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. 2011 Jul 8;12(8):863–870. doi: 10.1038/embor.2011.109

Reconstitution of the Mycobacterium tuberculosis pupylation pathway in Escherichia coli

Francisca A Cerda-Maira 1,*, Fiona McAllister 2,*, Nadine J Bode 1, Kristin E Burns 1, Steven P Gygi 2, K Heran Darwin 1,a
PMCID: PMC3147258  PMID: 21738222

Abstract

Prokaryotic ubiquitin-like protein (Pup) is a post-translational modifier that attaches to more than 50 proteins in Mycobacteria. Proteasome accessory factor A (PafA) is responsible for Pup conjugation to substrates, but the manner in which proteins are selected for pupylation is unknown. To address this issue, we reconstituted the pupylation of model Mycobacterium proteasome substrates in Escherichia coli, which does not encode Pup or PafA. Surprisingly, Pup and PafA were sufficient to pupylate at least 51 E. coli proteins in addition to the mycobacterial proteins. These data suggest that pupylation signals are intrinsic to targeted proteins and might not require Mycobacterium-specific cofactors for substrate recognition by PafA in vivo.

Keywords: Pup, Mycobacterium tuberculosis , proteasome, pupylation

Introduction

Protein-to-protein post-translational modifiers are essential for several aspects of eukaryotic biology (reviewed in Hochstrasser, 2000; Chen & Sun, 2009). In particular, the small protein ubiquitin (Ub) targets proteins for degradation by a compartmentalized protease called the proteasome (reviewed in Pickart & Cohen, 2004). Post-translational protein modifiers have recently been identified in bacteria (Pearce et al, 2008; Burns et al, 2009) and archaea (Humbard et al, 2010), but have not been characterized as extensively as their eukaryotic counterparts. In Mycobacterium species, a prokaryotic Ub-like protein (Pup) modifies proteins to target them for degradation by a bacterial proteasome (Pearce et al, 2008; Festa et al, 2010; Poulsen et al, 2010; Striebel et al, 2010; Watrous et al, 2010). Thus, Pup is functionally, if not biochemically, related to Ub.

Pup can have either a carboxy-terminal glutamine (PupGln) or glutamate (PupGlu) residue. Mycobacteria encode PupGln, which must be deamidated to PupGlu by deamidase of Pup (Dop) before substrate conjugation (Striebel et al, 2009). Proteasome accessory factor A (PafA) attaches PupGlu to substrate lysines (Guth et al, 2011), and the resulting product is a covalently linked complex with an isopeptide bond between Pup and the substrate (Pearce et al, 2008; Burns et al, 2009). Similar to ubiquitylation, pupylation is reversible. Dop can hydrolyse the Pup–substrate bond, and thus Dop is a depupylase in addition to functioning as a deamidase (Burns et al, 2010a; Imkamp et al, 2010b). Depupylation is not required for protein degradation in vitro (Striebel et al, 2010), but it seems to enable Pup recycling (Cerda-Maira et al, 2010). In addition, depupylation might facilitate proteasomal degradation of certain proteins in vivo (Burns et al, 2010a).

In eukaryotes, it is estimated that more than 1,000 E3 ligases determine which proteins are targeted for ubiquitylation (reviewed in Deshaies & Joazeiro, 2009; Rotin & Kumar, 2009). By contrast, Pup seems to require a single conjugating enzyme for all pupylation in Mycobacterium tuberculosis (Mtb; Pearce et al, 2008; Festa et al, 2010). In Mtb, pupylation favours 1 of 8 lysines in the proteasome substrate malonyl Co-A acyl carrier protein transacylase (FabD; Pearce et al, 2008), and 1 of 23 lysines in myo-inositol-1-phosphate synthetase (Ino1; Burns et al, 2009; Festa et al, 2010; Watrous et al, 2010). In vitro, PafA is sufficient to pupylate FabD with PupGlu, although at least two Pup∼FabD species are produced in this system (Striebel et al, 2009).

