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6 September 2019

Tyrosine Phosphorylation as a Widespread Regulatory Mechanism in Prokaryotes


Phosphorylation events modify bacterial and archaeal proteomes, imparting cells with rapid and reversible responses to specific environmental stimuli or niches. Phosphorylated proteins are generally modified at one or more serine, threonine, or tyrosine residues. Within the last ten years, increasing numbers of global phosphoproteomic surveys of prokaryote species have revealed an abundance of tyrosine-phosphorylated proteins. In some cases, novel phosphorylation-dependent regulatory paradigms for cell division, gene transcription, and protein translation have been identified, suggesting that a wide scope of prokaryotic physiology remains to be characterized. Recent observations of bacterial proteins with putative phosphotyrosine binding pockets or Src homology 2 (SH2)-like domains suggest the presence of phosphotyrosine-dependent protein interaction networks. Here in this minireview, we focus on protein tyrosine phosphorylation, a posttranslational modification once thought to be rare in prokaryotes but which has emerged as an important regulatory facet in microbial biology.


Recent phosphoproteomic data from some bacterial species contrast with early reports that detected very low levels of tyrosine-phosphorylated proteins (<1%) within bacterial proteomes (1, 2). Tyrosine phosphorylation of a small set of bacterial proteins was first linked to specialized cellular events, primarily with the synthesis and export of polysaccharides associated with lipopolysaccharide (LPS) and capsule biosynthesis (35). In contrast, eukaryotic tyrosine phosphorylation has long been considered a major tenet in signal transduction mechanisms, with a diverse array of tyrosine kinases and protein substrates (6). However, within the last 10 years, detailed bacterial and archaeal phosphoproteomic studies have resulted in a fresh view that compares closely to the eukaryotic condition, where tyrosine phosphorylation is a central paradigm. Improved phosphotyrosine peptide enrichment techniques combined with highly sensitive mass spectrometry now reveal that tyrosine phosphorylation is indeed widespread across archaeal and bacterial proteomes (713).


Within a cellular context, several factors are required for a phosphate group to be covalently linked to tyrosine. In brief, a kinase acts to mediate transfer of a phosphate group (PO4) to a protein substrate that has an accessible hydroxyl group on a tyrosine residue.

Tyrosine kinase activity.

The topic of bacterial tyrosine kinases (BY-kinases) has been extensively reviewed by many groups (1416), highlighting the diverse aspects of bacterial tyrosine kinase biology. Notably, most BY-kinases characterized to date reveal a structurally distinct class of proteins from the well-characterized “Hanks-type” eukaryotic tyrosine kinases (14). Furthermore, BY-kinases are also distinct from so-called “eukaryotic” serine/threonine kinases, which are more prevalent in bacterial genomes (17, 18). BY-kinases have two transmembrane domains and typical Walker A and B ATP binding motifs in their cytoplasmic catalytic domain. The ATP binding feature of these proteins supports their eventual autophosphorylation, most often observed on various clustered C-terminal tyrosine residues (1921).
The cell surface-exposed feature of BY-kinases is thought to facilitate extracellular signal integration, leading to cytoplasmic kinase activity and diverse physiological responses (Fig. 1). In the case of Firmicutes, the function of the membrane and cytoplasmic protein domains has been separated into two separate proteins (2224). Here, a membrane-located protein modulator interacts with a separate cytoplasmic protein that carries out kinase activity. It is not exactly clear why this bipartite arrangement has occurred, although separating the functions potentially allows for an expansion of cellular responses. For example, in Bacillus subtilis, TkmA, SalA, and MinD act as independent membrane association partners for the BY-kinase PtkA, which can then dissociate and target various cytoplasmic proteins for phosphorylation (25).
FIG 1 Selected phosphotyrosine-regulated processes in Gram-positive and Gram-negative bacterial cells. The two large boxes indicate a representative bacterial cell and the dashed line represents a cell division site (septum). Arrows indicate the pathway direction of the cellular process. Interrupted lines (in 3 and 5) indicate transcriptional inhibition. (1) Wzc autophosphorylation increases copolymerase activity in E. coli. (2) Tyrosine phosphorylation of DnaK by PtkA during heat shock enhances maintenance of misfolded proteins in Bacillus subtilis. (3) Tyrosine phosphorylation of SalA promotes DNA binding to prevent scoC transcription in B. subtilis. (4) S. pneumoniae CspD requires CpsC to be recruited to cell division site, leading to its autophosphorylation, which supports capsule production. (5) DNA binding and transcriptional repressor activity of Cra is blocked by its tyrosine phosphorylation, leading to LEE-1 gene expression in enterohemorrhagic E. coli. (6) Tyrosine phosphorylation of SSB enhances SSB affinity for DNA in B. subtilis. (7) Phosphorylation of CesT on Y152 results in enhanced effector secretion via the type III secretion system in enteropathogenic E. coli.
Another group of phosphorylating enzymes are known as dual-specificity protein kinases (DSPK) that catalyze the transfer of a phosphate group to serine, threonine, or tyrosine. DSPK have been identified in Bacillus spp., Salmonella enterica serovar Typhimurium, Chlamydophila pneumoniae, and Mycobacterium tuberculosis (11, 2628). While DSPK are common in yeasts, their specific roles in bacteria are modestly understood due to few characterization studies. Interestingly, Escherichia coli with genetic deletions for its two known BY-kinases (Etk and Wzc) was still capable of directing tyrosine phosphorylation for a substantial amount of its cellular proteins (7), suggesting that novel tyrosine kinases remain to be identified or perhaps that known serine/threonine kinases exhibit a dual specificity that includes tyrosine phosphorylation.

