A pseudoautosomal glycosylation disorder prompts the revision of dolichol biosynthesis.

Dolichol is a lipid critical for N-glycosylation as a carrier for activated sugars and nascent oligosaccharides. It is commonly thought to be directly produced from polyprenol by the enzyme SRD5A3. Instead, we found that dolichol synthesis requires a three-step detour involving additional metabolites, where SRD5A3 catalyzes only the second reaction. The first and third steps are performed by DHRSX, whose gene resides on the pseudoautosomal regions of the X and Y chromosomes. Accordingly, we report a pseudoautosomal-recessive disease presenting as a congenital disorder of glycosylation in patients with missense variants in DHRSX (DHRSX-CDG). Of note, DHRSX has a unique dual substrate and cofactor specificity, allowing it to act as a NAD+-dependent dehydrogenase and as a NADPH-dependent reductase in two non-consecutive steps. Thus, our work reveals unexpected complexity in the terminal steps of dolichol biosynthesis. Furthermore, we provide insights into the mechanism by which dolichol metabolism defects contribute to disease.

Lysine Trimethylation in Planktonic and Pellicle Modes of Growth in Acinetobacter baumannii.

Over the past 30 years, Acinetobacter baumannii has been described as an important nosocomial pathogen due to frequent ventilator-associated infections. Many biological processes of A. baumannii remain elusive, such as the formation of an air-liquid biofilm (pellicle). Several studies demonstrated the importance of post-translational modifications (PTMs) in A. baumannii physiology. Here, we investigated K-trimethylation in A. baumannii ATCC 17978 in planktonic and pellicle modes using proteomic analysis. To identify the most high-confidence K-trimethylated peptides, we compared different sample preparation methods (i.e., strong cation exchange, antibody-capture) and processing software (i.e., different database search engines). We identified, for the first time, 84 K-trimethylated proteins, many of which are involved in DNA and protein synthesis (HupB, RplK), transporters (Ata, AdeB), or lipid metabolism processes (FadB, FadD). In comparison with previous studies, several identical lysine residues were observed acetylated or trimethylated, indicating the presence of proteoforms and potential PTM cross-talks. This is the first large-scale proteomic study of trimethylation in A. baumannii and will be an important resource for the scientific community (availability in Pride repository under accession PXD035239).

Human cytosolic transaminases: side activities and patterns of discrimination towards physiologically available alternative substrates.

Transaminases play key roles in central metabolism, transferring the amino group from a donor substrate to an acceptor. These enzymes can often act, with low efficiency, on compounds different from the preferred substrates. To understand what might have shaped the substrate specificity of this class of enzymes, we examined the reactivity of six human cytosolic transaminases towards amino acids whose main degradative pathways do not include any transamination. We also tested whether sugars and sugar phosphates could serve as alternative amino group acceptors for these cytosolic enzymes. Each of the six aminotransferases reacted appreciably with at least three of the alternative amino acid substrates in vitro, albeit at usually feeble rates. Reactions with L-Thr, L-Arg, L-Lys and L-Asn were consistently very slow-a bias explained in part by the structural differences between these amino acids and the preferred substrates of the transaminases. On the other hand, L-His and L-Trp reacted more efficiently, particularly with GTK (glutamine transaminase K; also known as KYAT1). This points towards a role of GTK in the salvage of L-Trp (in cooperation with ω-amidase and possibly with the cytosolic malate dehydrogenase, MDH1, which efficiently reduced the product of L-Trp transamination). Finally, the transaminases were extremely ineffective at utilizing sugars and sugar derivatives, with the exception of the glycolytic intermediate dihydroxyacetone phosphate, which was slowly but appreciably transaminated by some of the enzymes to yield serinol phosphate. Evidence for the formation of this compound in a human cell line was also obtained. We discuss the biological and evolutionary implications of our results.

The metalloprotein YhcH is an anomerase providing N-acetylneuraminate aldolase with the open form of its substrate.

