The bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system: regulation by protein phosphorylation and phosphorylation-dependent protein-protein interactions

Abstract

The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components.

FIG 1

Schematic presentation of the phosphorylation cascade formed by the B. subtilis PTS components necessary for the uptake of fructose (PTS^Lev), mannitol (PTS^Mtl), and cellobiose (PTS^Lic). The two general PTS components EI and HPr phosphorylate the EIIAs, which are specific for a certain carbohydrate. B. subtilis contains nine entire PTSs, six PTSs lacking an EIIA component, and one PTS lacking EIIA and EIIB components (49). For the seven incomplete PTSs, EIIA and EIIB components of other PTS, most likely from the same family, probably complement the transport and phosphorylation functions. Nevertheless, the PTSs usually exhibit different sugar specificities. The P∼EIIAs transfer the phosphoryl group to their cognate EIIB, which finally phosphorylates the carbohydrate bound to the corresponding membrane-integral EIIC or, for the fructose-specific levan PTS, to EIIC and EIID. The phosphorylated carbohydrate is subsequently released into the cytoplasm. While the PTS phosphorylation cascades for cellobiose and fructose are formed by EI, HPr, and two distinct EIIA and EIIB proteins, the EIIB component of PTS^Mtl is fused to the C terminus of the EIIC domain and is therefore attached to the cytoplasmic side of the membrane. Shown are also the fructose-1,6-bisphosphate (FBP)-stimulated and ATP-requiring phosphorylation of HPr at Ser-46 as well as the dephosphorylation of P-Ser-HPr, which follows a phosphorolysis reaction with P-Ser-HPr and P_i being the substrates and HPr and pyrophosphate (PP_i) the products (22). The ATP-dependent phosphorylation of HPr occurs in firmicutes but also in many proteobacteria containing HprK/P and the HPr paralogue NPr.

FIG 2

Schematic presentation of the different regulatory domains in PRD-containing proteins. Shown are the B. subtilis antiterminator LicT (which binds to RNA) and several transcription activators (which bind to DNA), including the B. subtilis regulators MtlR and LevR. Antiterminators are composed of two PRDs fused to the RNA binding domain. The two PRDs usually contain four potential sites of PTS-mediated phosphorylation (73). Similarly, MtlR-like transcription activators contain two PRDs fused to the DNA binding and Mga-like domains (71). However, in MtlR-like transcription activators, the PRDs are followed by an EIIB^Gat-like domain and an EIIA^Mtl-like domain. B. subtilis MtlR needs to be activated both by phosphorylation at His-342 and by the interaction of its C-terminal EIIB^Gat- and EIIA^Mtl-like domains (marked with a bracket) with the EIIB^Mtl domain of the mannitol-specific PTS permease MtlA (146) (Fig. 1). The domain order in the B. subtilis transcription activator LevR is different from that in MtlR: the DNA binding and NtrC-like domains are followed by PRD1, EIIA^Man- and EIIB^Gat-like domains, and finally a truncated PRD2 (which contains only one conserved His). In all of the presented proteins the known stimulating phosphorylation sites (by P∼His-HPr) are indicated by red numbers, and inhibitory phosphorylation sites (by P∼EIIA or P∼EIIB) are written in blue (pale blue in MtlR indicates slight phosphorylation). Also presented in this figure is the LevR-like transcription activator ManR from L. monocytogenes, which, similar to the case for MtlR from B. subtilis, needs to be activated by both phosphorylation by P∼His-HPr at His-585 in the EIIA^Man-like domain (A. Zébré, E. Milohanic, and J. Deutscher, unpublished results) and interaction with the EIIB component MpoB (78). It should be noted that the phosphorylation sites are not always conserved. For example, ManR from L. innocua, which is almost identical to L. monocytogenes ManR, was reported to become phosphorylated by P∼His-HPr at His-506 in the PRD1 domain (77). Finally, in some LevR-like regulators of the order Clostridiales, the truncated PRD2 can be replaced with an EIIA^Mtl-like domain, as is the case for the C. beijerinckii protein with ID number YP_001309609.1.