Substrate and site specificity.

Kinase substrate specificity is thought to be dependent on the amino acid sequence surrounding the phosphorylation site (29). We are only beginning to understand the nature of bacterial phosphosite motifs, yet differences and some similarities with eukaryotic phosphosites are apparent (7, 8). As more examples of tyrosine-phosphorylated peptides are identified through mass spectrometry-based analyses, conserved motifs will be determined. Phosphotyrosine-specific motifs have recently been proposed for E. coli and Shigella flexneri; however, only two motifs are conserved across both species, YXXXK and YXXK (7, 8). It should be noted that these two motifs are not readily observed in other bacteria with validated phosphotyrosine proteins, suggesting that other mechanistic or temporal determinants could be involved in specific bacterial lineages. Continued work to further investigate the putative motifs and other possible determinants will be valuable for characterizing kinase-substrate specificity. Lastly, cocrystallization studies are required to elucidate structural details about how specific tyrosine kinases interact with conserved tyrosine motifs. These and other challenging approaches are needed to better understand mechanistic aspects of tyrosine phosphorylation.


Beyond the prototypic role in capsule and lipopolysaccharide (LPS) biogenesis, tyrosine phosphorylation in prokaryotic organisms has been functionally linked to regulatory roles in nutrient sensing, protein localization, stress responses, transcriptional regulation, and virulence. Many examples were initially discovered in Gram-positive bacteria, primarily in Bacillus subtilis (reviewed by Mijakovic and Deutscher [30]), with roles for tyrosine phosphorylation recently reported in Gram-negative bacteria.

Capsular and extracellular polysaccharide biogenesis.

Some of the first discoveries for the role of tyrosine phosphorylation in bacteria were based on studies of capsular polysaccharide biogenesis. The role of tyrosine phosphorylation in capsule formation has been a very challenging area of study, and the exact mechanisms involved remain elusive. Multiple lines of evidence clearly indicate that autophosphorylation of the BY-kinase is required. In an apparent dichotomy, dephosphorylation of the requisite BY-kinase, often performed by a cognate phosphatase, is also required for capsule biogenesis. Therefore, it appears likely that a dynamic, multistep process involving reversible tyrosine phosphorylation contributes to capsule biogenesis. Many complex models with additional protein partners have been proposed, and the reader is directed to those reports for specific details (21, 31).

Transcriptional regulation.

Phosphotyrosine modification of many different transcription regulators has been shown to impact DNA binding and, correspondingly, to affect gene expression. Here, tyrosine phosphorylation and dephosphorylation act as a reversible switch for activating or silencing gene expression, allowing a cell to rapidly respond to environmental conditions. The examples identified to date highlight phosphorylation-mediated steric hindrance and protein conformational changes as mechanisms of action.