To understand how proteins are selected for pupylation, we sought to develop a system using an organism that does not normally produce Pup, PafA or Dop. We reconstituted pupylation of Mtb proteasome substrates in a laboratory Escherichia coli K-12 strain. Unexpectedly, this revealed that pupylation does not require specificity factors that are functionally related to E3 ligases in eukaryotes.

Results And Discussion

Pupylation of Mtb proteasome substrates in E. coli

We have previously identified targets of pupylation and proteasomal degradation in Mtb, including FabD, ketopantoate hydroxymethyltransferase (PanB) and Ino1 (Pearce et al, 2006; Burns et al, 2010b). Although FabD and PanB can be pupylated in vitro (Striebel et al, 2009), reconstitution of pupylation in E. coli could enable more rapid characterization of Pup, PafA, Dop and their substrates. We expressed pupGln or pupGlu and pafA–his6 from pET24b(+) using the T7 polymerase system and fabD–his6 from the arabinose-inducible araBAD promoter on the compatible plasmid pBAD33 (see supplementary Table S1 online for all plasmids and strains). We also cloned Mtb dihydrolipoamide acyltransferase (dlaT) into pBAD33; DlaT is not a known target of pupylation or proteasomal degradation in mycobacteria (Pearce et al, 2006, 2008). Finally, dop–his6 was expressed from a T7 promoter in a third plasmid, pETDuet-1. As pET24b(+) and pETDuet-1 have the same replication origin, we monitored gene-product synthesis by immunoblotting for all relevant plasmid-encoded proteins produced by these strains.

PupGln and PafA could not pupylate FabD in the absence of Dop (Fig 1A, lane 1), presumably because PupGln was not deamidated. By contrast, pupylation of FabD was observed after induction of pupGlu and pafA (Fig 1A, lane 2). We observed two pupylated species of FabD: a prominent band of the expected molecular weight of approximately 48 kDa and a less-abundant species with a molecular weight slightly below. A point mutation that inactivates PafA in Mtb (Cerda-Maira et al, 2010) also abrogated FabD pupylation in E. coli (Fig 1A, lane 3). We also tested whether Mtb DlaT—which is not known to be pupylated in mycobacteria—could be pupylated in E. coli. DlaT–His6 was not pupylated in E. coli under the tested conditions (Fig 1A, lane 4). Expression of pupGln with dop and pafA resulted in FabD pupylation (Fig 1A, lane 5). The expression of dop with an inactivating mutation did not produce Pup∼FabD (Fig 1A, lane 6), which is consistent with reports from other systems (Cerda-Maira et al, 2010; Imkamp et al, 2010a).

Figure 1.

Figure 1

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Pupylation of Mtb proteasome substrates in Escherichia coli. (A) Immunoblot analysis of total cell lysates of E. coli harbouring plasmids encoding components of the pupylation system and test substrates. The recombinant proteins produced in these strains are listed on the left. E11A, glutamate 11 to alanine; E10A, glutamate 10 to alanine. These glutamates are predicted to coordinate Mg2+. Asterisks indicate the lower-molecular-weight Pup∼FabD species similar to that observed in vitro (Striebel et al, 2009). The polyclonal rabbit antibody used to detect a relevant protein is listed below each panel. (B) Top: mutation of Lys 173 in FabD changed the pupylation pattern of FabD. Arrow indicates Pup∼FabD of the molecular weight observed in mycobacteria. Bottom: summary of MS/MS analysis of wild-type FabD purified from E. coli. Numbers represent abundance on the basis of spectral counting. (C) The Mtb proteasome substrate Ino1 was pupylated in E. coli expressing pupGlu and pafA. Dot indicates a faint cross-reactive protein in the pupGln lane (probably PafA–His6) that co-migrates with Ino1–His6. For all panels, all proteins, except Pup, have carboxy-terminal His6 tags. Induction of expression and immunoblot analysis are described in the Methods section. All data are representative of at least two independent experiments. DlaT, dihydrolipoamide acyltransferase; Dop, deamidase of Pup; Ino1, myo-inositol-1-phosphate synthetase; MS/MS, tandem mass spectrometry; PafA, proteasome accessory factor A; Pup, prokaryotic ubiquitin-like protein.

