Polyamines Mitigate Antibiotic Inhibition of A.Actinomycetemcomitans Growth

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Polyamines Mitigate Antibiotic Inhibition of A.actinomycetemcomitans Growth

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Allan Wattimena

Graduate Program in Dentistry

The Ohio State University

2017

Master's Examination Committee:

Dr John Walters, Advisor

Dr Purnima Kumar

Dr Sara Palmer

Dr Shareef Dabdoub

Allan Wattimena

2017

Abstract

Polyamines are ubiquitous polycationic molecules that are present in all prokaryotic and eukaryotic cells. They are the breakdown products of amino acids and are important modulators of cell growth, stress and cell proliferation. Polyamines are present in higher concentrations in the periodontal pocket and may affect antibiotic resistance of bacterial biofilms. The effect of polyamines was investigated with amoxicillin (AMX), azithromycin

(AZM) and doxycycline (DOX) on the growth of Aggregatibacter actinomycetemcomitans

(A.a.) Y4 strain. Bacteria were grown in brain heart infusion broth under the following conditions: 1) A.a. only, 2) A.a. + antibiotic, 3) A.a. + antibiotic + polyamine mix (1.4mM putrescine, 0.4mM spermidine, 0.4mM spermine). Growth curve analysis, MIC determination and metatranscriptomic analysis were carried out. The presence of exogenous polyamines produced a small, but significant increase in growth of A.a.

Polyamines mitigated the inhibitory effect of AMX, AZM and DOX on A.a. growth.

Metatranscriptomic analysis revealed differing transcriptomic profiles when comparing

AMX and AZM in the presence of polyamines. Polyamines produced a transient mitigation of AMX inhibition, but did not have a significant effect on gene transcription. Many gene transcription changes were seen when polyamines were in the presence of AZM.

Polyamines in a periodontal pocket may help protect A.a. and contribute to antimicrobial resistance.

Dedication

This document is dedicated to my family.

iii

Acknowledgments

Thanks to Dr. Walters, Dr. Kumar, Dr. Dabdoub, Dr. Palmer and Dr. Sukirth Ganesan for guidance throughout the project.

Vita

2004...... St Joan of Arc CHS

2008...... B.S. Human Biology, University of Toronto

2014...... D.D.S., University of Minnesota

2017...... M.S., Dentistry, The Ohio State University

Fields of Study

Major Field: Dentistry

Table of Contents

Abstract ...... ii

Dedication ...... iii

Acknowledgments...... iv

Vita ...... v

Table of Contents ...... vi

List of Tables ...... viii

List of Figures ...... ix

Introduction ...... 1

Aim of study ...... 8

Hypotheses ...... 8

Materials and Methods ...... 8

Materials ...... 8

Bacterial culture ...... 9

Growth Curves ...... 9

Minimal Inhibitory Concentration (MIC) determinations ...... 10

Bacterial viability determinations ...... 11 vi

Metatranscriptomic analysis ...... 11

Results ...... 13

Effect of polyamines on inhibition of A.a. growth by AMX, AZM and DOX ...... 13

Effect of polyamines on MIC for A. actinomycetemcomitans ...... 14

Bacterial viability determinations ...... 15

Transcriptome analysis ...... 15

Effect of polyamines on gene expression in control cultures...... 15

Effect of polyamines on cultures inhibited by AMX and AZM ...... 16

Discussion ...... 17

Conclusion ...... 21

References ...... 22

Appendix A: Tables and Figures ...... 27

Appendix B: Relative abundances of SEED transcripts in A.a...... 45

vii

List of Tables

Table 1: Description of antibiotics used ...... 27

Table 2: The effect of polyamines on the MIC of AMX, AZM and DOX...... 28

Table 3: Summary of A.a. cDNA samples ...... 29

Table 4: Number of genes upregulated/downregulated >2 fold ...... 30

Table 5: Effect of polyamine mixture on gene transcription in A.a...... 31

Table 6: Effect of polyamines and AMX on gene transcription in A.a...... 32

Table 7: Effect of polyamines and AZM on gene transcription in A.a...... 33

viii

List of Figures

Figure 1: Polyamine synthesis pathway ...... 35

Figure 2: Growth curve: AMX ...... 36

Figure 3: Growth curve: AZM ...... 37

Figure 4: Growth curve: DOX ...... 38

Figure 5: Circle Plot: Polyamines vs Ctrl ...... 39

Figure 6: Circle Plot: AMX + Polyamines vs AMX...... 40

Figure 7: Circle Plot: AZM + Polyamines vs AZM ...... 41

Figure 8: KEGG Pathway Map: Polyamines vs Ctrl...... 42

Figure 9: KEGG Pathway Map: AMX + Polyamines vs AMX ...... 43

Figure 10: KEGG Pathway Map: AZM + Polyamines vs AZM ...... 44

Introduction

Periodontitis is characterized by the inflammatory destruction of tissues surrounding the tooth. Its cause is complex and thought to be due to host interactions with bacteria present in the oral cavity. The host response plays a significant role in determining the rate of destruction and attachment loss that occurs1.

About 0.5% of individuals are susceptible to a form of the disease called aggressive periodontitis2. Aggressive periodontitis has a rapid rate of disease progression and occurs in individuals who are otherwise healthy. The disease has a tendency for familial aggregation and typically affects those under 35 years of age3. The amount of periodontal tissue destruction is not consistent with the amount of local factors, as patients usually present with low plaque and calculus levels. There are two forms of the disease: a generalized and a localized form. The localized form affects at least two permanent teeth that are first molars and/or incisors and no more than two teeth other than first molars and incisors. The generalized form is defined as attachment loss affecting at least three teeth other than first molars and incisors. Approximately 35% of individuals with localized aggressive periodontitis progress into generalized aggressive periodontitis. These individuals may exhibit a poor serum antibody response to infecting agents4. The localized form appears to be self-limiting, whereas the generalized form can recur various times in a patient’s life. The bacterial profile of individuals with generalized

aggressive periodontitis appears to be similar to those with chronic periodontitis, though the level of destruction is considerably different5. This may be related to genetic predisposition, the host response to bacteria, or deficiencies in host defense mechanisms6.

Approximately 10% of individuals are highly susceptible to periodontitis, whereas 10% are highly resistant7. It is currently unclear which specific immune processes are responsible for aggressive periodontitis, but an excessive plasma cell infiltrate may show an individual’s inability to defend against periodontal pathogens and may be a predisposition to developing periodontitis. Problems with neutrophil function, chemotaxis, genetic polymorphisms (cathepsin C) have been associated with aggressive periodontitis, but none have been consistently seen in all cases6. The development of aggressive periodontitis is complex and results from a combination of genetic, host and microbial factors8.

Only some species of subgingival bacteria are pathogenic and cause a destructive host immune response due to their virulence factors9. The presence of pathogenic bacterial species does not necessarily lead to disease10 or to a specific type of periodontitis11. Aggregatibacter actinomycetemcomitans (A.a.) is an important bacterial species associated with aggressive periodontitis. It is a non-motile, facultative gram negative coccobacillus. An individual who tests positive for A.a. in the oral cavity has an increased risk of developing aggressive periodontitis10. A.a. is present in almost all individuals diagnosed with localized aggressive periodontitis12. A.a. is capable of invading host epithelial and connective tissue cells and lingering inside. Not all serotypes of A.a. are disease-associated, but the presence of the JP2 clone subtype in particular is

associated with attachment loss13 and increased leukocytic activity14. Leukotoxin, one of its virulence factors, is a protein that kills leukocytes and peripheral monocytes15. A.a. is able to alter and evade host defenses by producing PMN chemotactic inhibitors, immunosuppressive proteins and Fc-binding proteins. It’s tissue destructive proteins include cytotoxins, collagenases and lipopolysaccharides (LPS). LPS stimulates bone resorption, a characteristic feature of periodontitis. A.a. has also been shown to inhibit fibroblast proliferation and bone formation16. If A.a. is successfully removed from affected sites, therapy is correlated with a positive result17. All of these virulence factors and clinical findings are what make A.a. a likely candidate in the etiology of aggressive periodontitis.

Removal of all bacteria from the periodontal pocket is difficult to accomplish with nonsurgical therapy alone, since deep sites are difficult to debride and some species (e.g.

A.a., Porphyromonas gingivalis and Prevotella intermedia) can invade the soft tissue wall of the periodontal pocket18. The current approach for initial nonsurgical treatment of aggressive periodontitis includes the use of systemic antibiotics in combination with scaling and root planing (SRP). SRP is an effective method in removing or disrupting existing biofilms within shallow periodontal pockets, but its effectiveness decreases as the pocket depth increases19. SRP has limited effectiveness in removing plaque and calculus at sites deeper than 6.2mm PD20. The difficulties associated with eradicating pathogens from the entire oral cavity and increasing the proportion of host-compatible bacteria provides a rationale for incorporating systemic antibiotics into the treatment of periodontitis. Administration of antibiotics in conjunction with SRP can induce a rapid

and substantial reduction in bacterial levels, which facilitates the recolonization of the subgingival environment by host-compatible species21. The overall effectiveness of an antibiotic in treating periodontitis lies in its ability to alter the microbial ecology and promote a shift towards a non-pathogenic community. The combination of amoxicillin and metronidazole appears to be most effective in the treatment of aggressive periodontitis21. A.a. has been shown to be susceptible to various antibiotics including amoxicillin (AMX), azithromycin (AZM) and doxycycline (DOX) in vitro22.

AMX, AZM and DOX have different mechanisms of action. AMX is a β-lactam antibiotic that is a bactericidal and disrupts bacterial cell wall synthesis. It has a moderately broad spectrum and inhibits gram negative and gram positive bacteria. AZM is a bacteriostatic macrolide antibiotic that has a broad spectrum and inhibits gram positive and gram negative bacteria (including A.a.)23. Its elimination half life time of 68 hours permits administration as a single daily dose, which could increase patient compliance. It inhibits translation by binding to the 50s ribosomal subunit. Haas et al, investigated the use of AZM in treating aggressive periodontitis and observed statistically significant attachment level gains when AZM was used in conjunction with non-surgical periodontal therapy compared to non-surgical periodontal therapy alone24. AZM lacks bactericidal activity and may not be able to alter oral biofilm compositions as well as the bactericidal combination of AMX and metronidazole21. DOX is classified as a tetracycline. It is bacteriostatic and inhibits protein translation by binding to the 30s ribosomal subunit. Because of increased resistance, it is now mainly used for very specific cases25.

Many antibiotics have been shown to induce oxidative stress via generation of reactive oxygen species (ROS) including gentamicin26, roxithromycin27, levofloxacin28, ceftazidime, piperacillin and ciprofloxacin29. Directly or indirectly, all bactericidal

30 ― antibiotics promote the production of ROS . ROS include O2• (superoxide), H2O2

(hydrogen peroxide) and OH• (hydroxyl radical). They can cause single and double stranded DNA breakage via oxygen radicals. By-products of lipid peroxidation caused by

ROS can also damage DNA31. Metabolism via the tricarboxylic acid (TCA) cycle is one mechanism by which hydroxyl radicals are formed. The use of tobramycin (an aminoglycoside) against Pseudomonas aeruginosa with mutations in the TCA cycle and respiratory electron transport chain was found to be less effective due to the decreased production of ROS32. Rifampin and metronidazole were found to be more effective at killing E.coli with an impaired ROS defense mechanism than wild type33. Both these studies show the importance of ROS generation and antibiotic effectiveness.

ROS defense mechanisms have been well studied in Escherichia coli. In E. coli, the presence of ROS is detected by the OxyR and SoxRS systems, which activate downstream genes to up-regulate the formation of oxidative stress related proteins and enzymes. Enzymatic reactions reduce ROS to less reactive forms: Superoxide dismutase converts superoxide into either oxygen or hydrogen peroxide and catalase converts hydrogen peroxide into water and carbon dioxide. Peroxidases also convert H2O2 into water and a carbon group. H2O2 is able to oxidize proteins, forming disulfide bonds, but these can be reduced by bacterial thioredoxins and glutaredoxins. The oxidation of proteins and the importance of redoxins in ROS defenses currently remains unclear34.

Glutathione S-transferases are enzymes that utilize glutathione to inactivate secondary metabolites of ROS such as unsaturated aldehydes, epoxides, and hydroperoxides.

Glutathione on its own can act as a nonenzymatic scavenger of ROS. Glutathione reacts with hydrogen peroxide to form water and oxygen. Oxidized glutathione can be reduced back to glutathione by glutathione reductase35. Polyamines are another class of nonenzymatic scavengers present in all cells.

Polyamines

Polyamines are ubiquitous polycationic molecules that are involved in gene transcription, translation, stress (osmolarity, heat, ROS, UV, psychiatric) and cell proliferation. They are able to modulate the levels of many different proteins involved in cell growth, thus creating an environment suitable for cell growth.

Polyamines are breakdown products of amino acids (Figure 1). The biosynthesis of polyamines starts with decarboxylation of ornithine and S-adenosylmethionine by ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) respectively. The most commonly found polyamines are spermine, spermidine and putrescine. Putrescine is produced from ornithine by ODC and putrescine can then be converted to spermidine and spermidine to spermine through various enzymatic reactions

(Figure 1). Although exceptions exist with various species, putrescine, spermidine and to a lesser extent, cadaverine, predominate in prokaryotes, whereas in spermidine and spermine predominate in eukaryotes.

Due to its polycationic nature, polyamines can interact and act as electrostatic bridges with negatively charged molecules, including DNA, RNA, proteins and ROS.

They play a role in modulating both DNA and protein synthesis and are able to bind weakly to adenosine triphosphate (ATP). Polyamines act as both activators and repressors of gene transcription. Polyamines act indirectly to increase production of various mRNA products and it is thought that polyamines are able to repress transcription by stabilizing chromosomes in their condensed state36. Polyamines can affect protein synthesis by stimulating assembly of 30S ribosomal subunits37 and by binding to ribosomes to increase codon accuracy during protein synthesis38. Genomic analysis has found that transporters for uptake of extracellular polyamines exist in gram negative and gram positive bacteria, as well as archaea, yeast and protozoa. There is a very high degree of conservation that suggests an adaptive or survival advantage39 as well as the overall importance of polyamines in simple and complex organisms.

Polyamines have been implicated in modulating the virulence phenotype of many bacterial pathogens. In some bacterial species, they are able to trigger genes important for biofilm formation40. Polyamines occur in higher concentrations in GCF in inflamed and periodontitis sites compared to healthy sites41 which may enhance biofilm growth in these areas. Polyamines are also found in increased concentrations in areas with high cell proliferation and cell lysis. Polyamines have been shown to accelerate PMN apoptosis in a dose dependent manner42. Polyamines may impair phagocyte chemotaxis43, granule release and respiratory burst in PMNs44, further disrupting the body’s immune functions and altering the inflammatory response.

Polyamines bind to reactive oxygen species (ROS), thereby nullifying their lethal effects. In polyamine-deficient E. coli mutants, the presence of ROS was lethal, whereas in normal E.coli it was not, showing that the presence of polyamines is very important in neutralizing ROS45. They may also be able to mitigate the oxidative stress produced by antibiotics46.

Aim of study

The aim of this study is to characterize the effects of polyamines on the inhibitory actions of antibiotics used in periodontal antimicrobial chemotherapy on A. actinomycetemcomitans.

Hypotheses

The high concentration of exogenous polyamines in the periodontal pocket is a factor that mitigates bacterial stress and decreases inhibition of A. actinomycetemcomitans growth by antibiotics. Polyamine effects on gene expression play a role in decreasing its sensitivity to antibiotics.

Materials and Methods

Materials

Doxycycline (DOX), putrescine (PUT), spermidine (SPD) and spermine (SPM) were purchased from Sigma Chemical Company (St Louis, MO). Amoxicillin (AMX)

and azithromycin (AZM) were purchased from US Pharmacopeia (Rockville, MD).

Polyamine and AMX stock solutions were prepared in sterile water. AZM was prepared ethanol and DOX was prepared in dimethyl sulfoxide.

Bacterial culture

Aggregatibacter actinomycetemcomitans (A.a.) strain Y4 (ATCC 43718,

American Type Culture Collection, Manassas, VA) was grown in brain heart infusion

(BHI) broth (Difco, Becton, Dickinson and Co, Sparks, MD) at 37°C in a candle jar environment containing 9% O2 and 4% CO2.

Growth Curves

Fresh overnight cultures of A.a. were diluted in BHI to an absorbance at 600 nm of 0.1 and added to sterile culture tubes (6 ml per tube). Stock solutions of antibiotics and polyamines were added to produce the following experimental conditions:

A. Control (A.a. only, with no antibiotic or polyamine additions)

B. A.a. + Polyamine (1mM PUT, 0.4 mM SPD and 0.4 mM SPM)*

C. Negative control (A.a +Antibiotic**)

D. A.a. + Antibiotic** + Polyamine (1 mM PUT, 0.4 mM SPD and 0.4 mM

SPM)*

E. A.a. + Antibiotic** + Polyamine (1 mM SPD and 1 mM SPM)

**Antibiotic concentrations were 4 μg/mL AMX, 0.5 μg/mL AZM and 0.5 μg/mL

DOX. Antibiotic concentrations were selected to provide an intermediate level of

inhibition.

*Corresponds to levels observed in gingival crevicular fluid samples obtained

from patients with untreated periodontitis41. At 1.5 hour intervals up to 9 hours, a

1mL aliquot was removed from each tube for spectrophotometric analysis at 600

nm. At least five replicates were obtained for each condition. Statistics were

performed on final growth yield.

Minimal Inhibitory Concentration (MIC) determinations

Fresh overnight cultures of A.a. were diluted in BHI to an absorbance at 600 nm of 0.01 (approximately 106 CFU/ml) and added to sterile snapped-cap Falcon culture tubes (6 ml/tube). Stock solutions of antibiotics and polyamines were added to create the following conditions:

1. Control (Antibiotic* + A.a.)

2. A.a. + Antibiotic* + Polyamine (mixture of 1 mM PUT, 0.4 mM SPD and 0.4

mM SPM)

3. A.a. + Antibiotic* + Polyamine (mixture of 1 mM SPD and 1 mM SPM)

*Antibiotic = AMX (0.125, 0.25, 0.5, 0.75, 1.0, 2.0μg/mL) or AZM (0.125, 0.25,

0.5, 0.75, 1.0, 2.0μg/mL) or DOX (0.125, 0.25, 0.5, 0.75, 1.0, 2.0μg/mL)

The cultures were incubated overnight at 37°C in a candle jar environment containing 9% O2 and 4% CO2. The MIC was defined as the lowest concentration of each 10

antibiotic that completely inhibited the growth of the inoculum. Seven replicate experiments were obtained for each condition.

Bacterial viability determinations

An aliquot of overnight growth A.a. culture was diluted to an absorbance at 670 nm of ~0.03. Half of the bacterial suspension was separated and killed by exposure to heat (95°C for 10min). The two suspensions were mixed to create five different proportions of live:dead cells (100:0, 90:10, 50:50, 10:90, 0:100), which were used to standardize the bacterial viability assay. The assay (LIVE/DEAD BacLight assay,

ThermoFisher Scientific) was conducted by fluorescence spectroscopy according to the directions provided by the manufacturer.

Metatranscriptomic analysis

Fresh overnight cultures of A.a. were diluted in BHI to an absorbance at 600 nm of 0.1 and added to sterile culture tubes (2 ml per tube). Stock solutions of antibiotics and polyamines were added to produce the following experimental conditions:

A. Control (A.a. only, with no antibiotic or polyamine additions)

B. A.a. + Polyamines (1mM PUT, 0.4 mM SPD, 0.4 mM SPM)

CAMX. A.a. + AMX (4 μg/mL)

DAMX. A.a. + AMX (4 μg/mL) + Polyamines (1 mM PUT, 0.4 mM SPD, 0.4

mM SPM)

CAZM. A.a. + AZM (0.5 μg/mL)

DAZM. A.a. + AZM (0.5 μg/mL) + Polyamines (1 mM PUT, 0.4 mM SPD, 0.4

mM SPM)

Each sample was vortexed every 1.5 hr to resuspend cultures. Cultures were removed from the incubator after 6 hours of growth.