N-acetylneuraminate (Neu5Ac), an abundant sugar present in glycans in vertebrates and some bacteria, can be used as an energy source by several prokaryotes, including Escherichia coli. In solution, more than 99% of Neu5Ac is in cyclic form (≈92% beta-anomer and ≈7% alpha-anomer), whereas <0.5% is in the open form. The aldolase that initiates Neu5Ac metabolism in E. coli, NanA, has been reported to act on the alpha-anomer. Surprisingly, when we performed this reaction at pH 6 to minimize spontaneous anomerization, we found NanA and its human homolog NPL preferentially metabolize the open form of this substrate. We tested whether the E. coli Neu5Ac anomerase NanM could promote turnover, finding it stimulated the utilization of both beta and alpha-anomers by NanA in vitro. However, NanM is localized in the periplasmic space and cannot facilitate Neu5Ac metabolism by NanA in the cytoplasm in vivo. We discovered that YhcH, a cytoplasmic protein encoded by many Neu5Ac catabolic operons and belonging to a protein family of unknown function (DUF386), also facilitated Neu5Ac utilization by NanA and NPL and displayed Neu5Ac anomerase activity in vitro. YhcH contains Zn, and its accelerating effect on the aldolase reaction was inhibited by metal chelators. Remarkably, several transition metals accelerated Neu5Ac anomerization in the absence of enzyme. Experiments with E. coli mutants indicated that YhcH expression provides a selective advantage for growth on Neu5Ac. In conclusion, YhcH plays the unprecedented role of providing an aldolase with the preferred unstable open form of its substrate.

Two Novel Homozygous Mutations in Phosphoglucomutase 3 Leading to Severe Combined Immunodeficiency, Skeletal Dysplasia, and Malformations.

Phosphoglucomutase 3 (PGM3) deficiency is a rare congenital disorder of glycosylation. Most of patients with autosomal recessive hypomorphic mutations in PGM3 encoding for phosphoglucomutase 3 present with eczema, skin and lung infections, elevated serum IgE, as well as neurological and skeletal features. A few PGM3-deficient patients suffer from a more severe disease with nearly absent T cells and severe skeletal dysplasia. We performed targeted next-generation sequencing on two kindred to identify the underlying genetic etiology of a severe combined immunodeficiency with developmental defect. We report here two novel homozygous missense variants (p.Gly359Asp and p.Met423Thr) in PGM3 identified in three patients from two unrelated kindreds with severe combined immunodeficiency, neurological impairment, and skeletal dysplasia. Both variants segregated with the disease in the two families. They were predicted to be deleterious by in silico analysis. PGM3 enzymatic activity was found to be severely impaired in primary fibroblasts and Epstein-Barr virus immortalized B cells from the kindred carrying the p.Met423Thr variant. Our findings support the pathogenicity of these two novel variants in severe PGM3 deficiency.

Global Dynamic Proteome Study of a Pellicle-forming Acinetobacter baumannii Strain.

For several decades, many bacteria, among which A. baumannii, have shown their ability to colonize the upper surface of static liquids, forming a biofilm at the air-liquid interface named pellicle. Despite the ubiquity of these pellicles in both natural and artificial environments, few studies have investigated this biofilm type. The present data set provides the first description of the whole proteome of A. baumannii cells grown as pellicle, using a label-free mass spectrometry approach. Results are in accord with the general findings reporting that sessile bacteria are far more resistant to detrimental conditions than their planktonic counterparts, by the accumulation of stress proteins. The present investigation also confirmed previous studies suggesting a correlation between the pellicle forming ability and the bacterial virulence. Indeed, we showed the up-regulation of numerous virulence factors during the pellicle growth, e.g. phospholipases, adhesion factors, as well as those of the GacAS Two-Component System (TCS) and Type 6 Secretion System (T6SS). We also highlighted that Bam and Tam systems, both related to the OM insertion machinery, play a critical role during pellicle biogenesis. Moreover, sessile bacteria activate several pathways, e.g. iron, magnesium, phosphate pathways, which allows for increasing the panel of nutrient sources.

Protein lysine acetylation in bacteria: Current state of the art.

Post-translational modifications of proteins are key events in cellular metabolism and physiology regulation. Lysine acetylation is one of the best studied protein modifications in eukaryotes, but, until recently, ignored in bacteria. However, proteomic advances have highlighted the diversity of bacterial lysine-acetylated proteins. The current data support the implication of lysine acetylation in various metabolic pathways, adaptation and virulence. In this review, we present a broad overview of the current knowledge of lysine acetylation in bacteria. We emphasize particularly the significant contribution of proteomics in this field.