A previous study reported that two Pup∼FabD species were observed when pupylation was reconstituted in vitro; it was speculated that modification of a different residue resulted in the faster-migrating Pup∼FabD (Striebel et al, 2009). In Mtb, FabD is pupylated on Lys 173, and mutagenesis of this lysine stabilizes the protein (Pearce et al, 2008). Although it is possible that other FabD residues could be targeted for pupylation in Mycobacteria, these secondary sites must be used rarely, because we did not observe pupylated FabDK173A. To determine whether Lys 173 was a target for pupylation in E. coli, we expressed fabDK173A in pupylation-competent E. coli. This mutation reduced the level of the slower-migrating Pup∼FabD species (Fig 1B, upper panel, arrow) and increased the relative abundance of the faster-migrating species (Fig 1B, upper panel, asterisk). This suggests that in the absence of the preferred residue, PafA pupylated the next most accessible lysine. Similarly to in the Ub system, mutating a preferred lysine can result in another residue in the substrate being ubiquitylated.

As the slower-migrating species did not disappear, we hypothesized that a non-Lys 173-modified FabD also migrated at this position. We purified Pup∼FabD from E. coli and analysed both the higher- and lower-molecular-weight species by tandem mass spectrometry (MS/MS). FabD was identified with 96% sequence coverage in the two species. Lys 173 was the predominant modified residue in FabD purified from E. coli (Fig 1B, lower panel). Lys 122 and Lys 181 were also identified as pupylation targets. Two of the three pupylated species were found in both the lower and upper bands, probably due to cross-contamination of the closely migrating proteins. Thus, our data show that at least three lysines in Mtb FabD can be pupylated in E. coli. It also seems that only one residue per FabD polypeptide was pupylated at one time, because we did not observe higher-molecular-weight species (Fig 1A).

We speculate that the selection of FabD Lys 173 for pupylation in mycobacteria might be partly due to its accessibility to Pup and PafA. FabD is part of the multi-enzyme fatty acid synthesis II (FASII) pathway and has eight surface-exposed lysines (Kremer et al, 2001; Ghadbane et al, 2007). We propose that other proteins or macromolecules mask lysines or binding domains in FabD, thus preventing their pupylation under typical culture conditions in mycobacteria. For example, other FASII enzymes might prevent PafA from having access to most of the lysines in FabD, and Lys 173 is therefore preferred because it is the most exposed. In addition, Lys 173 might not always be accessible, which could determine the degree of FabD pupylation. This might suggest that pupylation is stochastic. These hypotheses could be tested in E. coli by examining FabD pupylation in the context of other Mtb FASII enzymes. Interestingly, every protein encoded in the Mycobacterium fabD/FASII operon is a target for pupylation, although they are not all robust proteasome substrates under routine culture conditions (Festa et al, 2010; Watrous et al, 2010).

We also examined Ino1, a target for pupylation in both Mycobacterium smegmatis (Msm) and Mtb (Festa et al, 2010; Watrous et al, 2010). Expression of Mtb ino1, pafA and pupGlu, but not pupGln, resulted in pupylated Ino1 in E. coli (Fig 1C). Only one pupylated species of Ino1 was observed, similar to that observed in Mycobacteria (Burns et al, 2010b). MS/MS analyses of Ino1 from Mtb and Msm identified a single, specific lysine for pupylation (Burns et al, 2009; Festa et al, 2010; Watrous et al, 2010). Although it is possible that different lysines are pupylated when Ino1 is produced in E. coli, it seems likely that the same residue is modified in all bacterial species.