Total RNA was isolated from A.a. cultures using the mirVana miRNA isolation kit (ThermoFisher Scientific). Bacteria were lysed and RNA was isolated according to the directions provided by the manufacturer. The concentration of total RNA was determined using the Qubit® RNA Assay Kit (Life Technologies).

To evaluate the quality of the total RNA, RNA integrity value (RIN) was determined using the Agilent RNA 6000 Nano Reagents and RNA Nano Chips in an

Agilent 2100 Bioanalyzer (Agilent Technologies). Total RNA (500 ng) was used to remove the DNA contamination using Baseline-ZERO™ DNase (Epicentre) according to the manufacturer’s instructions. RNA was then purified using RNA Clean &

Concentrator columns (Zymo Research). DNA-free RNA samples were used for rRNA removal by using Ribo-Zero™ Gold rRNA Removal Kit (Epidemiology; Illumina). Final purification was performed using RNA Clean & Concentrator columns (Zymo Research). rRNA-depleted samples were used for library preparation using the TruSeq™ RNA LT

Sample Preparation Kit (Illumina) according to the manufacturer’s instructions.

Following library preparation, the final concentration of all the libraries were measured using the Qubit® dsDNA HS Assay Kit (Life Technologies), and the average library size was determined using the Agilent 2100 Bioanalyzer (Agilent Technologies). The libraries

were then pooled in equimolar ratios of 2 nM, and 4 pM of the library pool was clustered using the cBot (Illumina) and sequenced paired end for 300 cycles using the HiSeq 2500 system (Illumina) (2x150bp PE).

Raw reads with >10% unknown nucleotides or with >50% low quality nucleotides

(quality value <20) were discarded. Microbial transcripts were quality filtered using

SolexaQA++, and aligned against the Human Oral Microbiome Database (HOMD)47 using DIAMOND48. Aligned sequences were annotated to the SEED database using

Megan 648 and metabolic pathways were visualized using KEGG. The metagenomic sequence classifier Kraken49 was used along with our helper tool, kraken-biom

(github.com/smdabdoub/kraken-biom) for taxonomic confirmation. A customized web- application, VORTEX, was used for data visualization50.

Results

Effect of polyamines on inhibition of A.a. growth by AMX, AZM and DOX

Over the course of the growth period, amoxicillin (4 µg/ml) inhibited bacterial growth yield to 26% of the untreated control (Figure 2). In the presence of a mixture of 1 mM PUT, 0.4 mM SPD and 0.4 mM SPM, amoxicillin inhibited growth yield to 57% of control (difference statistically significant, p<0.05). Addition of the polyamine mix to control cultures increased the growth yield slightly, but the effect was not statistically significant. 13

Under the same experimental conditions, AZM (0.5 µg/ml) decreased bacterial growth yield to 52% of control (Figure 3). In the presence of the polyamine mix, AZM inhibited growth yield to 84% of control (significant at p<0.05). As previously noted, addition of polyamines to control cultures increased the growth yield, but the effect was not statistically significant.

DOX (0.5 µg/ml) reduced bacterial growth yield to 56% of the untreated control

(Figure 4). In the presence of the polyamine mix, DOX inhibited growth yield to 69% of control (p<0.05). Again, addition of polyamines to control cultures increased growth yield, but the effect was not statistically significant. However, when data from all 15 replicates portrayed in Figures 2-4 were pooled, growth in the presence of polyamines was enhanced by 5% at 7.5 hrs (significant at p = 0.003).

Effect of polyamines on MIC for A. actinomycetemcomitans

Polyamines had essentially no effect on the MIC for growth inhibition by AMX, regardless of whether the assays were conducted in the presence of a mix of 1 mM PUT,

0.4 mM SPD and 0.4 mM SPM as well as a mix of 1 mM SPD and 1 mM SPM.

However, the MIC for bacterial inhibition by AZM was significantly increased under both conditions. Similarly, the MIC for inhibition by DOX was increased in the presence of polyamines, but the difference was statistically significant only the presence of a mixture of 1 mM SPD and 1 mM SPM (Table 2).

Bacterial viability determinations

Live/Dead fluorescence spectroscopy determined that 90% of the bacteria were live after 6 hours of growth (data not shown).

Transcriptome analysis

The number of mapped transcripts, 9.0–12.4 million per sample, totaled

65,831,882 bases of sequenced cDNAs. 840 functionally identifiable transcripts from the

SEED database were found and matched belonging to 30 functional families.

The level of gene expression was compared between groups by looking at fold change differences. The level of significance was set transcripts with >2 fold change.

Effect of polyamines on gene expression in control cultures

The addition of a mixture of 1 mM PUT and 0.4 mM SPD and SPM to growing bacteria resulted in a change in gene transcription of at least a 2 fold in twelve genes

(Table 4). Eleven of these genes were up-regulated and one gene was down-regulated

(Fig 5, Table 5). Five level 1 families were up-regulated, including carbohydrates, amino acids & derivatives, fatty acids, lipids & isoprenoids, stress response, and iron acquisition

& metabolism. One transcript related to respiration was down-regulated. Gene expression of oxidative stress related proteins, catalase and glutathione reductase, were upregulated

5.52 and 2.02 fold respectively. Superoxide dismutase and thioredoxin reductase were upregulated by 1.91 and 1.86 fold respectively (data not shown).

Effect of polyamines on cultures inhibited by AMX and AZM

The addition of the polyamine mixture to bacteria inhibited with AMX resulted in a 2 fold down-regulation of a single CRISPR gene compared to AMX alone (Fig 6, Table

6).

The addition of polyamines to bacteria inhibited with AZM altered the expression of 32 genes by at least a 2-fold when compared to bacteria treated with AZM alone.

Twelve genes were up-regulated and twenty were down-regulated (Fig 7, Table 7). Up- regulation occurred in genes related to amino acids, carbohydrates, cell wall & capsule,

DNA metabolism, glutathione, mitochondrial electron transport, nucleosides & nucleotides, stress response, and sulfur metabolism. Down-regulation occurred in genes related to amino acids, carbohydrates, cell wall & capsule, DNA metabolism, fatty acids, lipids & isoprenoids, nitrogen metabolism, regulation & cell signaling, and respiration.

Pertinent to oxidative stress related proteins, glutathione S-transferase, thiol peroxidase and nonspecific DNA binding protein dps were up-regulated by 2.79, 2.21 and 2.15 fold respectively. Six of the eight genes with the highest degree of down-regulation (up to

7.86 fold) were related to nitrogen metabolism. Four genes related to cytochrome c maturation also showed >2 fold down-regulation.

Discussion

Ornithine decarboxylase and spermidine synthase are important enzymes in the polyamine synthesis pathway. A.a. lacks these enzymes and is unable to produce its own supply of polyamines in the usual way, but it can take up exogenous PUT, SPD and SPM.

For this reason, exogenous polyamines may play a role in modulating antibiotic resistance, stress and cell growth. Our findings indicate that polyamines have a multitude of effects on planktonic A.a. in vitro.

In the presence of polyamines, the inhibitory effects of AMX, AZM and DOX on

A.a. growth were reduced significantly. Polyamines induced a significant increase in the

MIC for AZM and DOX, but had no apparent effect on the MIC value for AMX. It is possible that polyamine effects on reversing inhibition of A.a. growth by AMX are transient, occurring during the first few hours during the lag and log phases of bacterial growth. This may be explained by the mechanism by which AMX is transported into the cell. Small hydrophilic molecules (e.g., β-lactam antibiotics) enter the cell through porin channels in the cell membrane, whereas macrolides are hydrophobic and are able to diffuse through the cell membrane (Delcour, 2009). Polyamines have been shown to decrease outer membrane permeability by inhibiting porin-mediated influx in E. coli51

(Vega, 1996). Polyamines likely bind to an internal pore-exposed site and trigger channel closures52. By impairing AMX entry into the cell periplasm, polyamines could reduce its bactericidal activity. A wild type strain of P. aeruginosa showed increased resistance to imipenem in the presence of polyamines, but a mutant strain devoid of outer membrane

porin OprD was susceptible to the antibiotic53. Polyamine interaction with outer membrane porins could play a role in blocking influx of certain antibiotics into the intracellular space.

To help explain the mechanisms by which polyamines mitigate A.a. inhibition by antibiotics, transcriptomic studies were conducted to determine how polyamines alter gene transcription in bacteria inhibited by AMX or AZM. Polyamines had a more profound effect on reversing these two agents than they did with DOX. RNA was isolated from samples obtained after 6 hours of growth. Under these conditions, the bacteria were still in log phase and their viability was about 90%, suggesting that most bacteria were actively producing mRNA.

The addition of AMX resulted in an up-regulation of 162 genes and a down- regulation of 42 genes. When polyamines and AMX were added, there was an up- regulation of 171 genes and a down-regulation of 37 genes. The addition of polyamines to the AMX had only limited effects on gene transcription (Table 3) and essentially no change in the MIC of AMX for A a.

In the presence of PUT, SPD and SPM, bacterial growth yield was enhanced slightly. Evaluation of gene transccription found an up-regulation of catalase, suggesting that polyamines can modulate oxidative stress. Polyamines appear to modulate genes in the glutathione redox pathway, which plays a role in oxidative stress reduction.

Glutathione is oxidized by hydrogen peroxide to form water, while glutathione reductase

(which underwent a 2.02 fold increase) reduces the oxidized glutathione molecule35. A

2.18 fold increase in fructose-1,6-bisphosphatase transcription suggests that the pentose

phosphate pathway may also be affected. This pathway is involved in cellular metabolism and the production of NADPH, an important reducing agent in the glutathione redox pathway35. The reduction of oxidative stress allows the bacteria to better adapt to the environment and grow at an increased rate.

AZM inhibited A.a. growth yield by 48%, but in the presence of polyamines,

AZM inhibition was reduced to 16%. Significant gene expression changes in the presence of polyamines occurred with AZM. The addition of AZM resulted in an up-regulation of

190 genes and a down-regulation of 132 genes. When polyamines and AZM were added, there was an up-regulation of 124 genes and a down-regulation of 103 genes (Table 3).

The presence of polyamines reduced the total number of genes that were up-regulated or down-regulated by 35% and 22%, respectively. A study by Kohanski et al, did not find that bacteriostatic antibiotics induced oxidative stress30, but in our study, polyamines resulted in an up-regulation of oxidative stress related genes. The presence of glutathione

S-transferase (2.79 fold), thiol peroxidase (2.21 fold) and DNA binding protein dps (2.15 fold) indicate that the bacteria were likely experiencing oxidative stress. DNA binding protein HU-α (2.35 fold up-regulation) has a protective role in stress by acting like histones by wrapping DNA and stabilizing it in order to prevent denaturation during environmental stress. Up-regulation of citrate lyase (2.05-2.29 fold), an important enzyme in the reverse citric acid cycle, leads to production of acetyl-CoA which is a precursor in carbohydrate, protein and fatty acid metabolism. Multiple components responsible for cytochrome c maturation were down-regulated significantly. This down- regulation may play an important role in cell survival. The presence of antibiotics lead to

the production of ROS via the electron transport chain30, in which cytochromes play a prominent role. Reducing levels of cytochrome c could potentially reduce overall levels of ROS in the cell.

Conversely, multiple studies of P. aeruginosa by Kwon et al found that the MIC values of several antibiotics significantly decreased in the presence of polyamines53,54.

MIC values for β-lactams, chloramphenicol, nalidixic acid, and trimethoprim were decreased up to 64-fold in the presence of either PUT, SPD, SPM or cadaverine.

Concentrations used in Kwon’s study were significantly greater than those in our study

(20mM PUT, 20mM SPD vs 1.4mM PUT, 0.4mM SPD) and this may have played a role in the contrasting results, however the study also found that 1mM SPM resulted in a significantly decreased MIC. Polyamines may have different effects in different species.

Since this is a preliminary study, there are limitations related to oversimplification and sample size. A single strain of planktonic A.a. may not be representative of a naturally occurring biofilm. The effect of polyamines and antibiotics on A.a. in a biofilm may present with a different response. An open-ended transcriptomic approach gives us a global view of gene alterations in response to AMX or AZM, but these changes only represent a single time point. Our study included one sample per group, so quantitative significance testing was not possible. The experiments should be repeated with more replicates to increase the statistical power of the study. Once target genes are selected, quantitative real time PCR (qRT-PCR) could be performed to accurately quantify and verify gene expression changes between conditions. Further investigation into the effects

of polyamines is required in order to determine important pathways and mechanisms that may prevent or inhibit antibiotic effectiveness.

The increased concentration of exogenous polyamines in a diseased periodontal pocket may affect the antimicrobial resistance of A.a. and may have a negative effect on periodontal therapeutic outcomes.

Conclusion

The presence of exogenous polyamines mitigated the inhibitory effect of AMX,

AZM and DOX on A.a. Metatranscriptomic analysis found differing transcriptomic profiles when comparing AMX and AZM in the presence of polyamines. Polyamines produced a transient mitigation of AMX inhibition, but did not have a significant effect on gene transcription. Many gene expression changes were seen when polyamines were in the presence of AZM. Thus, polyamines, at concentrations found in periodontal pockets, may help protect A.a. and contribute to antimicrobial resistance. Future studies should verify gene expression changes using qRT-PCR. The effects of polyamines should be analyzed in other periopathogenic species as well as in a biofilm model.

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Appendix A: Tables and Figures

Antibiotic Classification Cidal/Static Mechanism of action Peak serum [ ]

AMX β-lactam Bactericidal Inhibition of cell wall 5-8μg/mL synthesis

AZM Macrolide Bactericidal/static Inhibition of 50s 0.4μg/mL ribosomal subunit

DOX Tetracycline Bacteriostatic Inhibition of 30s 2-4μg/mL ribosomal subunit Table 1: Description of antibiotics used.

MIC Antibiotic 1. Control 2. PUT + SPD + SPM 3. SPD + SPM

AMX 0.54 ± 0.06 0.50 ± 0.08 0.54 ± 0.07

AZM 0.54 ± 0.04 0.71 ± 0.04* 0.75 ± 0.05*

DOX 0.61 ± 0.11 0.79 ± 0.10 0.86 ± 0.09*

Table 2: The effect of polyamines on the MIC of AMX, AZM and DOX. Data are represented as mean and SEM of 7 experiments. PUT + SPD + SPM: 1.4 mM PUT + 0.4 mM SPD + 0.4 mM SPM. SPD + SPM: 1 mM SPD + 1 mM SPM. * Significantly different from control (P < 0.05, RM ANOVA and Holm-Sidak post-hoc test).

Functionally identifiable Median per Condition Total reads transcripts on SEED base coverage Sample A 11,387,107 6,404,896 (56.3%) 230 6 hr Ctrl B 10,586,652 6,020,610 (56.9%) 231 6 hr PolyA

CAMX 9,079,691 5,320,242 (58.6%) 260 6 hr AMX

DAMX 10,468,326 6,146,148 (58.7%) 301 6 hr AMX + PolyA

CAZM 12,386,294 7,190,457 (58.1%) 285 6 hr AZM

DAZM 11,923,812 6,901,598 (57.9%) 245 6 hr AZM + PolyA Table 3: Summary of A.a. cDNA samples

Condition # of genes # of genes Total % of genes % of genes upregulated downregulated identifiable upregulated downregulated fold transcripts on SEED

B 11 1 840 1.3% 0.1%

CAMX 162 42 840 19.3% 5.0%

DAMX 171 37 840 20.4% 4.4%

CAZM 190 132 840 22.6% 15.7%

DAZM 124 103 840 14.8% 12.3% Table 4: Number of genes upregulated/downregulated >2 fold Number of genes up-regulated or down-regulated >2 fold with the addition of antibiotics and/or polyamines in comparison to control group.

Level 1 Level 3 Level 4 Fold Change Carbohydrates Photorespiration Catalase (EC 1.11.1.6) 5.519 (oxidative C2 cycle) Carbohydrates Lactose and Galactose Galactose/methyl galactoside ABC 2.777 Uptake and Utilization transport system, ATP-binding protein MglA (EC 3.6.3.17) Iron acquisition Campylobacter Iron Periplasmic protein p19 involved in 2.629 and metabolism Metabolism high-affinity Fe2+ transport Carbohydrates Maltose and Maltose/maltodextrin transport ATP- 2.462 Maltodextrin binding protein MalK (EC 3.6.3.19) Utilization Carbohydrates Glycogen metabolism Maltodextrin phosphorylase 2.189 (EC 2.4.1.1) Carbohydrates Calvin-Benson cycle Fructose-1,6-bisphosphatase, type I 2.184 (EC 3.1.3.11) Carbohydrates Inositol catabolism 5-deoxy-glucuronate isomerase (EC 2.173 5.3.1.-) Stress Response Glutathione: Redox Glutathione reductase (EC 1.8.1.7) 2.024 cycle Amino Acids and Cysteine Biosynthesis Sulfate transporter, CysZ-type 2.019 Derivatives Fatty Acids, Acetyl-CoA Biotin carboxyl carrier protein of 2.016 Lipids, and carboxylase acetyl-CoA carboxylase Isoprenoids complexes in plants Amino Acids and Cysteine Biosynthesis Cysteine synthase (EC 2.5.1.47) 2.010 Derivatives Respiration Formate Formate dehydrogenase O alpha -2.818 dehydrogenase subunit (EC 1.2.1.2) Table 5: Effect of polyamine mixture on gene transcription in A.a.