(i) SalA.

In vitro studies using the B. subtilis transcriptional repressor SalA revealed that efficient binding to specific DNA was ATP dependent (32). Further protein interaction studies showed that SalA interacted with the BY-kinase PtkA, raising the possibility that it is subject to tyrosine phosphorylation. Indeed, mass spectrometric data revealed that SalA was tyrosine phosphorylated at residue 327 by PtkA. Interestingly, this region of SalA is not involved in DNA binding; however, when it is tyrosine phosphorylated, it supports an enhanced ability of the amino-terminal region of SalA to bind ATP, thus resulting in higher binding affinity to DNA (32). Therefore, tyrosine phosphorylation of SalA results in its conformational change, which increases its functionality as a DNA binding transcriptional repressor (Fig. 1).

(ii) Cra.

The Cra transcriptional regulator is a member of the LacI/GalR repressor family, and within enterohemorrhagic E. coli (EHEC), it negatively regulates the first genetic operon within the locus of enterocyte effacement (LEE) pathogenicity island (33). An EHEC phosphotyrosine proteomic study identified Cra Y47 as a phosphosite (7), raising the possibility that Cra modification could impact gene expression. The cognate tyrosine kinase for Cra is not known, and therefore an approach to incorporate a nonhydrolyzable phosphotyrosine structural analog into Cra (in a site-specific manner) was employed (34). Specifically, p-carboxymethylphenylalanine (pCmF) was incorporated into Cra at residue 47, using a heterologous expression system, to study the role of Cra Y47 phosphorylation. The purified Cra protein (Cra-pCmF47) was used for in vitro DNA binding assays, which revealed reduced Cra-pCmF47 binding to the LEE1 operon promoter region compared to that of wild-type Cra or phosphodeficient Cra (Y47F) (Fig. 1). These data suggest that tyrosine phosphorylation of Cra sterically hinders and reduces its DNA binding capacity, which permits LEE1 gene expression, leading to EHEC virulence (35).


Single-stranded DNA binding proteins and other DNA binding proteins.

Single-stranded DNA binding protein(s) (SSB) is found in all organisms and serves to modulate DNA repair, recombination, and replication. In eukaryotes, SSB and other DNA binding proteins are known to be phosphorylated on serine and threonine residues (3638), whereas studies on bacterial SSBs have revealed phosphorylation on tyrosine residues (39). Specifically, a Bacillus subtilis protein kinase, YwqB, was shown to phosphorylate SSB, and phosphorylation increased the DNA binding efficiency of SSB by 200-fold (Fig. 1). Furthermore, the data showed that the tyrosine phosphorylation status of SSB decreases during RecA-dependent DNA repair. It was postulated that DNA bound by SSB would inhibit the nucleation of RecA. Indeed, during the DNA damage response, stimulated by mitomycin, SSB tyrosine phosphorylation status declined, and in a ΔywqD mutant the cells survived at a lower rate than that of wild-type cells. It is likely that SSB is dephosphorylated during the DNA damage response, modulating its binding to DNA and allowing RecA-dependent DNA repair to occur. In another paradigm of DNA damage responses with Deinococcus radiodurans, phosphorylation of RecA at Tyr77 and additionally at Thr318 modifies the activity of RecA by increasing its affinity for double-stranded DNA (dsDNA) (40). It is unclear whether this occurs for RecA homologues in other bacteria, although the above examples of tyrosine phosphorylation of DNA binding proteins involved in DNA repair and recombination mechanisms warrant a fresh look at the dynamic nature of stress responses in bacteria. Future studies that assess phosphoproteomes under stress-inducing conditions will likely provide new discoveries.

DnaK modification upon heat shock.

DnaK is a chaperone protein associated with heat shock responses, and it works in concert with its cochaperones, DnaJ and GrpE, among other proteins, to maintain misfolded or denatured proteins (41). Mass spectrometry analyses with various PtkA-deficient and PtpA-deficient strains (kinase and phosphatase, respectively) demonstrated that B. subtilis DnaK was phosphorylated on two tyrosine residues, Y573 and Y601; however, only Y601 was linked to its ability to respond to heat shock (42). Specifically, it was shown that bacteria expressing DnaK with a Y601F substitution had diminished survival rates after heat shock, and DnaK(Y601F) was deficient for protein interactions with its cochaperones, DnaJ and GrpE. Compared to DnaK sequences in other bacteria, it was noted that some species contain a phenylalanine residue at position 601. The authors posit that some bacteria may have mutated the phenylalanine at position 601 to tyrosine, resulting in a more robust and PtkA kinase-inducible regulation mediated by DnaK.

