Chapter 1: Beyond the Classic Heat Shock Response: Novel Thermal Stress Regulators in the Thermophilic Archaea

Home , P1 phage, Sulfolobus solfataricus, VapBC

ABSTRACT

COOPER, CHARLOTTE RENÉE. VapBC Toxin-Antitoxin Loci in the Extreme Thermoacidophile Sulfolobus solfataricus: Regulation of and Functional Biochemical Roles during Thermal Stress Response. (Under the direction of Dr. Robert M. Kelly.)

The heat shock response is universal across all domains of life and includes conserved mechanisms for protein refolding and protein degradation necessary for organisms to survive thermal and other stresses. In bacteria, chaperones, such as DnaK, DnaJ, GroEL, GroES, and

GrpE, have been well characterized. However, the majority of archaea lack homologues for such chaperones, though most archaeal genomes encode a “thermosome” that is functionally similar to GroEL. Archaea also lack many of the RNA management tools found in eukaryotes and bacteria, such as RNA interference, siRNA, the bacterial Rho transcription termination factor, and bacterial degradasomes. Recently, it has been proposed that toxin- antitoxin (TA) loci could fill important roles in stress response, especially in the archaea. TA loci are ubiquitous in prokaryotic genomes and abundant in the archaea, especially in the thermophilic archaea.

Functional genomics analysis of model extreme thermoacidophile Sulfolobus solfataricus during heat shock (80oC to 90oC) revealed dynamic changes in novel heat shock regulators and in the chromosomally encoded VapBC family TA loci. Several of these genes were targeted for disruption and deletion mutations in S. solfataricus strain PBL2025. When transcriptional regulator tetR (SSO2506) was disrupted, the importance of the Sulfolobus heat shock regulator (Shr, SSO1589) was further implicated in the thermal stress response. When the most highly transcribed VapC-22 toxin (SSO3078) was disrupted, there were several

cognate and non-cognate VapB antitoxins responding to heat shock. This suggested that

VapC-22 plays a role in regulating the abundance of active versus silenced toxins in the cell

by mediating the levels of antitoxins present. Finally, when the most heat shock responsive

TA loci (VapBC-6, SSO1494 and SSO1493) was deleted, the VapBC-6 deficient mutant was

thermally labile. Furthermore, the VapC-6 deficient mutant was more like the VapC-22

mutant, as there was an abundance of VapB antitoxins present, even before heat shock,

including cognate VapB-6. This implicates VapC-6 in VapB regulations as well.

Careful analysis of the genome lead to the discovery that a VapC toxin overlaps with

the γ subunit of the translation initiation factor in S. solfataricus, and that the cognate VapB

is predicted to be part of aIF2γ. The fact that this construct is common in many archaeal

genomes led to a study of this particular toxin’s form and function. The S. solfataricus

VapCγ and VapC-18 toxins were demonstrated to be ribonucleases, though VapC-18 is

dimeric and VapCγ is monomeric. Deletion of VapCγ from S. solfataricus led to a non-viable

phenotype. Recombinant VapC-18 was shown to specifically “fish” its cognate antitoxin

(VapB-18) from recombinant E. coli cell extracts, forming either a heterohexameric or

heterooctameric complex. While the VapCγ did not bind to subunit γ or the aIF2

heterotrimeric complex in the same manner as VapBC-18, this was consistent with the

structural details of the aIF2 complex. It is likely that if VapCγ and aIF2 interact at all, it will

be unique from typical TA interactions. A unique role was proposed for VapCγ in RNA management in archaea, in which it is involved in freeing aIF2 bound to non-initiator tRNAs that have been esterfied by methionine or complexes bound to the 5’ end of mRNA

transcripts. The results of this study further emphasize the importance of TA loci as post- transcriptional regulators. It also sheds light on the complex, and still unresolved, thermal stress response network in archaea that involves interplay between transcriptional and post- transcriptional regulators and translation initiation machinery.

VapBC Toxin-Antitoxin Loci in the Extreme Thermoacidophile Sulfolobus solfataricus: Regulation of and Functional Biochemical Roles during Thermal Stress Response

by Charlotte Renée Cooper

A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Chemical Engineering

Raleigh, North Carolina

March 2011

APPROVED BY:

______David F. Ollis Balaji M. Rao

______Fred J. Fuller Robert M. Kelly Chair of Advisory Committee

DEDICATION

To my mother, for believing in me and my dreams (no matter how crazy) and always

showing me that anything is possible.

BIOGRAPHY

Charlotte Renée Cooper was born and raised in the small southern town of Easley, SC.

Throughout most of her childhood she could be found buried in a book or banging on the piano. In high school, Charlotte fell in love with Chemistry, particularly the labs. It was here that a wise teacher, Dr. Johnson, persuaded her to think about majoring in Chemical

Engineering. In 2001, she left for the University of South Carolina where she relished her years as a Gamecock. Before she knew it, she had earned her Bachelors of Science in

Engineering in Chemical Engineering. While at the University of South Carolina, she worked on many research projects including fuel cell studies and Alzheimer’s Disease. Her love of research prompted her to apply to graduate schools. After graduation in 2005, she headed northward to North Carolina State University where she has investigated the role of stress responsive genes in model extreme thermoacidophile Sulfolobus solfataricus.

Charlotte will next be joining Novozymes as a Fermentation Engineer.

iii

ACKNOWLEDGMENTS

They say it takes a village to raise a child, and it also takes one to prepare a

dissertation. I would like to thank Dr. Kelly for allowing me the opportunity to study under

his guidance and our collaborator Dr. Blum who made much of this work possible. I would

also like to thank fellow members of the Kelly group who have offered me so much. First, I

would like to thank Sabrina Tachdjian who was a wonderful mentor and patient teacher. In

addition, I would like to thank Derrick Lewis and Kate Auernik for their endless discussions

and masterful ideas; but most of all I would like to thank them for their genuine friendship.