Pupylation of E. coli proteins

We next wondered if any E. coli proteins could be pupylated by this reconstituted system. Surprisingly, several proteins were pupylated in E. coli producing PupGlu and PafA (Fig 2A), or PupGln, Dop and PafA (Fig 2B). Mutations that abrogate PafA or Dop activity in mycobacteria (Cerda-Maira et al, 2010) also inactivated function in E. coli (Fig 2A,B). To determine when pupylation was maximal in the reconstituted E. coli system, we examined the appearance of the pupylome over time. Pupylation in E. coli was apparent within 20 min of pupGlu–pafA induction and reached a plateau at around 100 min (Fig 2C, top panel). Under the conditions tested, the viability of pupylation-competent E. coli was not different from that of E. coli expressing pupGln–pafA (Fig 2C, lower panel).

Figure 2.

Figure 2

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Pupylation of E. coli proteins. (A,B) Immunoblot analysis of total cell lysates of E. coli strains harbouring plasmids encoding various components of the pupylation system. Mouse monoclonal antibodies to Pup (m-anti-Pup) were used to detect the E. coli pupylome. (C) Detection of pupylation over time in E. coli expressing pupGlu–pafA (top). Pupylation of E. coli proteins was not detrimental to bacterial viability up to 3 h after induction of pup–pafA expression (bottom). The addition of IPTG to cultures of strains containing the empty vector typically slows growth but does not kill the bacteria; therefore, we also enumerated CFUs from a culture of the same E. coli strain carrying empty pET24b(+). (D) Truncating PafA and Dop abrogated pupylation in E. coli. Left: immunoblot analysis of total cell lysates of E. coli expressing pupGlu and wild-type or mutant pafA. Wild-type PafA comprises 452 amino acids plus a His6 tag. 414, 430 and 441 indicate the last residue of the truncated PafA protein. Right: immunoblot analysis of total cell lysates of E. coli expressing pupGln–pafA and wild-type or mutant dop. Wild-type Dop comprises 505 amino acids plus a His6 tag. 423, 451, 468 and 479 indicate the last residue of the truncated Dop protein. None of the truncated proteins was epitope tagged. (E) Immunoblot analysis of total cell lysates of Mtb expressing different pafA or dop truncations. Induction of expression, lysate preparation and immunoblot analysis are described in the Methods section. These data are representative of two independent experiments. comp, complemented; CFU, colony-forming unit; Dop, deamidase of Pup; IPTG, isopropyl-β-D-thiogalactopyranoside; Mtb, Mycobacterium tuberculosis; Myc; PafA, proteasome accessory factor A; Pup, prokaryotic ubiquitin-like protein; WT, wild type.

We next tested if other mutations in PafA or Dop affected pupylation in E. coli. Modelling and mutational analysis of PafA and Dop predict that the active sites of these proteins are at the amino-termini and are similar to those found in glutamine synthetases (Iyer et al, 2008; Cerda-Maira et al, 2010). By contrast, the C-termini do not resemble any protein of known function. We previously showed that the deletion of 38 residues from the C-terminus of PafA (PafA414) resulted in no pupylation in Mtb (Cerda-Maira et al, 2010). This mutation also eliminated pupylation in E. coli (Fig 2D, left panels). We therefore tested smaller C-terminal deletions of PafA, as well as four truncations of Dop (supplementary Table S1 online). All but one of the truncated proteins were detected in E. coli; the largest deletion in dop resulted in no protein, suggesting that this mutant protein was unstable or that the use of two incompatible plasmids affected the production of recombinant protein (Fig 2D, top right panel). None of the truncated proteins catalysed pupylation in E. coli (Fig 2D, lower panels). Most importantly, the truncated forms of pafA and dop were unstable and failed to complement transposon mutations in Mtb pafA or dop, respectively (Fig 2E).

Taken together, these results suggest that the C-termini of PafA and Dop are required for the folding, stability or activity of these enzymes. This E. coli system also demonstrated that additional Mycobacterium-specific cofactors are not required for pupylation in vivo. Importantly, these results demonstrate that E. coli could be used as a surrogate system in which to study Mtb pupylation.

