2005;Canova et al., 2008). In support of the likely important role played by STPKs in M. tuberculosis, a mass spectrometry-based phosphoproteomic study identified more than 500 Ser/Thr phosphorylation sites on 300 proteins in M. tuberculosis (Prisic et al., 2010) and, more recently, a complementary study detected a number of Tyr phosphorylated proteins in M. tuberculosis (Kusebauch et al., 2014). Notably though, the physiological significance of these findings remains largely unexplored.
In the past years, the use of mycobacterial models such as M. smegmatis and M. bovis BCG have significantly contributed to our current understanding of M. tuberculosis biology and environmental adaptation (as reviewed by Shiloh and Champion, 2010). M. bovis BCG is an attenuated bovine tuberculosis bacillus by a serial passage in the laboratory (Calmette et al., 1921). This mycobacterium is a particular convenient model in part due to it is slow growth rate similar to that observed in M. tuberculosis. On other hand M smegmatis is a fast growing mycobacterial species (with a doubling time of approximately 4 h) that has been widely used to investigate different aspects of mycobacterial physiology (Barry, 2001;Reyrat and Kahn, 2001;Danilchanka et al., 2008). As part of a concerted program to associate protein phosphorylation in mycobacteria with subsequent macromolecular events which determine growth rate and eventual environmental adaption, we have carried out a phosphopeptide enrichment and high throughput mass spectrometry-based study to investigate and compare the phosphoproteome of two model mycobacterial organisms-the fast growing M. smegmatis and the slow growing M. bovis BCG-our goal being to begin to elucidate the phosphorylation events and subsequent signal transduction pathways coordinating differential mycobacterial growth rates, which may in due course lead to important insights into M. tuberculosis physiology during active and latent infection-including aspects of slow growth associated drug resistance.

Protein Extraction
Cells were harvested during the exponential phase (OD 600 ∼1.2 and 0.6 for M. smegmatis and M. bovis BCG, respectively) by centrifugation at 4000 g for 15 min at 4 • C, washed twice with Phosphate Buffered Saline pH (7.5) (PBS). Cells were snap frozen in liquid nitrogen and stored at -80 • C until needed. Frozen pellets were suspended in 800 µl lysis buffer [500 mM Tris-HCl, 0.1% (w/v) SDS, 0.15% sodium deoxycolate, 1× protease inhibitor cocktail, 1× phosphatase inhibitor cocktail (Roche, Mannheim Germany) and 50 µg/ml lysozyme (Repaske, 1956)]. Cells were disrupted by sonication at maximum power for six cycles of 30 s, with 1 min cooling on ice between cycles. Cellular debris was removed by centrifugation at 4000 g for 5 min and the lysate filtered through 20 µm pore size low-protein binding filters (Merck, NJ, USA). Proteins were precipitated using the chloroform-methanol precipitation method as previously described (Wessel and Flügge, 1984). The pellet was resuspended in urea buffer (6 M urea, 2 M thiourea and 10 mM Tris-HCl, pH 8). Protein concentration was determined using a modified Bradford assay as described by Ramagli (1999).

In-Solution Trypsin Digestion
Five milligram of total protein was reduced with 1 mM DTT for 1 h with agitation and then alkylated with 5.5 mM IAA for 1 h in the dark. Proteins were pre-digested with Lysyl Endopeptidase LysC (Waco, Neuss, Germany) at room temperature for 3 h and diluted 4× with HPLC grade water before adding sequencing grade modified trypsin (Promega, Madison, USA) (1:100 w/w). Proteolysis was carried out at room temperature for 14 h with agitation at 30 rpm. Proteolysis was quenched by addition of trifluoroacetic acid (TFA) (Sigma Aldrich, St Louis, USA).