Cell cycle regulation.

Capsule production in various Streptococcus pneumoniae serotypes is linked to the tyrosine phosphorylation state of CpsD (43), although the detailed mechanism of capsule expression remains elusive and likely includes CpsD dephosphorylation mediated by the phosphatase CpsB (44, 45). A challenging question relating to how S. pneumoniae coordinates capsule expression during cell division was addressed using mutant strains expressing fluorescently tagged CpsC and CpsD proteins (21). CpsD was shown to localize to the cell division site in a CpsC-dependent manner. Furthermore, localization at the cell division site triggered CpsD autophosphorylation and recruitment of CpsH, forming a component of the capsule polymerase machinery. Bacteria expressing nonphosphorylatable CpsD variants were further shown to be deficient for capsule production at the cell division site, thereby linking tyrosine phosphorylation to a temporal-spatial aspect of cell division. Lastly, elongated cells with putative cell division defects were observed for bacteria expressing nonphosphorylatable CpsD, leading to a model where CpsD phosphorylation coordinates proper cell division and daughter cell encapsulation (21). It will be interesting to learn whether this is a widespread mechanism for other capsule-expressing bacteria, including Gram-negative bacteria that contain a single BY-kinase protein structure.


Archaeal genomes typically encode multiple Hanks-type protein serine/threonine kinases, in addition to a variety of serine/threonine phosphatases (46). There are no known tyrosine kinases in the Archaea and there do not appear to be any BY-kinase homologues encoded in archaeal genomes (47). In contrast, specific protein tyrosine phosphatases (PTP) have been identified and characterized (reviewed by Kennelly [46]), suggesting that tyrosine-phosphorylated proteins (i.e., cellular substrates) likely exist. In support of that view, a phosphoproteomic study in the archaeon Sulfolobus solfataricus revealed extensive protein phosphorylation, with the Ser/Thr/Tyr ratio skewed heavily toward phosphotyrosine (25.8/20.6/53.6%) (12). This dramatic skew has not been observed in other archaeal phosphoproteomes to date and is surprising, given the apparent absence of tyrosine kinase homologues in archaeal genomes. For example, a study in Halobacterium salinarum revealed a stark absence of phosphotyrosine proteins, with serine and threonine modifications being well represented (13). It is not clear why the significant skew toward pTyr was observed in the Sulfolobus study, although differences in phosphopeptide enrichment and mass spectrometry methodologies might explain the disparity. The kinase or kinases involved with these modifications have not been functionally identified, and this remains an open set of questions for the field.
Some of the extensive S. solfataricus phosphotyrosine modifications were observed on early enzymes required for gluconeogenesis, pentose and hexose metabolism, and the tricarboxylic acid (TCA) cycle (12). This suggests that many of these enzymes may require tyrosine phosphorylation to regulate specific biochemical reactions in these pathways. Notably, this pattern of tyrosine phosphorylation in proteins linked to sugar metabolism and the TCA cycle compares very closely to the finding for the phosphotyrosine proteome of E. coli (7). This very intriguing similarity between bacterial and archaeal phosphoproteomes will need to be followed up with mechanistic studies to address whether tyrosine phosphorylation impacts protein function toward carbohydrate and cellular metabolism.


Bacterial pathogenesis.

LPS and capsule formation are well understood as cell surface polymers contributing to virulence; therefore, for many bacteria, their respective syntheses broadly associate tyrosine phosphorylation with virulence. Extending beyond that corollary, many proteomic and directed studies have reported reproducible site-specific tyrosine phosphorylation of proteins associated with bacterial pathogenesis. Some of the modified proteins are encoded from pathogenicity islands (PAI) or virulence plasmids and are associated with specialized secretion systems.

T3SS and tyrosine phosphorylation.