The N-glycosylation defect in Lec5 and Lec9 CHO cells is caused by absence of the DHRSX gene.

Glycosylation-deficient Chinese hamster ovary (CHO) cell lines have been instrumental in the discovery of N-glycosylation machinery. Yet, the molecular causes of the glycosylation defects in the Lec5 and Lec9 mutants have been elusive, even though for both cell lines a defect in dolichol formation from polyprenol was previously established. We recently found that dolichol synthesis from polyprenol occurs in three steps consisting of the conversion of polyprenol to polyprenal by DHRSX, the reduction of polyprenal to dolichal by SRD5A3 and the reduction of dolichal to dolichol, again by DHRSX. This led us to investigate defective dolichol synthesis in Lec5 and Lec9 cells. Both cell lines showed increased levels of polyprenol and its derivatives, concomitant with decreased levels of dolichol and derivatives, but no change in polyprenal levels, suggesting DHRSX deficiency. Accordingly, N-glycan synthesis and changes in polyisoprenoid levels were corrected by complementation with human DHRSX but not with SRD5A3. Furthermore, the typical polyprenol dehydrogenase and dolichal reductase activities of DHRSX were absent in membrane preparations derived from Lec5 and Lec9 cells, while the reduction of polyprenal to dolichal, catalyzed by SRD5A3, was unaffected. Long-read whole genome sequencing of Lec5 and Lec9 cells did not reveal mutations in the ORF of SRD5A3, but the genomic region containing DHRSX was absent. Lastly, we established the sequence of Chinese hamster DHRSX and validated that this protein has similar kinetic properties to the human enzyme. Our work therefore identifies the basis of the dolichol synthesis defect in CHO Lec5 and Lec9 cells.

Impact of Hypermannosylation on the Structure and Functionality of the ER and the Golgi Complex.

Proteins of the secretory pathway undergo glycosylation in the endoplasmic reticulum (ER) and the Golgi apparatus. Altered protein glycosylation can manifest in serious, sometimes fatal malfunctions. We recently showed that mutations in GDP-mannose pyrophosphorylase A (GMPPA) can cause a syndrome characterized by alacrima, achalasia, mental retardation, and myopathic alterations (AAMR syndrome). GMPPA acts as a feedback inhibitor of GDP-mannose pyrophosphorylase B (GMPPB), which provides GDP-mannose as a substrate for protein glycosylation. Loss of GMPPA thus enhances the incorporation of mannose into glycochains of various proteins, including α-dystroglycan (α-DG), a protein that links the extracellular matrix with the cytoskeleton. Here, we further characterized the consequences of loss of GMPPA for the secretory pathway. This includes a fragmentation of the Golgi apparatus, which comes along with a regulation of the abundance of several ER- and Golgi-resident proteins. We further show that the activity of the Golgi-associated endoprotease furin is reduced. Moreover, the fraction of α-DG, which is retained in the ER, is increased. Notably, WT cells cultured at a high mannose concentration display similar changes with increased retention of α-DG, altered structure of the Golgi apparatus, and a decrease in furin activity. In summary, our data underline the importance of a balanced mannose homeostasis for the secretory pathway.

Altered mannose metabolism in chronic stress and depression is rapidly reversed by vitamin B12.

GDP-Mannose Pyrophosphorylase B (GMPPB) is a key enzyme for glycosylation. Previous studies suggested a dysregulation of GMPBB and mannose in depression. Evidence, however, was sporadic and interventions to reverse these changes are unknown. Here, we show that GMPPB protein, but not RNA abundance is increased in the postmortem prefrontal cortex (PFC) of depressed patients and the chronic variable stress (CVS) mouse-model. This is accompanied by higher plasma mannose levels. Importantly, a single dose of intraperitoneally administered vitamin B12, which has previously been shown to rapidly reverse behavioral symptoms and molecular signatures of chronic stress in mice, normalized GMPPB plasma mannose levels and elevated GDP-mannose abundance. In summary, these data underline metabolic dysregulation in chronic stress and depression and provide further support for rapid effects of vitamin B12 on chronic stress.