Finally, I would like to recognize the contributions of Kelly group members past and present.

I cannot begin to describe the immeasurable thanks I owe to my family. To my

mother, I thank you for your unwavering support and for being my #1 cheerleader. I’m glad

Verizon has the family plan or else I would be really poor! And to my future husband Chris, thank you for keeping me sane through to the hectic end and helping me see the big picture; you are truly the love of my life. To the rest of my family (those I was given and those that

I’ve chosen), I love you all.

Finally, I would like to thank a dear friend and mentor Dr. George Roberts. Dr.

Roberts was the first person I talked to from NC State after my acceptance, my favorite dance partner at the Cardinal Club, and an invaluable support through many tough problems I

encountered in graduate school. Dr. Roberts, you are missed but never forgotten.

TABLE OF CONTENTS

LIST OF TABLES ...... x

LIST OF FIGURES ...... xi

Chapter 1: Beyond the classic heat shock response: novel thermal stress regulators in the thermophilic archaea...... 1

Introduction...... 2

Thermal Stress Response - Heat Shock ...... 2

Heat Shock in Bacteria and Archaea ...... 4

Beyond the classic heat shock response in Bacteria ...... 5

Novel thermal stress response elements from the thermophilic Archaea...... 6

Toxin-Antitoxin Loci...... 10

The ccdAB family ...... 12

The relBE family...... 12

The phd-doc family...... 14

The higBA family...... 14

The mazEF (chp) family ...... 15

The parDE family...... 16

The hicAB family ...... 17

The hipBA family...... 17

The ω−ε−ζ family...... 17

The vapBC family...... 18

TA loci as novel thermal stress regulators in Archaea ...... 20

References...... 22

Tables ...... 36

Figures...... 39

Chapter 2: Role of vapBC Toxin-Antitoxin loci in the thermal stress response of Sulfolobus solfataricus ...... 42

Abstract ...... 43

Introduction ...... 44

vapBC Toxin-Antitoxin Loci and Archaea ...... 45

Heat Shock Response of Hyperthermophilic Archaea ...... 46

Concluding Remarks ...... 48

Acknowledgements ...... 49

References ...... 50

Figures...... 55

Chapter 3: Sulfolobus solfataricus strain PBL2025 during heat shock implicates roles of transcriptional regulators and VapBC Toxin-Antitoxins Loci...... 57

Abstract ...... 58

Introduction ...... 60

Materials and Methods ...... 64

S. solfataricus PBL2025 and P2 genome comparison...... 64

Mutant strain creation ...... 65

Culture conditions...... 65

Growth and heat shock of S. solfataricus ...... 65

S. solfataricus PBL2025 transcriptomic analysis ...... 66

Results and Discussion...... 67

S. solfataricus P2 and PBL2025 comparison...... 67

Wild-type S. solfataricus PBL2025 and mutant strains under thermal stress...... 68

S. solfataricus PBL2025 tetR-like gene mutant under thermal stress...... 70

S. solfataricus PBL2025 vapC-22 toxin gene mutant under thermal stress 73

S. solfataricus PBL2025 vapB-6 and vapC-6 gene mutants under thermal stress...... 75

Summary ...... 77

Acknowledgements ...... 79

References ...... 80

Tables ...... 87

Figures...... 95

Appendicies...... 110

Appendix 3.A...... 111

Chapter 4: VapB Antitoxin is associated with the aIF2-γ subunit in archaeal genomes ...... 157

Abstract ...... 158

vii

Introduction ...... 160

Materials and Methods ...... 163

Comparative Genomic Analysis ...... 163

Cloning and Expression of Sso aIF2, VapBγ, VapCγ, and VapBC18 ...... 163

Protein Purification...... 164

Assembling the aIF2 complex ...... 165

VapCγ ribonuclease activity assay ...... 166

VapC “fishing” experiments...... 167

Determination of VapB, VapC, and VapBC molecular assembly...... 168

Results and Discussion...... 168

aIF2γ/vapBγ co-location is conserved in archaea...... 168

Role of VapC in S. solfataricus ...... 169

Assembly of S. solfataricus aIF-2 subunits α−β−γ...... 169

Cognate TA interactions ...... 170

Molecular assembly of toxins, antitoxins, and complexes ...... 170

Summary ...... 171

Acknowledgements ...... 173

References ...... 174

Tables ...... 178

Figures...... 180

viii

Appendicies...... 199

Appendix 4.A...... 200

LIST OF TABLES

Chapter 1

Table 1. Summary of genomics and proteomics studies of thermal stress response in Archaea…...... 36

Table 2. Summary of Current TA Families ...... 37

Table 3. TA loci grouped by gene families as of 2006 ...... 37

Table 4. TA loci grouped by gene families as of 2009...... 38

Chapter 3

Table 1. Summary of PBL2025 mutant strains...... 87

Table 2. Fold-change comparison of Sso P2 to Sso PBL2025 after heat shock ...... 88

Table 3. Heat shock elements ...... 89

Table 4. Metabolic elements ...... 90

Table 5. Toxin-Antitoxin Loci and PIN-domain proteins...... 94

Table S1. Log2 values of all genes significantly regulated 2-fold or more (log2≥1) ...... 111

Chapter 4

Table 1. Co-location and arrangement of eIF2γ and VapC in the Archaea ...... 178

Table 2. Primers used for cloning eIF2 and vapBC genes...... 179

Table S1. Detailed information on archaeal VapCγ/aIF2γ co-location...... 200

LIST OF FIGURES

Chapter 1

Figure 1. HSP60s from bacteria and archaea...... 39

Figure 2. Schematic representation of the organization and mechanism of a Toxin (T)- Antitoxin (A) locus ...... 40

Figure 3. 45 of out 60 TA loci in the M. tuberculosis chromosome are from the VapBC family ...... 41

Chapter 2

Figure 1. The number of chromosomally encoded vapBC TA loci increases with optimal growth temperature for genome-sequenced archaea...... 55