Level 1 Level 3 Level 4 Fold change DNA Metabolism CRISPRs CRISPR-associated protein, Csd1 family -2.028 Table 6: Effect of polyamines and AMX on gene transcription in A.a. Effect of polyamines on gene transcription in A.a. inhibited by 2 μg/ml AMX

Level 1 Level 3 Level 4 Fold change Prediction based on At3g50560 Glutathione S-transferase (EC 2.788 plant-prokaryote 2.5.1.18) comparative analysis Nucleosides and Purine de novo Ribonucleotide reductase of class 2.780 Nucleotides biosynthesis in plants Ia (aerobic), beta subunit (EC 1.17.4.1) DNA Metabolism DNA structural DNA-binding protein HU-alpha 2.352 proteins, bacterial Carbohydrates acinetobacter tca Citrate lyase beta chain (EC 2.289 4.1.3.6) Sulfur Metabolism Thioredoxin-disulfide Thiol peroxidase, Tpx-type (EC 2.213 reductase 1.11.1.15) Stress Response Oxidative stress Non-specific DNA-binding 2.150 protein Dps Mitochondrial electron F0F1-type ATP ATP synthase F0 sector subunit c 2.117 transport system in synthase in plants plants (mitochondrial) DNA Metabolism DNA Repair Base Exodeoxyribonuclease I (EC 2.090 Excision 3.1.11.1) Amino Acids and Methionine Methionine ABC transporter 2.076 Derivatives Biosynthesis substrate-binding protein Carbohydrates acinetobacter tca Citrate lyase alpha chain (EC 2.053 4.1.3.6) Nucleosides and Purine conversions 5'-nucleotidase (EC 3.1.3.5) 2.008 Nucleotides Cell Wall and Capsule Rhamnose containing Teichoic acid export ATP- 2.005 glycans binding protein TagH (EC 3.6.3.40) Respiration Biogenesis of c-type Cytochrome c heme lyase subunit -2.001 cytochromes CcmF Fatty Acids, Lipids, CDP-diacylglycerol Phosphatidate -2.009 and Isoprenoids biosynthesis in plants cytidylyltransferase (EC 2.7.7.41) Respiration Biogenesis of c-type Cytochrome c heme lyase subunit -2.010 cytochromes CcmL Cell Wall and Capsule Sialic Acid Sialic acid-induced -2.011 Metabolism transmembrane protein YjhT(NanM), possible mutarotase Respiration Biogenesis of c-type Cytochrome c heme lyase subunit -2.020 cytochromes CcmH Table 7: Effect of polyamines and AZM on gene transcription in A.a. Effect of polyamines on gene transcription in A. actinomycetemcomitans inhibited by 0.5 μg/ml AZM

33 continued

Table 7 continued Respiration Soluble cytochromes and Cytochrome C553 (soluble -2.065 functionally related cytochrome f) electron carriers DNA DNA replication, archaeal Ribonuclease HII (EC 3.1.26.4) -2.068 Metabolism Carbohydrates Glycogen metabolism Maltodextrin phosphorylase (EC -2.080 2.4.1.1) Amino Acids and Branched-Chain Amino Acetolactate synthase large subunit -2.081 Derivatives Acid Biosynthesis (EC 2.2.1.6) Carbohydrates L-ascorbate utilization (and Ascorbate-specific PTS system, -2.094 related gene clusters) EIIA component (EC 2.7.1.-) Regulation and Autoinducer 2 (AI-2) Autoinducer 2 (AI-2) ABC -2.105 Cell signaling transport and processing transport system, membrane (lsrACDBFGE operon) channel protein LsrC Cell Wall and Sialic Acid Metabolism TRAP-type transport system, large -2.219 Capsule permease component, predicted N- acetylneuraminate transporter Nitrogen Nitrate and nitrite NrfD protein -2.225 Metabolism ammonification Carbohydrates Inositol catabolism Inosose dehydratase (EC 4.2.1.44) -2.262 Nitrogen Nitrate and nitrite Cytochrome c-type protein NrfB -2.326 Metabolism ammonification precursor DNA CRISPRs CRISPR-associated protein, Csd1 -2.886 Metabolism family Nitrogen Nitrate and nitrite Nitrate reductase cytochrome -4.960 Metabolism ammonification c550-type subunit Nitrogen Nitrate and nitrite Ferredoxin-type protein NapG -5.007 Metabolism ammonification (periplasmic nitrate reductase) Nitrogen Nitrate and nitrite Polyferredoxin NapH (periplasmic -5.982 Metabolism ammonification nitrate reductase) Nitrogen Nitrate and nitrite Periplasmic nitrate reductase -7.864 Metabolism ammonification component NapD

Figure 1: Polyamine synthesis pathway. A) Putrescine synthesis pathway B) Spermidine synthesis pathway55

Figure 2: Growth curve: AMX. Effect of polyamines on inhibition of A. actinomycetemcomitans growth by amoxicillin (AMX, 4 µg/ml). Data are represented as mean and SEM of 5 experiments. The polyamines included 1.4 mM putrescine, 0.4 mM spermidine and 0.4 mM spermine, which approximate the mean concentrations found in gingival crevicular fluid at untreated periodontitis sites. Bacterial growth over the course of the experiment was enhanced in the presence of polyamines, but the effect was significant only in the presence of AMX (P < 0.05, repeated measures ANOVA with Holm-Sidak post-hoc test).

Figure 3: Growth curve: AZM Effect of polyamines (1.4 mM putrescine, 0.4 mM spermidine and 0.4 mM spermine) on inhibition of A. actinomycetemcomitans growth by azithromycin (AZM, 0.5µg/ml). The data are representative of 5 experiments. Bacterial growth over the course of the experiment was enhanced in the presence of polyamines, but the effect was statistically significant only in the presence of AZM (P < 0.05, repeated measures ANOVA with Holm-Sidak post-hoc test).

Figure 4: Growth curve: DOX Effect of polyamines (1.4 mM putrescine, 0.4 mM spermidine and 0.4 mM spermine) on inhibition of A. actinomycetemcomitans growth by doxycycline (DOX, 0.5µg/ml). The data are representative of 7 experiments. Bacterial growth over the course of the experiment was enhanced in the presence of polyamines, but the effect was significant only in the presence of DOX (P<0.05, repeated measures ANOVA with Holm-Sidak post-hoc test).

Figure 5: Circle Plot: Polyamines vs Ctrl Effects of a mixture of 1 mm PUT, 0.4 mm SPD and 0.4 mm SPM on gene transcripts in A. actinomycetemcomitans (comparison of groups B and A). Core transcripts are grouped into higher order functions. Orange circles indicate >2 fold up-regulation, blue circles indicate >2 fold down-regulation. Circles are sized based on fold change differences between the two groups.

Figure 6: Circle Plot: AMX + Polyamines vs AMX Effects of a mixture of 1 mm PUT, 0.4 mm SPD and 0.4 mm SPM on gene transcripts in A. actinomycetemcomitans treated with 4 µg/ml AMX (comparison of groups DAMX and CAMX). Core transcripts are grouped into higher order functions. Orange circles indicate >2 fold up-regulation, blue circles indicate >2 fold down-regulation. Circles are sized based on fold change differences between the two groups.

Figure 7: Circle Plot: AZM + Polyamines vs AZM Effects of a mixture of 1 mm PUT, 0.4 mm SPD and 0.4 mm SPM on gene transcripts in A. actinomycetemcomitans treated with 0.5 µg/ml AZM (comparison of groups DAZM and CAZM). Core transcripts are grouped into higher order functions. Orange circles indicate >2 fold up-regulation, blue circles indicate >2 fold down-regulation. Circles are sized based on fold change differences between the two groups.

Figure 8: KEGG Pathway Map: Polyamines vs Ctrl A map of the KEGG metabolic pathways comparing groups B and A. Overlaid in orange are the core pathways that have been up-regulated and overlaid in blue are those that have been down-regulated. The line widths of the core pathways are sized by fold changes of the occurrence of those pathways.

Figure 9: KEGG Pathway Map: AMX + Polyamines vs AMX

A map of the KEGG metabolic pathways comparing groups DAMX and CAMX. Overlaid in orange are the core pathways that have been up-regulated and overlaid in blue are those that have been down-regulated. The line widths of the core pathways are sized by fold changes of the occurrence of those pathways.

Figure 10: KEGG Pathway Map: AZM + Polyamines vs AZM

A map of the KEGG metabolic pathways comparing groups DAZM and CAZM. Overlaid in orange are the core pathways that have been up-regulated and overlaid in blue are those that have been down-regulated. The line widths of the core pathways are sized by fold changes of the occurrence of those pathways.

Appendix B: Relative abundances of SEED transcripts in A.a.

Level 1 Level 3 Level 4 A B CAMX DAMX CAZM DAZM A Hypothetical Protein Pyrroline-5-carboxylate reductase (EC 0.014 0.026 0.015 0.016 0.028 0.024 Related to Proline Metabolism 1.5.1.2) Alanine racemase, biosynthetic (EC 0.069 0.070 0.069 0.083 0.042 0.047 5.1.1.1) Branched-chain amino acid Alanine biosynthesis 0.071 0.080 0.053 0.053 0.115 0.096 aminotransferase (EC 2.6.1.42) Cysteine desulfurase (EC 2.8.1.7), IscS 0.632 0.707 0.349 0.382 0.271 0.325 subfamily D-3-phosphoglycerate dehydrogenase 0.067 0.115 0.100 0.101 0.045 0.042 (EC 1.1.1.95) Phosphoserine aminotransferase (EC 0.046 0.074 0.050 0.052 0.022 0.026 Alanine, serine, glycine 2.6.1.52) metabolism in plants Phosphoserine phosphatase (EC 3.1.3.3) 0.081 0.099 0.089 0.084 0.062 0.067 Serine hydroxymethyltransferase (EC 0.318 0.631 0.497 0.449 0.283 0.296 2.1.2.1) Diaminopimelate epimerase (EC 5.1.1.7) 0.035 0.045 0.048 0.049 0.041 0.034 Amino acid racemase Glutamate racemase (EC 5.1.1.3) 0.019 0.016 0.023 0.023 0.020 0.016 Amino Acids Arginine ABC transporter, periplasmic 0.033 0.026 0.046 0.068 0.022 0.021 and Derivatives arginine-binding protein ArtI

Arginine ABC transporter, permease 0.011 0.007 0.022 0.033 0.007 0.007 protein ArtM Arginine pathway regulatory protein 0.007 0.011 0.010 0.009 0.008 0.006 ArgR, repressor of arg regulon Arginine/ornithine antiporter ArcD 0.020 0.027 0.018 0.020 0.043 0.039 Arginine and Ornithine Degradation Carbamate kinase (EC 2.7.2.2) 0.005 0.006 0.003 0.004 0.004 0.003 NADP-specific glutamate 0.021 0.022 0.019 0.016 0.015 0.010 dehydrogenase (EC 1.4.1.4) Argininosuccinate lyase (EC 4.3.2.1) 0.074 0.091 0.067 0.065 0.046 0.039

Argininosuccinate synthase (EC 6.3.4.5) 0.060 0.055 0.087 0.088 0.092 0.069

N-succinyl-L,L-diaminopimelate 0.028 0.038 0.068 0.067 0.039 0.035 desuccinylase (EC 3.5.1.18) Asparaginyl-tRNA synthetase (EC Arginine deiminase and 0.241 0.300 0.177 0.191 0.207 0.234 6.1.1.22) agmatine deiminase pathways in Streptococci Aspartate aminotransferase (EC 2.6.1.1) 0.044 0.063 0.035 0.038 0.028 0.025 Branched-Chain Amino Acid Acetolactate synthase large subunit (EC 0.000 0.000 0.001 0.001 0.002 0.001 Biosynthesis 2.2.1.6)

45 continued Appendix B continued Ketol-acid reductoisomerase (EC 0.007 0.009 0.004 0.004 0.010 0.009 1.1.1.86) Branched-Chain Amino Acid Leucine-responsive regulatory protein, Biosynthesis regulator for leucine (or lrp) regulon and 0.037 0.038 0.039 0.043 0.039 0.039 high-affinity branched-chain amino acid transport system 3-dehydroquinate synthase (EC 4.2.3.4) 0.047 0.054 0.126 0.120 0.173 0.130 5-Enolpyruvylshikimate-3-phosphate 0.046 0.072 0.119 0.113 0.204 0.117 synthase (EC 2.5.1.19) Chorismate biosynthesis in Chorismate synthase (EC 4.2.3.5) 0.030 0.036 0.045 0.046 0.034 0.026 plants Shikimate 5-dehydrogenase I alpha (EC 0.015 0.025 0.022 0.022 0.015 0.014 1.1.1.25) Shikimate kinase I (EC 2.7.1.71) 0.030 0.037 0.044 0.041 0.074 0.073 2-keto-3-deoxy-D-arabino- Chorismate biosynthesis in heptulosonate-7-phosphate synthase I 0.122 0.111 0.056 0.059 0.043 0.056 Streptococci alpha (EC 2.5.1.54) 2-Keto-3-deoxy-D-manno-octulosonate- 0.045 0.054 0.063 0.058 0.185 0.157 8-phosphate synthase (EC 2.5.1.55) 3-dehydroquinate dehydratase II (EC Chorismate Synthesis 0.019 0.015 0.021 0.021 0.024 0.022 4.2.1.10) Shikimate 5-dehydrogenase I gamma 0.018 0.028 0.050 0.042 0.010 0.015 (EC 1.1.1.25) Acetaldehyde dehydrogenase (EC Cinnamic Acid Degradation 0.033 0.037 0.146 0.167 0.051 0.042 1.2.1.10)

Common Pathway For Transcriptional repressor protein TrpR 0.009 0.007 0.008 0.006 0.010 0.009 Synthesis of Aromatic Compounds (DAHP synthase Transcriptional repressor protein TyrR 0.021 0.019 0.028 0.025 0.057 0.041 to chorismate) Cys regulon transcriptional activator 0.022 0.019 0.051 0.046 0.095 0.089 CysB Cysteine synthase (EC 2.5.1.47) 0.091 0.183 0.073 0.083 0.048 0.043

Cysteine Biosynthesis Serine acetyltransferase (EC 2.3.1.30) 0.059 0.051 0.034 0.035 0.025 0.033 Sulfate and thiosulfate import ATP- 0.024 0.024 0.029 0.032 0.009 0.010 binding protein CysA (EC 3.6.3.25) Sulfate transporter, CysZ-type 0.014 0.027 0.022 0.023 0.026 0.018 Gamma-glutamyl phosphate reductase 0.010 0.009 0.014 0.013 0.017 0.015 (EC 1.2.1.41) Evolution of Proline Biosynthesis (for review by Glutamate 5-kinase (EC 2.7.2.11) 0.004 0.004 0.007 0.005 0.010 0.007 Fichman et al) Proline/sodium symporter PutP (TC 0.032 0.043 0.074 0.077 0.038 0.042 2.A.21.2.1)

Glutamine, Glutamate, Aspartate--ammonia ligase (EC 6.3.1.1) 0.055 0.053 0.049 0.039 0.025 0.022 Aspartate and Asparagine Biosynthesis L-asparaginase (EC 3.5.1.1) 0.056 0.079 0.027 0.038 0.016 0.018

Glycerate kinase (EC 2.7.1.31) 0.002 0.003 0.016 0.010 0.010 0.006

L-serine dehydratase (EC 4.3.1.17) 0.032 0.034 0.063 0.056 0.060 0.050 Glycine and Serine Utilization Serine transporter 0.046 0.042 0.037 0.040 0.025 0.029

Seryl-tRNA synthetase (EC 6.1.1.11) 0.145 0.176 0.113 0.117 0.101 0.086

46 continued

Appendix B continued Glycine cleavage system transcriptional Glycine cleavage system 0.015 0.015 0.013 0.015 0.024 0.018 activator GcvA Histidine Biosynthesis in Adenylosuccinate synthetase (EC 0.127 0.152 0.230 0.231 0.076 0.084 plants 6.3.4.4) Aspartate-semialdehyde dehydrogenase 0.239 0.280 0.111 0.111 0.081 0.089 (EC 1.2.1.11) Diaminopimelate decarboxylase (EC 0.033 0.047 0.028 0.032 0.020 0.020 Lysine and threonine 4.1.1.20) metabolism in plants Homoserine kinase (EC 2.7.1.39) 0.067 0.074 0.058 0.060 0.024 0.028

Threonine synthase (EC 4.2.3.1) 0.046 0.042 0.056 0.063 0.015 0.022 2,3,4,5-tetrahydropyridine-2,6- Lysine Biosynthesis DAP dicarboxylate N-succinyltransferase (EC 0.045 0.043 0.068 0.070 0.082 0.081 Pathway 2.3.1.117) Lysine degradation L-lysine permease 0.026 0.021 0.043 0.042 0.027 0.025

Acetate kinase (EC 2.7.2.1) 0.609 0.590 0.504 0.510 0.248 0.389 Lysine fermentation Acetyl-CoA acetyltransferase (EC 0.001 0.001 0.003 0.003 0.006 0.004 2.3.1.9) 5'-methylthioadenosine nucleosidase (EC 0.070 0.062 0.067 0.072 0.083 0.075 3.2.2.16) 5-methyltetrahydropteroyltriglutamate-- homocysteine methyltransferase (EC 0.183 0.121 0.100 0.096 0.058 0.081 Methionine and cysteine 2.1.1.14) metabolism in plants Cystathionine beta-lyase (EC 4.4.1.8) 0.083 0.092 0.045 0.050 0.041 0.038 S-adenosylmethionine synthetase (EC 0.019 0.023 0.023 0.025 0.025 0.022 2.5.1.6) Cystathionine gamma-synthase (EC 0.319 0.378 0.169 0.190 0.108 0.120 2.5.1.48) Homoserine O-acetyltransferase (EC 0.065 0.068 0.082 0.070 0.224 0.152 2.3.1.31) Methionine ABC transporter ATP- 0.306 0.180 0.086 0.135 0.066 0.114 binding protein Methionine ABC transporter permease Methionine Biosynthesis 0.046 0.036 0.017 0.026 0.010 0.015 protein Methionine ABC transporter substrate- 0.146 0.078 0.045 0.057 0.014 0.030 binding protein S-ribosylhomocysteine lyase (EC 0.065 0.058 0.032 0.029 0.027 0.037 4.4.1.21) Transcriptional activator MetR 0.018 0.028 0.031 0.032 0.027 0.023 Pyruvate dehydrogenase E1 component Methionine Degradation 2.256 2.403 1.529 1.749 0.495 0.771 (EC 1.2.4.1) Biosynthetic Aromatic amino acid 0.024 0.033 0.031 0.036 0.008 0.009 aminotransferase beta (EC 2.6.1.57) Phenylalanine and Tyrosine Branches from Chorismate Periplasmic aromatic amino acid aminotransferase beta precursor (EC 0.126 0.117 0.079 0.069 0.137 0.127 2.6.1.57) PII Superfamily Nitrogen regulatory protein P-II 0.015 0.018 0.009 0.012 0.011 0.010 Poly-gamma-glutamate Gamma-glutamyltranspeptidase (EC 0.004 0.006 0.005 0.005 0.007 0.005 biosynthesis 2.3.2.2) Putrescine transport ATP-binding protein Polyamine Metabolism 0.091 0.114 0.128 0.122 0.114 0.094 PotA (TC 3.A.1.11.1)

47 continued

Appendix B continued Spermidine Putrescine ABC transporter permease component potC 0.055 0.038 0.085 0.075 0.116 0.184 (TC_3.A.1.11.1) Threonine anaerobic Phosphate acetyltransferase (EC 2.3.1.8) 0.216 0.200 0.549 0.678 0.075 0.123 catabolism gene cluster Anthranilate phosphoribosyltransferase 0.011 0.011 0.018 0.018 0.043 0.039 (EC 2.4.2.18) Anthranilate synthase, amidotransferase 0.002 0.003 0.004 0.004 0.009 0.005 component (EC 4.1.3.27) Indole-3-glycerol phosphate synthase 0.059 0.056 0.056 0.063 0.185 0.108 Tryptophan biosynthesis in (EC 4.1.1.48) Streptococci Para-aminobenzoate synthase, aminase 0.014 0.017 0.011 0.011 0.010 0.008 component (EC 2.6.1.85) Tryptophan synthase alpha chain (EC 0.002 0.003 0.004 0.004 0.005 0.003 4.2.1.20) Tryptophan synthase beta chain (EC 0.003 0.005 0.005 0.007 0.006 0.004 4.2.1.20) Acetone Butanol Ethanol Alcohol dehydrogenase (EC 1.1.1.1) 0.007 0.014 0.005 0.005 0.006 0.004 Synthesis Dihydrolipoamide acetyltransferase Acetyl-CoA biosynthesis in component of pyruvate dehydrogenase 0.561 0.615 0.604 0.709 0.133 0.169 plants complex (EC 2.3.1.12) Citrate lyase alpha chain (EC 4.1.3.6) 0.067 0.048 0.162 0.206 0.017 0.036

Citrate lyase beta chain (EC 4.1.3.6) 0.079 0.053 0.096 0.130 0.014 0.031 Dihydrolipoamide succinyltransferase component (E2) of 2-oxoglutarate 0.020 0.018 0.038 0.044 0.023 0.015 dehydrogenase complex (EC 2.3.1.61) acinetobacter tca Fumarate hydratase class II (EC 4.2.1.2) 0.163 0.190 0.178 0.219 0.055 0.055 Succinate dehydrogenase flavoprotein 0.060 0.058 0.135 0.169 0.043 0.043 subunit (EC 1.3.99.1) Succinate dehydrogenase iron-sulfur 0.037 0.046 0.081 0.095 0.025 0.022 protein (EC 1.3.99.1) Succinyl-CoA ligase [ADP-forming] 0.017 0.026 0.028 0.034 0.027 0.022 beta chain (EC 6.2.1.5) Carbohydrates Nucleoside 5-triphosphatase RdgB alpha carboxysome (dHAPTP, dITP, XTP-specific) (EC 0.016 0.021 0.025 0.023 0.025 0.025 3.6.1.15) Maltose/maltodextrin ABC transporter, Alpha-Amylase locus in substrate binding periplasmic protein 0.015 0.021 0.015 0.017 0.009 0.008 Streptocococcus MalE beta carboxysome Carbonic anhydrase (EC 4.2.1.1) 0.018 0.019 0.013 0.011 0.026 0.019

