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Identification of Lysine Acetylation in Mycobacterium abscessus Using LC-MS/MS after Immunoprecipitation Jintao Guo, Changwei Wang, Yi Han, Zhiyong Liu, Tian Wu, Yan Liu, Yang Liu, Yaoju Tan, Xinshan Cai, Yuanyuan Cao, Bangxing Wang, Buchang Zhang, Chunping Liu, Shouyong Tan, and Tianyu Zhang J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00116 • Publication Date (Web): 20 Jun 2016 Downloaded from http://pubs.acs.org on June 21, 2016

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Journal of Proteome Research

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Identification of Lysine Acetylation in Mycobacterium abscessus Using LC-MS/MS after Immunoprecipitation Jintao Guo a, ‡, Changwei Wang a, ‡, Yi Han a, Zhiyong Liu a, Tian Wu a, Yan Liu a, Yang Liu a, c, Yaoju Tan b, Xinshan Cai b, Yuanyuan Cao a, c, Bangxing Wang a, c, Buchang Zhang c, Chunping Liu b, Shouyong Tan b and Tianyu Zhang a, * a

State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine

and Health (GIBH), Chinese Academy of Sciences (CAS), Guangzhou, China. b State Key Laboratory of Respiratory Disease, Department of Clinical Laboratory, The Guangzhou Chest Hospital, Guangzhou, Guangzhou, China. c School of Life Sciences, University of Anhui, Hefei, China.

Running title: Lysine acetylation in M. abscessus

Keywords:

Mycobacterium

abscessus

(M.

abscessus),

lysine

acetylation,

post-translational modifications (PTMs), mass spectrometry (MS)

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ABSTRACT

Mycobacterium abscessus (MAB), which manifests in the pulmonary system, is one of the neglected causes of nontuberculous mycobacteria (NTM) infection. Treatment against MAB is difficult, characterized by its intrinsic antibiotic drug-resistance. Lysine acetylation can alter the physiochemical property of proteins in living organisms. This study aimed to determine if this protein post-translational modification (PTM) exists in a clinical isolate M. abscessus GZ002. We used the anti-acetyl-lysine immunoprecipitation to enrich the low-abundant PTM proteins, followed by the LC-MS/MS analysis. The lysine acetylome of M. abscessus GZ002 was determined. There were 459 lysine acetylation sites found in 289 acetylated proteins. Lysine acetylation occurred in 5.87 % of the M. abscessus GZ002 proteome and at least 25 % of them were growth essential. Aerobic respiration and carbohydrate metabolic pathways of M. abscessus GZ002 were enriched with lysine acetylation. Through bioinformatics analysis, we identified four major acetyl motif logos (KacY, KacF, KacH and DKac). Further comparison of the reported M. tuberculosis (MTB) acetylomes and that of MAB GZ002 revealed several common features between these two species. The lysine residues of several antibiotic-resistance, virulence and persistence-related proteins were acetylated in both MAB GZ002 and MTB. There were 51 identical acetylation sites in 37 proteins found in common between MAB GZ002 and MTB. Overall, we demonstrate a profile of lysine acetylation in MAB



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GZ002 proteome that shares similarities with MTB. Interventions that target at these conserved sections may be valuable as anti-NTM and/or anti-TB therapies.

INTRODUCTION The nontuberculous mycobacteria (NTM) can cause a wide spectrum of infections, including the manifestation in the pulmonary system. Increasing reports in the literature suggest that the NTM cause a greater number of pulmonary infections than the Mycobacterium tuberculosis (MTB) complex.

1-2

The pulmonary infection caused

by the rapidly growing Mycobacterium abscessus (MAB) is difficult to cure due to its intrinsic resistance to the standard anti-tuberculosis drugs (reviewed in 3-4). As a result, increased prevalence of NTM infections is in contrast with a reduced number of tuberculosis (TB) infections.

5-7

New agents with enhanced activity against MAB and

other NTM are needed. Study of the protein post-translational modification (PTM) is imperative in understanding the biology of NTM and MTB to reveal potential new targets for disease prevention and treatment. Though hundreds of lysine-acetylated proteins were identified in MTB H37Rv and the attenuated strain MTB H37Ra,

8-9

the lysine

acetylome has not been determined for MAB. In general, the lysine acetylation and deacetylation can act as the molecular switches that initiate or inhibit protein activities. 10-12

Acetylation of the glyceraldehyde phosphate dehydrogenase kinase/phosphatase

in Salmonella enterica could shift the enzyme’s activity from gluconeogenesis to glycolysis.

13

Alternatively, lysine acetylation neutralizes the positively charged 3 ACS Paragon Plus Environment

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ε-amino group that prevents protein from interacting or binding with substrates. 10-14 It was also evident that cross-talk between various PTMs is a part of the global protein function regulatory network since co-occurring of acetylation and phosphorylation in the interaction interfaces of multifunctional proteins were frequently found in Mycoplasma pneumoniae.

15

Lysine acetylation in histones is another well-known

mechanism employed by the living organisms to regulate transcription and DNA repair. 16 The general control of amino acid synthesis protein 5 (GCN5)-related N-acetyltransferases (GNATs) catalyze the protein lysine acetylation by transferring an acetyl group from acetyl-coenzyme A to the ε-amine group of lysine (Nε-modification) or to the α-amine of protein N-terminus (Nα-modification).

8-9

The reverse reaction

can be carried out by deacetylases. The changes in protein structures by PTM may have major impacts on protein function and compartmentalization.

17-18

At least three

putative GNATs have been identified in the MAB genome. The MAB_3168c and MAB_4395 were associated with the aminoglycosides resistance, MAB_1725c might play a role in mycobacterial virulence.

20

3, 19

whereas the

The identifications of

these acetyltransferases suggest that lysine acetylation is functional in MAB. However, apart from the general associations with drug-resistance and virulence, the actual effects of lysine acetylation on cell physiology and which acetyltransferase is responsible

for

the

target

protein

modification

remains

exclusive.

The

PTM-dependent pathways that regulate the MAB disease progression are yet to be identified. 4 ACS Paragon Plus Environment


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In the current work we examined the lysine acetylome of a MAB clinical strain GZ002. In order to identify the low abundant lysine-acetylated protein, we used immunopurification with an anti-acetyl-lysine antibody followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The acetyl-lysines were widely distributed in the MAB GZ002 proteome. Proteins involved in the carbon metabolism pathway, tricarboxylic acid (TCA) cycle, and the aerobic respiration pathway were enriched with acetyl-lysines. We further compared the lysine acetylome of MAB GZ002 with those from the reported MTB strains, array of common acetylated proteins and identical amino acid sequences flanking the acetylation sites were identified in these two species. We hypothesized that these conserved lysine acetylation pathways might be potential targets for therapeutic avenues.