Phosphopeptide Enrichment with TiO 2 Chromatography
Acetonitrile (ACN) (Sigma Aldrich, St Louis, USA) at a final concentration of 30% was added to the tryptic peptides and the pH was adjusted to 2. Ten milligram of Titasphere TiO2 beads (MZ Analysentechnik, Mainz, Germany) in loading buffer [30 mg/ml 2,5 dihydrobenzoic acid (Sigma Aldrich, St Louis, USA), 80% ACN] were added to the sample and incubated at room temperature with rotation for 1 h. The beads were pelleted and the decanted supernatant was further incubated with a fresh batch of 5 mg of beads for 30 min. This step was repeated one further time, giving three fractions of enriched phosphoproteins bound to beads in total. Phosphopeptides bound to the beads were washed vigorously with shaking for 10 min in 1 ml of wash buffer 1 (30% acetonitrile, 3% trifluoroacetic acid) followed by an additional 10 min of vigorous wash with wash buffer 2 (80% acetonitrile, 0.1% trifluoroacetic acid). Phosphopeptides were loaded onto C8 stage tip, washed with wash buffer 2 and then eluted with 3 × 50 µl Elution buffer [40% Mass-spec grade NH 4 OH (aq, 25% NH 3 ; Sigma-Aldrich, St Louis, USA), 60% acetonitrile (pH 10.5 or higher)]. Solvent was removed in a speed drying vacuum at room temperature and peptides were resuspended in 20 µl Solvent A (2% ACN, 0.1% formic acid).

LC/MS/MS Analysis
Liquid chromatography separation was carried out using a precolumn (100 µm ID × 20 mm) connected to an analytical column (75 µm × 500 mm) packed with C18 Luna 5 µ 100 Å beads (phenomenex 04A-5452). The columns were connected to an Easy n-LC II system (Proxeon). 5 µl of resuspended phosphopeptides were loaded onto the column with starting mobile phase of 2% ACN, 0.1% formic acid. Peptides were eluted with the following gradient 5% ACN to 35% ACN for 120 min the up to 80% ACN over 5 min staying at 80% ACN for 10 min, this was followed by a column wash of 7 to 80% gradient for 30 min, and a column equilibration at 2% ACN for 2 min. The flow rate was held at 300 nL/min. Mass spectra were acquired on an Orbitrap Q Exactive mass spectrometer in a data-dependent manner, with automatic switching between MS and MS/MS scans using a "Top-10" method. MS spectra were acquired at a resolution of 70,000 with a target value of 3 × 10 6 ions or a maximum injection time of 250 ms. Peptide fragmentation was performed via higher-energy collision dissociation (HCD) with the energy set at 25 NCE. Intensity threshold for ions selection was set at 1.7e4 with charge exclusion of z = 1 and z > 5. The MS/MS spectra were acquired at a resolution of 17,500, with a target value of 2 × 10 5 ions or a maximum injection time of 120 ms. The scan range was limited to 300 to 1750 m/z and the isolation window at 4.0 m/z.

Data Processing
Maxquant software package version 1.5.0.3 with integrated Andromeda search engine was used to analyse the raw MS spectra acquired. M. smegmatis (taxonomy 246196) and M. bovis BCG Pasteur (taxonomy 410289) databases obtained from Uniprot were used as the target-decoy databases for the Andromeda search. LysC and Trypsin were defined as proteases and two missed cleavages were allowed. Phosphorylation on serine, threonine, and tyrosine residues, oxidation of methionines and Nterminal acetylation were specified as variable modifications. Carbamidomethylation on cysteines was defined as a fixed modification. We allowed a FTMS MS/MS match tolerance of 20 ppm. False discovery rate was set at 1% for identification of peptides and proteins without re-quantifying. Stringent filtering criteria were employed to validate phospho sites. Phosphopeptides were considered as high confidence phosphorylation events and taken for further analysis only if they had a localization probability of >0.75, had a posterior error probability (PEP) score of <0.001. We further manually validated the phospho sites on the Maxquant viewer for good b-and y-ion series coverage.

Bioinformatics
Predicted Gene Ontology (GO) cellular functions of identified phosphoproteins with high confidence from two mycobacterium strains were obtained from The Database for Annotation, Visualization and Integrated Discovery (DAVID, v6.7) and were grouped into functional categories. To compare the identified phosphoproteomes of these two mycobacterial species, we identified homologs of the M. smegmatis phosphorylated proteins in M. bovis BCG using the University of Cape Town's Computational Biology online bioinformatics tool found at http://galaxy-fen.uct.ac.za/root. For identification of tyrosinephosphorylation site motifs, we used the Motif-X algorithm (Schwartz, 2005). We defined a sequence window of +/-10 amino acids on each side of the tyrosine site and generated the sequence logo by Phosphosite logo generator using the algorithm PSP production (Cell signaling Technology). We aligned genomic sequences of both strains using the online tool, obtained from http://www.ebi.ac.uk/Tools/msa/clustalw2/ and respective fasta files were obtained from Uniprot.