Gaseous NO2 induces various envelope alterations in Pseudomonas fluorescens MFAF76a.

Anthropogenic atmospheric pollution and immune response regularly expose bacteria to toxic nitrogen oxides such as NO• and NO2. These reactive molecules can damage a wide variety of biomolecules such as DNA, proteins and lipids. Several components of the bacterial envelope are susceptible to be damaged by reactive nitrogen species. Furthermore, the hydrophobic core of the membranes favors the reactivity of nitrogen oxides with other molecules, making membranes an important factor in the chemistry of nitrosative stress. Since bacteria are often exposed to endogenous or exogenous nitrogen oxides, they have acquired protection mechanisms against the deleterious effects of these molecules. By exposing bacteria to gaseous NO2, this work aims to analyze the physiological effects of NO2 on the cell envelope of the airborne bacterium Pseudomonas fluorescens MFAF76a and its potential adaptive responses. Electron microscopy showed that exposure to NO2 leads to morphological alterations of the cell envelope. Furthermore, the proteomic profiling data revealed that these cell envelope alterations might be partly explained by modifications of the synthesis pathways of multiple cell envelope components, such as peptidoglycan, lipid A, and phospholipids. Together these results provide important insights into the potential adaptive responses to NO2 exposure in P. fluorescens MFAF76a needing further investigations.

GMPPA defects cause a neuromuscular disorder with α-dystroglycan hyperglycosylation.

GDP-mannose-pyrophosphorylase-B (GMPPB) facilitates the generation of GDP-mannose, a sugar donor required for glycosylation. GMPPB defects cause muscle disease due to hypoglycosylation of α-dystroglycan (α-DG). Alpha-DG is part of a protein complex, which links the extracellular matrix with the cytoskeleton, thus stabilizing myofibers. Mutations of the catalytically inactive homolog GMPPA cause alacrima, achalasia, and mental retardation syndrome (AAMR syndrome), which also involves muscle weakness. Here, we showed that Gmppa-KO mice recapitulated cognitive and motor deficits. As structural correlates, we found cortical layering defects, progressive neuron loss, and myopathic alterations. Increased GDP-mannose levels in skeletal muscle and in vitro assays identified GMPPA as an allosteric feedback inhibitor of GMPPB. Thus, its disruption enhanced mannose incorporation into glycoproteins, including α-DG in mice and humans. This increased α-DG turnover and thereby lowered α-DG abundance. In mice, dietary mannose restriction beginning after weaning corrected α-DG hyperglycosylation and abundance, normalized skeletal muscle morphology, and prevented neuron degeneration and the development of motor deficits. Cortical layering and cognitive performance, however, were not improved. We thus identified GMPPA defects as the first congenital disorder of glycosylation characterized by α-DG hyperglycosylation, to our knowledge, and we have unraveled underlying disease mechanisms and identified potential dietary treatment options.

The putative Escherichia coli dehydrogenase YjhC metabolises two dehydrated forms of N-acetylneuraminate produced by some sialidases.

Homologues of the putative dehydrogenase YjhC are found in operons involved in the metabolism of N-acetylneuraminate (Neu5Ac) or related compounds. We observed that purified recombinant YjhC forms Neu5Ac from two dehydrated forms of this compound, 2,7-anhydro-N-acetylneuraminate (2,7-AN) and 2-deoxy-2,3-didehydro-N-acetylneuraminate (2,3-EN) that are produced during the degradation of sialoconjugates by some sialidases. The conversion of 2,7-AN into Neu5Ac is reversible and reaches its equilibrium when the ratio of 2,7-AN to Neu5Ac is ≈1/6. The conversion of 2,3-EN is irreversible, leading to a mixture of Neu5Ac and 2,7-AN. NMR analysis of the reaction catalysed by YjhC on 2,3-EN indicated that Neu5Ac was produced as the α-anomer. All conversions require NAD+ as a cofactor, which is regenerated in the reaction. They appear to involve the formation of keto (presumably 4-keto) intermediates of 2,7-AN, 2,3-EN and Neu5Ac, which were detected by liquid chromatography-mass spectrometry (LC-MS). The proposed reaction mechanism is reminiscent of the one catalysed by family 4 ß-glycosidases, which also use NAD+ as a cofactor. Both 2,7-AN and 2,3-EN support the growth of Escherichia coli provided the repressor NanR, which negatively controls the expression of the yjhBC operons, has been inactivated. Inactivation of either YjhC or YjhB in NanR-deficient cells prevents the growth on 2,7-AN and 2,3-EN. This confirms the role of YjhC in 2,7-AN and 2,3-EN metabolism and indicates that transport of 2,7-AN and 2,3-EN is carried out by YjhB, which is homologous to the Neu5Ac transporter NanT.