EXPERIMENTAL PROCEDURES Bacterial Strain A full description of the speciation procedures for the MAB clinical isolate GZ002 obtained form the Guangzhou Chest Hospital (P. R. China) can be found in SI. Bacteria were grown in Middlebrook 7H9 broth (Difco), supplemented with 10% Oleic acid-bovine albumin-dextrose-catalase (OADC) (Becton Dickinson), 0.05% Tween-80 and 0.5% glycerol.

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Cultures were maintained at 37 °C. Three genes of the MAB

GZ002, including the 16S rRNA, the DNA gyrase subunit B (gyrB), and the DNA-directed RNA polymerase subunit β (rpoB)

21-22

were amplified by PCR using

primers listed in Table S-1. After gene sequencing (Beijing Genomics Institute), 5 ACS Paragon Plus Environment

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BLAST searches were performed to compare each of the MAB GZ002 gene sequence with its homologous gene in reference strain MAB ATCC 19977.

Extraction and Trypsin Digestion of Proteins Soluble proteins were extracted from mid-log phase bacteria using sonication as described.

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The bacteria were harvested from the same batch, washed twice in cold

PBS (pH 7.5) and sonicated in 8 M urea lysis buffer, supplemented with 1% (v/v) protease inhibitor cocktail (Protease Inhibitor Cocktail SetIII; Calbiochem), 1 mM dithiothreitol (DTT), 2 mM EDTA and deacetylase inhibitor mix (30 mM nicotinamide,

50 mM sodium butyrate and 3 µM trichostatin A). After centrifugation, the 2-D Quant kit (GE Healthcare) was used to quantify the protein content in clear supernatant according to the manufacturer’s instructions. Together, 12 mg of soluble proteins were used. The proteins were precipitated with 20% (w/v) trichloroacetic acid overnight at 4 oC. Then the precipitates were washed three times with cold acetone. Peptide reduction and alkylation were carried out using 5 mM dithiothreitol (at 56 °C for 45 min) and 15 mM iodoacetamide (at room temperature for 30 min, protected from light) respectively. The reactions were stopped by adding 15 mM cysteine and kept for 30 min at room temperature. Proteins were subjected to overnight trypsin (Promega) digestion in 100 mM NH4HCO3.

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The processed peptides received a

second trypsin digestion for 4 h to ensure complete digestion. The digested peptides were lyophilized in 8 cryotubes using a SpeedVac concentrator (Thermo Scientific) and stored at – 80 °C for further characterization. 6 ACS Paragon Plus Environment


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Immunoenrichment of Acetylated Peptides The lysine-acetylated peptides in 8 cryotubes were enriched using the anti-acetyl-lysine pan antibody-conjugated beads (PTM Biolabs) as described previously.

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Peptides were suspended in NETN buffer (50 mM Tris-HCl at pH 8.0,

100 mM NaCl, 1 mM EDTA and 0.5% v/v Nonidet P-40), then incubated with the antibody-conjugated beads for 8 h at 4 °C with rotary shaking. After centrifugation, the beads were washed in cold PBS and distilled water. The bound peptides were eluted in 0.1% (v/v) trifluoroacetic acid and subsequently lyophilized in a SpeedVac concentrator.

Mass Spectroscopy The immunoprecipitated peptides from each cryotube were re-suspended in buffer containing 2% (v/v) acetonitrile and 0.1% (v/v) formic acid (solvent A). After centrifugation, supernatant from each cryotube was loaded onto an Acclaim PepMap 100 C18 trap column (Dionex) by the EASY nLC1000 nanoUPLC (Thermo Scientific) and resolved on an Acclaim PepMap RSLC C18 analytical column (Dionex). Peptides were eluted at a linear gradient of 5-30% solvent B (80% v/v acetonitrile and 0.1% v/v formic acid) for 34 min at constant flow rate of 300 nL/min, followed by 2 min in 40% solvent B, then 2 min to 80% solvent B, and maintaining at 80% solvent B for 4 min. After separation, the peptides were ionized by the Nanospray Ionization (NSI) Source (Thermo Scientific) and analyzed using an online nanoflow LC–MS/MS on a Q 7 ACS Paragon Plus Environment

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Exactive quadrupole Orbitrap mass spectrometer (Thermo Scientific). For the MS/MS analysis, intact peptides with a resolution of 70000 or ion fragments with a resolution of 17500 were detected by the Orbitrap mass analyzer. They were then fragmented using 25% Normalized Collision Energy (NCE) with 4% stepped NCE. The data acquisition was performed in a data dependent manner whereas the 15 most intense precursor ions from each MS survey scan were selected for fragmentation. Full MS spectra were acquired from m/z 350 to 1800. The electrospray voltage applied was 1.8 kV. Automatic

gain control (AGC) was set so that the MS/MS were generated from 2 × 105 ion counts. Together 8 runs of LC–MS/MS were performed and data were pooled for further analysis.

Data Processing The pooled raw MS/MS data from 8 runs were analyzed by MaxQuant software with integrated Andromeda search engine (v. 1.3.0.5) as described. 8-9 Data were searched against the Uniprot M. abscessus ATCC19977/DSM44196 protein database (http://www.uniprot.org/proteomes/UP000007137) that contained 4940 protein sequences, the reverse decoy database and protein sequences of common contaminants. The search parameters in this study were: a) precursor mass ranged between 350 to 1800 Da; b) minimum peak count was set to 5; c) signal to noise threshold set to 1.5; d) trypsin was used as a protease and it was allowed up to 3 missed cleavages; e) precursor mass tolerance of 10 ppm and fragment tolerance of 0.02 Da; f) oxidation of methionine, acetylation on lysine and acetylation on protein N-terminus were set as 8 ACS Paragon Plus Environment


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variable modifications and carbamidomethylation of cysteine as fixed modification; and g) 1% false discovery rate. Minimum peptide length was set at 7. The identified lysine acetylation sites with localization probability less than 0.75 or acetyl-lysine sites from reverse or contaminant protein sequences were excluded from further analysis.