Results and Discussion
There is increasing evidence indicating that protein phosphorylation plays an essential role during mycobacterial cell division and environmental adaptation. Understanding the mechanistic connection between such regulatory signaling networks and mycobacterial growth rate requires a global view of the phosphorylation events taking place at a given time under defined conditions. Here, we have carried out a comparative phosphoproteomic analysis of two mycobacterial strainsone fast growing (M. smegmatis), the other slow growing (M. bovis BCG). Our analysis included three rounds of TiO 2 chromatography to enrich phosphorylated peptides (see Supplementary Figure S1), followed by subsequent analysis of phosphorylation-events using liquid chromatography coupled with state-of-the-art high resolution tandem mass spectrometry (LC/MS/MS) (Supplementary Figure S2a and S2b), in order to compare the phosphoproteomes of these two model mycobacterium species during exponential growth.
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   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).
The differences between the compared phosphoproteomes compelled us to investigate whether some of these dissimilarities could be explained by genomic events rather than posttranslational control. The multiple sequence alignment of 130 selected M. bovis BCG phosphoproteins with their respective M. smegmatis orthologs (Supplementary S3A) revealed that from 197 M. bovis BCG Ser/Thr/Tyr phosphorylated sites: 12 are conserved across the two mycobacterial species and were found to be phosphorylated in both species, while 94 conserved Ser/Thr/Tyr residues were found to be phosphorylated in M. bovis BCG only. Furthermore, whereas 91 M. bovis BCG phosphorylated residues were aligned with a different non-phosphorylated M. smegmatis residue, 72 of these were aligned with a non-phosphorylated amino acid (Supplementary S3A) and 19 were aligned with different nonphosphorylated Ser/Thr/Tyr residue. Conversely, the multiple sequence alignment of 64 M. smegmatis phosphoproteins with their respective M. bovis BCG orthologs showed that besides the 12 conserved Ser/Thr/Tyr residues phosphorylated in both species, 31 conserved residues were found to be phosphorylated in M. smegmatis only. In this case, while 43 M. smegmatis phosphorylated residues were aligned with a different non-phosphorylated M. bovis BCG amino-acid, 36 of these were aligned with a non-phosphorylated amino acid residue (Supplementary S3B) and seven were aligned with different non-phosphorylated Ser/Thr/Tyr residue (Supplementary S3B). These results point out that some of differences observed between the two phosphoproteomes can be explained by the absence of the corresponding amino acid residue, indicating that during exponential growth phase these two mycobacterial species present an inherently different sub-set of Ser/Thr/Tyr kinase substrates. Additionally, there are some interesting examples in which orthologous proteins were phosphorylated at different psites. This suggests that kinase specificities for a substrate could be intimately related with the actual site of phosphorylation. Finally, it is notable that occasions the Ser/Thr/Tyr residue was aligned with different Ser/Thr/Tyr residue (in most cases S for T and vice versa) in some punctual situation the respective residue was phosphorylated (e.g., PknB) but for the majority of the cases these were aligned with non-phosphorylated Ser/Thr/Tyr residue. This intriguing observation leaves open the possibility that Ser/Thr exchange could be a result of an evolutionary processes/environmental adaptation, in which the replacement for the respective residue would probably favor site phosphorylation and therefore the gain of an additional mechanism of protein functional regulation. Although speculative, it would be interesting to explore further in which conditions these sub set of Ser/Thr/Tyr sites are phosphoryalated.
Finally, the number of phosphoproteins/sites identified in M. smegmatis is comparable to those reported in other soil bacteria, e.g., E. coli (Macek et al., 2008;Soares et al., 2013), Bacillus subtilis (Shi et al., 2014) and Pseudomonas putida (Ravichandran et al., 2009), as well as in some pathogenic bacteria such as Pseudomonas aeruginosa (Ravichandran et al., 2009), Streptococcus pneumonia (Sun et al., 2009), Helicobacter pylori (Ge and Shan, 2011), and Klebsiella pneumonia (Lin et al., 2009). Whereas, the number of phosphorylated Ser/Thr/Tyr detected in M. bovis BCG phosphoproteome is comparable to that described in M. tuberculosis H37Rv (Prisic et al., 2010). It is of particular interest that the M. bovis BCG phosphoproteome shows a number of phosphoproteins/sites that are orthologous to those reported in M. tuberculosis H37Rv (Prisic et al., 2010) but also a number that are not conserved. Recently a comparison between the Ser/Thr/Tyr phosphoproteomes of Acinetobacter baumannii reference strain (ATCC17978) and a highly invasive, multidrug resistant clone (AbH12O-A2) demonstrated that, during stationary phase, the multidrug isolate showed twice as many phosphorylation-events as the reference strain (Soares et al., 2014). In contrast to reports on Pseudomonas species (Ravichandran et al., 2009), our current study supports the notion that bacteria within the same genus/species may utilize differing numbers of phosphoproteins.