Identification of the E. coli pupylome

We hypothesized that the E. coli pupylome could provide insight into how proteins are selected for pupylation. We affinity purified pupylated proteins from E. coli expressing pupGlu and pafA (see Methods section; Festa et al, 2010). Incubation of the E. coli pupylome with Mtb Dop resulted in the disappearance of the Pup signal when assayed by immunoblotting, whereas overall protein levels were unchanged (Fig 3A). This is consistent with the idea that Pup formed isopeptide linkages with E. coli proteins, similarly to in mycobacteria (Festa et al, 2010; Watrous et al, 2010; Burns et al, 2010a).

Figure 3.

Figure 3

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Analysis of the Escherichia coli pupylome. (A) The purified E. coli pupylome was sensitive to depupylation by Mtb Dop–His6. Samples were analysed by 12% SDS–PAGE and immunoblotting against Pup (left) or Coomassie blue staining (right). (B) E. coli Adk–His6 was pupylated in E. coli expressing pupGlu–pafA–his6. Ni-NTA-purified Adk–His6 was analysed by 12% SDS–PAGE and immunoblotting with polyclonal antibodies to Pup (top) or His5 (bottom). Question mark indicates possible species of Adk–His6 with several Pup molecules attached, or other pupylated proteins that copurified with Adk–His6. (C) E. coli PtsI–His6 was pupylated in Msm. The recombinant protein was purified from Msm using Ni-NTA chromatography and analysed by 10% SDS–PAGE. Molecular-weight markers and 15 μl of each sample were loaded onto the same gel twice. Proteins were transferred to a nitrocellulose membrane and the membrane was cut in half for immunoblotting with antibodies to His5 (upper panel) or Pup (lower panel). Question mark indicates possible species of PtsI–His6 with several Pup molecules attached. Adk, adenylate kinase; Dop, deamidase of Pup; Msm, Mycobacterium smegmatis; Mtb, Mycobacterium tuberculosis; MW, molecular weight; Ni-NTA; nickel-nitrilotriacetic acid; PafA, proteasome accessory factor A; PtsI, phosphoenolpyruvate-protein phosphotransferase I; Pup, prokaryotic ubiquitin-like protein; SDS–PAGE, SDS–polyacrylamide gel electrophoresis.

Proteomic analysis identified over 400 proteins in the E. coli pupylome, 51 of which had one or more lysines linked to PupGlu (Table 1; supplementary Table S2 online). Several E. coli proteins (AdhC, adenylate kinase (Adk), ProS, GroL and SodB) have homologues in Mtb that are also targets of pupylation (Festa et al, 2010). However, when the equivalent lysines were present they were not modified in both bacterial species. Furthermore, comparison of the peptides did not identify a consensus sequence surrounding the modified lysines.

Table 1. Escherichia coli proteins with pupylated lysines*.