Bioinformatics Analysis (i) Protein Functional Annotation The identified acetylated proteins were grouped in the Gene Ontology Annotation (UniProt-GOA) Database (www.http://www.ebi.ac.uk/GOA/) to cellular component, biological process and molecular function based on their Gene Ontology (GO) terms. If an acetyl peptide could not be annotated by the UniProt-GOA database, the peptide was assigned to a GO function using the InterProScan software

26

based on its peptide

sequence. (ii) Functional Enrichment Analysis To gain information on which pathways were enriched for acetyl-lysines, GO enrichment analysis was performed to annotate protein pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) online tools, the KEGG Automatic Annotation Server (KAAS) database and the KEGG mapper as described.

8, 23

The

InterPro database and InterProScan software were used to annotate protein domains and the protein’s subcellular localization was predicted using Cello database (http://cello.life.nctu.edu.tw/). In the enrichment analysis, fold enrichment was calculated using the formula: (a/c) / (b/d), where “a” was the number of acetylated 9 ACS Paragon Plus Environment

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proteins in each GO term; “b” was the number of proteins quantified in each GO term; “c” was the total number of acetylated proteins; and “d” was the total number of all proteins. Fisher’s exact test was applied to test if the fold enrichment of acetylated proteins in specific annotation term was significant. A GO term was defined as significantly enriched with acetylated proteins when the fold enrichment > 1 and the p-value < 0.05. (iii) Motif Analysis The acetyl-lysine motifs were analyzed using motif-X

27

to identify enrichment or

depletion of amino acids in the acetyl-21-mers (10 amino acids upstream and downstream of the site) compared to that of the non-acetyl-21-mers in an identical protein. The flanking sequence of 21-mers was examined to provide better coverage of the acetylation motif than the 13-mers. Fisher’s exact test was applied to test if the relative abundance of an amino acid was significant in a position of the acetyl-21-mers. The relative abundances were schematically represented as -log10 of the Fisher’s exact test p-values in an intensity map. The corresponding p-value < 0.05 was considered statistically significant. (iv) Motif-based Clustering Analysis. Clusters of acetylated proteins in each motif logo were subject to GO enrichment analysis as described. For the enrichment analysis of an individual logo in a specific GO term, the Fisher’s exact test p-value was −log10 transformed and it was given a Z score. The motif logos were then clustered to specific GO term using one-way hierarchical clustering (Euclidean distance, average linkage clustering) in Genesis. The 10 ACS Paragon Plus Environment


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clusters of motif logos in cellular component, biological process and molecular function were plotted in heat maps using the “heatmap.2” function from the ggplots plotting system in the R graphic package.

RESULTS Identification of MAB GZ002 Nucleotide sequence alignment showed that the full 16S rRNA gene sequence and, partial gyrB and rpoB gene sequences of the clinical isolate MAB GZ002 were identical with the reference strain MAB ATCC 19977 (Figure S-1). We therefore confirmed that the genetic background of this clinical isolate belonged to the MAB complex.

Identification of MAB GZ002 Lysine Acetylome There were 289 lysine-acetylated proteins and 459 acetylation sites in the MAB GZ002 harvested from 7H9 broth culture supplemented with glycerol and OADC. The total protein coverage rate of lysine acetylation in MAB GZ002 was 5.87% (Table 1). Figure 1A shows that 83% of the trypsinized peptides were within the range of trypsin cleavage sizes (8-20 peptides). 28-29 The accuracy of LC-MS/MS analysis was revealed by the mass error that measured the differences between the exact mass and the integer mass of the identified peptide. Majority of the identified peptides (81%) had mass errors of ± 2 ppm, below the mass tolerance of 10 ppm (Figure 1B). In the present study, a single lysine acetylation site was detected in approximately two-thirds (68%, 11 ACS Paragon Plus Environment

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199 proteins) of the lysine acetylome in MAB GZ002 (Figure 1C). The top three highly acetylated proteins were the 60 kDa chaperonin 2 (14 acetyl sites), the 70 kDa chaperone protein (10 acetyl sites) and the 2-oxoglutarate dehydrogenase (7 acetyl sites) (Table S-2). Overall, we found that at least 55% of the MAB GZ002 lysine acetylome (161 acetylated proteins) was in common with the lysine acetylome from MTB H37Rv

8

grown with OADC supplementations (Figure 2). Total 52 identical

acetylation sites in 37 proteins were identified in MAB GZ002 and MTB H37Rv,

8

including 18 identical acetylation sites in 10 proteins were also found in common with MTB H37Ra (Table 2). 9

GO Functional Annotation of Acetylated Protein We conducted GO annotation analysis to determine the functional domains of the MAB GZ002 lysine acetylome using the UniProt-GOA Database. Given the wide distribution of lysine acetylation in eukaryotic and prokaryotic cells,

11

we first

determined the distribution of acetylated proteins in three annotation categories, including the cellular component, biological process and molecular function. It was predicted that 9.5 % of the total proteins mapped to cellular component and biological processes were acetylated, while the acetylation rate in the molecular function category was 8.3 % (Table 3). Since the lysine acetylome profile of MAB GZ002 could be affected by the availabilities of carbon sources, the lysine acetylome identified here represented both carbohydrate and fatty acid metabolisms, as glycerol, oleic acid and dextrose supplementations were included in the culture media. 12 ACS Paragon Plus Environment


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Functional Enrichment Analysis GO enrichment analysis was applied to determine if certain cell functions or cellular components (GO terms) of the individual annotation category were in favor of lysine acetylation. We defined a GO term to be significantly enriched with acetyl-lysines if its respective Fisher’s exact test p-value was less than 0.05 (-log10 (p-value) > 1.3) with fold of enrichment greater than 1 (Figure 3 and Table S-3). In the cellular component category, lysine acetylation in the large ribosomal subunit (4 of 7 proteins acetylated in this GO term) was highly enriched (fold enrichment = 6) and statistically significant (-log10 (p-value) = 1.67) (Figure 3A). Two of the biological processes in MAB GZ002 were significantly enriched with acetyl-lysines, including the tricarboxylic acid cycle (8 of 14 proteins acetylated, fold enrichment = 6, -log10 (p-value) = 3.9) and the reactive oxygen species metabolic process (5 of 7 proteins acetylated, fold enrichment = 7.49, -log10 (p-value) = 2.66) (Figure 3A). In the molecular function category, the GO term translation factor activity (nucleic acid binding) that comprised of 12 proteins in which 6 were acetylated was significantly enriched with acetyl-lysines (fold enrichment = 6, -log10 (p-value) = 2.74) (Figure 3A). In addition, several cellular pathways and protein domains were also significantly enriched with acetyl-lysine. The abundance of acetyl-lines in carbon fixation pathways, the aerobic respiration (citrate cycle), carbon/lipid metabolism pathways, and the ATP-binding fold domain (Figure 3B and 3C) suggested this modification was involved in production and utilization of energy that were critical for bacterial survival. To further shed light on 13 ACS Paragon Plus Environment

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this hypothesis, we compared the list of proteins in the lysine acetylome of MAB GZ002 with the genomic regions in MTB that were essential for growth. About 25 % 30 to 37 % 31 of acetylated proteins in the MAB GZ002 were essential depending on the classification in different studies (Table S-2).