Conserved Ser/Thr Phosphorylated Sites
Macek et al. (2008) reported evidence of a possible high degree of conservation within potential bacterial phospho-sites although, as noted by those authors, the conservation of residues does not mean that they are phosphorylated in all species. In fact, as the number of bacterial phosphoproteomic studies increases, it is becoming clearer that the degree of conserved phosphosites among bacterial species is rather limited and certainly lower than reported within eukaryotic phosphoproteomes (e.g., Freschi et al., 2014). Here, a comparison between the M. smegmatis, M. bovis BCG, and M. tuberculosis H37Rv (Prisic et al., 2010;Kusebauch et al., 2014) phosphoproteomes revealed that these three mycobacterial species share a number of conserved phosphorylated sites ( Table 2). Interestingly, we found that M. bovis BCG and M. tuberculosis H37Rv phosphoproteomes share at least 32 Ser/Thr conserved phospho-sites on 27 proteins, of which three were conserved in all three species ( Table 2). As pointed out by Freschi et al. (2014), phosphorylation sites that are phosphorylated in different species are more likely to be functional and this conservation criterion could be used to prioritize phosphorylation events for additional characterization.
In the present study we have focused in particular on the STPKs PknB and PknA that have known or predicted functions in cell wall generation and growth in M. smegmatis, M. bovis BCG, and M. tuberculosis (Gee et al., 2012;Kusebauch et al., 2014). We found PknB to be phosphorylated in Thr 173 in all three species and in Thr 171 in M. smegmatis and M. bovis BCG ( Table 2). Previous in vitro assays demonstrated that both Thr 173 and Thr 171 are conserved auto-phosphorylated residues that lie in the activation loop of PknB (Boitel et al., 2003). Additionally, a M. tuberculosis double mutant Thr 171 /Thr 173 was 300-fold less active than respective wild-type PnkB, suggesting a combined effect of both Thr 171 and Thr 173 residues on kinase activity. Subsequent studies confirmed that the mutation of these residues had a strong effect not only on PknB kinase activity but also in the process of activation loop-mediated recruitment of its substrates (Villarino et al., 2005). Here we have provided evidence that Thr 173 and Thr 171 phosphorylation both occur in vivo during the exponential phase, at which PknB is most abundant and is thought be at its maximum activity. Thus, our data reinforces a previous hypothesis suggesting that in vivo this enzyme is regulated through an autophosphorylation mechanism involving the phosphorylation state of both Thr 173 and Thr 171 . Another proposed mechanism of PknB regulation relates to the maintenance of an inactive state via the interaction of the juxtamembrane region with the kinase domain. In this model, the auto-phosphorylation of specific residues in the juxtamembrane sequences releases the inhibition by making the sequence available for further interactions with domains of target proteins (Wybenga-Groot et al., 2001). However, previously it was not clear whether Thr 294 and/or Thr 309 were the target residues involved so it is notable that our data clearly demonstrate that PknB of M.bovis BCG is in fact phosphorylated on Thr 309 .
Our analyses indicate that PknA is phosphorylated in at least one conserved residue, Ser 309 /Ser 310 (see Table 2). Intriguingly, in this study M. bovis BCG PknA was found to be phosphorylated on seven different residues (three Ser and four Thr, respectively), all located in the juxtamembrane region. Unlike PknB, in PknA the juxtamembrane region, encompassing residue 269-338 is indispensable not only for auto-phosphorylation of PknA but also for its substrate phosphorylation ability (Thakur et al., 2008). STPks exhibit a wide variety of mechanisms for their regulation. Taking into account the degree of phosphorylation verified here in the juxtamembrane region of M. bovis BCG PknA compared to that observed in M. smegmatis PknA, it is tempting to speculate that this level of phosphorylation of the juxtamembrane region could be in fact limiting the access of PknA to its substrates and this way controlling the action of the enzyme. Importantly, as noted by Chawla et al. (2014), whilst the structure and mode of activation of PknB and PknA have been well established in vitro, the structure-function relationships of the various domains have yet to be investigated in the context of mycobacterial growth (Chawla et al., 2014). Here through a MS based phosphoproteomic approach we have established (at the phospho-site level) the phosphorylation state of different domains for both PknA and PknB in vivo during growth at exponential phase.

