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ScienceDirect Post-translational modifications as key regulators of bacterial metabolic fluxes Tippapha Pisithkul1,2, Nishaben M Patel2 and Daniel Amador-Noguez2 In order to survive and compete in natural settings, bacteria must excel at quickly adapting their metabolism to fluctuations in nutrient availability and other environmental variables. This necessitates fast-acting post-translational regulatory mechanisms, that is, allostery or covalent modification, to control metabolic flux. While allosteric regulation has long been a well-established strategy for regulating metabolic enzyme activity in bacteria, covalent post-translational modes of regulation, such as phosphorylation or acetylation, have previously been regarded as regulatory mechanisms employed primarily by eukaryotic organisms. Recent findings, however, have shifted this perception and point to a widespread role for covalent posttranslational modification in the regulation of metabolic enzymes and fluxes in bacteria. This review provides an outline of the exciting recent advances in this area. Addresses 1 Cellular and Molecular Biology, University of Wisconsin-Madison, United States 2 Department of Bacteriology, University of Wisconsin-Madison, United States Corresponding author: Amador-Noguez, Daniel ([email protected])

Current Opinion in Microbiology 2015, 24:29–37 This review comes from a themed issue on Cell regulation Edited by Carol Gross and Angelika Gru¨ndling

http://dx.doi.org/10.1016/j.mib.2014.12.006 1369-5274/# 2015 Elsevier Ltd. All rights reserved.

Introduction During the last decade we have witnessed a resurgence in metabolic research and a renewal of efforts toward understanding metabolism and the regulation of metabolic fluxes. This renewed interest, propelled by modern mass spectrometry and advances in computational modeling of metabolic networks, is hardly surprising given the central role of metabolism in human health, synthetic biology, and metabolic engineering [1]. The regulation of metabolic enzyme activity, which determines metabolic flux, occurs at different levels: www.sciencedirect.com

transcriptional, translational, and post-translational. In bacterial systems, a wealth of research has shown that regulation of metabolic fluxes is frequently extremely rapid, on the order of seconds to a few minutes [2–6]. This speedy response, which correlates well with the expected need of bacteria to rapidly respond to environmental perturbations, emphasizes the requirement of fast-acting post-translational regulatory mechanisms of metabolic flux control: that is, allosteric regulation and covalent protein modification [7]. The role of allostery in controlling metabolic fluxes in bacteria is well established and continues to be strengthened by exciting new research [7,8]. However, although the regulation of bacterial metabolic enzymes by posttranslational modification (PTM) has long been known via classical examples such as inhibition of glutamine synthetase (GS) by adenylylation [9] or isocitrate dehydrogenase (Icd) by phosphorylation [10], the widespread role that PTMs play in the regulation of metabolic fluxes has only started to come into full light in recent years.

Posttranslational covalent modifications are prevalent in bacteria Through the application of mass-spectrometry-based proteomics methods, it is now clear that PTMs, particularly acetylation and phosphorylation, are highly prevalent in bacteria [3,11]. Phosphoproteome and acetylome studies in an increasing number of bacteria, including Escherichia coli [12–15], Bacillus subtilis [16], Streptococcus pneumoniae [17], Mycobacterium tuberculosis [18], Salmonella enterica [19], Staphylococcus aureus [20], Thermus thermophilus [21,22], Mycoplasma pneumoniae [23], Rhodopseudomonas palustris [24], and Corynebacterium glutamicum [25], have shown that a significant fraction of their proteomes is subject to PTM. Although proteins with many types of functions are subject to PTM, a unifying trend in these studies has been the high prevalence of PTMs among metabolic enzymes (Figure 1 and Table 1).