Figure 2. Sulfolobus solfataricus P2 vapBC TA loci transcriptome before and after heat shock ...... 56

Chapter 3

Figure 1. Comparison of S. solfataricus P2 to PBL2025 ...... 95

Figure 2. Fermentor set-up...... 96

Figure 3. Optimum growth temperatures for PBL2025 and mutant strains...... 97

Figure 4. Experimental design for microarray hybridization...... 98

Figure 5. Wild-type growth curves...... 99

Figure 6. Wild-type Venn diagram of total genes significantly regulated ...... 100

Figure 7. Heat plots of TA loci and PIN-domain proteins ...... 101

Figure 8. Heat plot of Sso transcriptional regulators...... 102

Figure 9. ΔTetR mutant growth curve...... 103

Figure 10. ΔTetR mutant (strain PBL2026) Venn diagram of total genes significantly regulated ...... 104

Figure 11. ΔVapC-22 mutant growth curves...... 105

Figure 12. ΔVapC22 mutant (strain PBL2026) Venn diagram of total genes significantly regulated ...... 106

Figure 13. ΔVapBC-6 mutant growth curve ...... 107

Figure 14. ΔVapC-6 mutant growth curves...... 108

Figure 15. ΔVapBC-6 mutant (strain PBL2078) and ΔVapC-6 mutant (strain PBL2080) Venn diagrams of total genes significantly regulated...... 109

Chapter 4

Figure 1. Structure of the archaeal initiation factor aIF2γ from S. solfataricus (PDB: 2PLF) ...... 180

Figure 2. Co-location of of aIF2γ with vapCγ...... 181

Figure 3. RNase Alert ribonuclease activity assay...... 182

Figure 4. Assemby of the aIF2 complex ...... 183

Figure 5. Temperature dependence of α−β−γ complex formation...... 184

Figure 6. The effect of VapBγ on complex formation...... 185

Figure 7. Effect of VapCγ on α−β−γ complex formation ...... 186

Figure 8. Co-elution of VapB18 and VapC18-C...... 187

Figure 9. VapCγ association with aIF2γ...... 188

Figure 10. VapCγ association with α−β−γ...... 189

Figure 11. VapCγ C-His purification...... 190

Figure 12. VapCγ C-His association with aIF2γ...... 191

xii

Figure 13. VapCγ C-His association with α−β−γ...... 192

Figure 14. Native gel of VapBC18 complex...... 193

Figure 15. VapC18 C-His size estimation...... 194

Figure 16. VapB18 size estimation ...... 195

Figure 17. VapBC18 C-His size estimation...... 196

Figure 18. VapCγ size estimation ...... 197

Figure 19. Predicted mechanism for VapCγ/aIF2γ...... 198

xiii

CHAPTER 1

Beyond the classic heat shock response:

Novel thermal stress regulators in the thermophilic archaea

Charlotte R. Cooper and Robert M. Kelly

Department of Chemical and Biomolecular Engineering North Carolina State University Raleigh, NC 27695-7905

INTRODUCTION

While the majority of microorganisms studied to date exist at moderate temperatures

and near neutral pH, there is a subset that lives at extremes of temperature, pH, ionic

strength, metals concentration, and/or pressure (86, 107). Even though these so-called

“extremophiles” thrive in biologically-demanding niches, they too are subject to stresses and

their response to such stresses has been of great interest (3, 107). Here, the focus is on thermal stress response of archaea, especially the model extremely thermoacidophilic

Crenarchaeon Sulfolobus solfataricus (97). Of specific interest is the potential role that toxin- antitoxin (TA) loci play in the capacity of S. solfataricus to respond to and survive thermal stress.

Thermal Stress Response - Heat Shock

An organism will experience “heat shock” when the environmental temperature is

rapidly increased from optimal or even sub-optimal levels. The magnitude of the temperature

shift that drives a heat shock response varies from organism to organism. However, thermal

stress typically has certain universal features that are manifested in the cell’s transcriptome

and proteome, and characteristically involves representatives from seven universal classes of

heat shock genes/proteins (90).

• Class 1: molecular chaperones

• Class 2: the proteolytic system that is used to degrade misfolded or aggregated

proteins

• Class 3: RNA and DNA modifying enzymes necessary for DNA repair

• Class 4: metabolic enzymes

• Class 5: regulators, such as transcription factors

• Class 6: proteins involved in maintaining cellular structure such as the cytoskeleton in

eukaryotes

• Class 7: transport, detoxifying, and membrane-modulating proteins

Although extremophiles and non-extremophiles alike exhibit a heat shock response when

subjected to thermal stress, the already elevated temperatures characteristic of thermophilic

biotopes provides a whole new context (1). As such, hyperthermophiles, those extremophiles that grow optimally at or above 80°C, are intriguing to examine from the perspective of heat shock. To date, such studies have shown that these microorganisms have complex heat shock responses that implicate both known classes of heat shock proteins, as well as many hypothetical proteins yet to be defined in terms of their specific biochemical and physiological roles (13, 101).

Due to recent advances in DNA sequencing, microbial genomes can be completely decoded in days or even hours (9). This has greatly facilitated genome-wide examination of key physiological features, even those like the heat shock response, which can be a transient phenomenon. Focus on the dynamic heat shock transcriptome of prokaryotes has revealed new and interesting mechanisms for dealing with thermal stress, including those falling into the seven classes listed above, but also putative heat shock regulators like those encoding the toxin-antitoxin (TA) loci found in extreme thermoacidophile S. solfataricus (101). To

provide some perspective on how certain TA proteins play a role in thermal stress response

and regulation, some background on prokaryotic heat shock is first necessary.