Conserved Tyr Phosphorylated Sites
In our study we have identified nine Tyr p-sites (see Table 1), of which four were also found to be phosphorylated in M. tuberculosis (Kusebauch et al., 2014): FHA-domain-containing protein (Tyr 215 and Tyr 232 ), 60 kDa chaperonin 1 (Tyr 358 ) and conserved membrane protein mmpS3 (Tyr 70 ) ( Figure 1B). FHA-domain-containing protein is a substrate of numerous STPKs, including PknB. Phosphorylation of FHA by PnkB has implication in cell wall synthesis with a possible involvement in mycobacterial virulence (Gupta et al., 2009). Likewise both 60 kDa Chaperonin and conserved membrane protein mmpS3 have been implicated in mycobacterial virulence (Wells et al., 2013). Based on our data, we searched for possible conservation of these peptides across other bacterial species. A sequence motif derived from 60 kDa Chaperonin Tyr 358 (RQEIENSDSDYDREKLQERLA) using Seq2Logo revealed an overrepresentation of Tyr 358 (Figure 2). A conserved Tyr 360 residue on apparently conserved peptide (SDSDYDREKL) was found in three Gram negative pathogenic species, specifically Shigella spp., Klebsiella spp., and Salmonella spp., suggesting that this conserved Tyr phosphorylation site warrants further investigation for possible roles in bacterial pathogenesis.

Phosphoprotein Functional Enrichment
Gene ontology (GO) terms revealed that in both M. bovis BCG and M. smegmatis, the phosphorproteins/phosphosites were functionally enriched in nine distinct groups, (e.g., ATP binding, translation, kinase activity, cell division, see Figure 2). Of interest, a great deal of phosphorylated proteins in M. bovis BCG was clustered into the Transmembrane group (Figure 2). This included a considerable number of multiple phosphorylated proteins and some phosphorylated in internal as well as external regions, like BCG_3967, it is a probable trans-membrane protein and we found it to be phosphorylated four times, at position 10, which like on the flagellin domain and position 801 and 801, the kinase domain. This suggests that there are transmembrane proteins with a potential role in signal transduction. Additionally, it was visible that M bovis BCG phosphoproteome comprised a notable group of phosphoproteins involved in cell division, possible implications of this is discussed below.