Lysine Acetylation Motif To assess if there was significant enrichment of specific amino acids flanking the acetylation sites respect to the general amino acid composition of the entire MAB GZ002 proteome, we conducted motif analysis and generated a heat map to illustrate the relative abundance of amino acids flanking the acetylation sites. It was evident that the acetyl-lysine flanked by four amino acids exhibited high abundances that included the aspartic acid (D) at the -1 position; histidine (H) at the +1 position; tyrosine (Y) at the +1 position; and phenylalanine (F) at the -2 and +1 positions (-log10 (p-value) = 5, Figure 4A). Enrichment of arginine (R) was found in further distance from the acetyl-lysine at +5 position (-log10 (p-value) = 5, Figure 4A).

Motif of Lysine Acetylome Is Clustered to Distinct Cell Functions There were four highly conserved motif sequences (motif logos) in the lysine acetylome of MAB GZ002. The Y, F and H were most commonly found at the +1 position of the acetyl-lysine and the D was commonly found at the -1 position (Figure 4B and Table S-4). Similar patterns were also characterized in the acetyl-lysine substrates derived from the mitochondria in higher eukaryotes.

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The preference for 14

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hydrophilic Y at the +1 position (motif logo 1, KacY) was enriched in pup ligase/deamidase and DNA topoisomerase (type IIA−like domain) proteins (Figure 4C). The same motif logo was also found enriched in various binding activities of glycolysis or gluconeogenesis, carbon metabolism pathways and, to a lesser extent, in amino acids biosynthesis pathway (Figure 4D). Hydrophobic F at the +1 position of acetyl-lysine (motif logo 2, KacF) was more commonly found in the lysine-acetylated proteins annotated to the metabolic pathways of propanoate, β-alanine, fatty acid, and degradation of hydrophobic amino acids (Figure 4D). The motif sequence with a positively charged amino acid H at the +1 position (motif logo 3, KacH) was enriched in the ATP-citrate lyase, the succinyl-CoA ligase or synthetase-like protein domains, and in the TCA cycle of MAB GZ002 (Figure 4C and 4D). There was also a preference for negatively charged D at the -1 position (motif logo 4, DKac). Though it was not unique to the MAB GZ002, this pattern was not reported in MTB.

8-9

The motif logo 4 (DKac) could be commonly

found in the ATP-dependent Clp protease protein domain, as well as the transketolase-like protein, pyrimidine-binding protein and translocation-enhancing protein domains (Figure 4C). This motif was also enriched in diverse cellular pathways, including the RNA degradation and RNA polymerase pathways, and the purine metabolism pathway of mycobacteria (Figure 4D). The GO enrichment analysis further revealed clusters of metabolic processes and molecular functions for individual acetyl-lysine motif. Acetylated proteins containing motif KacY were significantly enriched in binding and isomerase activities (Figure 15 ACS Paragon Plus Environment

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S-2). It was also abundant in wide range of catabolic and metabolic processes as well as glycolysis process (Figure S-3). The motif KacF was mainly enriched in structural protein functions and had weak association with biological processes (Figure S-2 and S3). Consistence with the TCA cycle enriched characteristic, the motif KacH was more commonly found in the enzyme activities mediating acid-thiol and succinate-CoA formations, coenzyme A (CoA) activities of TCA cycle (Figure S-2), and in the cellular respiration processes (Figure S-3). Lastly the motif DKac sequence was abundant in the adenyl nucleotide and ATP binding activities other than the RNA polymerase activity (Figure S-2). The DKac was enriched in protein folding process, protein metabolic process and wide range of catabolic processes (Figure S-3). Taken together, the clusters of proteins that acted on distinctive processes or pathways were associated with the individual acetyl motif. Our results suggested that specific acetyltransferase(s) that recognized the respective motif might be responsible for the acetylation in different cellular pathways of MAB GZ002.

Lysine acetylome is associated with mycobacterial virulence and antibiotic resistance Several antibiotic resistance, virulence and bacterial persistence-related factors were identified in the lysine acetylome of MTB H37Rv, thus, it was speculated that the lysine acetylation might play a role in TB disease progression.

8

Searching from the

MTB drug-resistant genes listed in the TB Drug Resistance Mutation Database, 33 we identified 11 of 40 drug-resistance-related orthologous gene products in the lysine 16 ACS Paragon Plus Environment


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acetylome of MAB GZ002 (Table 4). In this list, 9 factors were commonly found in the lysine acetylome of MTB H37Rv, 8 and 3 factors were identified in the attenuated MTB H37Ra strain (Table 4). 9 The 3 highly conserved resistance-related factors in the lysine acetylomes of MAB GZ002, MTB H37Rv and MTB H37Ra were the DNA-directed RNA polymerase subunit β and β', and the 3-oxoacyl-[acyl-carrier-protein] reductase (Table 4). However the 3-oxoacyl-[acyl-carrier-protein] synthase 1 (MAB_4608) implicated in isoniazid (INH) resistance was found in the lysine acetylome of MAB GZ002 but not in MTB (Table 4). This may be important as the MTB but not the MAB was susceptible to INH. 34 We compared the MTB virulence factors

20, 35

with the lysine acetylome of MAB

GZ002 to show that 20 orthologous virulence-related factors were identified in the lysine acetylome of MAB GZ002, of which 16 were found in lysine acetylome of MTB H37Rv and 4 were identified in attenuated strain MTB H37Ra (Table 4). The 4 commonly acetylated virulence-related substrates in MAB GZ002 and the two strains of MTB were the glutamine synthetase, the sulfurtransferase, the isocitrate lyase and the long chain fatty acid CoA (Table 4). Since the actual molecular mechanisms underlying persistence infection of mycobacteria remain unclear, some genes that are considered as persistence-related 8, 36 might also play a role in the antibiotic resistance or virulence. We identified 6 persistence-related factors in the lysine acetylome of MAB GZ002, all of which were identified in the MTB H37Rv and 3 were found in the MTB H37Ra (Table 4). The long chain fatty acid CoA FadD26, the chaperone protein DnaK and the isocitrate lyase 17 ACS Paragon Plus Environment

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were the 3 commonly acetylated persistence-related factors (Table 4). The catalase-peroxidase that was required for activation of INH was substrate for lysine acetylation in MAB GZ002 and MTB H37Rv, but not in MTB H37Ra (Table 4).