Independent studies have reported that a variety of type III secretion system (T3SS)-associated proteins are modified by tyrosine phosphorylation (7, 8, 27, 48). In the cases of EHEC and Shigella, multiple data sets identified more than 10 different phosphotyrosine peptides, suggesting a role for this modification in T3SS-mediated virulence.
In EHEC, the T3SS multicargo chaperone CesT displayed tyrosine phosphorylation on residue Y152 or Y153 (7). Importantly, each independent modification was later linked to different virulence-associated outcomes within a related enteropathogenic Escherichia coli (EPEC) strain (48). CesT comprises 156 amino acids, so the targeted adjacent 152 or 153 residues are very close to the C terminus. Bacteria expressing nonphosphorylatable forms of CesT were severely attenuated for T3SS-mediated intestinal colonization in a mouse infection model, implicating CesT tyrosine phosphorylation as an important event leading to enteric pathogenesis (48). Furthermore, the CesT 152 tyrosine residue was strictly required for the protein expression of the type III effector NleA, revealing an unexpected site-specific regulation activity for a T3SS chaperone protein (further discussed below in context of pTyr binding).
In Shigella flexneri, VirB acts as a master transcriptional regulator of select T3SS genes. A phosphoproteomic study identified the transcriptional regulator VirB as tyrosine phosphorylated on residues Y100 and Y113 (8). Putative phosphomimetic substitutions (tyrosine to glutamic acid) at each corresponding residue resulted in reduced T3SS secretion levels, whereas phosphoablative substitutions had no effect. This led the authors to suggest that VirB tyrosine phosphorylation acts to limit VirB activity toward T3SS genes (8). A Shigella infection plaque assay on HeLa cells revealed a deficiency for bacteria expressing VirB Y100E and no difference for bacteria expressing VirB Y113E. Additional experiments will be required to determine if VirB tyrosine phosphorylation impacts DNA binding or another mechanistic aspect of T3SS transcriptional regulation.
There are other examples of tyrosine-phosphorylated T3SS proteins in EHEC, Shigella spp., and Chlamydia spp. that include conserved apparatus components and translocator proteins (7, 8, 27). Currently, it is unclear what functional roles tyrosine phosphorylation plays in T3SS function, although mechanisms of host sensing, secretory activation, and substrate hierarchy are all unresolved questions in the field (49). Tyrosine phosphorylation could represent a posttranslational reversible mechanism to regulate T3SS function, and thus further investigations will ideally address this and other questions. Notably, threonine phosphorylation serves to activate type six secretion in Pseudomonas aeruginosa and Agrobacterium tumefaciens (50, 51), so a paradigm, albeit an analogous comparison, exists for regulatory protein phosphorylation and secretion system function.

Mycobacterial tyrosine phosphorylation.

Several serine/threonine kinase enzymes (Pkn family, PknA-K) in Mycobacterium tuberculosis that play a functional role in cell growth (52) have been identified, with some associated with virulence (53). Notably, members of the Pkn family have been identified as phosphorylated on tyrosine, despite M. tuberculosis lacking traditional BY-kinases (11). Specifically, PknB, PknD, PknE, PknF, and PknG were shown to catalyze Tyr phosphorylation of protein substrates. Therefore, given these findings, these enzymes should be considered DSPK. These Pkn enzymes likely organize into a network for activation of one another, and protein phosphorylation status has been demonstrated to correspond to various growth conditions. PknA and PknB are both essential for M. tuberculosis replication, with PknB implicated as a key factor in latency regulation. In the case of PknG, several studies have linked it to regulating amino acid metabolism under nutrient-limiting conditions and latency (54, 55). Moreover, M. tuberculosis pknG mutants are defective for survival in macrophages (56, 57) and have been shown to exhibit reduced virulence in mouse and guinea pig infection models (54, 55). Therefore, it appears that members of the Pkn family of enzymes are involved in metabolic functions and, in the context of infection, at least PknG contributes to virulence. There remains the broader question of whether protein tyrosine phosphorylation mediated by Pkn family members contributes to virulence.