Global Profiling of Lysine Acetylation in Borrelia burgdorferi B31 Reveals Its Role in Central Metabolism.

The post-translational modification of proteins has been shown to be extremely important in prokaryotes. Using a highly sensitive mass spectrometry-based proteomics approach, we have characterized the acetylome of B. burgdorferi. As previously reported for other bacteria, a relatively low number (5%) of the potential genome-encoded proteins of B. burgdorferi were acetylated. Of these, the vast majority were involved in central metabolism and cellular information processing (transcription, translation, etc.). Interestingly, these critical cell functions were targeted during both ML (mid-log) and S (stationary) phases of growth. However, acetylation of target proteins in ML phase was limited to single lysine residues while these same proteins were acetylated at multiple sites during S phase. To determine the acetyl donor in B. burgdorferi, we used mutants that targeted the sole acetate metabolic/anabolic pathway in B. burgdorferi (lipid I synthesis). B. burgdorferi strains B31-A3, B31-A3 ΔackA (acetyl-P- and acetyl-CoA-) and B31-A3 Δpta (acetyl-P+ and acetyl-CoA-) were grown to S phase and the acetylation profiles were analyzed. While only two proteins were acetylated in the ΔackA mutant, 140 proteins were acetylated in the Δpta mutant suggesting that acetyl-P was the primary acetyl donor in B. burgdorferi. Using specific enzymatic assays, we were able to demonstrate that hyperacetylation of proteins in S phase appeared to play a role in decreasing the enzymatic activity of at least two glycolytic proteins. Currently, we hypothesize that acetylation is used to modulate enzyme activities during different stages of growth. This strategy would allow the bacteria to post-translationally stimulate the activity of key glycolytic enzymes by deacetylation rather than expending excessive energy synthesizing new proteins. This would be an appealing, low-energy strategy for a bacterium with limited metabolic capabilities. Future work focuses on identifying potential protein deacetylase(s) to complete our understanding of this important biological process.

The aliphatic amidase AmiE is involved in regulation of Pseudomonas aeruginosa virulence.

We have previously shown that the eukaryotic C-type natriuretic peptide hormone (CNP) regulates Pseudomonas aeruginosa virulence and biofilm formation after binding on the AmiC sensor, triggering the amiE transcription. Herein, the involvement of the aliphatic amidase AmiE in P. aeruginosa virulence regulation has been investigated. The proteome analysis of an AmiE over-producing strain (AmiE+) revealed an expression change for 138 proteins, including some that are involved in motility, synthesis of quorum sensing compounds and virulence regulation. We observed that the AmiE+ strain produced less biofilm compared to the wild type, and over-produced rhamnolipids. In the same line, AmiE is involved in P. aeruginosa motilities (swarming and twitching) and production of the quorum sensing molecules N-acyl homoserine lactones and Pseudomonas Quinolone Signal (PQS). We observed that AmiE overproduction reduced levels of HCN and pyocyanin causing a decreased virulence in different hosts (i.e. Dictyostelium discoideum and Caenorhabditis elegans). This phenotype was further confirmed in a mouse model of acute lung infection, in which AmiE overproduction resulted in an almost fully virulence decrease. Taken together, our data suggest that, in addition to its role in bacterial secondary metabolism, AmiE is involved in P. aeruginosa virulence regulation by modulating pilus synthesis and cell-to-cell communication.

Proteomic characterization of Nα- and Nε-acetylation in Acinetobacter baumannii.