N-Lysine acetylation and the regulation of acyl-CoA synthetases N-Lysine acetylation (KAc) is a reversible PTM best known for its role in controlling eukaryotic gene regulation through histone modification [26]. KAc is catalyzed by acetyltransferases and reversed by deacetylases, although it may also occur non-enzymatically [27]. The acyl group transferred onto lysine residues comes most frequently from acetyl-CoA [28]. KAc is prevalent in bacterial proteomes, and the acetylation of metabolic Current Opinion in Microbiology 2015, 24:29–37

30 Cell regulation

Figure 1

Glk

G1P

D-glucose

Pgm

G6P Pgi

Glycolytic enzymes

F6P

Gluconeogenic enzymes

Pfk

Fbp

TCA cycle enzymes

FBP Fba

Activating PTM Inhibitory PTM

Tpi

GAP

No functional evidence

DHAP

GapA

1,3BPG

Acetylated

Succinylated

Pupylated

Glutarylated

Phosphorylated

Pgk

Adenylylated

Uridylylated

3PG Gpm

Post-translational modifiers

2PG

Phosphorylation (+)

Eno

Pck

/

(–)

Protein-protein interaction

PEP Pyk

Pyruvate

AceK

PDH complex

Acetyl-CoA Ppc

CobB PatZ GltA

Oxaloacetate

Citrate

Malate

AceA

Fumarate AceA

Gdh

α-KG

Glu

Gln

(–)

Succinyl-CoA Lpd

Succinate

GS

Icd

SucAB

SdhA

PII AR UT

Isocitrate

Glyoxylate

FumA

UR AT PII

AcnA

Mdh

SdhBC

GS AceK

(–)

GOGAT GarA

(+)

SucCD Current Opinion in Microbiology

Post-translational modifications (PTMs) are ubiquitous in bacterial central metabolic enzymes. The PTMs shown here were identified in E. coli, S. enterica, R. palustris, M. smegmatis, M. tuberculosis, B. subtilis, T. thermophilus, and/or S. cerevisiae as referenced in the main text and in Table 1. The in vivo functionality of most PTMs remains to be elucidated (non-colored symbols); if known, it is indicated by green (activating effect) or red (inhibitory effect). For example: isocitrate dehydrogenase phosphatase/kinase (AceK) phosphorylates isocitrate dehydrogenase (Icd) to inhibit its function; in turn, AceK is negatively controlled by PatZ-mediated acetylation. As a second example: adenylyltransferase and adenylylremoving enzyme (AT/AR) regulates glutamine synthetase (GS) activity; PII, which is controlled by uridylyltransferase/uridylyl-removing enzyme (UT/UR), in turn determines the activity (adenylylation versus de-adenylylation) of AT/AR. See text for additional details. Metabolite abbreviations: 1,3BPG, 1,3-bisphosphoglycerate; a-KG, a-ketoglutarate; DHAP, dihydroxyacetone phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6bisphosphate; G1P, glucose-1-phosphate; G2P, 2-phosphoglycerate; G3P, 3-phosphoglycerate; G6P, glucose-6-phosphate; GAP, glyceraldehyde3-phosphate; Gln, glutamine; Glu, glutamate; PEP, phosphoenolpyruvate. Enzyme abbreviations: AceA, isocitrate lyase; AcnA, aconitate hydratase; Eno, enolase; Fba, FBP aldolase; Fbp, FBP phosphatase; FumA, fumarase; GapA, GAP dehydrogenase; Gdh, glutamate dehydrogenase; Glk, glucokinase; GltA, citrate synthase; Gpm, phosphoglycerate mutase; Lpd, a-KG dehydrogenase complex; Mdh, malate dehydrogenase; Pck, PEP carboxykinase; Pdh, pyruvate dehydrogenase; Pfk, 6-phosphofructokinase; Pgi, phosphoglucose isomerase; Pgk, phosphoglycerate kinase; Pgm, phosphoglucomutase; Ppc, PEP carboxylase; Pyk, pyruvate kinase; SdhABC, succinate dehydrogenase; SucAB, SucCD, succinyl-CoA synthetase; Tpi, triose phosphate isomerase. Current Opinion in Microbiology 2015, 24:29–37