Heat shock in Bacteria and Archaea

There is comprehensive literature on the heat shock response of bacteria (over 9500 citations in PubMed) and much has been discovered since the identification of some of the first thermal stress responsive proteins in bacteria (62). The classic heat shock response of bacteria usually includes the HSP100, HSP90, DnaK (HSP70), DnaJ (HSP40), GrpE

(HSP23), and HSP20 molecular chaperones and the GroEL/GroES chaperonin that are able to help fold errant proteins and can work cooperatively (37, 90). All of these fall into class one of the seven classes described above. The HSP100 and HSP90 are absent from nearly all archaea, and the HSP70 and HSP40 are missing from most thermophiles and hyperthermophiles (59). Homologues of the GroEL/GroES chaperonin (HSP60) are conserved in archaea and are also referred to as the “thermosome” and “rosettasome” (Figure

1) (51, 84). There are also small HSPs (sHSPs), which are chaperones. These are ubiquitous in eukaryotes, bacteria, and archaea, though they are the most diversified of the heat shock proteins in that their functions are conserved but sequences are divergent. Several archaeal

sHSPs have been characterized and all are from thermophiles (2, 11, 47, 58, 114). Dynamic

transcriptional response analysis of thermal shifts has revealed cellular strategies for stress

management that go beyond well-characterized folding and proteolysis pathways.

Beyond the classic heat shock response in Bacteria

First, whole-genome transcriptomic studies performed with model mesophilic bacteria. Under osmotic stress (0 Æ 0.3 M NaCl), thermal stress (30o Æ 43oC), or both, the

gram-negative bacterium Escherichia coli turns on oxidative stress regulators SoxRS and

OxyR (over 26 genes total) (39). This cross-protection seen between oxidative stress genes

and thermal stress has been seen previously in E. coli, but was not fully understood (45). It is

interesting that heat shock response genes encoding chaperones and proteases showed

transient induction, while the oxidative stress genes remained up-regulated, even when only

thermal stress was present, indicating that this may be a general stress response mechanism

for E. coli (39).

Bacillus subtilis, a gram-positive bacterium, also undergoes significant transient

changes in its transcriptome upon heat shock (37o Æ 48oC). In addition to the HSPs and

sHSP responding, the genes encoding the heat-inducible transcription repressor HrcA and heat shock regulator CstR are induced, along with TetR and MarR family regulators (44).

The complex heat shock response involves regulation at the both the transcript and protein levels.

The hyperthermophilic bacterium Thermotoga maritima shares several types of heat shock proteins with mesophilic bacteria, including DnaJ/DnaK, HrcA, GrpE, GroEL/GroES, and a sHSP, but also encodes unique heat shock regulators. Upon heat shock (80oÆ90oC), T.

maritima significantly and quickly induces the classic HSPs within the first 5 minutes, while later in the heat shock transcriptional regulators MarR, TetR, and GntR, among others, are strongly induced (89). There are two MarR regulators in T. maritima (TM0816 and

TM0710) but only TM0816 is significantly up-regulated (108-fold), while TM0710 has little response to heat shock. This indicates that TM0816 may be a novel heat shock regulator employed by this extremophile.

Novel thermal stress response elements from the thermophilic Archaea

Because Archaea lack certain features of heat shock response found in Bacteria and

Eukaryotes, the heat shock responses from model Archaea are crucial for identification of novel thermal stress elements, especially the thermophiles and hyperthermophiles (Table 1).

Thermophilic Factor 55 (TF55 or HSP60) is a molecular chaperonin first identified in the extreme thermoacidophile Sulfolobus shibatae that has similarity to a eukaryotic miotic spindle formation protein and is the archaeal equivalent of the GroEL/GroES (106). The functions are proposed to be similar, but the TF55 comes from a group of type II chaperonins that are only found in Archaea. TF55 has α, β, and γ subunits that come together to form

“rosettasomes” composed of 18 subunits (105). Upon heat shock (76oÆ86oC), S. shibatate increases the production of two of the subunits: TF55-α and β. The rosettasomes re-arrange from 1α:3β:1γ in cultures grown at 60oC to 2α:3β:0γ after heat shock at 86oC. The γ is more sensitive to thermal denaturation than the α or β, and it is the least conserved in other archaea, indicating that it may play a unique regulator role of TF55 composition in each organism (52). The rosettasomes are membrane-associated and the increase upon HS correlates with a decrease in membrane permeability, though it is not known if and how the change in composition of the rosettasomes affects this (52, 104).

The extremely thermoacidophilic archaeon, Metallosphaera sedula, grows optimally

at a pH of 2 and temperatures around 70oC. After heat shock (70Æ80o or 85oC) of nearly 4

hours, the proton motive force across the membrane decreases to nearly zero, the internal pH

rises to 4, and respiration essentially ceases (83). The fact that M. sedula is experiencing a

HS response is evident by induction of the M. sedula homologue of the HSP TF55. The

study also showed that while large temperature changes were detrimental to the organism,

small temperature shifts (73oÆ79oC) cause a chemical/biological synergistic effect that could

be beneficial to the organism if experienced in nature (83). This may help to explain the

transient nature of many of the heat shock inducible genes. Han et al. saw a similar

phenomenon when M. sedula was grown in continuous culture (41). By raising the growth

temperature in small increments, the heat shock response was initiated, as evidence by the

increase in the TF55 protein abundance, and the culture was able to grow at supra-optimal

temperatures, up to 81oC, in a “stressed phase”. Returning the culture to 74oC decreased the

TF55 protein levels and thermotolerance was relinquished as changing the temperature directly to 81oC was lethal. This implicated TF55 has a critical role in the regulation of

thermotolerance.