Nα- and Nε-acetylation represent a pivotal post-translational modification used by both eukaryotes and prokaryotes to modulate diverse biological processes. Acinetobacter baumannii has been described as an important nosocomial pathogen for the past 30 years, frequently involved in ventilator-associated pneumonia, bloodstream and urinary tract infections. Many aspects of the biology of A. baumannii remain elusive, in particular the extent and function of N-acetylation. We investigated here N-acetylation in A. baumannii strain ATCC 17978 by proteomic analysis, and we showed the usefulness of using different analytical approaches. Overall, we identified 525 N-acetylated proteins in which, 145 were Nα-acetylated and 411 were Nε-acetylated. Among them, 41 proteins carried both types of N-acetylation. We found that N-acetylation may play a role in biofilm formation, bacterial virulence (e.g. in several iron acquisition pathways), as well as a number of phenotypes, such as, stress adaptation and drug resistance. BIOLOGICAL SIGNIFICANCE: This study is the first to perform the N-acetylome of A. baumannii using different analytical approaches. Each analytical tool permitted to characterize distinctive modified peptides. The combination of all these methods allowed us to identify 145 and 411 Nα- and Nε-acetylated proteins. Besides the fact that acetylation was involved in central metabolism as previously described in other bacteria, some N-acetylated proteins showed interesting role in bacterial virulence (iron acquisition), biofilm formation, stress adaptation and drug resistance of A. baumannii.

Skin-bacteria communication: Involvement of the neurohormone Calcitonin Gene Related Peptide (CGRP) in the regulation of Staphylococcus epidermidis virulence.

Staphylococci can sense Substance P (SP) in skin, but this molecule is generally released by nerve terminals along with another neuropeptide, Calcitonin Gene Related Peptide (CGRP). In this study, we investigated the effects of αCGRP on Staphylococci. CGRP induced a strong stimulation of Staphylococcus epidermidis virulence with a low threshold (<10-12 M) whereas Staphylococcus aureus was insensitive to CGRP. We observed that CGRP-treated S. epidermidis induced interleukin 8 release by keratinocytes. This effect was associated with an increase in cathelicidin LL37 secretion. S. epidermidis displayed no change in virulence factors secretion but showed marked differences in surface properties. After exposure to CGRP, the adherence of S. epidermidis to keratinocytes increased, whereas its internalization and biofilm formation activity were reduced. These effects were correlated with an increase in surface hydrophobicity. The DnaK chaperone was identified as the S. epidermidis CGRP-binding protein. We further showed that the effects of CGRP were blocked by gadolinium chloride (GdCl3), an inhibitor of MscL mechanosensitive channels. In addition, GdCl3 inhibited the membrane translocation of EfTu, the Substance P sensor. This work reveals that through interaction with specific sensors S. epidermidis integrates different skin signals and consequently adapts its virulence.

Transport and Catabolism of Pentitols by Listeria monocytogenes.

Transposon insertion into Listeria monocytogenes lmo2665, which encodes an EIIC of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), was found to prevent D-arabitol utilization. We confirm this result with a deletion mutant and show that Lmo2665 is also required for D-xylitol utilization. We therefore called this protein EIICAxl. Both pentitols are probably catabolized via the pentose phosphate pathway (PPP) because lmo2665 belongs to an operon, which encodes the three PTSAxl components, two sugar-P dehydrogenases, and most PPP enzymes. The two dehydrogenases oxidize the pentitol-phosphates produced during PTS-catalyzed transport to the PPP intermediate xylulose-5-P. L. monocytogenes contains another PTS, which exhibits significant sequence identity to PTSAxl. Its genes are also part of an operon encoding PPP enzymes. Deletion of the EIIC-encoding gene (lmo0508) affected neither D-arabitol nor D-xylitol utilization, although D-arabitol induces the expression of this operon. Both operons are controlled by MtlR/LicR-type transcription activators (Lmo2668 and Lmo0501, respectively). Phosphorylation of Lmo0501 by the soluble PTSAxl components probably explains why D-arabitol also induces the second pentitol operon. Listerial virulence genes are submitted to strong repression by PTS sugars, such as glucose. However, D-arabitol inhibited virulence gene expression only at high concentrations, probably owing to its less efficient utilization compared to glucose.

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

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.