Phosphorylation Events Observed in Proteins that Regulate Mechanisms of Growth and Cell Division
Both PknA and PknB are encoded by genes (pknA and pknB, respectively) located on the same operon as protein phosphatase PstP, RodA (implicated in cell shape control) and PbpA (implicated in peptidoglycan synthesis) (Cole et al., 1998). This locus includes also two FHA (forkhead-associated) domaincontaining proteins and in mycobacteria is found near the origin of replication. In M. bovis BCG, all proteins referred to above FIGURE 3 | Seq2Logo alignment analysis derived from 60 kDa chaperonin revelead an overrepresentation of Tyr 358 . Seq2Logo analysis indicate that a conserved Tyr 358−360 is found in additional three pathogenic species, specifically Shigella spp., Klebsiella spp., and Salmonella ssp.
Frontiers in Microbiology | www.frontiersin.org (except PbpA) were found phosphorylated at a total of 15 p-sites: PknA (7); PknB (3); RodA (1); PstP (1) and FHA domain containing protein (3) (see Supplementary Table S1A). In M. smegmatis, however, only a few proteins were found phosphorylated at a total of 5 p-sites: PknA (2); PknB (2) FHA domain containing protein (1) (see Supplementary Table S1B). These observations suggest that the slow growth of M. bovis BCG preserves this central set of cell division proteins under a tight regulatory network in which key elements are intimately inter-related by an important series of functional (de)phosphorylation events. For example, PknA and PknB are regulated by PstP-mediated phosphorylation (Boitel et al., 2003;Chopra et al., 2003); additionally, recently it has been shown that both PknA and PknB phosphorylate PstP (Sajid et al., 2011). As discussed above, our results showed that both M. smegmatis and M. bovis BCG PknB has conserved phosphorylation on Thr 171 and Thr 173 -both of which sites are known to be substrates for PstP-thus suggesting that in both cases PstP is at least partially inactive. This would make sense considering that during exponential phase PknA and PknB are likely to be at their peak of activity. PstP has been reported to be phosphorylated by PknB on Thr 173 , Thr 141 , Thr 290 , and Thr 137 in its cytosolic domain and on Thr 174 by PknA (Sajid et al., 2011). Curiously phosphorylated PstP has been reported to be more active than its unphosphorylated form (Sajid et al., 2011). Here, our results indicate that PstP of M. bovis BCG is phosphorylated in vivo on the high confidence p-site, Ser 155 (see Supplementary Table S1A). Interestingly, PstP contains three metal-binding centers in its structure (Pullen et al., 2004), sharing the fold and two-metal center of human PP2Cα whilst having a third Mn 2+ in a site created by a large shift in a flap domain next to the active site; this Mn 2+ occurs at the position of Ser 160 so it is plausible that phosphorylation of Ser 155 may directly interfere with PstP activity, thus accounting for our deduction here of reduced PstP activity during exponential phase growth.
Overall, M. bovis BCG has 3 times more STPks and nearly 4 times respective p-sites compared to M. smegmatis. Apart from PknA and PknB, the M. bovis BCG phosphoproteome is comprised of PknG (Thr 95 ), PknH (Thr 174 ), PknE (Ser 304 and Ser 326 ) PknF (Thr 287 ). Some of these enzymes have previously been directly implicated in mycobacterial growth [e.g., PknG (Fiuza et al., 2008), PknH (Zheng et al., 2007), PknE and PknF (Gupta et al., 2014)] and it is therefore conceivable that some of the proteins comprising the M. bovis BCG phosphoproteome are in fact substrate of some of these phosphorylated protein kinases (vide infra). It is worth noting that our study has also identified several Two Component sensory signal transduction proteins as phosphoproteins (e.g., Two component sensory transduction protein regX3 (Thr 151 and Thr 153 ), Two component sensor histidine kinase ppr (Ser 446 ), Two component transcriptional regulatory pprA (Ser 20 ). These results are reminiscient of those previously described in B. subtilis (Jers et al., 2011) and suggest that in M bovis BCG there may be cross talk between Ser/Thr/Tyr phosphorylation and Two component systems, which would add extra complexity to the overall protein phosphorylation signal transduction pathways regulating exponential growth of M. bovis BCG cells.