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PTMs regulate bacterial metabolic fluxes Pisithkul, Patel and Amador-Noguez 31

Table 1 Post-translational modifications (PTMs) in bacterial metabolic enzymes Type of PTM Lysine acetylation

Phosphorylation

Succinylation

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Major findings AcP is a critical regulator of acetylation: mutants unable to produce AcP had reduced levels of acetylation. AcP may acetylate proteins non-enzymatically. Acs is post-translationally regulated by lysine acetylation, resulting in inactivation of adenylate intermediate synthesis. Acs Activation is achieved by CobB-mediated deacetylation. AcuA acetyltransferase, NAD+-dependentSrtN deacetylase, and NAD+-independent AcuC regulate Acs activity via reversible lysine acetylation Lysine acetylation mediated by AcuA-like acetyltransferase and SrtN-like deacetylase regulates Acs activity. Expression of lysine acetyltransferase gene patZ and acs gene is upregulated by cAMP, indicating that multiple regulatory mechanisms are interconnected and interdependent. Many different AMP-forming acyl-CoA synthetases are negatively regulated by lysine acetylation. Enzymes regulating central metabolism are reversibly acetylated mainly by acetyltransferase, Pat, and deacetylase, CobB. Dependent on cAMP, MsPat acetylates lysine residue on and inactivates Acs. Acetate metabolism is regulated by reversible acetylation of Acs by MsPat and deacetylase, MsSrtN. cAMP allosterically activates MsPatA. Identified 332 lysine acetylation sites on 185 proteins. Most acetylated proteins are involved in central metabolism and protein synthesis Glycolytic enzymes, Pgm and Pyk, are differentially phosphorylated during glucose starvation. FBP aldolase, FdaB, and glyoxalase III, HchA, are differentially phosphorylated during nitrosative stress. Multiple phosphorylation of SahH negatively affects its enzyme activity. SahH phosphorylation plays a key role in regulating intracellular concentrations of SAH, adenosine, and L-homocysteine. Phosphorylation inhibits SahH by decreasing its affinity for NAD+. Non-phosphorylated GarA inhibits TCA cycle and favors glutamate synthesis. Threonyl-phosphorylation of PPDK leads to increased lipid production. HPrK/P phosphorylates Crh in vivo. The HPrK/P-catalyzed phosphorylation plays a key role in carbon catabolite repression. Crh is differentially phosphorylated over the course of carbon utilization. Succinylation is globally regulated by succinyl-CoA concentration. Overlap exists between succinylation and acetylation.

PTMs effects

Organisms

References

N/A

E. coli

[39]

Lysine acetylation inactivates Acs.

S. enterica

[35]

Lysine acetylation inactivates Acs.

B. subtilis

[31]

Lysine acetylation inactivates Acs.

S. erythraea

[66]

cAMP upregulates pat and acs expression

E. coli

[36]

Lysine acetylation inactivates acylCoA synthetases

R. palustris

[24]

Acetylation increases GapA activity and decreases AceA and AceK activity

S. enterica

[19]

cAMP allosterically activates MsPat, which in turn inactivates Acs

M. tuberculosis

[33]

M. smegmatis

[32]

N/A

B. subtilis

[31]

N/A

S. aureus

[41]

Phoshorylation reduces SahH activity

M. tuberculosis

[44]

Phosphorylation decreases SahH ability to bind NAD+ Phosphorylation inhibits GarA binding to its target enzymes N/A

M. tuberculosis

[45]

M. tuberculosis

[56]

R. palustris

[46]

Non-phosphorylated Crh inhibits MgsA in the methylglyoxal bypass while CrhP represses GapA activity.