Butanol Biosynthesis Pyruvate formate-lyase (EC 2.3.1.54) 1.176 1.519 0.964 1.100 1.038 1.197 Fructose-1,6-bisphosphatase, GlpX type 0.011 0.018 0.011 0.008 0.015 0.014 (EC 3.1.3.11) Fructose-1,6-bisphosphatase, type I (EC 0.087 0.189 0.059 0.054 0.063 0.067 3.1.3.11) Calvin-Benson cycle Fructose-bisphosphate aldolase class II 0.260 0.384 0.391 0.451 0.071 0.096 (EC 4.1.2.13) NAD-dependent glyceraldehyde-3- 1.437 1.580 0.935 0.948 0.619 0.876 phosphate dehydrogenase (EC 1.2.1.12) Calvin-Benson cycle Phosphoglycerate kinase (EC 2.7.2.3) 0.251 0.346 0.321 0.420 0.098 0.120

48 continued

Appendix B continued Ribose 5-phosphate isomerase A (EC 0.020 0.026 0.018 0.019 0.013 0.015 5.3.1.6) Ribulose-phosphate 3-epimerase (EC 0.035 0.061 0.045 0.060 0.032 0.034 5.1.3.1) Triosephosphate isomerase (EC 5.3.1.1) 0.433 0.541 0.185 0.198 0.082 0.106

Carboxysome NADH dehydrogenase (EC 1.6.99.3) 0.162 0.186 0.111 0.092 0.155 0.143

Beta-hexosaminidase (EC 3.2.1.52) 0.170 0.137 0.067 0.058 0.029 0.049 Glucosamine-6-phosphate deaminase 0.010 0.011 0.013 0.016 0.017 0.011 (EC 3.5.99.6) N-acetylglucosamine-6-phosphate 0.066 0.094 0.083 0.079 0.081 0.073 Chitin and N- deacetylase (EC 3.5.1.25) acetylglucosamine utilization N-acetylglucosamine-6P-responsive transcriptional repressor NagC, ROK 0.041 0.046 0.062 0.065 0.074 0.051 family PTS system, N-acetylglucosamine- 0.032 0.039 0.057 0.065 0.120 0.089 specific IIB component (EC 2.7.1.69) Citrate Metabolism KE Transaldolase (EC 2.2.1.2) 0.467 0.643 0.240 0.254 0.106 0.159 NADP-dependent malic enzyme (EC Citrate Metabolism KE3 0.085 0.113 0.147 0.185 0.058 0.056 1.1.1.40) D-glycerate transporter (predicted) 0.004 0.004 0.020 0.014 0.011 0.007 D-galactarate, D-glucarate and D-glycerate catabolism Sugar diacid utilization regulator SdaR 0.006 0.006 0.005 0.004 0.012 0.008 2-dehydro-3-deoxygluconate kinase (EC 0.003 0.004 0.004 0.005 0.007 0.008 2.7.1.45) 2-dehydro-3-deoxyphosphogluconate 0.005 0.008 0.006 0.006 0.005 0.004 aldolase (EC 4.1.2.14)

Alpha-glucosidase (EC 3.2.1.20) 0.010 0.009 0.008 0.008 0.007 0.008 D-mannonate oxidoreductase (EC 0.004 0.004 0.004 0.003 0.004 0.004 D-Galacturonate and D- 1.1.1.57) Glucuronate Utilization Gluconokinase (EC 2.7.1.12) 0.005 0.007 0.005 0.004 0.009 0.006

Hexuronate utilization operon 0.022 0.022 0.009 0.010 0.012 0.016 transcriptional repressor ExuR Mannonate dehydratase (EC 4.2.1.8) 0.037 0.033 0.019 0.022 0.022 0.028

Uronate isomerase (EC 5.3.1.12) 0.010 0.013 0.010 0.013 0.014 0.013 Gluconate utilization system Gnt-I 0.026 0.025 0.024 0.023 0.040 0.035 D-gluconate and transcriptional repressor ketogluconates metabolism Low-affinity gluconate/H+ symporter 0.007 0.006 0.014 0.013 0.013 0.007 GntU Ribokinase (EC 2.7.1.15) 0.009 0.007 0.019 0.015 0.029 0.020 Ribose ABC transport system, ATP- 0.007 0.008 0.009 0.010 0.011 0.007 binding protein RbsA (TC 3.A.1.2.1) D-ribose utilization Ribose ABC transport system, periplasmic ribose-binding protein RbsB 0.011 0.011 0.013 0.016 0.012 0.009 (TC 3.A.1.2.1) Ribose ABC transport system, permease 0.005 0.005 0.007 0.007 0.009 0.005 protein RbsC (TC 3.A.1.2.1) 6-phosphofructokinase (EC 2.7.1.11) 0.074 0.103 0.105 0.106 0.105 0.100

49 continued

Appendix B continued D-Tagatose and Galactitol Tagatose 1,6-bisphosphate aldolase (EC 0.002 0.001 0.005 0.003 0.038 0.022 Utilization 4.1.2.40) Deoxyribose operon repressor, DeoR 0.018 0.024 0.026 0.026 0.021 0.016 family Deoxyribose-phosphate aldolase (EC 0.016 0.031 0.036 0.033 0.023 0.020 Deoxyribose and 4.1.2.4) Deoxynucleoside Catabolism Purine nucleoside phosphorylase (EC 0.215 0.276 0.111 0.129 0.035 0.055 2.4.2.1) Putative deoxyribonuclease YjjV 0.026 0.023 0.045 0.044 0.047 0.037

Aldose 1-epimerase (EC 5.1.3.3) 0.003 0.004 0.005 0.006 0.006 0.005 EC 5.1.3.- Racemases and L-ribulose-5-phosphate 4-epimerase (EC epimerases acting on 0.015 0.021 0.026 0.030 0.013 0.010 5.1.3.4) carbohydrates and derivatives UDP-glucose 4-epimerase (EC 5.1.3.2) 0.145 0.231 0.156 0.170 0.117 0.124 6-phosphogluconolactonase (EC 0.029 0.025 0.028 0.034 0.011 0.013 3.1.1.31), eukaryotic type Entner-Doudoroff Pathway Enolase (EC 4.2.1.11) 0.379 0.375 0.335 0.351 0.148 0.188

Pyruvate kinase (EC 2.7.1.40) 0.211 0.271 0.508 0.551 0.156 0.164

D-lactate dehydrogenase (EC 1.1.1.28) 0.737 0.708 0.599 0.563 0.942 0.939 Formate efflux transporter (TC 2.A.44 0.210 0.232 0.129 0.147 0.234 0.216 family) NAD(P) transhydrogenase alpha subunit 0.571 0.862 0.409 0.415 0.101 0.146 Fermentations in Streptococci (EC 1.6.1.2) NAD(P) transhydrogenase subunit beta 0.434 0.593 0.405 0.396 0.119 0.160 (EC 1.6.1.2) Pyruvate formate-lyase activating 0.011 0.013 0.021 0.023 0.050 0.026 enzyme (EC 1.97.1.4) Sugar/maltose fermentation stimulation Fermentations: Mixed acid 0.019 0.022 0.013 0.011 0.012 0.011 protein homolog 5-formyltetrahydrofolate cyclo-ligase 0.046 0.040 0.037 0.029 0.036 0.026 (EC 6.3.3.2) Dihydrofolate reductase (EC 1.5.1.3) 0.013 0.021 0.018 0.020 0.013 0.010 Folate-mediated one-carbon Formyltetrahydrofolate deformylase (EC 0.012 0.010 0.019 0.016 0.107 0.074 metabolism in plants 3.5.1.10) Methenyltetrahydrofolate cyclohydrolase 0.057 0.080 0.040 0.032 0.040 0.041 (EC 3.5.4.9) Thymidylate synthase (EC 2.1.1.45) 0.070 0.070 0.091 0.104 0.025 0.035 Glucosamine--fructose-6-phosphate Formaldehyde assimilation: aminotransferase [isomerizing] (EC 0.072 0.069 0.256 0.196 0.913 0.776 Ribulose monophosphate 2.6.1.16) pathway Glucose-6-phosphate isomerase (EC 0.247 0.331 0.244 0.280 0.078 0.083 5.3.1.9) 1-phosphofructokinase (EC 2.7.1.56) 0.056 0.055 0.107 0.145 0.022 0.033 Fructose utilization Fructose repressor FruR, LacI family 0.094 0.094 0.048 0.050 0.073 0.071 0. Phosphocarrier protein of PTS system 0.042 0.042 0.037 0.041 0.043 045

PTS system, fructose-specific IIB 0.096 0.108 0.189 0.236 0.034 0.049 component (EC 2.7.1.69)

50 continued

Appendix B continued Galactose degradation in Galactose-1-phosphate 0.002 0.002 0.004 0.003 0.005 0.003 plants uridylyltransferase (EC 2.7.7.10) Hydroxypyruvate reductase (EC Glycerate metabolism 0.029 0.037 0.030 0.034 0.015 0.016 1.1.1.81) 1,4-alpha-glucan (glycogen) branching 0.039 0.062 0.031 0.040 0.023 0.019 enzyme, GH-13-type (EC 2.4.1.18) 4-alpha-glucanotransferase 0.031 0.045 0.032 0.031 0.053 0.037 (amylomaltase) (EC 2.4.1.25) Glycogen debranching enzyme (EC 0.009 0.012 0.008 0.010 0.009 0.006 Glycogen metabolism 3.2.1.-) Glycogen phosphorylase (EC 2.4.1.1) 0.018 0.013 0.036 0.031 0.080 0.050

Glycogen synthase, ADP-glucose 0.029 0.029 0.025 0.026 0.033 0.027 transglucosylase (EC 2.4.1.21) Maltodextrin phosphorylase (EC 2.4.1.1) 0.005 0.012 0.008 0.008 0.013 0.006 Glycolate, glyoxylate Phosphoglycolate phosphatase (EC 0.039 0.049 0.048 0.058 0.044 0.046 interconversions 3.1.3.18) Glycolysis and Phosphoenolpyruvate carboxykinase 0.164 0.175 0.636 0.814 0.244 0.168 Gluconeogenesis in plants [ATP] (EC 4.1.1.49) 5-deoxy-glucuronate isomerase (EC 0.008 0.018 0.005 0.007 0.004 0.004 5.3.1.-) 5-keto-2-deoxygluconokinase (EC 0.007 0.009 0.007 0.007 0.010 0.007 2.7.1.92) Epi-inositol hydrolase (EC 3.7.1.-) 0.001 0.002 0.003 0.003 0.002 0.002 Inositol transport system permease 0.003 0.003 0.003 0.003 0.005 0.004 protein Inositol transport system sugar-binding 0.002 0.002 0.001 0.001 0.004 0.002 protein Inositol catabolism Inosose dehydratase (EC 4.2.1.44) 0.001 0.001 0.001 0.001 0.001 0.000 Methylmalonate-semialdehyde 0.009 0.012 0.009 0.011 0.011 0.008 dehydrogenase [inositol] (EC 1.2.1.27) Myo-inositol 2-dehydrogenase 1 (EC 0.001 0.002 0.002 0.003 0.002 0.001 1.1.1.18) Myo-inositol 2-dehydrogenase 2 (EC 0.008 0.008 0.008 0.006 0.010 0.008 1.1.1.18) Predicted transcriptional regulator of the 0.026 0.029 0.013 0.013 0.011 0.010 myo-inositol catabolic operon 3-keto-L-gulonate 6-phosphate 0.003 0.003 0.007 0.007 0.004 0.003 decarboxylase Ascorbate-specific PTS system, EIIA 0.001 0.001 0.003 0.003 0.003 0.001 component (EC 2.7.1.-) L-ascorbate utilization (and Ascorbate-specific PTS system, EIIC 0.003 0.003 0.009 0.011 0.007 0.006 related gene clusters) component L-xylulose 5-phosphate 3-epimerase (EC 0.022 0.028 0.022 0.024 0.019 0.016 5.1.3.-) L-xylulose/3-keto-L-gulonate kinase (EC 0.017 0.014 0.021 0.023 0.021 0.014 2.7.1.-) Probable L-ascorbate-6-phosphate lactonase UlaG (EC 3.1.1.-) (L-ascorbate 0.059 0.104 0.039 0.041 0.025 0.022 utilization protein G)

L-rhamnose utilization L-lactate dehydrogenase (EC 1.1.2.3) 0.527 0.496 0.268 0.267 0.216 0.294

Lactate utilization L-lactate permease 0.080 0.058 0.068 0.063 0.058 0.081

51 continued

Appendix B continued Beta-galactosidase (EC 3.2.1.23) 0.000 0.000 0.000 0.000 0.001 0.001 Lactose and Galactose Uptake and Utilization Galactose operon repressor, GalR-LacI 0.008 0.010 0.006 0.007 0.008 0.007 family of transcriptional regulators Galactose/methyl galactoside ABC transport system, ATP-binding protein 0.001 0.003 0.001 0.002 0.003 0.002 MglA (EC 3.6.3.17) Galactose/methyl galactoside ABC Lactose and Galactose Uptake transport system, D-galactose-binding 0.015 0.019 0.016 0.017 0.006 0.010 and Utilization periplasmic protein MglB (TC 3.A.1.2.3) Galactose/methyl galactoside ABC transport system, permease protein MglC 0.012 0.012 0.019 0.020 0.019 0.013 (TC 3.A.1.2.3) Galactoside O-acetyltransferase (EC Lactose utilization 0.020 0.029 0.023 0.023 0.018 0.015 2.3.1.18) Maltose operon periplasmic protein 0.004 0.004 0.007 0.006 0.004 0.004 MalM Maltose/maltodextrin ABC transporter, 0.003 0.006 0.004 0.005 0.004 0.002 permease protein MalF Maltose/maltodextrin ABC transporter, 0.001 0.002 0.003 0.004 0.001 0.001 permease protein MalG Maltose and Maltodextrin Maltose/maltodextrin transport ATP- Utilization 0.002 0.004 0.002 0.003 0.002 0.001 binding protein MalK (EC 3.6.3.19) Mlc, transcriptional repressor of MalT (the transcriptional activator of maltose 0.032 0.036 0.031 0.031 0.038 0.036 regulon) and manXYZ operon Periplasmic alpha-amylase (EC 3.2.1.1) 0.006 0.006 0.012 0.014 0.009 0.006

Mannitol operon repressor 0.016 0.012 0.016 0.018 0.009 0.007 Mannitol-1-phosphate 5-dehydrogenase 0.057 0.053 0.055 0.066 0.025 0.029 Mannitol Utilization (EC 1.1.1.17) PTS system, mannitol-specific IIB 0.077 0.069 0.070 0.083 0.044 0.040 component (EC 2.7.1.69) Phosphomannomutase (EC 5.4.2.8) 0.155 0.105 0.214 0.187 0.362 0.257 Mannose Metabolism PTS system, mannose-specific IIB 0.056 0.079 0.074 0.101 0.034 0.041 component (EC 2.7.1.69) Hydroxyacylglutathione hydrolase (EC 0.036 0.041 0.030 0.036 0.031 0.032 3.1.2.6) Methylglyoxal Metabolism Methylglyoxal synthase (EC 4.2.3.3) 0.013 0.014 0.017 0.016 0.014 0.012 Photorespiration (oxidative C2 Catalase (EC 1.11.1.6) 0.022 0.120 0.095 0.096 0.049 0.034 cycle) Oxaloacetate decarboxylase alpha chain 0.011 0.014 0.049 0.063 0.042 0.026 Pyruvate metabolism I: (EC 4.1.1.3) anaplerotic reactions, PEP Oxaloacetate decarboxylase beta chain 0.011 0.012 0.047 0.046 0.078 0.042 (EC 4.1.1.3) Pyruvate metabolism II: Acylphosphate phosphohydrolase (EC acetyl-CoA, acetogenesis from 0.007 0.008 0.005 0.005 0.005 0. 005 3.6.1.7), putative pyruvate Xylose isomerase (EC 5.3.1.5) 0.001 0.002 0.002 0.002 0.003 0.002 Sugar utilization in Thermotogales Xylulose kinase (EC 2.7.1.17) 0.005 0.005 0.009 0.008 0.010 0.009 Hypothetical protein VC0266 (sugar VC0266 0.016 0.018 0.039 0.034 0.046 0.034 utilization related?)

52 continued

Appendix B continued D-xylose transport ATP-binding protein 0.004 0.004 0.003 0.003 0.007 0.004 XylG Xylose ABC transporter, periplasmic Xylose utilization 0.002 0.002 0.002 0.001 0.002 0.001 xylose-binding protein XylF Xylose activator XylR (AraC family) 0.017 0.017 0.017 0.017 0.013 0.011 Cell division protein DivIC (FtsB), 0.009 0.013 0.009 0.008 0.012 0.012 stabilizes FtsL against RasP cleavage Cell division protein FtsA 0.227 0.194 0.254 0.240 0.337 0.254

Cell division protein FtsK 0.144 0.128 0.207 0.222 0.168 0.137

Cell division protein FtsQ 0.146 0.131 0.149 0.150 0.174 0.141

Cell division protein FtsW 0.029 0.031 0.064 0.059 0.045 0.030

Cell division protein FtsZ (EC 3.4.24.-) 0.642 0.592 0.723 0.632 1.207 0.835 Bacterial Cytoskeleton Rod shape-determining protein MreB 0.161 0.222 0.222 0.214 0.101 0.114

Rod shape-determining protein MreC 0.018 0.019 0.050 0.044 0.042 0.030

Rod shape-determining protein MreD 0.015 0.010 0.025 0.021 0.031 0.028

Rod shape-determining protein RodA 0.053 0.047 0.055 0.054 0.051 0.044

Septum site-determining protein MinC 0.054 0.059 0.056 0.053 0.112 0.089

Z-ring-associated protein ZapA 0.017 0.025 0.015 0.014 0.016 0.023 Control of cell elongation - Cell Division Endonuclease III (EC 4.2.99.18) 0.019 0.017 0.016 0.019 0.010 0.008 and Cell Cycle division cycle in Bacilli Phosphoglucosamine mutase (EC Diadenylate cyclase cluster 0.269 0.371 0.547 0.624 0.271 0.215 5.4.2.10) DNA primase (EC 2.7.7.-) 0.073 0.046 0.136 0.119 0.493 0.262

RNA polymerase sigma factor RpoD 0.261 0.204 0.338 0.318 0.665 0.490 Macromolecular synthesis operon SSU ribosomal protein S21p 0.159 0.166 0.171 0.160 0.233 0.294 TsaD/Kae1/Qri7 protein, required for threonylcarbamoyladenosine t(6)A37 0.014 0.016 0.032 0.034 0.043 0.040 formation in tRNA Chromosome partition protein MukB 0.321 0.261 0.248 0.292 0.053 0.101 MukBEF Chromosome Chromosome partition protein MukE 0.040 0.039 0.036 0.041 0.009 0.013 Condensation Chromosome partition protein MukF 0.082 0.090 0.100 0.100 0.070 0.060

Two cell division clusters Ribonuclease III (EC 3.1.26.3) 0.019 0.017 0.059 0.054 0.039 0.031 relating to chromosome Signal recognition particle, subunit Ffh partitioning 0.030 0.043 0.069 0.068 0.065 0.060 SRP54 (TC 3.A.5.1.1) TsaB protein, required for YgjD and YeaZ threonylcarbamoyladenosine (t(6)A) 0.032 0.036 0.053 0.050 0.039 0.035 formation in tRNA D-glycero-D-manno-heptose 1,7- 0.041 0.037 0.017 0.018 0.019 0.029 Cell Wall and bisphosphate phosphatase (EC 3.1.1.-) Capsular heptose biosynthesis Capsule Phosphoheptose isomerase (EC 5.3.1.-) 0.028 0.019 0.035 0.035 0.031 0.028