Transcriptional profiling has led to the discovery of several novel archaeal heat shock

regulators in extremophiles such as Pyrococcus furiosus. P. furiosus, a hyperthermophilic

archaeon, grows anaerobically at temperatures of 90oC and above (30). Like most archaea, it

lacks several of the bacterial heat shock response genes, such as the bacterial HSPs DnaK,

DnaJ, and GrpE (66). However, when shifted from 90o Æ 105oC, a targeted DNA

microarray revealed that genes encoding the “thermosome” (PF1974) and the HSP20 sHSP

(PF1833) were strongly induced (98). The “thermosome” is a molecular chaperone

comprised of α, β, and γ subunits and is in the same family as the TF55 “rosettasome” but

differs in the number of subunits in the complex (16 in the thermosome versus 18 in the

roesttasome) (64, 84, 105). Additionally, two molecular chaperones, VAT-like, were induced

(PF1882 and PF0963). The VAT chaperones are homologues of eukaryotic VCP-like

ATPases found in Thermoplasma, and they are predicted to be unfoldases, as seen in

Thermoplasma acidophilum (35).

Of the seven classes of heat shock genes, the least is probably known about the

regulators; however, a novel regulator from P. furiosus (Phr) has been identified using

proteomics and transcriptomics (113). Phr (PF1790) has been implicated in the regulation of

a sHSP hsp20 and an aaa+ ATPase, as well as the phr gene itself (55, 113). Surprisingly,

while heat shock from 95Æ103oC dramatically induced the transcript level of phr, the

protein production was only slightly induced indicating that the heat shock response is

complex and likely involves multiple regulators.

In a similar experiment, hyperthermophilic archaeon Archaeoglobus fulgidus was

heat shocked from 78oÆ89oC (93). A whole-genome transcriptomic analysis revealed that

around 10% of the genome was significantly regulated and that 11 genes were up 5-fold or

more. These genes included two HSP20 sHSPs (AF1296 and AF1971), the thermosome α

and β subunits (AF2238 and AF1451), one known heat shock responsive genes (a cell

division and control protein AF1297), two previously unknown stress responsive genes (a

group II decarboxylase AF1323 and a TBP-interacting protein AF1813), and four

hypothetical proteins (AF1298, AF0172, AF1526, and AF1835). One of the hypothetical

proteins, AF1298, encodes a putative A. fulgidus heat shock regulator (HSR1) and was

shown to self-regulate, though other genes it potentially regulates are unknown.

Methanococcus jannaschii is a hyperthermophilc methanarchaeon that grows

optimally around 85oC (49). Heat shock to 95oC was lethal to a small subset of the cell population, causing up to 10% of the total population of cells to die (13). A whole-genome

transcriptomic analysis revealed that during heat shock response, 76 genes were significantly

changing, including: around 30 hypothetical proteins, a sHSP (MJ0285), the thermosome

(MJ0999), a putative protease regulatory subunit (MJ1494), prefoldin subunit α (MJ0648), and several CRISPR-associated genes (13). In S. solfataricus, prefoldin is a heterohexameric ATP-independent chaperon that is predicted to interact with the thermosome and assist in protein folding, but the mechanism is still debated (25). CRIPSR-associated (from clustered regularly interspaced short palindromic repeats) proteins are associated with DNA

helicases and other DNA processing proteins, and can protect archaea and bacteria against

mobile genetic elements (48, 108). 39% of the significantly regulated genes are hypothetical

proteins and it is likely that within these genes lie more clues to the mystery of the heat shock

response. Unfortunately, this is common observation with all archaea as many genes in these

genomes are still not assigned function.

The heat shock response of Sulfolobus solfataricus, a model hyperthermophilic

crenarchaeon, has been well characterized. Like other archaea, upon HS (80oÆ90oC), sHSPs

(SSO2427 and SSO2603), universal stress proteins (UPS) (SSO0529, SSO1865, SSO2778,

and SSO3183), and the thermosome respond (α (SSO0862) and β (SSO0282) subunits are

high throughout heat shock and γ (SSO3000) is down-regulated); and a whole-genome

transcriptomic analysis revealed that the HS effect on the organism is dramatic with over 1/3

of the genome significantly affected (101). Nearly 37% of the genes regulated 5 minutes

after heat shock are mobile elements (insertion sequences, transposases, and resolvases). S.

solfataricus has a large number of mobile elements (~400) and over 60% are significantly

regulated at some point during the dynamic heat shock indicating that an increased rate of

mutation caused by these mobile elements may be a thermal stress survival mechanism

employed by the thermoacidophile. Two of the most highly transcribed genes after heat

shock are transcriptional regulators TetR (SSO2506) and GntR (SSO1589). Another

particularly interesting finding was the observation that several Toxin-Antitoxin loci from the

VapBC family (as annotated by (80)) were identified as thermal stress responsive elements

(101). In S. solfataricus, there are at least 22 pairs of VapBC TA loci and one solitary toxin

(80). Some of these cognate protein pairs are greatly induced by stress, especially heat shock, indicating that they may represent a novel class of thermal stress regulators.

Toxin-Antitoxin Loci

Toxin-Antitoxin loci, also known as plasmid addiction systems or poison/antidote

systems, were first identified as plasmid maintenance systems that activated post-

segregational killing (PSK) in plasmid-free progeny (34). These loci encode a two-

component protein system (or protein/RNA system) composed of a labile antitoxin (A) and

stable toxin (T). We now know that TA loci are also chromosomally-encoded and ubiquitous

in free-living prokaryotes (80).

TA loci are typically arranged in operons in which the antitoxin gene comes before

the toxin gene (except for hicBA and higBA where the toxin precedes the antitoxin) (Figure

2). The function of toxin-antitoxin as pairs appears to be consistent across microbial biology, although there is still only limited evidence along these lines. As long as the antitoxin is present, it interacts with the toxin to silence its activity. There is recent evidence that non- cognate toxins and antitoxins from the same organism can interact functionally, even if the toxin and antitoxin are from different families (119, 123). However when the antitoxins are lost in plasmid-free progeny or proteolytically degraded in chromosomally-encoded cases, the toxins are activated. The details of TA function are unique to specific types (Table 2).