B. subtilis

[51,52]

N/A

E. coli

[59]

Current Opinion in Microbiology 2015, 24:29–37

32 Cell regulation

Table 1 (Continued ) Type of PTM Pupylation

Glutarylation

Major findings

PTMs effects

Identified pupylation sites on proteins, numbers of which are involved in metabolism. Most of the pupylated proteins in this proteasome-lacking organism are involved in metabolism and translation. 23 glutarylated proteins were identified in E. coli, several of which were central metabolic enzymes.

Organisms

References

N/A

M. tuberculosis

[62]

N/A

C. glutamicum

[25]

N/A

E. coli

[58]

enzymes appears as a common feature across all tested bacteria [13,19,21,29–33]. For example, a large fraction of E. coli enzymes involved in sugar, amino acid and nucleotide metabolism can be lysine-acetylated [34], as well as nearly all central metabolic enzymes in S. enterica and B. subtilis [19,31]. The role of KAc in controlling bacterial metabolic enzymes is perhaps best demonstrated by the regulation of acyl-CoA synthetases (Table 1 and Figure 2a). This was first revealed in S. enterica, where acetyl-CoA synthetase (Acs) is inactivated by KAc near its active site [35]. Re-activation of the acetylated enzyme requires the NAD+-dependent deacetylase CobB, whose deletion impairs growth in acetate as a carbon source [35]. In some bacteria, Acs acetylation may be controlled by intracellular metabolic signals. In E. coli, for example, the expression of acetyltransferase ( patZ) and acetyl-CoA synthetase (acs) are concomitantly upregulated by cAMP, a second messenger metabolite that accumulates during glucose starvation [36] (Figure 2b). Although the simultaneous transcriptional upregulation of both Acs and the acetylase that inactivates it may seem paradoxical, it can be understood as a nuanced system for fine-tuning Acs

activity in which acetylation by patZ serves as a second regulatory mechanism that acts on top of transcription. The transcriptional co-regulation of Acs and its cognate deacetylase also occurs in B. subtilis [37] (Figure 2b). Such multilayered systems highlight the interdependence between transcriptional and post-transcriptional control mechanisms that takes place in the regulation on metabolic pathways. An example of a more direct regulation of Acs acetylation by cAMP has been recently described in mycobacteria, where acetyltransferases are allosterically activated by cAMP [32,38]. In M. tuberculosis, cAMP level increases rapidly in cells engulfed by macrophages [38]; the cAMP-mediated acetyltransferase activation and the ensuing inactivation of Acs have been suggested to play an important role in its pathogenicity [33]. An additional level of complexity in Acs regulation by KAc is seen in B. subtilis, where two deacetylases are found: a NAD+-independent deacetylase, AcuC, and a sirtuin-type NAD+-dependent deacetylase, SrtN [31] (Figure 2a). These deacetylases may respond to different indicators of metabolic status. The inactivation of Acs

Figure 2

(a)

(b)

Acetyl-CoA

Acs

CoA PatZ/AcuA

Acetate

Acs CoA

NAD+

OAADPr

AcuC CobB/SrtN

Low glucose: MP P cA cAM

CRPCRP

acs

MP P cA cAM

CRPCRP

Acetyl-CoA Acetylation

E. coli

patZ

High glucose: CcpA

CcpA

acsA

acuA

B. subtilis Current Opinion in Microbiology

Post-translational and transcriptional regulation of acetyl-CoA synthase in E. coli and B. subtilis. (a) Acetylation of acetyl-CoA synthetase (Acs) by PatZ (in E. coli) or AcuA (in B. subtilis) negatively regulates its activity. The NAD+-dependent de-acetylation and reactivation of Acs is mediated by CobB (in E. coli) or SrtN (in B. subtilis). In addition, B. subtilis also posses a NAD+-independent deacetylase, AcuC. (b) In E. coli, during glucose starvation or growth in acetate, the expression of patZ and acs is concomitantly up-regulated by cAMP-CRP [35,36]. In B. subtilis, when glucose is abundant, CcpA transcriptionally inhibits both acsA and acuA [11,37]. Abbreviations. AcuA, acetoin utilization protein A; AcuC, acetoin utilization protein C; cAMP, cyclic adenosine monophosphate; CcpA, catabolite control protein A; CobB, NAD+-dependent lysine deacetylase; CRP, cAMP receptor protein; NAD+, nicotinamide adenine dinucleotide; OAADPr, 20 -O-acetyl-ADP-ribose; SrtN, sirtuin (silent information regulator 2 (Sir2)) NAD+-dependent deacetylase. Current Opinion in Microbiology 2015, 24:29–37