53 continued

Appendix B continued Capsular heptose biosynthesis Phosphoheptose isomerase 1 (EC 5.3.1.-) 0.100 0.133 0.056 0.056 0.055 0.081 dTDP-glucose 4,6-dehydratase (EC dTDP-rhamnose synthesis 0.205 0.182 0.146 0.144 0.177 0.183 4.2.1.46) Undecaprenyl-phosphate Exopolysaccharide galactosephosphotransferase (EC 0.016 0.009 0.018 0.018 0.014 0.015 Biosynthesis 2.7.8.6) 3-deoxy-D-manno-octulosonate 8- 0.004 0.004 0.006 0.007 0.008 0.006 phosphate phosphatase (EC 3.1.3.45) 3-deoxy-manno-octulosonate 0.010 0.012 0.020 0.020 0.005 0.008 cytidylyltransferase (EC 2.7.7.38) Acyl-[acyl-carrier-protein]--UDP-N- acetylglucosamine O-acyltransferase 0.213 0.183 0.221 0.216 0.214 0.200 (EC 2.3.1.129) Arabinose 5-phosphate isomerase (EC 0.011 0.020 0.021 0.024 0.022 0.019 5.3.1.13) Lipid A biosynthesis (KDO) 2-(lauroyl)- 0.017 0.018 0.022 0.027 0.014 0.013 lipid IVA acyltransferase (EC 2.3.1.-) Lipid A biosynthesis lauroyl 0.025 0.026 0.022 0.023 0.029 0.029 acyltransferase (EC 2.3.1.-) KDO2-Lipid A biosynthesis Lipid A export ATP-binding/permease 0.032 0.045 0.051 0.055 0.041 0.038 protein MsbA Lipid-A-disaccharide synthase (EC 0.046 0.033 0.179 0.101 0.419 0.221 2.4.1.182) Lipopolysaccharide ABC transporter, 0.067 0.081 0.070 0.072 0.086 0.079 ATP-binding protein LptB LPS-assembly lipoprotein RlpB 0.026 0.031 0.039 0.039 0.033 0.032 precursor (Rare lipoprotein B) LptA, protein essential for LPS transport 0.052 0.057 0.051 0.050 0.062 0.060 across the periplasm UDP-3-O-[3-hydroxymyristoyl] N- acetylglucosamine deacetylase (EC 0.283 0.301 0.327 0.273 0.560 0.455 3.5.1.108) Outer membrane chaperone Skp (OmpH) 0.330 0.238 0.158 0.177 0.070 0.110 precursor Outer membrane lipoprotein LolB 0.011 0.008 0.021 0.025 0.007 0.008 precursor Outer membrane protein NlpB, lipoprotein component of the protein 0.042 0.045 0.041 0.041 0.029 0.024 assembly complex (forms a complex Lipopolysaccharide assembly with YaeT, YfiO, and YfgL) Survival protein SurA precursor (Peptidyl-prolyl cis-trans isomerase 0.194 0.216 0.252 0.228 0.540 0.457 SurA) (EC 5.2.1.8) Uncharacterized ABC transporter, 0.199 0.145 0.176 0.185 0.132 0.136 periplasmic component YrbD Uncharacterized protein YrbK clustered 0.070 0.079 0.051 0.049 0.080 0.080 with lipopolysaccharide transporters ADP-heptose--lipooligosaccharide 0.036 0.043 0.094 0.084 0.110 0.081 heptosyltransferase II (EC 2.4.1.-) ADP-L-glycero-D-manno-heptose-6- 0.074 0.089 0.046 0.053 0.034 0.034 LOS core oligosaccharide epimerase (EC 5.1.3.20) biosynthesis Beta-1,4-galactosyltransferase 0.021 0.018 0.038 0.038 0.075 0.052 D-glycero-beta-D-manno-heptose 7- 0.072 0.108 0.092 0.075 0.096 0.081 phosphate kinase

54 continued

Appendix B continued Major Outer Membrane Outer membrane protein A precursor 5.853 5.838 4.716 4.022 2.385 3.376 Proteins Beta N-acetyl-glucosaminidase (EC 0.016 0.017 0.043 0.042 0.021 0.016 3.2.1.52) D-alanyl-D-alanine carboxypeptidase 0.114 0.095 0.160 0.155 0.135 0.138 (EC 3.4.16.4) Membrane-bound lytic murein Murein Hydrolases 0.034 0.031 0.026 0.030 0.029 0.024 transglycosylase B precursor (EC 3.2.1.-) Membrane-bound lytic murein 0.104 0.090 0.094 0.095 0.138 0.122 transglycosylase C precursor (EC 3.2.1.-) Soluble lytic murein transglycosylase 0.036 0.029 0.048 0.044 0.055 0.039 precursor (EC 3.2.1.-) 3-oxoacyl-[acyl-carrier protein] 0.191 0.173 0.177 0.199 0.131 0.128 reductase (EC 1.1.1.100) Acyl carrier protein 0.087 0.106 0.104 0.094 0.064 0.109 mycolic acid synthesis Enoyl-[acyl-carrier-protein] reductase 0.112 0.122 0.128 0.122 0.131 0.161 [NADH] (EC 1.3.1.9) Malonyl CoA-acyl carrier protein 0.146 0.151 0.152 0.155 0.193 0.165 transacylase (EC 2.3.1.39) Monofunctional biosynthetic peptidoglycan transglycosylase (EC 0.021 0.015 0.026 0.024 0.060 0.043 2.4.2.-) Multimodular transpeptidase- transglycosylase (EC 2.4.1.129) (EC 0.236 0.240 0.350 0.344 0.337 0.268 3.4.-.-) Murein-DD-endopeptidase (EC 3.4.99.-) 0.072 0.071 0.053 0.050 0.059 0.049 N-acetylglucosamine-1-phosphate 0.043 0.058 0.190 0.161 0.313 0.222 uridyltransferase (EC 2.7.7.23) Penicillin-binding protein 2 (PBP-2) 0.044 0.033 0.078 0.078 0.078 0.059 Phospho-N-acetylmuramoyl- 0.043 0.052 0.078 0.077 0.083 0.077 pentapeptide-transferase (EC 2.7.8.13)

Peptidoglycan Biosynthesis Rare lipoprotein A precursor 0.058 0.051 0.066 0.065 0.068 0.053 UDP-N-acetylenolpyruvoylglucosamine 0.042 0.055 0.044 0.043 0.031 0.026 reductase (EC 1.1.1.158) UDP-N-acetylglucosamine 1- 0.161 0.127 0.325 0.342 0.161 0.121 carboxyvinyltransferase (EC 2.5.1.7) UDP-N-acetylglucosamine--N- acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N- 0.030 0.034 0.064 0.067 0.036 0.028 acetylglucosamine transferase (EC 2.4.1.227) UDP-N-acetylmuramoylalanine--D- 0.054 0.077 0.121 0.123 0.104 0.082 glutamate ligase (EC 6.3.2.9) UDP-N-acetylmuramoylalanyl-D- glutamate--2,6-diaminopimelate ligase 0.091 0.092 0.166 0.161 0.163 0.110 (EC 6.3.2.13) Recycling of Peptidoglycan Aminoacyl-histidine dipeptidase 0.130 0.227 0.105 0.118 0.056 0.052 Amino Acids (Peptidase D) (EC 3.4.13.3) Teichoic acid export ATP-binding Rhamnose containing glycans 0.039 0.030 0.021 0.022 0.014 0.028 protein TagH (EC 3.6.3.40) N-acetylmannosamine kinase (EC 0.016 0.022 0.034 0.034 0.043 0.026 2.7.1.60) Sialic Acid Metabolism N-acetylneuraminate lyase (EC 4.1.3.3) 0.030 0.037 0.036 0.038 0.035 0.027 55 continued

Appendix B continued Putative sugar isomerase involved in 0.029 0.041 0.019 0.020 0.015 0.017 processing of exogenous sialic acid Sialic acid utilization regulator, RpiR 0.011 0.014 0.019 0.022 0.037 0.023 family Sialic acid-induced transmembrane protein YjhT(NanM), possible 0.002 0.003 0.006 0.005 0.015 0.007 mutarotase TRAP-type transport system, large permease component, predicted N- 0.008 0.009 0.012 0.012 0.032 0.015 acetylneuraminate transporter Sialic Acid Metabolism TRAP-type transport system, periplasmic component, predicted N- 0.011 0.011 0.007 0.008 0.013 0.011 acetylneuraminate-binding protein TRAP-type transport system, small permease component, predicted N- 0.003 0.003 0.002 0.002 0.002 0.003 acetylneuraminate transporter 2-C-methyl-D-erythritol 4-phosphate 0.015 0.017 0.024 0.024 0.023 0.021 cytidylyltransferase (EC 2.7.7.60) Teichoic and lipoteichoic acids biosynthesis Undecaprenyl-phosphate N- acetylglucosaminyl 1-phosphate 0.063 0.074 0.041 0.045 0.060 0.066 transferase (EC 2.7.8.-) TsaE protein, required for YjeE threonylcarbamoyladenosine t(6)A37 0.040 0.040 0.026 0.028 0.025 0.031 formation in tRNA Central EC 6.4.1.- Ligases that form Acetyl-coenzyme A carboxyl transferase 0.101 0.126 0.069 0.071 0.043 0.049 metabolism carbon-carbon bonds alpha chain (EC 6.4.1.2) 5-FCL-like-OF 5-FCL-like protein 3.249 3.781 2.791 3.072 1.099 1.374 B12 Biosynthesis (Tavares Uroporphyrinogen-III methyltransferase 0.216 0.191 0.157 0.176 0.211 0.157 copy1) (EC 2.1.1.107) B12 duf71 Queuosine Biosynthesis QueC ATPase 0.012 0.015 0.012 0.014 0.019 0.017

Lipoate synthase 0.068 0.095 0.123 0.109 0.095 0.080 Octanoate-[acyl-carrier-protein]-protein- 0.015 0.014 0.027 0.028 0.026 0.018 N-octanoyltransferase Proposed lipoate regulatory protein BEY LIP 0.008 0.008 0.010 0.013 0.011 0.008 YbeD

Pyridoxal kinase (EC 2.7.1.35) 0.215 0.190 0.103 0.111 0.054 0.082 Cofactors, Vitamins, Pyridoxamine 5'-phosphate oxidase (EC 0.006 0.008 0.005 0.006 0.005 0.004 Prosthetic 1.4.3.5) Groups, 8-amino-7-oxononanoate synthase (EC Pigments 0.035 0.029 0.037 0.033 0.035 0.028 2.3.1.47) Adenosylmethionine-8-amino-7- oxononanoate aminotransferase (EC 0.061 0.059 0.053 0.054 0.059 0.053 2.6.1.62) Biotin biosynthesis Biotin synthase (EC 2.8.1.6) 0.069 0.088 0.109 0.119 0.059 0.057

Dethiobiotin synthetase (EC 6.3.3.3) 0.022 0.023 0.031 0.031 0.041 0.031

Long-chain-fatty-acid--CoA ligase (EC 0.279 0.300 0.295 0.308 0.163 0.160 6.2.1.3) Biotin carboxylase of acetyl-CoA Biotin synthesis & utilization 0.053 0.051 0.157 0.142 0.179 0.129 carboxylase (EC 6.3.4.14) 1-deoxy-D-xylulose 5-phosphate CLO thiaminPP biosynthesis 0.041 0.040 0.072 0.075 0.069 0.060 synthase (EC 2.2.1.7)

56 continued

Appendix B continued Thiamin ABC transporter, ATPase 0.015 0.017 0.034 0.036 0.019 0.017 component Thiamin ABC transporter, substrate- 0.132 0.144 0.079 0.093 0.082 0.098 binding component Dephospho-CoA kinase (EC 2.7.1.24) 0.019 0.020 0.010 0.013 0.008 0.009

Pantothenate kinase (EC 2.7.1.33) 0.032 0.035 0.027 0.025 0.029 0.031 Pantothenate:Na+ symporter (TC 0.064 0.041 0.077 0.070 0.118 0.075 CoA Pantothenate HMP 2.A.21.1.1) Phosphopantetheine adenylyltransferase 0.011 0.012 0.017 0.016 0.014 0.012 (EC 2.7.7.3) Phosphopantothenoylcysteine synthetase 0.044 0.060 0.097 0.103 0.060 0.056 (EC 6.3.2.5) 2-amino-4-hydroxy-6- coA-FolK hydroxymethyldihydropteridine 0.010 0.007 0.022 0.019 0.017 0.017 pyrophosphokinase (EC 2.7.6.3) Core tetrapyrrole biosynthesis Glutamate-1-semialdehyde 0.040 0.053 0.078 0.077 0.064 0.062 in plants aminotransferase (EC 5.4.3.8) Glutamyl-tRNA synthetase (EC 0.097 0.115 0.083 0.091 0.084 0.071 6.1.1.17) dephospho- tetrahydromethanopterin Dihydroneopterin aldolase (EC 4.1.2.25) 0.014 0.016 0.014 0.013 0.016 0.019 biosynthesis MYousef GTP cyclohydrolase I (EC 3.5.4.16) type 0.029 0.034 0.038 0.032 0.079 0.078 1 Queuosine biosynthesis QueD, PTPS-I 0.010 0.014 0.013 0.015 0.012 0.011 FIG000605: protein co-occurring with 0.009 0.007 0.004 0.004 0.004 0.004 transport systems (COG1739) Hypothetical, related to broad specificity Experimental tye 0.024 0.017 0.025 0.024 0.021 0.025 phosphatases COG0406 Radical SAM family enzyme, similar to coproporphyrinogen III oxidase, oxygen- 0.008 0.008 0.033 0.029 0.025 0.019 independent, clustered with nucleoside- triphosphatase RdgB Ribonuclease D (EC 3.1.26.3) 0.013 0.012 0.029 0.026 0.053 0.031

Chaperone protein HscA 0.115 0.129 0.216 0.230 0.074 0.082

Chaperone protein HscB 0.085 0.079 0.075 0.078 0.040 0.045 CsdL (EC-YgdL) protein of the HesA/MoeB/ThiF family, part of the 0.020 0.031 0.031 0.030 0.030 0.027 Fe-S cluster assembly CsdA-E-L sulfur transfer pathway Cysteine desulfurase CsdA-CsdE, sulfur 0.006 0.005 0.019 0.014 0.034 0.019 acceptor protein CsdE Iron-sulfur cluster assembly scaffold 0.171 0.186 0.097 0.109 0.050 0.076 protein IscU

Iron-sulfur cluster regulator IscR 0.101 0.163 0.052 0.053 0.040 0.048

Flavodoxin 1 0.158 0.171 0.095 0.086 0.195 0.235

Flavodoxin Flavodoxin 2 0.136 0.127 0.076 0.090 0.085 0.087

Flavoprotein MioC 0.028 0.037 0.020 0.020 0.020 0.026

Folate Biosynthesis Dihydropteroate synthase (EC 2.5.1.15) 0.035 0.042 0.096 0.094 0.101 0.062 57 continued

Appendix B continued Folylpolyglutamate synthase (EC 0.021 0.014 0.035 0.034 0.056 0.049 6.3.2.17) GTP cyclohydrolase II (EC 3.5.4.25) 0.064 0.104 0.036 0.033 0.057 0.073

Methionyl-tRNA formyltransferase (EC Folate Biosynthesis 0.075 0.063 0.105 0.101 0.087 0.064 2.1.2.9) 2-succinyl-5-enolpyruvyl-6-hydroxy-3- cyclohexene-1-carboxylic-acid synthase 0.059 0.078 0.113 0.114 0.088 0.078 (EC 2.2.1.9) 2-succinyl-6-hydroxy-2,4- cyclohexadiene-1-carboxylate synthase 0.005 0.005 0.010 0.009 0.014 0.009 (EC 4.2.99.20) Menaquinone and Menaquinone-specific isochorismate 0.023 0.034 0.064 0.067 0.063 0.045 Phylloquinone Biosynthesis synthase (EC 5.4.4.2) Naphthoate synthase (EC 4.1.3.36) 0.062 0.099 0.108 0.117 0.082 0.071 O-succinylbenzoate synthase (EC 0.050 0.043 0.077 0.070 0.067 0.058 4.2.1.113) O-succinylbenzoic acid--CoA ligase (EC 0.030 0.026 0.031 0.033 0.039 0.032 6.2.1.26) Inosine-5'-monophosphate Methanofuran 0.036 0.043 0.086 0.080 0.137 0.090 dehydrogenase (EC 1.1.1.205) Cyclic pyranopterin phosphate synthase 0.054 0.074 0.056 0.076 0.091 0.071 (MoaA) (EC 4.1.99.18) Molybdate-binding domain of ModE 0.058 0.068 0.047 0.048 0.063 0.072

Molybdenum ABC transporter, periplasmic molybdenum-binding 0.006 0.005 0.003 0.004 0.004 0.005 protein ModA (TC 3.A.1.8.1) Molybdenum cofactor biosynthesis 0.007 0.009 0.009 0.011 0.017 0.010 protein MoaD Molybdenum cofactor biosynthesis 0.012 0.017 0.015 0.017 0.030 0.018 protein MoaE Molybdenum transport ATP-binding 0.013 0.015 0.017 0.017 0.017 0.017 Molybdenum cofactor protein ModC (TC 3.A.1.8.1) biosynthesis Molybdenum transport system protein 0.006 0.004 0.008 0.006 0.015 0.008 ModD Molybdopterin biosynthesis Mog 0.116 0.071 0.436 0.294 1.105 0.650 protein, molybdochelatase Molybdopterin biosynthesis protein 0.037 0.045 0.049 0.051 0.023 0.023 MoeA Molybdopterin biosynthesis protein 0.017 0.022 0.026 0.025 0.012 0.011 MoeB Molybdopterin-guanine dinucleotide 0.016 0.021 0.023 0.020 0.035 0.032 biosynthesis protein MobA Molybdopterin-guanine dinucleotide 0.007 0.011 0.014 0.011 0.021 0.013 biosynthesis protein MobB ADP-ribose pyrophosphatase (EC 0.010 0.013 0.010 0.011 0.012 0.011 3.6.1.13) DNA ligase (EC 6.5.1.2) 0.034 0.036 0.071 0.070 0.042 0.033 NAD and NADP cofactor biosynthesis global NAD kinase (EC 2.7.1.23) 0.032 0.039 0.033 0.032 0.049 0.043

Nicotinamide-nucleotide adenylyltransferase, NadR family (EC 0.066 0.068 0.052 0.052 0.078 0.075 2.7.7.1)

58 continued

Appendix B continued NMN 5'-nucleotidase, extracellular (EC 0.277 0.331 0.106 0.121 0.086 0.123 3.1.3.5) NMN phosphatase (EC 3.1.3.5) 0.015 0.019 0.014 0.019 0.005 0.006

Ribosyl nicotinamide transporter, PnuC- 0.049 0.064 0.033 0.035 0.034 0.038 like Queuosine Biosynthesis QueE Radical 0.005 0.005 0.008 0.009 0.007 0.005 SAM S-adenosylmethionine:tRNA Queuosine HMP 0.014 0.018 0.036 0.030 0.081 0.053 ribosyltransferase-isomerase (EC 5.-.-.-) tRNA-guanine transglycosylase (EC 0.042 0.050 0.066 0.078 0.045 0.045 2.4.2.29) 3,4-dihydroxy-2-butanone 4-phosphate 0.030 0.034 0.076 0.060 0.098 0.084 synthase (EC 4.1.99.12) 6,7-dimethyl-8-ribityllumazine synthase 0.067 0.080 0.086 0.089 0.066 0.076 (EC 2.5.1.78) Riboflavin HMP Riboflavin kinase (EC 2.7.1.26) 0.027 0.038 0.047 0.047 0.049 0.041 Riboflavin synthase 0.055 0.060 0.022 0.023 0.043 0.058 eubacterial/eukaryotic (EC 2.5.1.9) 5-amino-6-(5- phosphoribosylamino)uracil reductase 0.028 0.032 0.057 0.058 0.053 0.044 (EC 1.1.1.193) Multi antimicrobial extrusion protein (Na(+)/drug antiporter), MATE family of 0.035 0.040 0.092 0.076 0.086 0.065 Riboflavin, FMN and FAD MDR efflux pumps biosynthesis in plants tRNA (5-methylaminomethyl-2- thiouridylate)-methyltransferase (EC 0.038 0.046 0.052 0.048 0.023 0.020 2.1.1.61) tRNA pseudouridine synthase B (EC 0.039 0.040 0.047 0.044 0.073 0.061 4.2.1.70) tRNA-specific adenosine-34 deaminase Test - Riboflavin 0.008 0.007 0.017 0.016 0.004 0.005 (EC 3.5.4.-) Test - Thiamin Thiamine biosynthesis protein thiI 0.007 0.008 0.023 0.020 0.048 0.031 Dihydroorotate dehydrogenase (EC 0.014 0.013 0.015 0.015 0.009 0.009 1.3.3.1) Ferredoxin 0.004 0.005 0.005 0.005 0.007 0.006