DISCUSSION One of the advantages of using fresh clinical isolate rather than the laboratory reference strains was that we could limit the functional impairments due to significant genomic variations after long-history of cultivation in vitro. 37 The use of MAB GZ002 isolate could therefore provide more clinically relevant data. Using the anti-acetyl-lysine immunoprecipitation method, we were able to identify a vast array of acetylated proteins in MAB GZ002. The numbers of lysine-acetylated proteins in the metabolic pathways of MAB GZ002 were abundant, suggesting a role of lysine acetylation in regulating energy metabolism.

13, 38

Several key factors of the aerobic

respiration pathway were commonly found in the lysine acetylomes of MAB GZ002 and MTB. 8-9 These included the two subunits of pyruvate dehydrogenase (E1 and E2) that convert pyruvate to acetyl-CoA in the glycolysis cycle, the acetyl-CoA synthetase that ligates acetate and CoA to produce acetyl-CoA and the citrate synthase catalyzes the first reaction in the TCA cycle (Table S-5). 39 Therefore, lysine acetylation may be a common mechanism for metabolic regulations in mycobacteria. More intriguingly, 52 identical acetylation sites in 37 proteins were determined in MAB GZ002 and MTB H37Rv, 8 including an identical acetylation site (_VYKacNYDPR_) of citrate synthase (Table 2). This suggests that one or several acetyltransferases that recognize the same 18 ACS Paragon Plus Environment


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acetylation sites are evolutionary conserved between MAB GZ002 and MTB H37Rv, though these sites might also be the targets for non-enzymatic acetylation. 40 The lysine acetylation profile of mycobacteria can be influenced by numerous factors including the availability of energy sources. To generate comparable data, the MAB GZOO2 used in the present study was grown in a similar way as the previous MTBs studies,

8-9

of which the immunoprecipitation of the lysine acetylated proteins

was also done in a comparable fashion. It should be emphasized that glycerol and OADC supplements were included in the culture media. As shown in Table S-5, the lysine acetylome of MAB GZ002 contained at least 36 % (14 of 39) of the proteins in the glycolytic pathway, 29 % (14 of 48) in pyruvate metabolism pathway, and 48 % (16 of 33) in the TCA cycle. Our data therfore match the wide distribution of of lysine acetylation in central metabolism of MTB strains. 8-9 The key findings that stimulated interest in relationship between lysine acetylation and the anti-tuberculosis drug-resistance were the identifications of resistance-related factors in the lysine acetylome of MTB H37Rv

8

and the involvement of MAB

acetyltransferases in the aminoglycosides resistance. 3, 19 However, comparisons of the lysine acetylomes in MAB GZ002 and the two MTB strains revealed no specific correlations between lysine acetylation and drug-resistant or pathogenic strains (Table 4). Despite that, the abundances of lysine acetylation in drug-resistance- and virulence-related proteins suggests that acetylation may play a role in regulating some of these proteins.


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Persistent mycobacteria that have limited accesses to oxygen and carbohydrates can utilize the host cholesterol to survive. 41 In order to switch from carbohydrate to fatty acid metabolism, mycobacteria up-regulate isocitrate lyase

42

to promote the

productions of glyoxylate and succinate from the fatty acid-derived acetyl-CoA, and replenish the CO2-generating steps of TCA cycle that are catalyzed by the isocitrate dehydrogenase. 43 Both isocitrate lyase and isocitrate dehydrogenase were identified in the lysine acetylomes of MAB GZ002 (Table S-5), MTB H37Rv and H37Ra.

8-9

We

therefore speculated that the lysine acetylation might also play a role in regulation of the carbon flux between the TCA and glyoxylate cycle. To further support this notion, 45% of the proteins (15 of 33) in the glyoxylate cycle were lysine-acetylated in MAB GZ002 (Table S-5). Another requirement for the mycobacteria to persist in host cell is the ability to survive under hypoxia conditions.

44

The gene products of MAB_3008,

MAB_4254c, tpiA, MAB_2779c, gpmA and MAB_2109 in the glycolysis pathway; and the three subunits of succinate dehydrogenase, MAB_4421, MAB_4422 and MAB_4423 in the TCA pathway were considered as essential factors for the survival in hypoxia that had also been identified in the lysine acetylome of MAB GZ002 (Table S-2). The attenuated MTB H37Ra 9 might have lost the ability to acetylate certain number of virulence and persistence-related proteins. For example, the catalase-peroxidase (katG) that neutralized the reactive oxygen species produced by the macrophage

45

could be found in the lysine acetylomes of MAB GZ002 and MTB H37Rv, 8 but not in the MTB H37Ra (Table 4). 9 The chaperone protein (DnaK), the membrane lipoprotein (LprG) and cytoplasmic 60 kDa chaperonin proteins that mediated the toll-like receptor 20 ACS Paragon Plus Environment


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(TLR) signaling in macrophages and/or dendritic cells 46-48 were identified in the lysine acetylomes of MAB GZ002 and MTB H37Rv, 8 but not in MTB H37Ra (Table 4).