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PTMs regulate bacterial metabolic fluxes Pisithkul, Patel and Amador-Noguez 33

by acetylation is thought to be favored by high acetyl-CoA levels, which are indicative of an adequate intracellular supply of carbon, and can be therefore understood as a negative feedback mechanism that prevents uncontrolled acetyl-CoA production and ATP depletion. AcuC-mediated deacetylation of Acs may occur in response to a low acetyl-CoA/CoA ratio, helping maintain acetyl-CoA levels. SrtN-mediated deacetylation may instead take place in response to a low NADH/NAD+ ratio, working as part of a regulatory mechanism that helps maintain an adequate supply of both carbon and reducing power. Acetyl-CoA and NAD+ may not be the only metabolic triggers for KAc in bacteria. A recent study found that global KAc levels in E. coli increase during stationary phase in a manner that is dependent on the formation of acetyl-phosphate (AcP) through glycolysis. Mutant cells defective in the production of AcP showed significantly reduced global acetylation levels, while mutant cells that accumulate AcP displayed significantly elevated acetylation levels [39]. It was further shown that AcP could chemically acetylate lysine residues in vitro, suggesting that AcP may acetylate proteins non-enzymatically within cells, with potential global effects on enzymatic activity and metabolism [39]. Although KAc of metabolic enzymes is widespread in bacteria, its potential role in regulating other enzymes beside acyl-CoA synthetases is still unclear. An attempt at understanding the global role of KAc was performed in S. enterica [19]. The authors examined KAc status under glycolysis and gluconeogenesis conditions. Using metabolic flux profiling, they found that the glycolysis/ gluconeogenesis flux ratio was higher in DcobB (deacetylase) mutants compared to wildtype in the presence of glucose. Consistently, this flux ratio was lower in Dpat (acetyltransferase) mutants compared to wildtype. These observations strongly suggested that acetylated enzymes favor glycolysis. Furthermore, in vitro studies demonstrated that Pat treatment increases glyceraldehyde-3-phosphate dehydrogenase (GapA) activity in glycolysis while reducing its activity in gluconeogenesis [19]. However, a subsequent report could not reproduce some of the key results in this study and additional studies are needed to properly address how KAc regulates flux in glycolysis/ gluconeogenesis [24].

The diverse roles of phosphorylation in metabolic regulation Although protein phosphorylation is best known for its role in eukaryotic signal transduction, a growing body of evidence now demonstrates that this PTM has key regulatory functions in bacteria as well. Phosphoproteomic studies on diverse bacterial species have identified large numbers of phosphorylation events on Ser, Thr, and Tyr residues, many of them in metabolic enzymes [14,16,22,40–43]. Hansen et al. investigated the prevalence www.sciencedirect.com