Ferric uptake regulation protein FUR 0.144 0.147 0.071 0.067 0.162 0.187 YgfZ Orotate phosphoribosyltransferase (EC 0.033 0.036 0.048 0.048 0.059 0.057 2.4.2.10) S-formylglutathione hydrolase (EC 0.135 0.167 0.087 0.094 0.064 0.062 3.1.2.12) Thiol:disulfide interchange protein DsbC 0.197 0.183 0.106 0.106 0.193 0.245

tRNA-i(6)A37 methylthiotransferase 0.021 0.042 0.066 0.050 0.099 0.074

Twin-arginine translocation protein TatA 0.034 0.041 0.024 0.021 0.032 0.053

2-phosphoglycolate salvage Putative phosphatase YqaB 0.024 0.041 0.022 0.024 0.019 0.015

CRISPR-associated protein Cas1 0.007 0.006 0.006 0.005 0.010 0.007 DNA Metabolism CRISPRs CRISPR-associated protein Cas2 0.003 0.003 0.002 0.003 0.005 0.004

CRISPR-associated protein, Csd1 family 0.000 0.000 0.000 0.000 0.000 0.000 59 continued

Appendix B continued CRISPR-associated protein, Csd2/Csh2 0.001 0.001 0.001 0.001 0.000 0.001 family DNA topoisomerase III (EC 5.99.1.2) 0.024 0.022 0.081 0.079 0.102 0.062

Protein involved in catabolism of DNA processing cluster 0.031 0.024 0.038 0.037 0.053 0.038 external DNA Recombination protein RecR 0.021 0.029 0.059 0.058 0.073 0.054

DNA polymerase I (EC 2.7.7.7) 0.105 0.138 0.219 0.229 0.087 0.084 DNA-3-methyladenine glycosylase (EC 0.017 0.016 0.016 0.017 0.016 0.013 3.2.2.20)

DNA Repair Base Excision Exodeoxyribonuclease I (EC 3.1.11.1) 0.039 0.031 0.036 0.040 0.011 0.024 Formamidopyrimidine-DNA glycosylase 0.027 0.017 0.029 0.027 0.044 0.033 (EC 3.2.2.23) Single-stranded-DNA-specific 0.262 0.204 0.259 0.263 0.407 0.350 exonuclease RecJ (EC 3.1.-.-) DNA mismatch repair endonuclease 0.019 0.018 0.014 0.017 0.014 0.013 MutH DNA recombination-dependent growth 0.054 0.051 0.033 0.033 0.038 0.049 factor C DNA repair protein RecN 0.093 0.101 0.196 0.166 0.121 0.097 DNA repair, bacterial Exodeoxyribonuclease VII small subunit 0.012 0.020 0.017 0.017 0.017 0.016 (EC 3.1.11.6) RecA protein 0.191 0.211 0.503 0.373 0.936 0.634 SOS-response repressor and protease 0.067 0.072 0.098 0.087 0.178 0.103 LexA (EC 3.4.21.88) DNA mismatch repair protein MutL 0.049 0.056 0.075 0.079 0.028 0.025 DNA repair, bacterial MutL- MutS system DNA mismatch repair protein MutS 0.072 0.085 0.080 0.080 0.076 0.060 Exodeoxyribonuclease V alpha chain 0.038 0.023 0.061 0.065 0.058 0.038 (EC 3.1.11.5) DNA repair, bacterial Exodeoxyribonuclease V beta chain (EC 0.058 0.051 0.122 0.111 0.147 0.094 RecBCD pathway 3.1.11.5) Exodeoxyribonuclease V gamma chain 0.094 0.109 0.114 0.111 0.101 0.073 (EC 3.1.11.5) ATP-dependent DNA helicase RecQ 0.046 0.046 0.097 0.087 0.056 0.052 DNA recombination and repair protein DNA repair, bacterial 0.006 0.006 0.031 0.022 0.021 0.013 RecFOR pathway RecF DNA recombination and repair protein 0.062 0.058 0.037 0.057 0.052 0.075 RecO DNA repair, bacterial UvrD and related helicases ATP-dependent DNA helicase Rep 0.039 0.029 0.101 0.086 0.118 0.076

DNA repair, bacterial UvrD ATP-dependent DNA helicase 0.101 0.098 0.150 0.153 0.238 0.164 and related helicases UvrD/PcrA Excinuclease ABC subunit A 0.098 0.121 0.211 0.201 0.084 0.074

DNA repair, UvrABC system Excinuclease ABC subunit B 0.080 0.076 0.063 0.072 0.062 0.053

Excinuclease ABC subunit C 0.008 0.007 0.021 0.021 0.005 0.005

DNA replication, archaeal Ribonuclease HII (EC 3.1.26.4) 0.024 0.016 0.121 0.067 0.318 0.154 60 continued

Appendix B continued DNA-binding protein Fis 0.031 0.020 0.078 0.054 0.156 0.127

DNA-binding protein HU-alpha 0.982 0.954 0.459 0.412 0.219 0.516 DNA structural proteins, bacterial Integration host factor alpha subunit 0.072 0.073 0.089 0.088 0.093 0.091

Integration host factor beta subunit 0.350 0.251 0.174 0.160 0.238 0.312 DNA topoisomerases, Type I, DNA topoisomerase I (EC 5.99.1.2) 0.173 0.183 0.187 0.182 0.099 0.099 ATP-independent DNA gyrase subunit A (EC 5.99.1.3) 0.136 0.166 0.223 0.245 0.225 0.165

DNA topoisomerases, Type II, DNA gyrase subunit B (EC 5.99.1.3) 0.272 0.320 0.371 0.369 0.228 0.222 ATP-dependent Topoisomerase IV subunit A (EC 0.021 0.016 0.054 0.053 0.072 0.048 5.99.1.-) ATP-dependent DNA helicase RecG 0.045 0.037 0.101 0.093 0.060 0.040 (EC 3.6.1.-) Chromosomal replication initiator 0.107 0.091 0.110 0.124 0.094 0.079 protein DnaA Crossover junction endodeoxyribonuclease RuvC (EC 0.124 0.096 0.086 0.082 0.144 0.114 3.1.22.4) DNA polymerase III beta subunit (EC 0.082 0.099 0.118 0.121 0.067 0.063 2.7.7.7) DNA polymerase III chi subunit (EC 0.022 0.026 0.032 0.029 0.035 0.031 DNA-replication 2.7.7.7) DNA polymerase III epsilon subunit (EC 0.026 0.040 0.025 0.025 0.019 0.019 2.7.7.7) Holliday junction DNA helicase RuvA 0.032 0.028 0.061 0.055 0.120 0.082

Holliday junction DNA helicase RuvB 0.037 0.035 0.097 0.079 0.177 0.115

Primosomal replication protein N 0.364 0.300 0.328 0.306 0.434 0.477

Transcription-repair coupling factor 0.147 0.185 0.272 0.249 0.121 0.097 Putative DNA-binding protein in cluster Restriction-Modification with Type I restriction-modification 0.009 0.009 0.006 0.006 0.011 0.009 System system YcfH Putative deoxyribonuclease YcfH 0.034 0.031 0.041 0.041 0.047 0.035 Ribosome-associated heat shock protein Dormancy and Sporulation Cluster implicated in the recycling of the 50S 0.006 0.010 0.007 0.007 0.005 0. 004 Sporulation subunit (S4 paralog) Sporulation-associated proteins with broader Peptidyl-tRNA hydrolase (EC 3.1.1.29) 0.009 0.010 0.033 0.024 0.069 0.043 functions Acetyl-CoA carboxylase Biotin carboxyl carrier protein of acetyl- 0.018 0.037 0.051 0.050 0.061 0.044 complexes in plants CoA carboxylase Acyl-CoA thioesterase II Acyl-CoA thioesterase II (EC 3.1.2.-) 0.007 0.006 0.018 0.019 0.024 0.016

Fatty Acids, Cardiolipin synthetase (EC 2.7.8.-) 0.031 0.030 0.059 0.062 0.056 0.043 Lipids, and Isoprenoids CDP-diacylglycerol--glycerol-3- Cardiolipin biosynthesis in phosphate 3-phosphatidyltransferase (EC 0.034 0.035 0.034 0.036 0.024 0.027 plants 2.7.8.5) Phosphatidylglycerophosphatase A (EC 0.013 0.011 0.034 0.032 0.020 0.016 3.1.3.27)

61 continued

Appendix B continued 1-acyl-sn-glycerol-3-phosphate 0.069 0.058 0.223 0.131 0.475 0.247 acyltransferase (EC 2.3.1.51) CDP-diacylglycerol Glycerol-3-phosphate acyltransferase 0.174 0.232 0.184 0.213 0.164 0.126 biosynthesis in plants (EC 2.3.1.15) Phosphatidate cytidylyltransferase (EC 0.130 0.074 0.501 0.289 1.247 0.621 2.7.7.41) 3-hydroxyacyl-[acyl-carrier-protein] 0.106 0.111 0.098 0.095 0.090 0.099 dehydratase, FabZ form (EC 4.2.1.59) Fatty Acid Biosynthesis FASII 3-oxoacyl-[acyl-carrier-protein] 0.527 0.867 0.526 0.512 0.366 0.500 synthase, KASI (EC 2.3.1.41) 4'-phosphopantetheinyl transferase (EC Fatty Acid Biosynthesis FASII 0.002 0.003 0.006 0.004 0.002 0.002 2.7.8.-) Acyl-phosphate:glycerol-3-phosphate O- 0.056 0.074 0.049 0.048 0.054 0.062 acyltransferase PlsY Glycerolipid and CDP-diacylglycerol--serine O- Glycerophospholipid 0.089 0.103 0.071 0.077 0.059 0.058 phosphatidyltransferase (EC 2.7.8.8) Metabolism in Bacteria Phosphatidylserine decarboxylase (EC 0.040 0.058 0.068 0.064 0.033 0.028 4.1.1.65) 1-deoxy-D-xylulose 5-phosphate 0.097 0.094 0.119 0.118 0.118 0.106 reductoisomerase (EC 1.1.1.267) 1-hydroxy-2-methyl-2-(E)-butenyl 4- 0.028 0.032 0.054 0.057 0.023 0.021 diphosphate synthase (EC 1.17.7.1) Isoprenoid Biosynthesis 2-C-methyl-D-erythritol 2,4- 0.015 0.018 0.024 0.028 0.012 0.014 cyclodiphosphate synthase (EC 4.6.1.12) 4-hydroxy-3-methylbut-2-enyl 0.018 0.016 0.072 0.067 0.107 0.065 diphosphate reductase (EC 1.17.1.2) 3-oxoacyl-[ACP] synthase (EC 2.3.1.41) 0.031 0.030 0.068 0.081 0.024 0.025 FabV like Acyl carrier protein (ACP1) 0.010 0.006 0.013 0.014 0.017 0.012

Acyl carrier protein (ACP2) 0.012 0.009 0.016 0.019 0.022 0.015 FIG002571: 4-hydroxybenzoyl-CoA 0.011 0.010 0.014 0.015 0.004 0.006 thioesterase domain protein Phospholipid and Fatty acid FIG018329: 1-acyl-sn-glycerol-3- 0.021 0.016 0.042 0.045 0.053 0.036 biosynthesis related cluster phosphate acyltransferase

FIG021862: membrane protein, exporter 0.014 0.013 0.042 0.047 0.012 0.010 FIG027190: Putative transmembrane 0.014 0.011 0.021 0.023 0.009 0.008 protein FIG085779: Lipoprotein 0.019 0.021 0.024 0.025 0.012 0.013

FIG138576: 3-oxoacyl-[ACP] synthase 0.020 0.022 0.046 0.045 0.015 0.014 (EC 2.3.1.41) Lysophospholipase (EC 3.1.1.5) 0.009 0.009 0.006 0.005 0.008 0.007 Triacylglycerol metabolism Lysophospholipase L2 (EC 3.1.1.5) 0.019 0.019 0.031 0.029 0.015 0.013 General Stress Response and RpoS Regulators SG1 Protein yihD 0.027 0.032 0.021 0.016 0.028 0.027 Stationary Phase Response Ferric iron ABC transporter, iron- 0.912 1.173 0.229 0.233 0.119 0.170 Iron acquisition Campylobacter Iron binding protein and metabolism Metabolism Ferric iron ABC transporter, permease 0.066 0.061 0.062 0.073 0.027 0.034 protein

62 continued

Appendix B continued Ferric siderophore transport system, 0.057 0.086 0.041 0.036 0.043 0.043 biopolymer transport protein ExbB Ferric siderophore transport system, 0.067 0.067 0.053 0.043 0.061 0.058 periplasmic binding protein TonB Magnesium and cobalt transport protein 0.050 0.058 0.072 0.068 0.293 0.231 CorA Periplasmic protein p19 involved in 0.077 0.203 0.077 0.088 0.008 0.015 high-affinity Fe2+ transport Ferrous iron transporter Ferrous iron transport permease EfeU 0.018 0.026 0.021 0.022 0.009 0.007 EfeUOB, low-pH-induced Outer membrane receptor proteins, Hemin transport system 0.694 0.867 0.423 0.303 0.220 0.273 mostly Fe transport Iron acquisition in Ferric iron ABC transporter, ATP- 0.024 0.024 0.015 0.014 0.017 0.012 Streptococcus binding protein Iron acquisition in Vibrio TonB-dependent receptor 0.047 0.047 0.075 0.066 0.220 0.135 Iron(III) dicitrate transport system, Iron(III) dicitrate transport periplasmic iron-binding protein FecB 0.053 0.101 0.060 0.055 0.015 0.016 system Fec (TC 3.A.1.14.1) Cytochrome c551 peroxidase (EC RCJ pfr 0.061 0.068 0.083 0.075 0.057 0.054 1.11.1.5) Possible ABC transporter, periplasmic 0.003 0.002 0.004 0.004 0.002 0.002 ABC transporter of unknown substrate X binding protein precursor substrate X Putative ABC transporter of substrate X, 0.003 0.004 0.003 0.003 0.005 0.003 ATP-binding subunit Oligopeptide ABC transporter, periplasmic oligopeptide-binding protein 0.371 0.429 0.173 0.191 0.122 0.120 OppA (TC 3.A.1.5.1) Oligopeptide transport ATP-binding 0.005 0.005 0.004 0.004 0.005 0.005 protein OppD (TC 3.A.1.5.1) ABC transporter oligopeptide Oligopeptide transport ATP-binding (TC 3.A.1.5.1) 0.007 0.007 0.006 0.006 0.008 0.008 protein OppF (TC 3.A.1.5.1) Oligopeptide transport system permease 0.007 0.009 0.008 0.008 0.006 0.004 protein OppB (TC 3.A.1.5.1) Oligopeptide transport system permease 0.002 0.002 0.003 0.003 0.006 0.004 protein OppC (TC 3.A.1.5.1) ATPase component NikO of energizing 0.035 0.042 0.053 0.073 0.023 0.021 module of nickel ECF transporter Membrane ECF class transporters Substrate-specific component NikM of Transport 0.035 0.044 0.044 0.062 0.018 0.018 nickel ECF transporter Preprotein translocase subunit SecG (TC HtrA and Sec secretion 3.A.5.1.1) 0.015 0.019 0.022 0.014 0.039 0.036

Protein export cytoplasm chaperone HtrA and Sec secretion protein (SecB, maintains protein to be 0.246 0.224 0.090 0.096 0.128 0.210 exported in unfolded state) Peptide transport periplasmic protein 0.057 0.057 0.058 0.059 0.054 0.048 SapA Peptide ABC transport system Peptide transport system ATP-binding 0.009 0.008 0.023 0.021 0.051 0.033 Sap protein SapF Peptide transport system permease 0.021 0.018 0.028 0.026 0.033 0.021 protein SapC Phosphoglycerate transport system 0.001 0.001 0.001 0.001 0.008 0.005 Phosphoglycerate transport sensor protein PgtB (EC 2.7.3.-) system Phosphoglycerate transport system 0.002 0.002 0.005 0.003 0.009 0.006 transcriptional regulatory protein PgtA

63 continued

Appendix B continued T6SS BR ClpB protein 0.060 0.067 0.305 0.373 0.037 0.034 4-hydroxybenzoyl-CoA thioesterase 0.058 0.054 0.072 0.069 0.119 0.116 family active site Biopolymer transport protein ExbD/TolR 0.079 0.092 0.051 0.046 0.059 0.058 MotA/TolQ/ExbB proton channel family 0.066 0.055 0.089 0.092 0.150 0.155 protein Peptidoglycan-associated lipoprotein 1.136 1.301 0.654 0.575 0.396 0.734 Ton and Tol transport systems precursor Tol biopolymer transport system, TolR 0.020 0.017 0.033 0.031 0.047 0.030 protein TolA protein 0.038 0.026 0.049 0.050 0.060 0.038 tolB protein precursor, periplasmic protein involved in the tonb-independent 0.360 0.327 0.354 0.374 0.278 0.289 uptake of group A colicins Additional component NikL of nickel 0.042 0.066 0.056 0.080 0.023 0.022 Transport of Nickel and ECF transporter Cobalt Additional periplasmic component NikK 0.180 0.198 0.127 0.192 0.045 0.060 of nickel ECF transporter Twin-arginine translocation protein TatB 0.045 0.064 0.058 0.058 0.053 0.051 Twin-arginine translocation system Twin-arginine translocation protein TatC 0.014 0.016 0.034 0.035 0.021 0.016

Flp pilus assembly protein RcpB 0.007 0.006 0.005 0.004 0.008 0.008

Flp pilus assembly protein RcpC/CpaB 0.007 0.007 0.005 0.005 0.010 0.012

Flp pilus assembly protein TadB 0.016 0.014 0.010 0.011 0.022 0.022 Widespread colonization island Flp pilus assembly protein, pilin Flp 0.001 0.003 0.001 0.001 0.001 0.002

Protein TadG, associated with Flp pilus 0.374 0.330 0.150 0.138 0.146 0.212 assembly Type II/IV secretion system ATP hydrolase TadA/VirB11/CpaF, TadA 0.023 0.024 0.018 0.019 0.036 0.032 subfamily Type II/IV secretion system ATPase TadZ/CpaE, associated with Flp pilus 0.021 0.021 0.013 0.014 0.025 0.027 assembly Type II/IV secretion system protein TadC, associated with Flp pilus 0.011 0.010 0.007 0.008 0.015 0.015 assembly Metabolite damage and its Bis(5'-nucleosyl)-tetraphosphatase RidA1 subgroup 0.013 0.022 0.011 0.013 0.007 0.007 repair or (asymmetrical) (EC 3.6.1.17) mitigation tRNA nucleotidyltransferase (EC At2g33980 At1g28960 0.022 0.016 0.038 0.035 0.014 0.016 2.7.7.21) (EC 2.7.7.25) Thiol peroxidase, Bcp-type (EC At5g04520 AT1G06240 0.018 0.018 0.010 0.010 0.016 0.017 1.11.1.15) Miscellaneous UPF0234 protein YajQ 0.072 0.072 0.050 0.055 0.037 0.043 Broadly distributed proteins UPF0265 protein YeeX 0.102 0.117 0.038 0.040 0.071 0.104 not in subsystems YciL protein 0.023 0.021 0.016 0.015 0.008 0.012