Locus Gene Position of modified lysines (protein MW) Locus Gene Position of modified lysines (protein MW)
b0014 dnaK 635, 637 (638) b2185 rplY 34 (94)
b0026 ileS 814 (938) b2400 gltX 44 (471)
b0115 aceF 161 (630) b2416 ptsI 174 (575)
b0118 acnB 77 (865) b2507 guaA 437 (525)
b0166 dapD 100 (274) b2614 grpE 43, 66 (197)
b0194 proS 426, 495, 571 (572) b2687 luxS 163 (171)
b0356 adhC 13 (369) b2891 prfB 162 (365)
b0474 adk 47, 50, 136, 141 (214) b2935 tktA 316, 347 (663)
b0492 ybbN 184, 268 (296) b2954 rdgB 3 (197)
b0525 ppiB 60 (164) b3011 yqhD 43 (387)
b0578 nfnB 21, 62 (217) b3296 rpsD 156, 167 (206)
b0812 dps 105 (167) b3301 rplO 141 (144)
b0965 yccU 97 (164) b3341 rpsG 56, 131 (179)
b1059 solA 357 (372) b3559 glyS 624 (689)
b1107 nagZ 283 (341) b3640 dut 15 (151)
b1136 icd 4, 273 (416) b3644 yicC 86 (287)
b1215 kdsA 79 (284) b3708 tnaA 161 (476)
b1656 sodB 44 (193) b3866 yihI 158 (169)
b1667 ydhR 60, 74 (101) b3924 fpr 135 (248)
b1749 xthA 141 (268) b3960 argH 95 (457)
b1779 gapA 249 (331) b4113 basR 70, 164 (222)
b1823 cspC 9 (69) b4143 groL 65, 168, 321 (548)
b1854 pykA 60, 93 (480) b4200 rpsF 35 (131)
b1864 yebC 192 (246) b4207 fklB 146 (259)
b2040 rfbD 245 (299) b4280 yjhC 79 (377)
b2146 yeiT 364 (412)      
MW, molecular weight.
*Four additional proteins with pupylated peptides but no locus identifiers were observed by tandem mass spectrometry (supplementary Table S2 online).
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We next tested whether E. coli proteins could be pupylated in Mycobacteria. We chose two proteins: phosphoenolpyruvate-protein phosphotransferase I (PtsI)—one of the most highly represented proteins in the E. coli pupylome (supplementary Table S3 online)—and Adk, which has an Mtb homologue that is a target for pupylation (Festa et al, 2010). In addition, E. coli Adk had four lysines that could be pupylated (Table 1). Before examining pupylation in Mycobacteria, we cloned adk and ptsI with his6 tags into pBAD33 to assess whether these recombinant proteins could still be pupylated in E. coli. Adk–His6 seemed to be pupylated when purified from E. coli expressing pupGlu–pafA, but not pupGln–pafA (Fig 3B). Additional higher-molecular-weight bands were also detected using antibodies to Pup, which might indicate that Adk–His6 was polypupylated in E. coli. It is also possible that these larger Pup-reactive species are different pupylated proteins that copurified with Adk–His6. PtsI was also pupylated in E. coli expressing pupGlu–pafA, as shown by immunoblotting with antibodies to His5 or Pup (Fig 3C). Additional higher-molecular-weight species were also detected with both antibodies, suggesting that PtsI could have more than one Pup attached. Taken together, these results suggest that addition of the His6 tag did not hinder pupylation of these proteins in E. coli.

Finally, we expressed E. coli adk–his6 and ptsI–his6 in Msm (supplementary Table S1 online) and purified the recombinant proteins using nickel affinity chromatography. Although Adk–His6 was robustly produced in Msm, we could not detect a pupylated species (data not shown). Interestingly, Mtb Adk—a target of pupylation as detected by MS/MS in Mtb—could not be purified in the pupylated form from Mycobacteria (Festa et al, 2010). In contrast to Adk, PtsI–His6 was pupylated in Msm, as detected with antibodies to Pup (Fig 3C, lower panel, last lane). We detected Pup∼PtsI–His6 using antibodies to His5 only if the film was overexposed (not shown); however, we have previously observed that His5 antibodies are not always sensitive enough to detect pupylated proteins (Festa et al, 2010). PtsI does not have a homologue in mycobacteria, and thus a completely foreign protein could be targeted by the native Mycobacterium pupylation system.

Perhaps it is not surprising that E. coli proteins could be pupylated, as a motif is lacking in over 50 pupylated proteins purified from either Mtb or Msm (Festa et al, 2010; Watrous et al, 2010). Although pupylation seems to be widespread, not just any protein or lysine can be pupylated. For example, Mtb DlaT has never been demonstrated to be a target of pupylation, despite having 29 lysines (Fig 1; Pearce et al, 2008). This suggests that proteins require additional chemical signatures, such as localized changes in charge, phosphorylation, oxidation, misfolding, or even other proteins such as chaperones, to target them for pupylation.