FIG 3

PTS-catalyzed glucose uptake and the HPr/Crh “regulon” in firmicutes. HPr, the central regulator of carbon metabolism in firmicutes, exists in four different forms: unphosphorylated, phosphorylated at His-15, phosphorylated at Ser-46, and doubly phosphorylated. B. subtilis contains in addition the HPr paralogue Crh, which lacks His-15 and therefore can be phosphorylated only at Ser-46. Histidyl-phosphorylated HPr prevails when a less favorable carbon source is utilized, whereas P-Ser-HPr and P-Ser-Crh are formed when glucose or other preferred sugars are metabolized. The utilization of glucose leads to an increase of the concentration of the glycolytic intermediate FBP, which stimulates the kinase function of HprK/P. Either the different forms of HPr (and B. subtilis Crh) interact with their target proteins (YesS, MG synthase, CcpA, and MalK) or P∼His-HPr phosphorylates them (glycerol kinase, PRD-containing transcription activators and antiterminators, and LacS). The following regulatory functions of HPr and Crh and their various phosphorylated forms are presented. (i) Unphosphorylated Crh of B. subtilis interacts with methylglyoxal synthase (MG synthase) and inhibits its activity. Methylglyoxal synthase is an enzyme at the entry point of the methylglyoxal bypass of glycolysis that catalyzes the transformation of dihydroxyacetone-P into methylglyoxal. (ii) P-Ser-HPr as well as P-Ser-Crh interacts with the B. subtilis glycolytic enzyme glyceraldehyde-3-P DH (GAP DH) and inhibits its activity. (iii) P-Ser-HPr and in B. subtilis also P-Ser-Crh interact with CcpA and stimulate its repressor function for CCR by binding to the cre operator sites of numerous catabolic genes. (iv) P-Ser-HPr of lactobacilli and lactococci inhibits maltose uptake by an inducer exclusion mechanism by probably directly interacting with a component of the ABC transporter. (v) P∼His-HPr interacts with and stimulates the B. subtilis transcription activator YesS, which controls the expression of the pectin/rhamnogalacturonan genes. (vi) P∼His-HPr phosphorylates and activates several PRD-containing antiterminators (AT) and transcription activators (TA), and the absence of their phosphorylation during glucose metabolism is used as a CcpA-independent CCR mechanism. (vii) P∼His-HPr also phosphorylates and activates glycerol kinase (GlpK). The absence of GlpK phosphorylation leads to inducer exclusion. (viii) Finally, in streptococci, P∼His-HPr phosphorylates the EIIA^Glc-like domain of LacS and stimulates the lactose/galactose exchange reaction catalyzed by this protein.

FIG 4

Gene arrangements around the hprR gene, encoding a σ⁵⁴-dependent regulator, in different organisms. The hprR gene is represented by the yellow arrows; when it is fused to ptsH, we call the gene ptsH-hprR and the protein accordingly PtsH-HprR. The hprR gene fused to ptsH was discovered in C. acetobutylicum, where a ptsI gene oriented in opposite direction is located upstream from ptsH-hprR. The ptsI gene is expressed from a σ⁵⁴-dependent promoter and therefore probably controlled by PtsH-HprR (CA_C3088). In other organisms, ptsH-hprR is part of the dhaMKL (A. woodii, Awo_c08990) or dhaKLM (C. beijerinckii, Cbei_2147) operon. The latter order of the dha genes is more common and is also found in H. modesticaldum and B. cereus. However, HprR of these two organisms (HM1_0841 and II5_05639, respectively) does not contain an HPr domain fused to its N terminus. Instead, a separate ptsH gene is located downstream from the dhaKLM genes, which is followed by a ptsI gene in H. modesticaldum and by a glpF-like gene in B. cereus. In the latter organism, a gene encoding a glycerol dehydrogenase-like protein (gldA) is located upstream from hprR and oriented in the opposite direction.

FIG 5

PTS-catalyzed glucose uptake and the EIIA^Glc inducer exclusion “regulon” of E. coli. Unphosphorylated EIIA^Glc but not phosphorylated EIIA^Glc interacts with several transport proteins or catabolic enzymes and inhibits their activity. Direct interactions have been shown for the catabolic enzyme glycerol kinase GlpK (88), the ATP binding subunit MalK of the maltose/maltodextrin-specific ABC transport system (89, 90), and the lactose permease LacY (91, 92). The crystal structures of the complexes formed between EIIA^Glc and the ATP-hydrolyzing MalK protein (106) as well as EIIA^Glc and GlpK, which catalyzes the ATP-dependent phosphorylation of glycerol to glycerol-3-P, have been solved (85). The uptake of the other four carbohydrates, l-arabinose, d-galactose, melibiose, and raffinose, is also subject to inducer exclusion, which is prevented when the EIIA^Glc-encoding crr gene is deleted. For the transporters labeled with a question mark, a direct interaction with EIIA^Glc is suggested by genetic data but has so far not been established by using biochemical or immunological methods.