64 continued

Appendix B continued YpfJ protein, zinc metalloprotease 0.052 0.073 0.057 0.057 0.039 0.039 superfamily Translation initiation factor SUI1-related COG2016 0.022 0.023 0.014 0.012 0.016 0.013 protein

Competence or DNA damage- Poly(A) polymerase (EC 2.7.7.19) 0.019 0.012 0.057 0.049 0.036 0.028 inducible protein CinA and related protein families UPF0125 protein yfjF 0.008 0.008 0.005 0.006 0.012 0.012 EC 6.3.4.- Ligases that form CTP synthase (EC 6.3.4.2) 0.079 0.111 0.163 0.178 0.147 0.113 carbon-nitrogen bonds Adenylate cyclase (EC 4.6.1.1) 0.236 0.198 0.188 0.186 0.130 0.128

Inorganic pyrophosphatase (EC 3.6.1.1) 0.372 0.363 0.151 0.159 0.072 0.138 IojapClusters LSU ribosomal protein L21p 0.252 0.305 0.281 0.212 0.555 0.630

LSU ribosomal protein L27p 0.156 0.197 0.180 0.150 0.552 0.665

5-nucleotidase SurE (EC 3.1.3.5) 0.025 0.027 0.060 0.064 0.047 0.038 Deoxyuridine 5'-triphosphate 0.032 0.032 0.036 0.038 0.015 0.021 nucleotidohydrolase (EC 3.6.1.23) Metabolite repair Hydrogen peroxide-inducible genes 0.260 0.249 0.169 0.182 0.131 0.144 activator Nucleotidase YfbR, HD superfamily 0.013 0.009 0.018 0.015 0.024 0.019 GTP-binding and nucleic acid-binding 0.019 0.021 0.071 0.057 0.138 0.096 protein YchF YebC Threonyl-tRNA synthetase (EC 6.1.1.3) 0.150 0.166 0.229 0.231 0.250 0.202 Sigma factor RpoE regulatory protein YgfZ-Fe-S 0.011 0.006 0.030 0.029 0.066 0.038 RseC ATP synthase alpha chain (EC 3.6.3.14) 0.535 0.352 0.406 0.468 0.169 0.307

ATP synthase beta chain (EC 3.6.3.14) 0.470 0.353 0.325 0.355 0.119 0.201 ATP synthase epsilon chain (EC Mitochondrial 0.138 0.106 0.100 0.104 0.041 0.074 F0F1-type ATP synthase in 3.6.3.14) electron transport plants (mitochondrial) system in plants ATP synthase F0 sector subunit b 0.153 0.097 0.106 0.122 0.062 0.106

ATP synthase F0 sector subunit c 0.070 0.052 0.062 0.068 0.023 0.048 ATP synthase gamma chain (EC 0.259 0.154 0.177 0.203 0.066 0.125 3.6.3.14) Cytochrome c-type heme lyase subunit 0.013 0.010 0.022 0.025 0.034 0.020 nrfE, nitrite reductase complex assembly Cytochrome c-type heme lyase subunit 0.010 0.007 0.010 0.012 0.017 0.011 nrfF, nitrite reductase complex assembly Cytochrome c-type protein NrfB 0.001 0.002 0.006 0.011 0.015 0.006 precursor Nitrogen Nitrate and nitrite Ferredoxin-type protein NapG 0.002 0.003 0.017 0.026 0.017 0.003 Metabolism ammonification (periplasmic nitrate reductase) Nitrate reductase cytochrome c550-type 0.001 0.001 0.004 0.006 0.006 0.001 subunit Nitrate/nitrite response regulator protein 0.027 0.027 0.028 0.028 0.017 0.016

NrfD protein 0.004 0.006 0.013 0.015 0.018 0.008

65 continued

Appendix B continued Periplasmic nitrate reductase component 0.000 0.001 0.002 0.004 0.005 0.001 NapD Polyferredoxin NapH (periplasmic 0.001 0.002 0.008 0.013 0.011 0.002 nitrate reductase) Putative thiol:disulfide oxidoreductase, 0.004 0.003 0.004 0.006 0.007 0.005 nitrite reductase complex assembly Uracil permease 0.028 0.023 0.038 0.037 0.040 0.037 De Novo Pyrimidine Synthesis Uracil phosphoribosyltransferase (EC 0.099 0.105 0.059 0.063 0.043 0.050 2.4.2.9) Purine conversions 5'-nucleotidase (EC 3.1.3.5) 0.403 0.373 0.178 0.211 0.068 0.137 Adenine phosphoribosyltransferase (EC 0.030 0.035 0.041 0.038 0.044 0.041 2.4.2.7) Adenylate kinase (EC 2.7.4.3) 0.027 0.037 0.038 0.033 0.057 0.052 GMP synthase [glutamine-hydrolyzing] 0.025 0.027 0.110 0.096 0.265 0.150 (EC 6.3.5.2) Purine conversions Guanylate kinase (EC 2.7.4.8) 0.030 0.030 0.020 0.020 0.022 0.021 Hypoxanthine-guanine 0.012 0.018 0.009 0.007 0.013 0.009 phosphoribosyltransferase (EC 2.4.2.8) Nucleoside diphosphate kinase (EC 0.060 0.078 0.031 0.027 0.008 0.013 2.7.4.6) Ribonucleotide reductase of class Ia 0.278 0.404 0.491 0.558 0.086 0.115 Purine de novo biosynthesis in (aerobic), alpha subunit (EC 1.17.4.1) plants Ribonucleotide reductase of class Ia Nucleosides and 0.101 0.088 0.093 0.100 0.024 0.067 (aerobic), beta subunit (EC 1.17.4.1) Nucleotides Purine nucleotide synthesis Purine nucleotide synthesis repressor 0.026 0.042 0.022 0.023 0.031 0.029 regulator Purine Utilization Guanine-hypoxanthine permease 0.032 0.030 0.047 0.039 0.043 0.048

Cytidine deaminase (EC 3.5.4.5) 0.083 0.090 0.031 0.035 0.026 0.025

Cytidylate kinase (EC 2.7.4.25) 0.138 0.138 0.146 0.131 0.131 0.167 Deoxycytidine triphosphate deaminase 0.008 0.009 0.020 0.024 0.023 0.015 pyrimidine conversions (EC 3.5.4.13) Thioredoxin reductase (EC 1.8.1.9) 0.202 0.374 0.284 0.317 0.106 0.135 Thymidylate kinase (EC 2.7.4.9) 0.039 0.039 0.051 0.052 0.054 0.046

Ribonucleotide reductase of class III (anaerobic), activating protein (EC 0.007 0.009 0.021 0.022 0.026 0.018 1.97.1.4) Ribonucleotide reduction Ribonucleotide reductase of class III 0.153 0.296 0.345 0.444 0.100 0.094 (anaerobic), large subunit (EC 1.17.4.2) Ribonucleotide reductase transcriptional 0.023 0.024 0.027 0.027 0.024 0.027 regulator NrdR Heat shock protein 60 family chaperone 0.561 0.400 1.419 1.754 0.113 0.129 Phages, GroEL Prophages, Staphylococcal pathogenicity Transposable islands SaPI SSU ribosomal protein S18p 0.209 0.183 0.199 0.158 0.275 0.305 elements, Plasmids tmRNA-binding protein SmpB 0.038 0.041 0.021 0.022 0.028 0.028

66 continued

Appendix B continued Alkylphosphonate utilization operon Alkylphosphonate utilization 0.011 0.015 0.012 0.010 0.012 0.015 protein PhnA Apolipoprotein N-acyltransferase (EC 0.011 0.008 0.022 0.019 0.095 0.061 2.3.1.-) Exopolyphosphatase (EC 3.6.1.11) 0.065 0.061 0.062 0.060 0.032 0.035 Phosphorus Metabolism Magnesium and cobalt efflux protein 0.030 0.033 0.036 0.034 0.029 0.025 Phosphate metabolism CorC Phosphate transport regulator (distant 0.127 0.124 0.056 0.068 0.042 0.072 homolog of PhoU) Probable low-affinity inorganic 0.129 0.096 0.083 0.088 0.057 0.091 phosphate transporter

Glutathione-regulated Cobalt-zinc-cadmium resistance protein 0.030 0.030 0.045 0.044 0.047 0.033 potassium-efflux system and associated functions Cyclic AMP receptor protein 0.084 0.093 0.058 0.054 0.052 0.055 FKBP-type peptidyl-prolyl cis-trans 0.306 0.246 0.231 0.206 0.156 0.206 isomerase FkpA precursor (EC 5.2.1.8) FKBP-type peptidyl-prolyl cis-trans Potassium 0.203 0.146 0.147 0.158 0.236 0.273 metabolism isomerase SlyD (EC 5.2.1.8) Glutathione-regulated Glutathione-regulated potassium-efflux potassium-efflux system and 0.027 0.030 0.076 0.062 0.054 0.043 system ATP-binding protein associated functions Large-conductance mechanosensitive 0.174 0.209 0.153 0.148 0.233 0.366 channel Trk system potassium uptake protein 0.067 0.047 0.072 0.066 0.074 0.057 TrkA LSU ribosomal protein L34p 0.065 0.072 0.064 0.051 0.135 0.151 At1g14345 Ribonuclease P protein component (EC 0.021 0.017 0.032 0.029 0.105 0.059 3.1.26.5) DNA-directed RNA polymerase alpha 1.485 1.104 1.444 1.254 2.055 2.589 Predictions subunit (EC 2.7.7.6) based on plant- Peptide chain release factor 1 0.043 0.056 0.118 0.095 0.284 0.171 prokaryote At2g23840 comparative Periplasmic thiol:disulfide analysis oxidoreductase DsbB, required for DsbA 0.174 0.169 0.111 0.101 0.062 0.086 reoxidation At3g50560 Glutathione S-transferase (EC 2.5.1.18) 0.564 0.662 0.240 0.238 0.069 0.193 At4g10620 At3g57180 SSU ribosomal protein S20p 0.028 0.023 0.050 0.035 0.081 0.101 At3g47450 Alpha-aspartyl dipeptidase Peptidase E EC 3.4.13.- Dipeptidases 0.003 0.004 0.003 0.003 0.007 0.005 (EC 3.4.13.21) Alanyl-tRNA synthetase (EC 6.1.1.7) 0.379 0.374 0.415 0.398 0.662 0.558

Arginyl-tRNA synthetase (EC 6.1.1.19) 0.054 0.089 0.129 0.111 0.127 0.089

Aspartyl-tRNA synthetase (EC 6.1.1.12) 0.355 0.376 0.179 0.200 0.152 0.152 Protein EC 6.1.1.- Ligases forming Glutaminyl-tRNA synthetase (EC Metabolism 0.172 0.188 0.164 0.155 0.214 0.216 aminoacyl-tRNA and related 6.1.1.18) compounds Glycyl-tRNA synthetase alpha chain (EC 0.077 0.085 0.078 0.085 0.086 0.086 6.1.1.14) Phenylalanyl-tRNA synthetase alpha 0.033 0.055 0.060 0.067 0.067 0.049 chain (EC 6.1.1.20) Phenylalanyl-tRNA synthetase beta 0.357 0.332 0.429 0.453 0.464 0.398 chain (EC 6.1.1.20)

67 continued

Appendix B continued Tryptophanyl-tRNA synthetase (EC 0.035 0.034 0.056 0.058 0.057 0.049 6.1.1.2) Heat shock protein 60 family co- GroEL GroES 0.072 0.051 0.188 0.274 0.013 0.016 chaperone GroES Lipoprotein signal peptidase (EC Lipoprotein Biosynthesis 0.007 0.008 0.019 0.019 0.017 0.012 3.4.23.36) Cytochrome c-type biogenesis protein Peptide methionine sulfoxide CcdA homolog, associated with MetSO 0.029 0.035 0.020 0.024 0.016 0.022 reductase reductase FKBP-type peptidyl-prolyl cis-trans 0.033 0.038 0.074 0.059 0.024 0.021 Peptidyl-prolyl cis-trans isomerase fklB (EC 5.2.1.8) isomerase Peptidyl-prolyl cis-trans isomerase PpiB 0.075 0.114 0.055 0.059 0.049 0.056 (EC 5.2.1.8) Cytochrome c-type biogenesis protein CcmG/DsbE, thiol:disulfide 0.013 0.008 0.015 0.013 0.043 0.022 oxidoreductase Periplasmic disulfide Cytochrome c-type biogenesis protein interchange DsbD, protein-disulfide reductase (EC 0.131 0.137 0.094 0.096 0.088 0.092 1.8.1.8) Periplasmic thiol:disulfide interchange 0.067 0.061 0.064 0.057 0.087 0.081 protein DsbA Programmed frameshift Peptide chain release factor 2 0.046 0.053 0.067 0.070 0.086 0.067

Copper sensory histidine kinase CpxA 0.034 0.027 0.038 0.034 0.017 0.026 Prolyl-tRNA synthetase associated editing enzymes Copper-translocating P-type ATPase (EC 0.154 0.218 0.153 0.159 0.048 0.057 3.6.3.4) ATP-dependent Clp protease ATP- 0.024 0.023 0.085 0.078 0.060 0.046 binding subunit ClpX ATP-dependent hsl protease ATP- 0.048 0.044 0.199 0.226 0.020 0.019 binding subunit HslU ATP-dependent protease HslV (EC Proteasome bacterial 0.008 0.010 0.039 0.057 0.003 0.004 3.4.25.-) ATP-dependent protease La (EC 0.191 0.243 0.488 0.485 0.305 0.196 3.4.21.53) Type I ATP-dependent protease La (EC 0.048 0.040 0.037 0.036 0.032 0.035 3.4.21.53) Type II Chaperone protein DnaJ 0.051 0.057 0.086 0.080 0.173 0.116

Protein chaperones Chaperone protein HtpG 0.035 0.031 0.196 0.248 0.024 0.023

DnaJ-class molecular chaperone CbpA 0.023 0.022 0.036 0.052 0.019 0.019

Protein chaperones Heat shock protein GrpE 0.024 0.026 0.082 0.104 0.021 0.018

Protein degradation Oligopeptidase A (EC 3.4.24.70) 0.233 0.243 0.206 0.260 0.067 0.068 Outer membrane stress sensor protease 0.191 0.134 0.421 0.495 0.276 0.225 Proteolysis in bacteria, ATP- DegQ, serine protease dependent Outer membrane stress sensor protease 0.016 0.016 0.035 0.030 0.035 0.025 DegS Ribosomal protein S12p Asp88 (E. coli) 0.025 0.026 0.067 0.061 0.097 0.068 Ribosomal protein S12p Asp methylthiotransferase methylthiotransferase SSU ribosomal protein S12p (S23e) 0.550 0.576 0.391 0.338 0.368 0.441 Ribosomal protein S5p SSU ribosomal protein S5p (S2e) 0.652 0.404 0.563 0.532 1.258 1.531 acylation

68 continued

Appendix B continued LSU ribosomal protein L28p 0.184 0.143 0.144 0.119 0.292 0.328

LSU ribosomal protein L31p 0.272 0.345 0.136 0.119 0.233 0.332 Ribosomal proteins, zinc requirement LSU ribosomal protein L33p, zinc- 0.207 0.178 0.197 0.145 0.217 0.275 independent SSU ribosomal protein S14p (S29e) 0.565 0.431 0.494 0.501 0.316 0.409

Ribosome activity modulation Ribosome hibernation protein YfiA 0.072 0.070 0.025 0.020 0.008 0.008 Ribosomal large subunit pseudouridine 0.029 0.035 0.042 0.036 0.060 0.046 synthase A (EC 4.2.1.70) Ribosomal large subunit pseudouridine 0.014 0.018 0.029 0.023 0.059 0.048 synthase C (EC 4.2.1.70) Ribosomal large subunit pseudouridine 0.025 0.035 0.039 0.044 0.032 0.028 synthase D (EC 4.2.1.70) Ribosome biogenesis bacterial Ribosomal protein L11 0.063 0.039 0.052 0.050 0.077 0.060 methyltransferase (EC 2.1.1.-) Ribosomal-protein-S18p-alanine 0.006 0.007 0.008 0.008 0.008 0.006 acetyltransferase (EC 2.3.1.-) tRNA (Guanine37-N1) - 0.985 0.591 1.031 0.871 1.151 0.963 methyltransferase (EC 2.1.1.31) LSU ribosomal protein L10p (P0) 0.457 0.367 0.368 0.366 0.762 1.075

LSU ribosomal protein L11p (L12e) 0.336 0.319 0.284 0.242 0.373 0.320

LSU ribosomal protein L13p (L13Ae) 0.410 0.324 0.511 0.438 0.449 0.630

LSU ribosomal protein L14p (L23e) 0.754 0.683 0.636 0.628 0.535 0.553

LSU ribosomal protein L15p (L27Ae) 0.385 0.231 0.320 0.314 0.794 0.949

LSU ribosomal protein L16p (L10e) 0.473 0.316 0.299 0.302 0.787 0.653

LSU ribosomal protein L18p (L5e) 0.437 0.291 0.379 0.338 0.548 0.858

LSU ribosomal protein L19p 0.343 0.200 0.388 0.335 0.485 0.436

LSU ribosomal protein L1p (L10Ae) 0.550 0.466 0.437 0.392 0.672 0.544

Ribosome LSU bacterial LSU ribosomal protein L20p 0.179 0.168 0.170 0.159 0.190 0.271

LSU ribosomal protein L22p (L17e) 0.348 0.214 0.267 0.264 0.746 0.586

LSU ribosomal protein L23p (L23Ae) 0.190 0.103 0.186 0.196 0.315 0.435

LSU ribosomal protein L24p (L26e) 0.605 0.544 0.457 0.418 0.253 0.322

LSU ribosomal protein L25p 0.085 0.086 0.082 0.076 0.216 0.270

LSU ribosomal protein L29p (L35e) 0.266 0.185 0.148 0.135 0.243 0.396

LSU ribosomal protein L2p (L8e) 0.526 0.324 0.503 0.517 1.312 1.044

LSU ribosomal protein L30p (L7e) 0.246 0.150 0.222 0.229 0.224 0.277

LSU ribosomal protein L35p 0.065 0.058 0.051 0.042 0.071 0.100

LSU ribosomal protein L3p (L3e) 0.523 0.316 0.492 0.503 1.104 1.089

69 continued

Appendix B continued LSU ribosomal protein L4p (L1e) 0.387 0.235 0.404 0.416 1.117 0.971

LSU ribosomal protein L6p (L9e) 0.555 0.413 0.568 0.536 1.254 1.519

LSU ribosomal protein L7/L12 (P1/P2) 0.245 0.194 0.164 0.151 0.371 0.559

LSU ribosomal protein L9p 0.291 0.243 0.332 0.290 0.403 0.455

SSU ribosomal protein S10p (S20e) 0.271 0.166 0.237 0.254 0.596 0.558

SSU ribosomal protein S11p (S14e) 0.565 0.475 0.552 0.478 0.739 0.891

SSU ribosomal protein S15p (S13e) 0.337 0.362 0.258 0.203 0.220 0.381

SSU ribosomal protein S16p 0.298 0.193 0.309 0.281 0.322 0.348 Ribosome SSU bacterial SSU ribosomal protein S17p (S11e) 0.224 0.186 0.183 0.165 0.352 0.354

SSU ribosomal protein S19p (S15e) 0.255 0.142 0.189 0.201 0.542 0.424

SSU ribosomal protein S1p 2.285 1.596 1.707 1.635 1.913 2.684

SSU ribosomal protein S2p (SAe) 0.591 0.502 0.429 0.387 0.669 0.735

SSU ribosomal protein S3p (S3e) 0.948 0.560 0.658 0.710 1.271 1.314

SSU ribosomal protein S4p (S9e) 0.790 0.568 0.797 0.735 1.158 1.407

Ribosome SSU bacterial SSU ribosomal protein S6p 0.347 0.315 0.331 0.264 0.434 0.485