Phosphorylation Events Observed in Proteins that Regulates Mechanisms of Cell Elongation and Division
In mycobacteria, cell elongation is regulated by a macrocomplex that regulates peptidoglycan remodeling during growth by means of hydrolytic and synthetic roles (as reviewed by (Kieser and Rubin, 2014)). Our data indicate that in M. bovis BCG, three important proteins of the macromolecular elongation complex are phosphorylated during exponential growth, namely Wag31 (Ser 245 ), CwsA (Thr 77 ) and a putative hydrolase (BCG_0021 involved in peptidoglycan catabolic process) (Thr 43 ). In mycobacteria, Wag31 is phosphorylated by PknA and is essential for correct polar localization and biosynthesis (Jani et al., 2010;Lee et al., 2014); in addition, Wag31 is stabilized by the cell wall protein CwsA. Wag31 is thought to be phosphorylated during exponential phase and remains non-or lowlyphosphorylated during stationary phase (Kang et al., 2005;Park et al., 2008). Interestingly, Wag31, CwsA and the putative peptidoglycan hydrolase were not found amongst the M. smegmatis phosphorylated proteins in the present study. Whilst we cannot rule out that our assay did not isolate phosphorylated M. smegmatis Wag31, it is perhaps more likely that in the fast growing M. smegmatis the elongation complex is regulated by alternative non-phosphorylated mechanism.
Another macromolecular complex, named divisome, is responsible for mycobacterial cell division. Assembly and disassembly of this complex is regulated by protein phosphorylation (Kieser and Rubin, 2014). According to the our data, in M. bovis BCG there are five divisome proteins which are phosphorylated, including cell division FtsQ (Thr 24 ), FtsW-like protein (Thr 29 ), CwsA (Thr 77 ) as well as other additional phosphorylated cell division proteins such as RodA (Thr 463 ), cell division transmembrane protein FtsK (Thr 325 ; Thr 642 ) and FtsY (Thr 72 ), strongly suggesting that divisome assembly and indeed cell division in M. bovis BCG is subject to a high level of phosphorylation.
Of interest, in our analysis we have detected Hup, a conserved histidine-like protein, phosphorylated on three different p-sites (Thr 45 , Thr 65 , and Ser 90 ). In Mycobacterium sp., the homolog of HU (Mhpl) is implicated in bacterial adaptation to stress response conditions, possible inhibition of cellular metabolism and reduction of bacterial growth rate through nucleoid reorganization (Lee et al., 1998;Matsumoto et al., 1999;Katsube et al., 2007). Apparently, is expressed in exponentially growing cells of M. tuberculosis H37Ra and it is shown to be maximally expressed during stationary phase, while Hup kinases (PknE, PknF, and PknB) were found to be constitutively expressed during exponential phase (Gupta et al., 2014). It has been suggested that the phosphorylation of HupB during the exponential phase by the referred kinases would limit the interaction with DNA (Gupta et al., 2014). In our M. bovis BCG data we have identified all the intervenient proteins involved in the described posttranslational regulation mechanism, including the phosphorylation of phosphosite Hup Thr 65 . It is therefore appropriate to assume that the same mechanism takes place in M. bovis BCG cells during exponential growth, and although under limited action it remains possible that the rate of unphosphorylated HupB would have an impact on the overall growth rate. In contrast, we did not find any evidence to indicate that similar mechanisms operate in M. smegmatis cells during exponential growth.

Stress Related Proteins
In rich broth during exponential phase, bacteria experience nearly optimal conditions of growth where there is excess nutrients and little accumulation of by products, in addition to scarce competition between bacterial cells. Surprisingly, under these conditions we observed an unexpected number of chaperones and stress related proteins in the M. bovis BCG phosphoproteome: For instance hyperosmotic and heat shock related proteins such as the chaperon protein DnaJ (Thr 120 ) chaperon protein DnaK (Ser 558 ) and GrpE (Ser 12 and Thr 2 ), multiply phosphorylated 60 kDa chaperonin, 10 kDa chaperonin, and Copper-sensing transcriptional repressor CsoR (Thr 93 ). On the other hand none of these stress related proteins were found in the M. smegmatis phosphoproteome, which suggests that even under optimal environmental conditions, slow growing mycobacteria such as M. bovis BCG maintain a preventive basal level of stressrelated proteins that may act as frontline defense barrier to ensure adequate and prompt response to any sudden change in local environmental conditions. In this scenario protein phosphorylation would keep most of these proteins in an inactive state, whereby dephosphorylation could then immediately recruit these proteins when environmental conditions become unfavorable. A convenient and versatile regulatory mechanism such as this could in fact be a determinant for the survival and persistence of some bacteria.

Conclusion and Prespectives
This study clearly demonstrated that there are major differences between a fast growing and a slow growing mycobacterial phosphoproteome. The M. smegmatis phosphoproteome observed here is in many aspects similar to those reported in other soil-dwelling bacterial models and can be viewed as a minimalist phosphoproteome compared to that of M. bovis BCG. This latter organism presents a much more complex and sophisticated protein phosphorylation network, regulating important cellular cycle events such as cell wall biosynthesis, elongation, and cell division, as well as apparently being involved in regulating response to stress, which over all would allow a quick cellular response to abrupt environmental changes. However, this regulatory advantage might be associated with a cost, reflected by reduced metabolic fitness and slower growth rate.
This study demonstrates M. bovis BCG is a good model to study aspects of mycobacterial phospho-dependent signal transduction pathways, including those involved in persistence and slow growth, including that associated with drug resistance. By contrast, the substantial differences reported here in the phosphoproteomes of M. smegmatis and M. bovis BCG suggest that exponentially growing M. smegmatis cells in vitro are of limited relevance when modeling phosphorylation networks and phospho-regulation events likely to occur in M. tuberculosis at the site of disease during an infection.