The reconstitution of pupylation in E. coli is important because it will simplify the structure–function analysis of enzymes of a less-genetically-tractable organism. Researchers in the Mtb field can now easily test if a protein is a target of pupylation. It is also striking that so many proteins can be pupylated in E. coli, in comparison to ubiquitylation in eukaryotes. Several Ub ligases determine which proteins are modified at a particular place and time in the cell. Despite this fact, there is precedent for the modification of proteins by heterologous systems. Proteins injected from pathogenic Gram-negative bacteria can be ubiquitylated in mammalian cells (Knodler et al, 2009; Patel et al, 2009). The Ub-like protein interferon-stimulated gene 15 targets viral as well as host proteins (Durfee et al, 2010). Thus, protein modifiers might sometimes rely on overproduction to increase their chances of finding their targets. Pupylation is enzymatically, if not functionally, distinct from ubiquitylation, and it now seems that new differences will be identified in the manner in which substrates are selected for modification.

Methods

Strains, plasmids, primers and culture conditions. Strains and plasmids are listed in supplementary Table S1 online. Primer sequences are available on request. E. coli strains were grown in Luria–Bertani broth or on Luria–Bertani-agar plates (Difco). Mycobacteria strains were grown in Middlebrook 7H9 broth or on 7H10 agar (Difco) with supplementation (0.2% glycerol, 0.05% Tween-80, 0.5% bovine serum albumin, 0.2% dextrose and 0.085% sodium chloride for broth; or BBL Middlebrook OADC for agar) when necessary. Antibiotic concentrations were as follows for E. coli and Mycobacteria, respectively: 150 or 50 μg ml−1 hygromycin and 100 or 50 μg ml−1 kanamycin. Chloramphenicol was used at 25 μg ml−1 for E. coli. Isopropyl-β-D-thiogalactopyranoside was used at 0.1 mM.

pup and pafA derivatives were sequentially cloned into pET24b(+). Briefly, pafA (wild-type or mutant alleles) were first cloned into the NdeI–HindIII sites of pET24b(+). pup was amplified from pET24b+pup-STOP, digested with BglII and NheI, and cloned into the BglII and XbaI sites of the pET24b(+)-pafA plasmids. The dop derivatives were cloned into pETDuet-1 multiple cloning site 2 NdeI and PacI sites. dlaT, ino1, adk, ptsI and fabD were cloned into the KpnI or XbaI and SphI sites of pBAD33. For Mtb complementation analysis, pafA and dop alleles were cloned with their native promoters into pMV306, using KpnI and ClaI, or XbaI and HindIII, respectively. For expression in Msm, adk and ptsI were cloned into pMN402 using PacI and BamHI. All plasmids were sequenced to confirm the veracity of the cloned sequences (GENEWIZ Inc.). Transformation of bacteria was performed as described previously (Sambrook et al, 1989; Hatfull & Jacobs, 2000).

For analysis of Mtb lysates, 5 ml cultures were grown to an optical density at 580 nm of approximately 1.0–1.5. Bacteria were collected and processed as described previously (Cerda-Maira et al, 2010). Total cell lysates were analysed by immunoblotting using antibodies to PafA, Dop or Pup.

SDS–PAGE and immunoblot analysis. For immunoblotting analysis, proteins were separated on 7% (anti-PafA and anti-Dop), 10 or 12% (anti-His5), or 15% (anti-Pup) SDS–polyacrylamide gel electrophoresis (PAGE) gels (Ausubel et al, 2002). Proteins were transferred onto 0.22 μm nitrocellulose (Schleicher and Schuell) using a semi-dry transfer system (Bio-Rad), and incubated with antibodies to PafA-His6, Dop-His6, FabD-His6, Ino1-His6, Pup-His6 or His5 (Qiagen), as described previously (Festa et al, 2007, 2010; Pearce et al, 2008; Cerda-Maira et al, 2010). Horseradish peroxidase-coupled rabbit secondary antibodies were used according to the manufacturer's instructions (Thermo Scientific). Horseradish peroxidase was detected using either the SuperSignal West Pico or West Femto Chemiluminescent Substrate (Thermo Scientific).