9

Our data therefore suggest that certain aspects of the lysine acetylation network are evolutionary conserved in the MAB GZ002 and MTB H37Rv. Comparison of the lysine acetylomes in MAB GZ002, MTB H37Rv 8 and MTB H37Ra

9

revealed 52 sites in 37 proteins were identical in MAB GZ002 and MTB

H37Rv, including 18 acetylation sites in 10 proteins were also conserved in MTB H37Ra (Table 2). Motif KacY was harbored in 8 of 52 identical acetylation sites followed by the motifs KacF and DKac that were found in 4 of 52 identical sites (Table 2). The KacY and KacF motifs were seen in the 18 identical acetylation sites conserved in MAB GZ002, MTB H37Rv 8 and MTB H37Ra

9

without a clear cluster pattern

(Table 2). Despite the discovery of these conserved elements in lysine acetylation of MAB GZ002 and MTB strains, it is still largely unknown whether these identical acetylation sites are the targets of specific acetyltransferases or non-enzymatic acetylations. The preferences for F, H and Y amino acid residues at the +1 position and D at -1 position of the acetyl-lyisne (Figure 4A) may suggest a preference for linear motif binding in MAB GZ002 if the lysine acetylation is carried out by the acetyltransferases. Recognition of H and Y at the +1 position of modified lysine was also observed in the lysine acetylomes of mouse mitochondrial fractions, 32 E. coli

49

and MAB H37Rv. 8 Functional enrichment analysis of the acetyl-lysine motifs revealed enrichments of KacH motif in aerobic respiration process and KacY in various 21 ACS Paragon Plus Environment

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carbohydrate metabolic processes that were consistent with the functions of mitochondria in eukaryotic cells (Figure S-3). Our findings therefore support the hypothesis that the acetylation motifs (KacH and KacY) and the possible related acetyltransferase(s) that recognize these motifs are evolutionally conserved. Additionally, the prefer recognition of amino acids at the ±1 position of the acetyl-lysine suggests that a small subset of acetyltransferases, similar to those identified in E. coli and mitochondria,

32, 49

might be responsible for the lysine

acetylation in MAB GZ002. However, E.coli acetylation can also be catalyzed by non-enzymatic reactions depending on the availability of acetyl phosphate and the accessibility of target lysine in the protein.

40, 50

D at the -1 position was seen more

enriched in an ackA mutant that accumulated acetyl phosphate in E. coli than in the wild type bacteria.

40, 50

Therefore, at least for the D at the -1 position, the specificity of

acetylation motif in target proteins can also be due to non-enzymatic acetylation.

CONCLUSIONS

Here we demonstrate a profile of lysine acetylation in the proteome of rapid-growing strain MAB GZ002. Pathways enriched for this PTM are carbon metabolism, aerobic respiration and TCA cycle. Several drug-resistant, virulence and persistence-related factors were acetylated in MAB GZ002 and MTB strains. An array of identical acetylation sites in MAB GZ002 and MTB strains was also identified. Though the contribution and specificity of acetyltransferases and the non-enzymatic 22 ACS Paragon Plus Environment


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Page 24 of 45

acetylation to the acetylation profile in MAB GZ002 is unclear, the involvement of acetylated proteins in MAB pathogenesis and drug-resistance suggests that they are good

targets

for

future

study.

A

better

understanding

of

the

lysine

acetylation-dependent mechanisms in MAB disease progression may lead to potential avenues for more effective treatments.

Figure Caption Figure 1. Profiling lysine acetylome of MAB GZ002. Acetylation covered 5.87 % of total proteins (289 over 4920 proteins) in MAB GZ002. (A) Distribution of trypsin-cleaved peptide length. (B) Distribution of peptide score and mass error of identified acetyl peptides. (C) Number of acetylation sites in lysine acetylated proteins.

Figure 2. Comparison of the lysine acetylomes of MAB GZ002 and the MTB H37Rv adopted from Xie et al. 8

Figure 3. Functional enrichment analysis of lysine acetylome. (A) Enrichment analysis of acetyl protein based on annotations of acetyl-lysine sites for cellular component, biological processes and molecular function. (B) KEGG pathway


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enrichment analysis for lysine acetylated proteins. (C) InterPro protein domain enrichment analysis for lysine acetylated proteins. Data was presented as –log10 of Fisher exact test p value. A GO term was defined as significantly enriched with acetyl-lysine when –log10 (Fisher’s test p-value) > 1.3 with fold enrichment > 1. All examples shown meet both the p-value and fold-enrichment threshold significance.

Figure 4. Bioinformational analysis of the lysine acetylation sites and functional enrichment analysis of the motif logos. (A) The intensity map showed the relative abundance of amino acids flanking the acetyl-lysine at the acetyl-21-mers (10 amino acids upstream and downstream of the site). The relative abundance was represented as the –log10 (Fisher’s test p-value) of an amino acid in a position from the acetyl-lysine. Red, amino acid was most abundant. Green, amino acid was least abundant. (B) The four most abundant acetylated peptide motif logos. The * symbol represented random amino acid. (C) Heat map representing the InterPro protein domain enrichment analysis for the motif logos. (D) Heat map representing the KEGG pathway enrichment analysis of the motif logos. For each GO term, the –log10 (Fisher’s test p-value) was calculated and the degree of enrichment was estimated according to defined criteria based on Z scores (Zs). Motif logo was significantly enriched when Zs = -1, indicated as red.



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Page 26 of 45

Table 1. Lysine acetylation in Mycobacterium species. Genome

No. of

No. of

No. of

％

size

total

acetylated

acetylation

Proteome

(Mb)

proteins

proteins

sites

acetylated*

GZ002

5.09

4920

289

459

5.87

M. tuberculosis

a

4.41

4018

658

1128

16.38

M. tuberculosis

b

4034

137

226

3.40

Species M. abscessus

Strain

H37Rv H37Ra

a

4.42 8

Data adopted from Xie et al. Data adopted from Liu et al. 9 * % Proteome acetylated = (No. of acetylated proteins / No. of total protein)*100 b

Table 2. Identical lysine acetylation sites in MAB GZ002 and MTB H37Rv. Common lysine acetylated protein with MTB H37Rv 8

Identical lysine acetylatation motif #

Functional category *

DNA gyrase subunit A

_TKacTDLYR_

Information pathways

DNA-directed RNA polymerase subunit beta

_LHHLVDDKacIHAR_


DNA-directed RNA polymerase subunit beta'

_TLKPEKacDGLFCEK_#


RNA polymerase sigma factor SigA

_AVEKacFDYTK_


Elongation factor G

_QADKacYDVPR_


DNA-binding protein HU homolog (Histone-like)

_AELIDVLTQKacLGSDR_


Translation initiation factor IF-3

_IDDHDYETKacK_


50S ribosomal protein L19

_AKacLYYLR_


50S ribosomal protein L5

_SIAQFKacLR_


30S ribosomal protein S16

_ITGDWQKacFK_


Ribonuclease 3

_SEGDLAKacLR_


DNA polymerase III subunit beta

_LLDAEFPKacFR_#


50S ribosomal protein L7/L12

_DLVDGAPKacPLLEK_#



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_TTLTAAITKacVLHDK_# Elongation factor Tu