Some TA operons are apparently regulated by the binding of the N-terminus of the antitoxin to the promoter region (54, 69). Toxins may also function as co-repressors, since their binding to the antitoxin appears to increase the affinity of the latter for the promoter region

(Figure 2) (33).

There are three types of TA systems: Type I where the antitoxin is antisense RNA that is completely complementary to the toxin mRNA and binds to prevent translation and degradation of the transcript; Type II where the antitoxin is a protein and silences the toxin through protein-protein interaction; and Type III where the RNA antitoxin actually interferes with toxin activity rather than expression.

One of the first comprehensive reviews on Type II TA loci, published in 2005, reported 7 major TA families (80). In 2006, a new family was proposed (Table 3) (70).

Then, the distribution of members of the eight TA families in 218 prokaryotic genomes surveyed in showed that over 1472 TA loci could be identified, with an additional 63 solitary

toxins or antitoxins found. By 2010, new software tools developed to identify TA loci in sequenced genomes predicted that there were upwards of 20 TA families and thousands of putative TA pairs (Table 4), though the 8 major families, plus 2 new families, still prevail in the literature (71). One of the most striking features of the surveys is the distribution of loci in microbial genomes. Some organisms, like Mycobacterium smegmatis, have one TA loci, whereas M. tuberculosis has 60 or more (Figure 3). The most studied families are arguably relBE and mazEF, as they are abundant in mesophilic bacteria, although functional assessments of all 10 toxin families have been reported (Table 1) (109, 110).

The ccdAB family. The ccd (control cell death or division) system, the first TA pair discovered, is located on the F plasmid of E. coli. CcdA is a bifunctional antitoxin that auto- represses the loci’s expression and antagonizes the toxin (67). When the plasmid encoding

CcdA is lost, CcdB is a lethal DNA topoisomerase II poison (10). This system is only present in Gram-negative bacteria and is the least prevalent TA system (80). The chromosomally encoded Vibrio fischeri CcdB toxin is a dimer in solution (99) and the gyrase and peptide binding sites were identified.

The relBE family. The relBE operon of E. coli encodes the RelB antitoxin and RelE toxin that together form a non-toxic complex (36). The RelB antitoxin is a substrate of the Lon protease (20). The RelE toxin is an endoribonuclease that inhibits protein synthesis by cleaving mRNA codons at the ribosomal A site. Cleavage is highly codon-specific and occurs between the second and third ribonucleotides (82). After cleavage, the ribosome is

then bound to an mRNA transcript lacking a stop codon and remains stalled in this position unless rescued by tmRNA (19). However, in archaea such as Pyrococcus furiosus that contain RelBE TA loci but no known tmRNA system, the endoribonuclease may play a significant role in RNA management. Recently, it was shown that ectopic overexpression of

RelE is not bacteriocidal, but rather bacteriostatic. Production of the cognate antitoxin relieved the bacteriostatic state and re-started transcription (81). Based on the crystal structure for RelBE from the hyperthermophilic archaeon Pyrococcus horikoshii (102), it was proposed that the sheer size of the RelE/RelB complex prevents RelE from entering the ribosomal A site (116).

E. coli K-12 has two relBE homologs: dinJ-yafQ (relBE-2) and yefM-yoeB (relBE-3).

YafQ reduces protein synthesis by inhibiting [35S]-methionine incorporation but does not affect DNA or RNA synthesis. Structural modeling and comparison to known structures suggests that this toxin may act as a sequence-specific endoribonuclease (76). The YefM-

YoeB TA locus is found in major pathogens, such as Staphylococcus aureus, Streptococcus pneumoniae, and Mycobacterium tuberculosis. The YefM antitoxin is natively unfolded, making it a good target for anti-bacterial therapy (16). The YoeB toxin forms a tight polypeptide complex with YefM (17). The YefM-YoeB complex is active in modulating its own synthesis: YefM is a transcriptional repressor and the binding of YoeB to YefM enhances the repression, making YoeB a repressor enhancer (54). The yoeB-yefM loci of E. coli and S. pneumoniae have been studied from the standpoint of cross-talk between the species. While YoeBSpn overproduction is toxic to E. coli, only YefMSpn can relieve the

toxicity. Interestingly, the YoeB and YoeBSpn toxins have high sequence homology, while the cognate antitoxins appear to be designed for their specific bacterial loci (78, 79).

RelBE is one of the largest, and arguably the best understood, TA families. There are three RelBE loci in Mycobacterium tuberculosis H37Rv, the pathogenic strain that causes tuberculosis in humans (80). There is recent evidence that non-cognate TA interactions can occur between the RelBE, RelFG, and RelFJ of M. tuberculosis (119). It is interesting, that while RelE could functionally replace RelG in neutralizing toxicity, RelF increased the

toxicity of RelE and RelB did the same with RelK.

The phd-doc family. The phd (prevent host death)-doc (death on curing) locus is encoded

on the genetically stable extra-chromosomal plasmid Prophage P1 (60). The Phd antitoxin

neutralizes the Doc toxin (60, 61). As with other TA systems, phd-doc is an autorepressor of

its transcription; the Phd antitoxin binds to the promoter region, and with Phd/Doc in complex, the expression is further auto-regulated (69). The antitoxin is degraded by the

ClpXP bacterial protease (60). There is conflicting evidence on the mechanism of Doc toxicity. Some studies suggests that, coupled with MazF, Doc inhibits protein synthesis (43)

and that it does so by interaction with the 30S ribosome subunit (65). Another study

implicated Doc in mRNA cleavage (32). Arbing et al. showed the crystal structure of Phd-

Doc as a heterotetramer (5). They also noted that it may exist in a smaller 2:1 complex.