In the case of eukaryotes, it is well established that phosphotyrosine modification creates high-affinity binding sites for protein interactions, often supporting regulatory signaling cascades (6). Proteins that bind phosphotyrosine typically have structural polypeptide folds, and common examples include the Src homology 2 (SH2) domain and the protein tyrosine binding (PTB) domain (58). While the current evidence for bacterial pTyr binding domains is scarce, we hypothesize, based on the large amount of phosphotyrosine-modified proteins in bacteria, that prokaryotes have proteins that contextually bind phosphotyrosine, akin to the eukaryotic paradigm.

A putative pTyr binding pocket within CsrA for CesT.

The carbon storage regulator A protein (CsrA) is a well-studied RNA binding protein involved in posttranscription gene regulation in many bacteria (59). It has been reported that the EPEC type III secretion chaperone CesT competitively interacts with CsrA, displacing it from nleA mRNA, thus resulting in type III effector NleA translation to support EPEC pathogenesis (60). A CsrA-CesT cocrystal structure revealed that CesT tyrosine 152 participates in weak hydrogen-bonding interactions with a helical domain of CsrA that typically participates in RNA binding interactions (61). Our studies in EPEC have shown that CesT Y152 is strictly conserved (invariant) and subject to phosphorylation (48). Moreover, mutation of CesT residue 152 to a nonphosphorylatable phenylalanine strictly abrogated NleA effector expression, suggesting a possible role for O-linked phosphorylation in this mechanism. Molecular dynamic simulation modeling with tyrosine phosphorylation of CesT Y152 reveals PO3-mediated tripartite H-bonding coordinated by a contiguous “pocket” encompassed by His43, Val42, and Ser41 of CsrA (Fig. 2). Intriguingly, this exact region is where CsrA binds contextual mRNA “GGA” motifs (59, 62); thus, perhaps, this reveals molecular competition for this localized binding region. While additional work is required to elucidate the CesT-CsrA binding mechanism, it is notable that eukaryotic pTyr binding pockets tend to contain His and Arg residues that provide accessible hydrogen atoms for coordinating H-bonding with protein domains containing phosphate groups (58). Lastly, in the case of EHEC and K-12 E. coli, multiple proteomic data sets demonstrated that CsrA was tyrosine phosphorylated at residue 49, although a functional link was not investigated (7). Given that CsrA is a global regulator of gene expression in many bacteria, we speculate that proteins with appropriate contextual tyrosine phosphorylation modifications could participate in high-affinity CsrA binding interactions with profound regulatory consequences.
FIG 2 Molecular dynamic simulation of a CsrA-CesT pY152 interaction over a 125-ns period. Relevant time frames were extracted to show polar contacts between the pY152 and neighboring CsrA protein domain. The cocrystal within coordinate file PDB entry 5Z38 for the CesT-CsrA dimer of dimers was used for the analyses. Note the tripartite polar contacts formed by CesT pY152 and CsrA residues H43, Val42, and Ser41.

Src homology 2 (SH2) phosphotyrosine binding domains in bacterial species.

Eukaryotic proteins containing SH2 protein domains exhibit high binding affinity for specific phosphotyrosine peptide motifs (63). Such SH2 domains typically encompass 100 amino acids and are identifiable by a characteristic structural protein fold. SH2 domain-mediated binding to a phosphotyrosine in a given polypeptide is dependent on contextually positioned positively charged amino acids within the SH2 domain that provide a binding pocket to coordinate phosphotyrosine binding via electrostatic interactions (64). Furthermore, neighboring amino acids that surround the modified tyrosine within the polypeptide may also interact with allosteric SH2 domain amino acids, thereby providing specificity for these protein-protein interactions. In most cases, bioinformatic searches of bacterial genomes do not identify SH2 domain-encoding genes. In the case of Legionella species, putative SH2-like domains had been detected in a few early studies, although their functional relevance was elusive (65, 66). Given the predominantly intracellular lifestyle of Legionella bacteria within protozoan hosts, it was hypothesized that SH2 domain-encoding genes were acquired through horizontal gene transfer events. With many more genome sequences available, bioinformatic searches of multiple Legionella genomes have revealed 93 SH2 domains within 84 proteins, indicating that the presence of these proteins is not rare and perhaps has biological consequences (67). Notably, some of these Legionella SH2 domain proteins are translocated into host cells via the type IV secretion system (T4SS), raising the intriguing possibility of a role in subversion of pTyr-dependent host signaling cascades. In vitro studies with selected Legionella SH2 domain proteins revealed high binding affinity to a variety of synthetic pTyr-containing peptides with amino acid sequences corresponding to known eukaryotic protein targets (67). Interestingly, the Legionella SH2 domains bound to some pTyr peptides with affinities that exceeded that of the cognate eukaryotic SH2 domain partner. These observations suggested that the Legionella SH2 domain proteins exhibit high binding affinities for phosphotyrosine with relaxed specificity for the overall peptide architecture. Remarkably, one Legionella SH2 protein was found to exhibit higher binding affinity to phosphotyrosine than a rationally designed (i.e., recombinantly engineered) SH2 domain pTyr “superbinder” (68). Crystallography experiments revealed the spatial conformation of a peptide-associated pTyr side chain within the Legionella SH2 domain binding pocket as a major factor contributing to greater binding affinity. Therefore, certain Legionella SH2 domain proteins make extensive electrostatic interactions with pTyr, which collectively contribute to high binding affinity. The detailed functional and infection relevance of these Legionella SH2 domain-pTyr interactions remains elusive; however, their discovery and characterization open a new direction for host-pathogen interaction biology.