Pupylation in E. coli and Msm. To examine the pupylation of proteins in E. coli, strains were incubated at 37°C with shaking until an optical density of 0.6 at 600 nm was reached. When strains with pBAD33 derivatives were used, arabinose was added to a final concentration of 0.02–0.2% w/v and incubated for 1 h. Cultures were then induced with 0.1 mM IPTG and incubated for one more hour. If dop activity was tested, cultures were incubated for 2 h. A volume of 0.5 ml of the induced cultures was collected and bacteria were isolated by centrifugation for 1 min at top speed in microfuge tubes. Bacteria were resuspended in 400 μl of lysis buffer, mixed with 4 × sample buffer and boiled for 10 min before analysis by SDS–PAGE and immunoblotting.

To determine whether E. coli proteins were pupylated in Msm, 100 ml saturated overnight cultures of Msm producing recombinant protein were processed, as described previously for Mtb (Festa et al, 2010). Nickel-nitrilotriacetic acid purification (Qiagen) was used to isolate His6-tagged proteins.

Proteomic analysis. A 250-ml culture of EHD1295 (supplementary Table S1 online) was grown as described above. Cells were collected by centrifugation, resuspended in denaturing lysis buffer B (The QIAexpressionist manual), and TAP was performed as described in detail elsewhere (Festa et al, 2010). Purified proteins were visualized on a 12% SDS–PAGE gel and excised from the gel for MS/MS analysis. The gel slice was divided into 10 bands, each of which was cut into cubes of approximately 1 mm and transferred into 1.5 ml Eppendorf tubes. In-gel digestion was done as described previously (Li et al, 2007). Peptides were eluted from the StageTips (Rappsilber et al, 2003) using 40% acetonitrile and 5% formic acid, followed by 80% acetonitrile and 5% formic acid into glass inserts, and dried in a Speed-Vac. The resulting peptides were analysed as described previously (Festa et al, 2010). Protein hits were filtered at the peptide level to a 0.1% false discovery rate using the target-decoy strategy (Elias & Gygi, 2007) using in-house software and filtering on the basis of ΔCn, XCorr, p.p.m., charge state and peptides per protein. Proteins were also filtered at the protein level to a 1% false discovery rate using linear discriminate analysis based on Xcorr, ΔCn, precursor mass error and charge state, as described previously (Huttlin et al, 2010). For all pupylome MS/MS data, see supplementary Tables S2 and S3 online.

To identify the pupylated sites in Mtb FabD produced in E. coli, FabD–His6 was purified under native conditions from 250 ml EHD1288 cultured as described above. Protein was separated on 10% SDS–PAGE and the Pup∼FabD bands were excised for MS/MS analysis, as described previously.

Depupylation assay. The depupylation reaction was done essentially as described previously (Burns et al, 2010a). Briefly, 15 μg of Ni-NTA-purified proteins were incubated with 2.5 mM ATP, 20 mM MgCl2, 50 mM NaCl, 1 mM dithiothreitol and 2.5 μg Mtb Dop–His6 in 50 mM Tris–HCl, pH 8, in a final volume of 100 μl. Dop was purified using Ni-NTA from Msm, as described previously (Burns et al, 2010a). Aliquots were removed at the indicated time points, and the reaction was stopped with the addition of protein sample buffer.

Supplementary information is available at EMBO reports online (http://www.emboreports.org).

Supplementary Material

Supplementary Information
embor2011109s1.pdf (202KB, pdf)
Review Process File
embor2011109s2.pdf (202KB, pdf)

Acknowledgments

We thank A. Darwin and R. Festa for critical review of draft versions of this manuscript. We are grateful to T. Huang and K. Hofmann for helpful discussions. This work was supported by National Institutes of Health grants AI065437 and HL92774 awarded to K.H.D and HG3456 to S.P.G. K.H.D. is supported by the Irma T. Hirschl Trust and holds an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund.

Author contributions F.C.-M., F.M. and K.H.D. designed and conducted the experiments, and wrote the manuscript. N.J.B. and K.E.B. conducted the experiments. S.P.G. designed the experiments.

Footnotes

The authors declare that they have no conflict of interest.

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embor2011109s1.pdf (202KB, pdf)
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embor2011109s2.pdf (202KB, pdf)

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