of tyrosine-phosphorylation (pTyr) in E. coli and identified 512 pTyr sites on 342 proteins, including glycolytic and tricarboxylic acid (TCA) cycle enzymes such as FbaAB (fructose-1,6-bisphosphate aldolase), GapA (glyceraldehyde-3-phosphate dehydrogenase), PgK (phosphoglycerate kinase), Eno (enolase), AceA (isocitrate lyase), and FumA (fumarase) (Figure 1). Although the authors demonstrated the physiological relevance of pTyr for virulence, the effect of pTyr on the activity of these metabolic enzymes was not addressed [12]. In the human pathogen S. aureus, the phosphorylation status of the glycolytic enzymes Pgm (phosphoglucomutase) and Pyk (pyruvate kinase) was affected by glucose starvation while that of fructose-1,6-bisphosphate aldolase, FdaB, and glyoxalase III, HchA, was modulated under nitric oxide stress. A more direct link for the role of phosphorylation in metabolic regulation can be found in the pathogen M. tuberculosis, in which the enzyme S-adenosyl-L-homocysteine hydrolase (SahH) can be phosphorylated at multiple sites by Ser/Thr protein kinases negatively affecting its activity [44]. SahH catalyzes the hydrolysis of S-adenosyl-L-homocysteine (SAH), a by-product of SAM-dependent methyltransferase reactions, into free adenosine and L-homocysteine. Methyltransferase reactions are involved in a large number of cellular processes, including DNA replication and repair, cofactor and lipid biosynthesis, methionine metabolism, and others. The findings in this study suggest that phosphorylation of SahH plays a critical role in regulating the SAH/SAM balance in bacteria, with potentially widespread repercussions for cellular methylation processes [44]. Expanding upon these results, a subsequent study reported that the mechanism by which SahH phosphorylation inhibits its activity may be by decreasing its affinity toward the essential cofactor NAD+ [45]. Another example of the regulation of metabolic enzymes by phosphorylation is provided by a recent phosphoproteomic analysis of the phototrophic bacterium R. palustris. This study demonstrated that the activity of pyruvate phosphate dikinase (PPDK), a key enzyme involved in glycolysis/gluconeogenesis and CO2 assimilation, is positively regulated by phosphorylation [46]. The authors demonstrated that lipid production during photoheterotrophic growth is enhanced by threonyl-phosphorylation of PPDK. It was hypothesized that increased activity of phosphorylated PPDK leads to high intracellular phosphoenolpyruvate levels with a concomitant increase in the rate of lipid biosynthesis. Phosphorylation exerted on a non-metabolic enzyme can also have an effect on metabolism. One of the best-known examples is given by the regulatory role that histidine protein (HPr), a phosphocarrier in the sugar phosphotransferase system (PTS), plays in carbon catabolite repression (CCR) (Figure 3). In the presence of a preferred Current Opinion in Microbiology 2015, 24:29–37

34 Cell regulation

Figure 3

HPr

D-glucose

Crh

Allosteric activation

HPrK/P

Crh (–)

FBP

GAP

DHAP (–)

GapA

HPr (–)

CcpA

by inhibiting the activity of the first enzyme in the methylglyoxal bypass, methylglyoxal synthase (MgsA) [51]. However, in the presence of an abundant, rapidly metabolizable carbon source, phosphorylated-Crh inhibits the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GapA) [52], thereby redirecting flux toward the methylglyoxal bypass (Figure 3).

MgsA

1,3BPG HPr

Methylglyoxal

CcpA Pyruvate Carbon catabolite repression genes Current Opinion in Microbiology

Regulatory interactions between phosphorylation, allostery, and transcription in the methylglyoxal bypass of glycolysis. During carbon limiting conditions, the non-phosphorylated form of carbon-flux regulating histidine protein (Crh) directly inhibits methylglyoxal synthase (MgsA) via protein–protein interaction, thereby favoring carbon flux through glycolysis over the methylglyoxal bypass [52]. In the presence of excess glucose, however, HPr kinase/phosphorylase (HPrK/P) is allosterically activated by high fructose-1,6-bisphosphate (FBP) concentrations [50]. HPrK/P then serine-phosphorylates histidine protein (HPr) and the carbon-flux regulating histidine protein (Crh). Phosphorylation of Crh prevents inhibition of MgsA and at the same time, the serine-phosphorylated forms HPr and Crh now inhibit glyceraldehyde 3-phosphate dehydrogenase (GapA) in glycolysis; a situation that favors carbon flux through the methylglyoxal bypass versus glycolysis. Finally, serine-phosphorylated HPr also interacts with the transcription regulator CcpA (catabolite control protein A) to control expression of carbon catabolite repression genes [47,49,51,52].