Information pathways _VSALKacALEGDA_#

30S ribosomal protein S3

_ADLEKacLTGK_


Ribonucleoside-diphosphate reductase

_SKacYASGEFFDK_


Sulfurtransferase

_DFVDQQQFSKacLLSDK_

Intermediary metabolism and respiration

_DEPHADAEKacYR_ Pup-protein ligase

_IAQLDLAYHDIKacR_


_TVLCKacDPFR_ Phosphoenolpyruvate carboxykinase [GTP]

_LPK VFFVNWFR_

Putative oxidoreductase /3-ketosteroid-9-alpha-hydrox ylase reductase subunit

_ELAAKacYPDR_

Adenosylhomocysteinase

_FDNKacYGTR_

Deoxycytidine triphosphate deaminase

_LEGKacSSLGR_

Succinyl-diaminopimelate desuccinylase

_AKacYGWTDVSR_

ATP-dependent Clp protease proteolytic subunit

_ESNPYNKacLFEER_

Imidazoleglycerol-phosphate dehydratase

_DPHHITEAQYKacAVAR_

Citrate synthase

_VYKacNYDPR_

ac

#

_HFFVHYKacDLEPGK_# Inorganic pyrophosphatase _NKacYEVDHETGR_# Transaldolase

_VDTEIDKacR_

Probable ATP-dependent Clp protease ATP-binding subunit

_YIEKacDAALER_

Probable mannose-6-phosphate isomerase ManA

_GGLTPKacHVDVPELLR_

Intermediary metabolism and respiration Intermediary metabolism and respiration Intermediary metabolism and respiration Intermediary metabolism and respiration Intermediary metabolism and respiration Intermediary metabolism and respiration Intermediary metabolism and respiration Intermediary metabolism and respiration Intermediary metabolism and respiration Intermediary metabolism and respiration Intermediary metabolism and respiration Intermediary metabolism and respiration



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Probable phosphoglucomutase PgmA


_PSGTEDVYKacIYAESF_ _DKacMAMQR_

Virulence, detoxification, adaptation

Chaperone protein DnaK _NTTIPTKacR_ _AVNIKacPLEDK_# 10 kDa chaperonin

Page 28 of 45


ac

_YGGTEIK YNGEEYLILS AR_# _LAKacLAGGVAVIK_# _AVEKacVTETLLK_# _GIEKacAVEK_ _IGAELVKacEVAK_#

60 kDa chaperonin _KacWGAPTITNDGVSIAK_#


_WGAPTITNDGVSIAKacEIE LEDPYEK_ _NVAAGANPLGLKacR_# _NVVLEKacK_# _KacHSEIMGTINQQR_ Antigen 84

_PLTPADVHNVAFSKacPPI GK_#

Cell wall and cell processes

Probable acyl-CoA thiolase FadA (MAB_0850)

_FQKacDLNIPDEK_

Lipid metabolism

FHA domain-containing protein

_EPVDSAVLANGDEVQIG KacFR_#

Uncharacterized

, Identical acetylation site also in lysine acetylome of MTB H37Ra 9 *, Classifications were based on the Tuberculist database (http://tuberculist.epfl.ch/) #

Table 3. Percentage of lysine acetylation of MAB GZ002 in 3 gene ontology categories. No. of total proteins

No. of acetylated

mapped per

proteins per

category

category

Cell component

880

84

9.5

Biological processes

1836

175

9.5

% Acetylation per category


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2808

Molecular function

233

8.3

Table 4. Comparison of the antibiotic resistance, virulence and persistence-related factors identified in this and previous studies. Lysine acetylation Protein description

Antibiotic resistance

MAB GZ002 isolate*

MTB H37Ra**

MTB H37Rv***

3-oxoacyl-[acyl-carrier-protein] synthase 1

YES

-

-

Enoyl-[acyl-carrier-protein] reductase [NADH]

YES

-

YES

Catalase-peroxidase

YES

-

YES

3-oxoacyl-[acyl-carrier-protein] reductase

YES

YES

YES

DNA gyrase subunit A

YES

-

YES

DNA gyrase subunit B

YES

-

YES

DNA-directed RNA polymerase subunit beta

YES

YES

YES

DNA-directed RNA polymerase subunit beta'

YES

YES

YES

Enhanced intracellular survival protein

YES

-

-

Polyribonucleotide nucleotidyltransferase

YES

-

YES

Putative aminoglycoside phosphotransferase

YES

-

YES

Superoxide dismutase

YES

-

YES

Protein translocase subunit

YES

-

YES

Iron-dependent repressor

YES

-

YES

Glutamine synthetase

YES

YES

YES

Sulfurtransferase

YES

YES

YES

RNA polymerase sigma factor sigA

YES

-

YES

Virulence



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Persistence

Page 30 of 45

Enhanced intracellular survival protein

YES

-

-

Putative cytochrome P450, steroid C27-monooxygenase

YES

-

YES

Nucleoside diphosphate kinase

YES

-

-

Possible Mg2+ transport P-type ATPase C

YES

-

-

Isocitrate lyase

YES

YES

YES

O-succinylbenzoate-CoA ligase

YES

-

YES

Long chain fatty acid CoA FadD26

YES

YES

YES

Lipoprotein lprG

YES

-

YES

Catalase-peroxidase

YES

-

YES

Pup-protein ligase

YES

-

YES

Iron-dependent repressor IdeR

YES

-

YES

Phosphoenolpyruvate carboxykinase [GTP]

YES

-

YES

60 kDa chaperonin

YES

-

YES

Superoxide dismutase [Cu-Zn]

YES

-

-

Long chain fatty acid CoA FadD26

YES

YES

YES

Chaperone protein DnaK

YES

YES

YES

Isocitrate lyase

YES

YES

YES

Catalase-peroxidase

YES

-

YES

3-ketosteroid-9-alpha-hydroxyla se reductase subunit

YES

-

YES

-

YES

ABC transporter ATP-binding YES protein *, MAB GZ002 acetylome dataset from this study **, MTB H37Ra acetylome dataset from Liu et al. 2014. 9 ***, MTB H37Rv acetylome dataset from Xie et al. 2015. 8

ASSOCIATED CONTENT


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Supporting Information Supporting Method; Figure S-1. Nucleotide sequence alignment of partial sequences of the (A) 16S rRNA, (B) gryB and (C) rpoB genes; Figure S-2. Functional enrichment analysis of the motif logos based on annotations of acetyl-lysine sites for molecular function; Figure S-3. Functional enrichment analysis of the motif logos based on annotations of acetyl-lysine sites for biological process; Table S-1. Primers used for PCR; Table S-2. Acetylated protein annotation summary; Table S-3. Acetyl protein functional enrichment analysis; Table S-4. Acetyl-lysine motif functional enrichment analysis; Table S-5. Lysine acetylated proteins in metabolic processes; MS-MS spectra of lysine acetylated peptides in MAB GZ002. All the supporting methods, figures and tables are available free of charge via the Internet at http://pubs.acs.org.