In mammalian cells, a central dogma is that kinase-mediated protein phosphorylation contributes to regulated cell signaling. Over 30% of a cell’s proteins can be modified at any given time (6, 69, 70). Critically, dysregulated kinase activity is associated with many diseases, including cancer, and for that reason kinases are widely recognized as being “druggable” targets for cancer treatments. In fact, over 20 tyrosine kinase inhibitor drugs, mostly large antibody complexes, are approved for clinical use, and many more are in the development pipeline (71, 72). Coincidently, phosphorylation studies are continually being pursued to investigate aggressive human cancers (38, 7376), with a goal of improved health care outcomes and novel kinase inhibitor therapies. We and others reason that similar approaches are needed to elucidate tyrosine kinase and phosphorylation aspects of bacterial physiology, with a long-term goal of developing novel bacterial kinase inhibitors (14, 77).
Currently, there are no specific inhibitors that block the function of BY-kinases. BY-kinases are expected to have ATPase activity; thus, it is surprising that known ATPase inhibitors are ineffective against this family of enzymes. This might be due to the slightly different architecture of the Walker box motifs in BY-kinases from that of canonical ATPases (78), although this has not been investigated. Nonetheless, it is worth exploring strategies to inhibit bacterial tyrosine phosphorylation, perhaps leading to novel antimicrobials. The fact that most BY-kinases are multidomain membrane-associated proteins poses a challenge for large inhibitory antibodies to penetrate thick cell envelopes and hydrophobic barriers. A solution might be found by performing small-molecule drug library screening. As with all drug library screens, robust bacterial phenotypes are required, and there is generally a strong preference to target a conserved mechanism for broad-spectrum bacterial inhibition. Therefore, this highlights the need to explore further phosphoproteomic studies with clinically relevant pathogens, toward a goal to identify biologically and structurally conserved tyrosine phosphorylation substrates across diverse bacterial species.