carbon source such as glucose, HPr is serine-phosphorylated by HPr kinase/phosphorylase (HPrK/P) in response to high fructose-1,6-bisphosphate concentrations. Serinephosphorylated HPr then acts as a co-repressor by binding to CcpA (catabolite control protein A). This interaction allows CcpA to regulate the expression of hundreds of genes, enabling a coordinated response toward the availability of preferred carbon sources [47–50]. In B. subtilis, the carbon-flux regulating histidine protein (Crh), a paralogue of HPr, is also serine-phosphorylated by HPrK/P in response to high fructose-1,6-bisphosphate concentrations [51,52]. However, unlike HPr, Crh acts as a metabolic switch that controls carbon flux through the methylglyoxal bypass of glycolysis, a pathway hypothesized to prevent the accumulation of phosphorylated sugars and increase phosphate turnover during carbon overflow conditions. Under carbon-limiting conditions that favor a low HPrK/P kinase activity, the non-phosphorylated form of Crh directs carbons flux into glycolysis Current Opinion in Microbiology 2015, 24:29–37

Another interesting example highlighting that the phosphorylation of a non-metabolic enzyme can modulate metabolism pertains the regulation of the TCA cycle and glutamate synthesis by the regulatory protein GarA in M. tuberculosis (Figure 1). Through direct binding, nonphosphorylated GarA inactivates a-ketoglutarate dehydrogenase (KDH complex) and glutamate dehydrogenase (GDH) but activates glutamate synthase. These regulatory interactions have the net effect of inhibiting the TCA cycle while promoting glutamate production. When phosphorylated, GarA is no longer able to bind any of these enzymes, and the inhibition of the TCA cycle and activation of glutamate synthesis cease [53–55]. A recent report used targeted gene disruptions and site-directed mutagenesis to investigate the physiological role of GarA in M. tuberculosis. GarA was found to be essential for intracellular growth and survival during infection of human fibroblasts. The authors suggested that the biochemical effect of GarA deletion is the inability to properly regulate and inactivate KDH and GDH, resulting in glutamate and glutamine depletion that leads to growth inhibition [56]. Given that protein acetylation and phosphorylation are widespread in bacteria, it is possible that regulatory interactions may exist between these two PTMs. Wang et al. showed that when acetylated, isocitrate dehydrogenase phosphatase/kinase (AceK) loses its ability to phosphorylate, and therefore activate, isocitrate dehydrogenase (Icd) [10,19] (Figure 1). This orchestration is vital because, together with the acetyl-regulated isocitrate lyase (AceA), the phospho-regulated Icd controls carbon flux partitioning between the TCA cycle and the glyoxylate bypass [57]. In some instances, metabolic enzymes can be regulated by both acetylation and phosphorylation. For example, in a M. pneumonia study comparing wildtype versus kinase/ phosphatase and acetyltransferase/deacetylase mutants, Pgk activity was found to be influenced by both acetylation and phosphorylation. Here, it was also suggested that the two PTMs often co-occur on the same proteins [23]. It remains unclear, however, whether acetylation status can induce phosphorylation of the same enzyme or vice versa, and whether the two PTMs are exerted on the same enzyme molecules.