The higBA family. The higBA (host inhibition of growth) locus encodes the HigB antitoxin

and HigA toxin. This family was the first family identified where the toxin-encoding gene is

located upstream of the antitoxin gene (103). Studies in Vibrio cholerae, which contains two higBA loci in its super-integron, revealed that HigB toxins inhibit translation by mRNA

cleavage (18). Up to a certain point in time, the bacteriostatic effects of HigB can be

reversed by HigA. However, past that “point of no return”, HigB appears to be bacteriocidal

(14). The HigB toxin of Proteus vulgaris was shown to cleave at A-rich sites, 100% of the

time at AAA and 20% of the time at AA in vitro (46). M. tuberculosis has one HigBA loci

that has been shown to be a functional TA pair (40). The higA gene was shown to not only

serve as an antitoxin, but also serve as a specific binder and repressor of higB transcription,

as has been suggested for other TA families (31). The crystal structure of HigBA is not

known because crystals of HigA were the only ones to form (5). The crystal structure of

HigA shows the N-terminal DNA-binding domain and the C-terminal toxin neutralizing

domain.

The mazEF (chp) family. Along with relBE, mazEF is one of the most studied TA families.

The mazEF TA locus is a chromosomal homolog of pem (plasmid emergency maintenance)

loci that encode a Kid (killing determinant) toxin and Kis (killing suppression) antitoxin.

Kid cleaves single-stranded and double-stranded RNA preferentially at UAA or UAC sites

(77). The mazEF loci encode a MazE antitoxin and a MazF toxin. This TA loci appears on

the plasmids of all VRE strains, making it an attractive target for pharmacological agents

(75). It is also found within the chromosome of pathogens, such as Leptospira interrogans

(85). The crystal structure for a MazE/MazF heterohexamer was solved, shedding light on

the tight interaction of the complex (27). There are proposed dual substrate binding sites in

the MazF homodimeric structure, each of which is capable of interacting with a MazE

antitoxin at its C-terminal end (63). The MazF toxin is an endoribonuclease that specifically

cleaves single-stranded mRNAs at ACA sequences (121). The MazF of Staphylococcus

aureus can specifically cleave at UACAU pentads, which are abundant in genes for pathogenic factors (123). Activation of the mRNA interferase by degradation of the MazE antitoxin has been linked to stress response, in cases such as high temperatures, DNA

damage, and oxidative stress (42). MazEF was originally thought of in terms of a “suicide

model” that initiated programmed cell death (PCD) (28, 29). However, MazE

overexpression can rescue cells in the presence of active toxins (81). Overexpression of

tmRNA can rescue stalled ribosomes on cleaved mRNA, thus restoring translation (21).

Other studies have suggested MazEF-mediated cell death in response to antibiotics

(rifampicin, chloramphenicol, and spectinomycin), serine hydroxamate, high temperature,

and thymine starvation (56, 94, 95). There is recent speculation that MazG, a putative

pyrophosphohydrolase located in the mazEF operon, may also be involved in regulating

MazEF-dependent PCD (38).

The parDE family. The parDE family can be plasmid encoded and maintains plasmid

stability by inhibiting the growth of plasmid-free cells (91). Homologs of parDE are found

in both Gram-negative and Gram-positive bacteria (33). ParE was shown to generate double-

strand breaks in DNA and poison DNA gyrase (110). Vibrio cholerae has two chromosomes

and has three ParDE loci encoded within the super-integron on Chromosome II (120). If

Chromosome II is lost in progeny, Chromosome I is degraded by ParE, killing the cells.

The hicAB family. The hicAB family one of the newer additions to the TA systems (70). It is found widely distributed in both archaea and bacteria. HicA is the toxin and overproduction in E. coli K-12 is bacteriostatic (50). HicA severely limits translation, but

HicB can rescue the cells and restore cellular function; however, HicB can be cleaved by the

Lon protease, releasing the HicA toxin. Ectopic expression of HicA shows that it can cleave both mRNA and tmRNA.

The hipBA family. HipA has long been linked to persister cell formation in E. coli (57).

The effect is exacerbated by ectopic overexpression HipA and HipA mutants. HipA inhibits the synthesis of macromolecules and affects incorporation of precursors into both protein and

RNA, yet it does not cleave mRNA in the same manner as RelE or MazF. Correia et al. identified the activity of HipA as a protein kinase and determined that HipA was crucial to producing multi-drug resistant E. coli (24). The HipA can phosphorylate the bacterial translation factor Ef-Tu, and the recognition site is shown in the solved structure (96). The predicted complex is a dimer of HipB antitoxins silencing two HipA toxins.

The ω-ε−ζ family. To date, the ω−ε−ζ family is the only three-component TA family. The antitoxin ε is different from the autoregulator ω. The toxin ζ from Bacillus subtilis is found on a plasmid and maintains plasmid stability (15). The Streptococcus pyogenes ε−ζ forms

2:2 and 1:2 ratios as crystals (73). Though the specific function of the toxin is not known, it is speculated that the ζ has phosphotransferase activity (110). Expression of toxin ζ is

bactericidal for the gram-positive B. subtilis and bacteriostatic for the gram-negative E. coli

(73).

The vapBC family. When the TA were first annotated, the vapBC (virulence associated

protein) family was the most abundant TA system among prokaryotic genomes, representing

~40% of all TA loci (80). When the search for TA loci was expanded by Markova et al., the

toxins and antitoxins were paired into around 20 families and identification of the 8 major

families commonly recognized in the literature became tricky (71). Looking at the “PINs”

which are most likely VapCs, they still represent around 25% of all TA loci, though the antitoxins vary beyond VapB. M. tuberculosis has 60 known TA loci with 45 of them being

VapBCs (Figure 3) (80). VapBC TAs were first discovered on virulence plasmids in

Salmonella Dublin (87). After, a vap module was found encoded in the chromosome of the obligate anaerobe Dichelobacter nodosus implicated in foot rot in sheep (53). The ntrPR operon of Sinorhizobium meliloti functions as a TA system and has been shown to modulate transcription under symbiotic and stressful conditions (12, 88). Similarily, the fitAB (fast intracellular trafficking) operon found in Neisseria gonorrhoeae controls traits associated with gonococcus replication and transcytosis (or host invasion) (72, 115). VapBC loci are also encoded in the chromosome of the etiologic agent Leptospria interrogans (122).

