Recent site-specific phosphoproteome studies have revealed the presence of numerous phosphorylated serines, threonines, and tyrosines in bacterial cells (Macek & Mijakovic, 2011). First reported site-specific phosphoproteomes were those of the model bacteria B. subtilis (Macek et al., 2007) and Escherichia coli (Macek et al., 2008). Their phosphoproteomes turned out to be of average size, with about 100 phosphorylated proteins per cell. The smallest phosphoproteomes were detected in the genus Pseudomonas with 56–57 detected phosphopeptides per species (Ravichandran et al., 2009) and Mycoplasma pneumoniae with 63 reported phosphorylated proteins (Schmidl et al., 2010). The largest dataset so far was reported for M. tuberculosis, with over 300 phosphoproteins (Prisic et al., 2010). The intracellular pathogen M. tuberculosis possesses 11 Hanks-type kinases, which is a comparatively large set with respect to most of the sequenced bacterial species. Consequently, it is the best studied bacterial model with respect to their physiological roles. Hanks-type kinases of M. tuberculosis have been shown to participate in growth regulation, by influencing division and envelope synthesis (Molle & Kremer, 2010). A major player in this respect is the peptidoglycan-responsive kinase PknB (Mir et al., 2011), that activates the peptidoglycan assembly by phosphorylating the key biosynthetic protein MviN (Gee et al., 2012). PknB has other proteins substrates, some of which contain forkhead-associated domains. Regarding its substrate Rv0020c, it has been shown that the interaction between the kinase and the substrate can be modulated by the extent and pattern of substrate phosphorylation (Roumestand et al., 2011). Another Hanks-type kinase from M. tuberculosis, PknE, is involved in adaptive stress response (Kumar et al., 2013). It also promotes intracellular survival by antagonizing the apoptotic pathway of macrophages (Kumar & Narayanan, 2012). This exemplifies the participation of kinases in biochemical warfare between intracellular pathogens and the human host that involves scrambling the signaling pathways of the ‘adversary’. In addition to evidence of single kinase phosphorylating multiple substrates, a number of M. tuberculosis proteins can be phosphorylated by several Hanks-type kinases. For example, the cyclopropane synthase PcaA was phosphorylated in vitro by purified M. tuberculosis kinases PknD, PknF, PknH, and PknE, but not by PknA and PknB (Corrales et al., 2012).Similarly, HadAB and HadBC dehydratases from M. tuberculosis have been reported as substrates of PknA, PknB, PknD, PknE, PknF, PknH, and PknL (Slama et al., 2011). Because the serine/threonine kinases of M. tuberculosis figure so prominently in the physiology of this pathogen, research on M. tuberculosis is also leading the way in exploiting kinase-specific inhibitors as potential antimicrobial agents. PknB, the kinase involved in cell division and regulation of growth has been singled out as a promising target (Lougheed et al., 2011). Hanks-type kinases have also been extensively studied in Staphylococcus aureus (Ohlsen & Donat, 2010) and streptococci. Most important new insights from S. aureus include the structural studies on the catalytic domain of PknB (Rakette et al., 2012) and the peptidoglycan-binding domain of PrkC (Ruggiero et al., 2010), which contribute to the understanding of activation mechanisms for these kinases. In S. aureus, it has also been reported that Hanks-type kinases can be involved in regulation of quorum sensing (Cluzel et al., 2010) and carbon catabolite repression by phosphorylation of the major regulatory protein CcpA (Leiba et al., 2012). In Streptococcus pyogenes, the kinase Stk was shown to activate genes for virulence factors, osmoregulation, metabolism of α-glucans, fatty acid biosynthesis, as well as genes affecting cell-wall synthesis (Bugrysheva et al., 2011). In Streptococcus pneumoniae, the kinase StkP was found to participate in cell cycle control and cell division (Beilharz et al., 2012; Fleurie et al., 2012). It localizes to mid-cell, and controls correct septum progression and closure. Cells mutated for stkP display elongated morphologies. Because the septal localization of StkP depends on its penicillin-binding domains, the authors argue that its role is to transmit information about the cell-wall status to key players of cell division. The phenotype in cell division has been associated to phosphorylation of the division protein DivIVA by StkP (Fleurie et al., 2012). A similar growth-regulating role has been reported for the Hanks-type kinase AfsK that localizes to cell poles in Streptomyces. It is activated by the arrest of cell-wall synthesis and by consequence phosphorylates the division protein DivIVA (Hempel et al., 2012). Again, the image of coordination between different cellular processes emerges. The recurrent theme in the field is that new Hanks-type kinases get identified and characterized as being involved in one particular process, by phosphorylating one particular substrate. However, sooner or later, alternative substrates for each kinase get discovered, and this connects the kinases to multiple cellular roles. An exhaustive list of known substrates for some well-characterized bacterial serine/threonines kinases can be found in Pereira et al. (2011).
The same principle of kinase ‘promiscuity’ holds true for BY-kinases (Grangeasse et al., 2007). This family of tyrosine kinases specific for bacteria is phylogenetically related to Walker motif ATPases, and not to Hanks-type kinases, but their overall architecture is similar (Jadeau et al., 2008; Grangeasse et al., 2012). They also possess extracellular sensing domains, and catalytic cytosolic domains, with the exception that the two domains can be encoded by separate genes in Firmicutes (Grangeasse et al., 2007). First cellular substrates of BY-kinases have been identified in B. subtilis (Mijakovic et al., 2003) and in E. coli (Grangeasse et al., 2003). In both cases, BY-kinases were found to phosphorylate UDP-glucose dehydrogenases, thus increasing the activity of these enzymes. Soon thereafter, new substrates for the same enzymes were found. Besides phosphorylating UDP-glucose dehydrogenase Ugd (Mijakovic et al., 2003), B. subtilis BY-kinase PtkA was found to phosphorylate single-stranded DNA-binding proteins (Mijakovic et al., 2006). Accordingly, the inactivation of the ptkA gene led to a pleiotropic phenotype, with a pronounced defect in cell cycle and DNA replication (Petranovic et al., 2007). Soon thereafter, several tyrosine phosphorylated proteins detected in the B. subtilis phosphoproteome have been identified as PtkA substrates. PtkA-dependent phosphorylation has been shown to regulate the enzyme activity of some of them, and intracellular localization of others (Jers et al., 2010). Finally, a role for the kinase PtkA and its cognate phosphatase PtpZ in B. subtilis was recently proposed in biofilm formation (Kiley & Stanley-Wall, 2010). These findings support the view that BY-kinases, just like Hanks-type serine/threonine kinases may in fact constitute signal integration nodes in a complex regulatory network based on bacterial protein phosphorylation, and as such may coordinate different cellular processes. The list of bacterial functions known to be controlled by tyrosine phosphorylation has expanded considerably in recent years. To cite some notable examples: Tyrosine phosphorylation has been found to control sporulation in Myxococcus xanthus (Kimura et al., 2011) and phage resistance in Lysteria monocytogenes (Nir-Paz et al., 2012). In Caulobacter crescentus, the gene encoding the phosphotyrosine-protein phosphatase CtpA was found to be essential. This phosphotyrosine-protein phosphatase regulates cell separation, outer membrane integrity, and morphology in C. crescentus (Shapland et al., 2011), thus confirming the link between tyrosine phosphorylation and the bacterial cell cycle observed in B. subtilis. Finally, a very exciting discovery came from Bacillus anthracis, where the first dual specificity serine/threonine and tyrosine kinases PrkD and PrkG have been described (Arora et al., 2012). A possible role for these kinases in cell growth and development has been suggested.