SSU ribosomal protein S8p (S15Ae) 0.367 0.282 0.348 0.316 0.867 1.041

SSU ribosomal protein S9p (S16e) 0.254 0.218 0.336 0.257 0.295 0.437 L-seryl-tRNA(Sec) selenium transferase 0.023 0.027 0.027 0.033 0.016 0.014 (EC 2.9.1.1)

Selenide,water dikinase (EC 2.7.9.3) 0.130 0.156 0.070 0.075 0.067 0.065 Selenocysteine metabolism selenocysteine-containing 0.039 0.035 0.107 0.101 0.047 0.037 Selenocysteine-specific translation 0.056 0.069 0.047 0.058 0.022 0.020 elongation factor Translation elongation factor Translation elongation factor G 1.937 1.549 1.954 2.015 1.423 1.195 G family Translation elongation factors Translation elongation factor Ts 0.551 0.404 0.377 0.355 0.565 0.540 bacterial Ribosome-binding factor A 0.022 0.025 0.021 0.020 0.030 0.030 Translation initiation factor 1 0.049 0.048 0.045 0.034 0.126 0.157 Translation initiation factors bacterial Translation initiation factor 2 0.192 0.152 0.378 0.385 0.397 0.282

Translation initiation factor 3 0.323 0.280 0.456 0.382 0.532 0.646

GTP-binding protein Era 0.019 0.015 0.082 0.072 0.058 0.048

GTP-binding protein HflX 0.019 0.013 0.019 0.017 0.029 0.024 Universal GTPases GTP-binding protein TypA/BipA 0.047 0.055 0.232 0.216 0.143 0.109 GTPase and tRNA-U34 5-formylation 0.016 0.010 0.033 0.031 0.072 0.042 enzyme TrmE

70 continued

Appendix B continued Ribosome small subunit-stimulated 0.056 0.060 0.045 0.041 0.057 0.044 GTPase EngC Conserved protein YcjX with nucleoside A conserved operon linked to 0.024 0.027 0.021 0.020 0.040 0.029 triphosphate hydrolase domain TyrR and possibly involved in virulence Membrane protein YcjF 0.016 0.017 0.016 0.015 0.023 0.017 Autoinducer 2 (AI-2) ABC transport system, fused AI2 transporter subunits 0.002 0.002 0.002 0.003 0.005 0.003 and ATP-binding component Autoinducer 2 (AI-2) ABC transport 0.001 0.001 0.001 0.002 0.003 0.001 system, membrane channel protein LsrC Autoinducer 2 (AI-2) ABC transport 0.001 0.001 0.003 0.003 0.003 0.002 system, membrane channel protein LsrD Autoinducer 2 (AI-2) transport Autoinducer 2 (AI-2) ABC transport and processing (lsrACDBFGE system, periplasmic AI-2 binding protein 0.001 0.001 0.001 0.001 0.000 0.000 operon) LsrB Autoinducer 2 (AI-2) aldolase LsrF (EC 0.002 0.002 0.004 0.004 0.005 0.003 4.2.1.-) Autoinducer 2 (AI-2) kinase LsrK (EC 0.003 0.003 0.011 0.011 0.028 0.015 2.7.1.-) Autoinducer 2 (AI-2) modifying protein 0.001 0.001 0.002 0.002 0.004 0.002 LsrG Autoinducer 2 (AI-2) transport LsrR, transcriptional repressor of lsr and processing (lsrACDBFGE 0.004 0.005 0.004 0.006 0.009 0.006 operon operon) Regulation and 3',5'-cyclic-nucleotide phosphodiesterase Cell signaling cAMP signaling in bacteria 0.013 0.017 0.023 0.022 0.029 0.023 (EC 3.1.4.17) MazEF toxin-antitoxing (programmed cell death) Programmed cell death toxin ChpB 0.085 0.110 0.030 0.032 0.054 0.072 system Murein hydrolase regulation LrgA-associated membrane protein LrgB 0.030 0.040 0.038 0.035 0.029 0.029 and cell death Copper-sensing two-component system 0.030 0.032 0.019 0.019 0.015 0.018 response regulator CpxR DNA transformation protein TfoX 0.057 0.113 0.041 0.028 0.060 0.052 Orphan regulatory proteins Sensory histidine kinase QseC 0.006 0.007 0.010 0.011 0.016 0.010 Two-component system response 0.004 0.005 0.004 0.003 0.006 0.006 regulator QseB Plastidial (p)ppGpp-mediated GTP pyrophosphokinase (EC 2.7.6.5), response in plants 0.124 0.160 0.168 0.220 0.116 0.115 (p)ppGpp synthetase I

Stringent Response, (p)ppGpp GTP pyrophosphokinase (EC 2.7.6.5), 0.163 0.180 0.205 0.204 0.206 0.160 metabolism (p)ppGpp synthetase II StbD replicon stabilization protein 0.014 0.016 0.014 0.012 0.050 0.034 Toxin-antitoxin replicon (antitoxin to StbE) stabilization systems StbE replicon stabilization toxin 0.014 0.017 0.012 0.010 0.030 0.023

Arsenate reductase (EC 1.20.4.1) 0.025 0.024 0.012 0.012 0.010 0.012 Anaerobic respiratory reductases Formate dehydrogenase -O, gamma 0.002 0.002 0.002 0.002 0.003 0.003 subunit (EC 1.2.1.2) Respiration ABC transporter involved in cytochrome 0.009 0.011 0.032 0.027 0.116 0.067 Biogenesis of c-type c biogenesis, ATPase component CcmA cytochromes ABC transporter involved in cytochrome 0.006 0.006 0.019 0.017 0.059 0.032 c biogenesis, CcmB subunit

71 continued

Appendix B continued Cytochrome c heme lyase subunit CcmF 0.025 0.021 0.074 0.075 0.194 0.097

Cytochrome c heme lyase subunit CcmH 0.015 0.014 0.023 0.023 0.098 0.049

Cytochrome c heme lyase subunit CcmL 0.014 0.010 0.015 0.015 0.052 0.026 Cytochrome c-type biogenesis protein 0.008 0.007 0.018 0.019 0.077 0.044 CcmC, putative heme lyase for CcmE Cytochrome c-type biogenesis protein 0.008 0.007 0.021 0.020 0.066 0.038 CcmE, heme chaperone Ferredoxin--NADP(+) reductase (EC 0.040 0.062 0.039 0.040 0.024 0.024 1.18.1.2) Biogenesis of cytochrome c oxidases Frataxin homolog CyaY, facilitates iron supply for heme A synthesis or Fe-S 0.043 0.042 0.028 0.028 0.036 0.039 cluster assembly Coenzyme F420 hydrogenase maturation Coenzyme F420 hydrogenase 0.005 0.003 0.003 0.005 0.002 0.001 protease (EC 3.4.24.-) Formate dehydrogenase O alpha subunit 0.377 0.134 0.331 0.309 0.259 0.259 (EC 1.2.1.2) Formate dehydrogenase Formate dehydrogenase O beta subunit 0.000 0.000 0.001 0.001 0.001 0.001 (EC 1.2.1.2) Formate dehydrogenase chain D (EC 0.021 0.020 0.014 0.012 0.017 0.016 1.2.1.2) formate dehydrogenase formation 0.011 0.015 0.025 0.024 0.012 0.010 protein FdhE Formate hydrogenlyase subunit 3 0.013 0.017 0.025 0.033 0.010 0.008

Formate hydrogenlyase subunit 5 0.052 0.039 0.026 0.038 0.010 0.009 Formate hydrogenase Formate hydrogenlyase subunit 6 0.013 0.012 0.008 0.011 0.003 0.003

Formate hydrogenlyase subunit 7 0.013 0.014 0.011 0.016 0.004 0.003 Formate hydrogenlyase transcriptional 0.004 0.004 0.003 0.004 0.002 0.002 activator Hydrogenase-4 component C 0.007 0.008 0.015 0.014 0.004 0.003

Electron transport complex protein RnfA 0.011 0.015 0.014 0.014 0.019 0.021

Electron transport complex protein RnfB 0.006 0.007 0.014 0.015 0.013 0.012

Electron transport complex protein RnfD 0.017 0.016 0.032 0.034 0.028 0.023

Electron transport complex protein RnfE 0.016 0.017 0.022 0.026 0.017 0.014

Electron transport complex protein RnfG 0.017 0.016 0.020 0.023 0.021 0.016 Na(+)-translocating NADH- Na(+)-translocating NADH-quinone quinone oxidoreductase and 0.077 0.091 0.109 0.117 0.152 0.112 rnf-like group of electron reductase subunit A (EC 1.6.5.-) transport complexes Na(+)-translocating NADH-quinone 0.094 0.072 0.139 0.144 0.186 0.124 reductase subunit B (EC 1.6.5.-) Na(+)-translocating NADH-quinone 0.076 0.052 0.090 0.091 0.138 0.101 reductase subunit C (EC 1.6.5.-) Na(+)-translocating NADH-quinone 0.061 0.049 0.074 0.079 0.102 0.083 reductase subunit D (EC 1.6.5.-) Na(+)-translocating NADH-quinone 0.065 0.048 0.070 0.072 0.095 0.074 reductase subunit E (EC 1.6.5.-) Na(+)-translocating NADH-quinone 0.154 0.119 0.152 0.159 0.147 0.124 reductase subunit F (EC 1.6.5.-) 72 continued

Appendix B continued Probable exported or periplasmic protein 0.009 0.007 0.008 0.010 0.007 0.007 in ApbE locus [NiFe] hydrogenase metallocenter 0.007 0.008 0.013 0.011 0.026 0.025 assembly protein HybG [NiFe] hydrogenase metallocenter 0.010 0.011 0.025 0.033 0.007 0.006 assembly protein HypD [NiFe] hydrogenase metallocenter NiFe hydrogenase maturation 0.006 0.006 0.021 0.027 0.008 0.005 assembly protein HypE [NiFe] hydrogenase metallocenter 0.006 0.005 0.013 0.012 0.009 0.007 assembly protein HypF [NiFe] hydrogenase nickel incorporation 0.010 0.015 0.023 0.026 0.016 0.014 protein HybF Cytochrome d ubiquinol oxidase subunit 0.269 0.196 0.213 0.222 0.567 0.491 II (EC 1.10.3.-) Respiration / HGM Fumarate reductase subunit C 0.013 0.020 0.025 0.025 0.008 0.007

Fumarate reductase subunit D 0.017 0.029 0.037 0.032 0.010 0.008 Soluble cytochromes and Cytochrome C553 (soluble cytochrome functionally related electron 0.001 0.002 0.002 0.002 0.001 0.001 f) carriers Transport ATP-binding protein CydC 0.012 0.010 0.028 0.026 0.042 0.024 Terminal cytochrome d ubiquinol oxidases Transport ATP-binding protein CydD 0.159 0.169 0.142 0.133 0.084 0.089

ATP-dependent RNA helicase RhlB 0.032 0.038 0.047 0.047 0.039 0.035 ATP-dependent RNA helicases, bacterial ATP-dependent RNA helicase SrmB 0.025 0.036 0.061 0.061 0.044 0.038 Polyribonucleotide 0.690 0.781 1.014 0.915 1.129 0.883 nucleotidyltransferase (EC 2.7.7.8) Polyadenylation bacterial RNA-binding protein Hfq 0.364 0.314 0.160 0.139 0.126 0.198 Permease of the drug/metabolite 0.074 0.081 0.079 0.074 0.147 0.109 transporter (DMT) superfamily Queuosine exploration RZ Putative preQ0 transporter 0.071 0.092 0.040 0.038 0.092 0.093

Ribonuclease H Ribonuclease HI (EC 3.1.26.4) 0.005 0.007 0.005 0.005 0.010 0.007 DNA-directed RNA polymerase beta 0.657 0.546 0.973 1.138 0.340 0.345 subunit (EC 2.7.7.6) RNA DNA-directed RNA polymerase beta' RNA polymerase bacterial 0.866 0.930 1.167 1.364 0.356 0.393 Metabolism subunit (EC 2.7.7.6) DNA-directed RNA polymerase omega 0.037 0.039 0.029 0.028 0.038 0.044 subunit (EC 2.7.7.6) 3'-to-5' exoribonuclease RNase R 0.155 0.181 0.197 0.211 0.105 0.096

3'-to-5' oligoribonuclease (orn) 0.018 0.027 0.015 0.014 0.019 0.015 RNA processing and Exoribonuclease II (EC 3.1.13.1) 0.029 0.025 0.061 0.050 0.090 0.068 degradation, bacterial Ribonuclease E inhibitor RraA 0.140 0.175 0.096 0.092 0.082 0.094

Ribonuclease E inhibitor RraB 0.104 0.085 0.087 0.087 0.164 0.202 tRNA pseudouridine synthase A (EC RNA pseudouridine syntheses 0.033 0.032 0.046 0.035 0.141 0.094 4.2.1.70) rRNA modification Archaea tRNA:Cm32/Um32 methyltransferase 0.240 0.328 0.142 0.141 0.146 0.185

73 continued

Appendix B continued Ribosomal RNA small subunit 0.035 0.040 0.031 0.031 0.040 0.028 methyltransferase E (EC 2.1.1.-) rRNA modification bacteria rRNA small subunit methyltransferase I 0.013 0.018 0.015 0.019 0.012 0.011

TsaC protein (YrdC domain) required for t(6)A synthesis in Archaea threonylcarbamoyladenosine t(6)A37 0.011 0.012 0.020 0.022 0.010 0.008 and Eukaryotes modification in tRNA Transcription antitermination protein 0.167 0.192 0.252 0.198 0.428 0.321 NusG Transcription elongation factor GreA 0.007 0.009 0.010 0.009 0.029 0.028

Transcription factors bacterial Transcription elongation factor GreB 0.024 0.026 0.021 0.019 0.038 0.036

Transcription termination protein NusA 0.151 0.122 0.164 0.180 0.264 0.229

Transcription termination protein NusB 0.051 0.048 0.069 0.070 0.052 0.050

RNA polymerase sigma factor RpoE 0.390 0.300 0.425 0.481 0.527 0.447

RNA polymerase sigma factor RpoH 0.164 0.173 0.293 0.263 0.430 0.288 Transcription initiation, Sigma factor RpoE negative regulatory bacterial sigma factors 0.350 0.276 0.458 0.503 0.565 0.545 protein RseA Sigma factor RpoE negative regulatory 0.044 0.033 0.114 0.114 0.219 0.133 protein RseB precursor Predicted P-loop ATPase fused to an 0.054 0.054 0.137 0.119 0.138 0.094 acetyltransferase COG1444 tRNA pseudouridine 13 synthase (EC tRNA modification Archaea 0.030 0.030 0.058 0.062 0.036 0.031 4.2.1.-) tRNA(Cytosine32)-2-thiocytidine 0.011 0.014 0.034 0.022 0.086 0.058 synthetase Ribonuclease BN (EC 3.1.-.-) 0.006 0.006 0.010 0.011 0.009 0.006 tRNA processing Ribonuclease T (EC 3.1.13.-) 0.029 0.027 0.022 0.021 0.029 0.029 COG0613, Predicted metal-dependent 0.014 0.015 0.014 0.014 0.016 0.012 phosphoesterases (PHP family) YrdC-YciO Hypothetical YciO protein, TsaC/YrdC 0.036 0.040 0.061 0.055 0.075 0.064 paralog Secondary Steroid sulfates Arylsulfatase (EC 3.1.6.1) 0.030 0.018 0.021 0.020 0.008 0.015 Metabolism Carbon Starvation Stringent starvation protein B 0.043 0.041 0.064 0.051 0.172 0.162 L-proline glycine betaine ABC transport system permease protein ProV (TC 0.025 0.027 0.028 0.029 0.037 0.030 Choline and Betaine Uptake 3.A.1.12.1) and Betaine Biosynthesis L-proline glycine betaine binding ABC 0.032 0.031 0.027 0.026 0.033 0.028 Stress Response transporter protein ProX (TC 3.A.1.12.1) Glutathione: Biosynthesis and Glutathione biosynthesis bifunctional 0.282 0.255 0.127 0.146 0.111 0.111 gamma-glutamyl cycle protein gshF (EC 6.3.2.2)(EC 6.3.2.3) Glutathione: Non-redox FIG005121: SAM-dependent 0.008 0.010 0.007 0.006 0.011 0.009 reactions methyltransferase (EC 2.1.1.-) Glutathione: Redox cycle Glutathione reductase (EC 1.8.1.7) 0.146 0.296 0.182 0.184 0.157 0.145

HflC protein 0.069 0.057 0.091 0.096 0.049 0.052 Hfl operon HflK protein 0.052 0.065 0.138 0.151 0.065 0.056

74 continued

Appendix B continued Adenosine (5')-pentaphospho-(5'')- adenosine pyrophosphohydrolase (EC 0.091 0.113 0.071 0.062 0.055 0.060 3.6.1.-) Nudix KE ADP compounds hydrolase NudE (EC 0.030 0.029 0.013 0.014 0.008 0.010 3.6.1.-) Mutator mutT protein (7,8-dihydro-8- 0.012 0.013 0.021 0.022 0.012 0.012 oxoguanine-triphosphatase) (EC 3.6.1.-) Fumarate and nitrate reduction 0.123 0.096 0.059 0.064 0.122 0.123 regulatory protein

Non-specific DNA-binding protein Dps 0.169 0.270 0.116 0.097 0.097 0.209 Oxidative stress Paraquat-inducible protein B 0.058 0.039 0.082 0.080 0.049 0.044 Superoxide dismutase [Cu-Zn] precursor 0.516 0.983 0.533 0.571 0.116 0.152 (EC 1.15.1.1) Synthesis of osmoregulated Phosphoglycerol transferase I (EC 0.015 0.016 0.070 0.078 0.014 0.011 periplasmic glucans 2.7.8.20) Tellurite resistance: Tellurite resistance protein TehB 0.102 0.161 0.059 0.050 0.045 0.050 Chromosomal determinants Universal stress protein A 0.067 0.082 0.022 0.025 0.011 0.016 Universal stress protein family Universal stress protein E 0.250 0.311 0.082 0.096 0.059 0.091 At5g37530 (CsdL protein tRNA-specific 2-thiouridylase MnmA 0.047 0.043 0.042 0.045 0.033 0.026 Sulfur family) Metabolism Thioredoxin-disulfide Thiol peroxidase, Tpx-type (EC 0.175 0.178 0.094 0.102 0.023 0.052 reductase 1.11.1.15) Copper homeostasis Copper chaperone 0.033 0.043 0.020 0.017 0.005 0.009 Multidrug efflux pump in Campylobacter jejuni RND efflux system, outer membrane 0.242 0.225 0.135 0.152 0.101 0.145 (CmeABC operon) lipoprotein CmeC

Acriflavin resistance protein 0.166 0.142 0.286 0.285 0.320 0.204 Multidrug Resistance Efflux Macrolide export ATP-binding/permease 0.081 0.082 0.050 0.055 0.051 0.053 Pumps protein MacB (EC 3.6.3.-) Virulence Macrolide-specific efflux protein MacA 0.094 0.109 0.055 0.052 0.038 0.040 Membrane fusion component of tripartite 0.029 0.026 0.018 0.016 0.029 0.024 Multidrug Resistance, multidrug resistance system Tripartite Systems Found in Outer membrane component of tripartite Gram Negative Bacteria 0.007 0.012 0.017 0.021 0.003 0.003 multidrug resistance system Mycobacterium virulence operon involved in an Protein YidD 0.006 0.005 0.011 0.010 0.030 0.017 unknown function with a Jag Protein and YidC and YidD Virulence, Cytolethal distending toxin of Disease and Cytolethal distending toxin subunit B 0.040 0.046 0.019 0.024 0.016 0.022 Campylobacter jejuni Defense

75 continued