As phosphotyrosine proteomic studies continue to be reported, the field has several challenges ahead to address a widespread yet modestly understood aspect of prokaryotic biology.
A critical set of tasks is to decipher shared and conserved phosphotyrosine mechanisms among species. As most proteomic experiments typically represent a single growth condition or time point, they are essentially snapshots of highly dynamic reversible processes, and hence might only embody part of the picture. Additionally, the issue of reproducibility between experiments and sorting through false positives in phosphotyrosine data sets is yet another hurdle. Lastly, attributing functional mechanisms to tyrosine phosphorylation can be very challenging, especially if the cognate kinase and phosphatase are not known for the given substrate. The use of putative phosphomimetic substitutions (e.g., glutamic acid, aspartic acid) can be informative to restore negatively charged electrostatic interactions, although these residues do not accurately recapitulate the spatial and ionic bonding features uniquely presented by phosphotyrosine. Some researchers have employed innovative synthetic approaches to better mimic phosphotyrosine in proteins (34) or to generate de novo pTyr-containing proteins with engineered E. coli expression strains (79, 80). These and other approaches will be useful for future work addressing phosphotyrosine-dependent mechanisms.
The development of quality bench-side reagents to isolate and detect tyrosine phosphorylation (prior to mass spectrometry) is beneficial when studying protein phosphorylation. The use of commercially available antiphosphotyrosine antibodies has been a successful approach; however, these antibodies are imperfect, as their binding to targets is often amino acid sequence (context) specific, thus introducing unintended bias in some studies. Modifications to established approaches include the use of a “Phos-tag” compound in standard SDS-PAGE to impart protein mobilization differences (81) or the use of phospho-specific protein stains that detect down to 1 to 10 ng of a given phosphoprotein (82). While not phosphotyrosine specific, these detection strategies are cost-effective approaches for most laboratories and are thus valuable to assess workflow and sample content prior to more laborious mass spectrometry analyses. A recent development has been the recombinant engineering and/or cloning of so-called SH2 domain superbinder protein domains (83). It has been shown that these and other superbinders bind to phosphotyrosine with high affinity and reduced contextual sequence bias (67, 83).
After efficient phosphoprotein and phosphopeptide enrichment, the next major advance for the field is quantitative and time-resolved phosphoproteomics, which will help to address temporal aspects of microbial growth, cell signaling, and bacterial infection. Phosphotyrosine-dependent cell physiology occurring on a defined time scale can only be elucidated with time course-based experiments, ideally coupled to quantitative detection of phosphoprotein species. Isobaric peptide labeling approaches such as isobaric tag for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMT) can be applied to experimental workflows prior to mass spectrometry, allowing for quantitative differential measurements of distinct peptides in enriched samples (84). Another approach is stable isotope labeling by amino acids in cell culture (SILAC), which allows for relative quantification of proteins within a sample (85). SILAC has an advantage of labeling proteins in situ; however, it requires auxotrophs for analyses and has a limited labeling capacity based on the amino acid makeup of a given protein.
The inherently reversible nature of tyrosine phosphorylation (mediated by kinases and phosphatases) makes quantitative phosphoproteomics a difficult area to study. Furthermore, steady-state levels of most bacterial phosphoproteins are typically low, especially in bacterial infection samples where mammalian or plant proteins dominate the sample’s protein content. Therefore, innovative quantitative approaches combined with phosphoprotein enrichment techniques will likely be required to elucidate how phosphotyrosine protein modifications globally contribute to prokaryotic cell physiology and regulatory events.
Tyrosine phosphorylation has emerged as a widespread occurrence in bacteria and archaea that in some cases rivals the high levels seen in mammalian systems. As prokaryotic tyrosine phosphorylation has long been understudied, it remains modestly understood. Preliminary studies suggest that other novel enzymes with tyrosine kinase activity exist, suggesting that there remain many phosphotyrosine-dependent cellular mechanisms to discover. Further studies will redefine views around prokaryotic physiology and bacterial pathogenesis. We envision the development of bacterial tyrosine kinase inhibitors as novel approaches to limit bacterial growth during infections or to be used as an adjunct antibiotic therapy in the treatment of challenging bacterial infections.


Research in the Thomas laboratory is supported by an operating grant (RGPIN/05807-2019) from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Dalhousie Medical Research Foundation (DMRF). Research in the Rainey laboratory is supported by NSERC operating grant RGPIN/05907-2017.
The funders had no role in interpretation or the decision to submit the work for publication.


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Published In

cover image Journal of Bacteriology
Journal of Bacteriology
Volume 201Number 191 October 2019
eLocator: 10.1128/jb.00205-19
Editor: William Margolin, McGovern Medical School


Published online: 6 September 2019


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  1. SH2 domain
  2. cell biology
  3. pathogenesis
  4. phosphorylation
  5. protein chaperone
  6. protein tyrosine binding domain
  7. proteomics
  8. transcriptional regulation
  9. tyrosine kinase



Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada
Cameron S. Runte
Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada
Jan K. Rainey
Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada
Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada
Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada
Department of Medicine, Division of Infectious Diseases, Dalhousie University, Halifax, Nova Scotia, Canada


William Margolin
McGovern Medical School


Address correspondence to Nikhil A. Thomas, [email protected].

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