Raw

MS-MS

spectra

are

available

publicly

via

the

ProteomeXchange Datasets with dataset identifier number PXD004038.

AUTHOR INFORMATION

Corresponding Author *Tianyu Zhang: Room A132, 190 Kaiyuan Road, Science Park, Guangzhou, Guangdong,

China

510530;

Tel:

(+86)-20-32015270;

Email:

[email protected]

Author Contributions 30 ACS Paragon Plus Environment


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‡

Page 32 of 45

Jintao Guo and Changwei Wang contributed equally to this work.

ACKNOWLEDGMENT This work was supported by One Hundred Talents Program of the Chinese Academy of Sciences (Category A, to T.Z.), by the Open Project Grant (2014SKLRD-O06) and the Key Project Grant (SKLRD2016ZJ003) from the State Key Lab of Respiratory Disease, Guangzhou Institute of Respiratory Disease, First Affiliated Hospital of Guangzhou Medical University, by the PhD Start-up Fund of Natural Science Foundation of Guangdong Province, China (2016A030310123 to J.G.), by the Chinese Academy of Sciences-Commonwealth Scientific and Industrial Research Organization Joint Grant (154144KYSB20150045) and partially supported by Guangzhou Municipal Industry and Research Collaborative Innovation Program (201508020248) and Guangzhou Municipal Clinical Medical Center Program (155700012).

ABBREVIATIONS USED GNATs,

general

control

of

amino

acid

synthesis

protein

5

-related

N-acetyltransferases; GO, gene ontology; INH, isoniazid; InhA, enoyl-acyl carrier protein

reductase;

KatG,

catalase-peroxidase;

LC-MS/MS,

liquid

chromatography-tandem mass spectrometry; LprG, membrane lipoprotein; MAB, Mycobacterium abscessus; MTB, Mycobacterium tuberculosis; NTM, nontuberculous 31 ACS Paragon Plus Environment

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mycobacteria;

OADC,

Oleic

acid-bovine

albumin-dextrose-catalase;

PTMs,

post-translational modifications; rpoB, DNA-directed RNA polymerase subunit β; TB, tuberculosis; TCA, tricarboxylic acid.

REFERENCES

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

for TOC only Carbon metabolism

M. abscessus Protein extraction

TCA cycle Trypsin diges,on An,-acetyl-lysine precipita,on LC-MS/MS

Protein Lysine Acetylation

Aerobic respiration Antibiotic resistance Virulence

Bioinforma,cs analysis

Persistence


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Page 43 of 45

Figure 1 A

60

B

83%

400

81%

Peptide Score

No. of peptides

50 40 30 20

300

200

100

10 0

5

10

15

20

25

30

35

40

45

C

-6

-4

-2

0

2

4

200 150 100 50 10 5 0 1

2

3

4

5

6

7

8

6

Mass error (ppm)

Peptide length

No. of proteins

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


9 10 10+

No. of acetylation sites


8

10


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2 Lysine acetylated proteins

M. tuberculosis H37Rv

497

161

128 M. abscessus GZ002


Page 44 of 45

Page 45 of 45

A

Cellular component

Figure 3 Cytoplasmic part

[5.41]

Non-membrane-bounded organelle

[4.79] [4.32] [4.42] [2.17] [1.67] [1.97] [2.59]

Ribonucleoprotein complex Ribosome Ribosomal subunit Large ribosomal subunit Hexose catabolic process

Biological processes

DNA replication Protein folding Monosaccharide metabolic process Hexose metabolic process Glucose metabolic process NADP metabolic process Aerobic respiration Tricarboxylic acid cycle Reactive oxygen species metabolic process Purine nucleotide binding

Molecular function

Ligase activity RNA binding Ligase activity (carbon-nitrogen bonds) Oxidoreductase activity (CH-OH donors) rRNA binding Oxidoreductase activity (NAD or NADP acceptor) Structural molecule activity Structural constituent of ribosome Translation factor activity, nucleic acid binding

[1.76] [3.46] [3.60] [3.28] [1.55] [2.94] [3.93] [2.66] [3.39] [3.77] [3.42] [2.12] [3.19] [2.44] [3.02] [4.91] [5.01] [2.74]

0 2 4 6 8 Fold of enrichment and the [-log10 (p-value)]

Pathway enrichment

B

Pyrimidine metabolism

[2.07]

Propanoate metabolism

[2.76]

Pyruvate metabolism

[2.69]

Glyoxylate and dicarboxylate metabolism

[2.10]

Carbon metabolism

[8.89]

Pentose phosphate pathway

[1.57]

Citrate cycle (TCA cycle)

[3.63]

Carbon fixation pathways in prokaryotes

[3.95]

RNA degradation

[1.53] [1.53]

PPAR signaling pathway


C

Domain enrichment

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ClpP/crotonase-like domain

[1.43]

Rossmann-like alpha/beta/alpha sandwich fold

[1.95]

Pre-ATP-grasp domain

[1.39]

Ribosomal protein S5 domain 2-type fold

[1.95]

ATP-grasp fold, subdomain 2

[2.07]

Aldehyde dehydrogenase, cysteine active site

[1.61]

ATP-grasp fold, subdomain 1

[2.21]

ATP-grasp fold

[2.21]




Figure 4 B

A

Motif Logo 4 Motif Logo 4

Motif Logo 2

Motif Logo 3 Motif Logo 3

D

Motif Logo 2

C

Motif Logo 1

-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 +1+2+3 +4+5 +6 +7+8+9+10 A C D E F G H I K L M N P Q R S T V W Y

Motif Logo 1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 45

Motif

1

**********KY*********

16

3.98

2

**********KF*********

12

3.60

3

**********KH*********

8

3.50

4

*********DK**********

6

2.28

Z-score (log10 [Fisher p-value]) 1

0

-1

Z-score (log10 [Fisher p-value]) 1

0

Motif Fold score increase

Logo

-1


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