Other PTMs may be important in metabolic regulation In addition to acetylation, lysine residues can also be posttranscriptionally modified by succinylation, propionylation, www.sciencedirect.com

PTMs regulate bacterial metabolic fluxes Pisithkul, Patel and Amador-Noguez 35

butyrylation or glutarylation [58,59,60–62]. In a recent succinylation study, over 2000 sites were identified in 1000 E. coli proteins, with a surprisingly large overlap between succinylation and acetylation sites. Deletion of cobB, the sole deacetylase in E. coli, did not affect global succinylation; but similarly to how acetyl-CoA influences acetylation, succinylation levels were globally affected by succinyl-CoA concentration [59]. Another recent study identified 23 glutarylated proteins in E. coli, including GapA, AceA, AceE (pyruvate dehydrogenase E1 component), and SucB (dihydrolipoamide succinyltransferase) [58]. The regulatory mechanisms of succinylation and glutarylation, and their potential effects on metabolism are yet to be revealed. In eukaryotes, ubiquitination marks proteins for degradation. Although not a reversible PTM, ubiquitination is pivotal to metabolic flux regulation through its effects on enzyme levels. In bacterial systems, recent studies have shown that prokaryotic ubiquitin-like protein (Pup) plays an analogous role to ubiquitin [62]. A study in M. tuberculosis revealed the presence of pupylation sites on ATP synthase and enzymes involved in citrate, ornithine, nucleotide, and amino acid biosynthesis [62]. A study in C. glutamicum revealed 66 pupylation sites on 55 proteins, many of which are involved in central metabolism and translation [25]. How pupylation affects the stability and abundance of these enzymes and what effects this may have on metabolism remain to be addressed. Finally, it is interesting to note that a central metabolic intermediate, 1,3-bisphosphoglycerate, was recently shown to react with select lysine residues in proteins to form 3-phosphoglyceryl-lysine (pgK). This type of PTM was found to be predominant near the active sites of glycolytic enzymes and does not require enzyme catalysis; but it is proposed to rely on the intrinsic chemical reactivity of 1,3-bisphosphoglycerate [63]. pgK modifications inhibit glycolytic enzymes and it has been hypothesized that its accumulation in cells exposed to high glucose may serve as a regulatory feedback mechanism that contributes to the buildup and redirection of glycolytic intermediates to alternate pathways [63].

estimations [65], the authors identified 35 enzymes with phosphorylation changes in different metabolic pathways: glycolysis, gluconeogenesis, respiration, and fermentation. In particular, they identified 4 key metabolic enzymes hitherto unknown to be phosphorylated — Fba1, Gpd1, Gpd2, and Pfk2. Site-directed mutagenesis combined with metabolic flux profiling and phosphoproteomics analysis identified an inhibitory effect of phosphorylation on a subunit of phosphofructose-1-kinase complex, Pfk2, and glycerol-3-phosphate dehydrogenase, Gpd1. Such systematic functional studies have rarely been performed in bacterial systems. The post-translational modification of metabolic enzymes appears nearly ubiquitous in bacteria. The challenge ahead is to identify its in vivo functionality and the metabolic or environmental signals that regulate posttranslational modification enzymes. This task will likely require holistic studies that combine proteomics and metabolic flux profiling with classical genetics and biochemistry approaches. We anticipate many exciting findings in the upcoming years that will lead to a better understanding of this important layer in metabolic flux regulation.

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Chubukov V, Uhr M, Le Chat L, Kleijn RJ, Jules M, Link H, Aymerich S, Stelling J, Sauer U: Transcriptional regulation is insufficient to explain substrate-induced flux changes in Bacillus subtilis. Mol Syst Biol 2013, 9 Article Number: 709.

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The need for more PTMs functional studies in bacteria In all likelihood, the identification of bacterial metabolic enzymes subject to PTM will continue to increase rapidly in coming years. This creates both a challenge and an opportunity for understanding the physiological role of these modifications and how they might regulate metabolic fluxes. Recent studies in Saccharomyces cerevisiae may provide inspiration about how to tackle these questions in bacteria. For example, a functional study of phosphorylation in yeast measured its proteome and phosphoproteome under different nutritional conditions [64]. By combining proteomics data with metabolic flux www.sciencedirect.com

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36 Cell regulation

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