Phylogenetic studies show that for a given vapBC loci, the toxin and the antitoxin do not always share the same origin and may have been acquired separately. The hypothesis of independent origination as well as the possible existence of a trans-acting mechanism between two vapBC systems has been proposed (122).

The VapC toxins of the prokaryotic vapBC family are characterized by a PIN domain

(a domain homologous to the N-terminal domain of the pilin biogenesis protein PilT) (4). In

eukaryotes, PIN domain proteins are ribonucleases involved in nonsense-mediated mRNA

decay and RNAi (22). The PIN domains yield evidence about the cellular targets of VapC toxins. They exhibit endonuclease activity in mycobacterium and exonuclease activity in the

crenarchaeon Pyrobaculum aerophilum (6, 8). A VapC toxin from Haemophilus influenzae

was determined to be a ribonuclease, degrading free RNA in vitro (26). Ectopic

overexpression of VapC toxins inhibits RNA translation in both Enterobacteria and

Mycobacterium smegmatis (92, 117). The crystal structure of a VapC from M. tuberculosis

was solved in complex with the C-terminal fragment of VapB; ribonuclease activity was confirmed for the toxin in vitro and found to be Mg+2 dependent (74).

Most VapB antitoxins contain a SpoVT_AbrB DNA binding domain. Such proteins belong to the superfamily of transcriptional regulators of the same name. AbrB, which has been studied extensively in Bacillus subtilis and B. anthracis, is a transition state regulator

(100, 111, 112). Efforts were unsuccessful in finding a consensus binding sequence in the promoter regions of the many genes controlled by AbrB in Bacillus. Structural studies showed that the binding versatility of this regulator is caused by a binding mechanism that is based on the recognition of a specific three-dimensional DNA topology, rather than detection of a specific nucleotide sequence (118). As a consequence, it is difficult to predict TA loci promoters in prokaryotic genomes.

Like other TA families, Vaps are able to self-regulate their transcription by binding to

their promoter. The mechanism by which this occurs was elucidated in Mycobacterium

smegmatis (23, 92).

Though VapBC is one of the largest families in both bacteria and archaea, it is

arguably one of the least understood. As mentioned above, heat shock studies have

implicated the pairs as stress responsive in model extreme thermoacidophile Sulfolobus

solfataricus (101), and that knocking out a specific thermally responsive loci VapBC-6 in S.

solfataricus rendered the organism thermally labile upon heat shock from 80oÆ90oC (23).

Recently, there have been a few reported targets of VapC-6 indicating that it preferentially degrades heat shock responsive genes and it’s own cognate antitoxin’s transcripts (68). Yet, there have been no plausible explanations for the abundance of the Vaps in non-pathogenic microorganisms like S. solfataricus. After 25 years and much research effort, the Vaps remain largely a mystery (7).

TA loci as novel thermal stress regulators in Archaea

The heat shock response is far from being completely unraveled in bacteria or archaea as new thermally responsive elements and regulators are still being discovered. The implication of several VapBC TA loci as thermal stress regulators in extreme thermoacidophile S. solfataricus is non-trivial. It appears that the abundance of Vap TA loci is not merely the consequence of lateral gene transfer but that the TAs have been acquired or evolved to serve specific functions, most likely in stress response. Transcriptomic analysis on a genome-wide scale facilitates the process of pinpointing when the Vaps are being

employed and also yields clues as to why. The Vaps are predicted ribonucleases and as such could play a critical role in RNA management. The fact that several Vap TA loci are highly induced by thermal stress coupled with the induction of TetR and GntR transcriptional regulators indicated that there may be a complex thermal stress response involving interplay between transcriptional and post-transcriptional regulators.

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TABLES

Table 1. Summary of genomics and proteomics studies of thermal stress response in Archaea

Distinguishing growth Temperature Organism characteristics Type of study shift (°C) References o Archaeoglobus fulgidus Topt = 83 C Transcriptome 78 Æ 89 (95) Halobacterium NRC-1 3.5-4.5 M NaCl Transcriptome 42 Æ 49 (24) Halobacterium NRC-1 3.5-4.5 M NaCl Proteome 42 Æ 49 (101) o Methanococcus jannaschii Topt = 85 C Transcriptome 85 Æ 95 (14) Methanosarcina barkerii Methane producing Transcriptome 30 Æ 45 (125) o Pyrococcus furiosus Topt = 100 C Transcriptome 90 Æ 105 (100) o Sulfolobus solfataricus Topt = 80-87 C, pH 2-4 Transcriptome 80 Æ 90 (105)

Table 2. Summary of Current TA Families

Antitoxin Toxin Toxin Activity Features CcdA CcdB DNA gyrase poison Plasmid and chromosome encoded RelB RelE Endoribonuclease Cuts at ribosomal A site Non-cognate TA interactions Phd Doc mRNA cleavage Multimeric complexs Inhibits protein synthesis HigA HigB mRNA cleavage Cuts at A-rich sites MazE MazF mRNA cleavage Cuts at UACAU ParD ParE DNA gyrase poison Plasmid and chromosome encoded Generates DS breaks in DNA HicB HicA mRNA cleavage Cuts mRNA and tmRNA HipB HipA Protein kinase Phosphorylates Ef-Tu ε ζ Putative phosphotransferase Three-component system with regulator ω VapB VapC Ribonuclease Conserved PIN-domain in toxins Adapted from (113, 114)

Table 3. TA loci grouped by gene families as of 2006

mazEF TA Family ccdAB relBE Phd-doc higBA parDE hicAB vapBC (or chpAB) # Loci in microbial 10 274 41 143 119 91 562 genomes 232

# Loci in bacteria 10 243 38 143 117 91 354 186

# Loci in archaea 0 31 3 0 2 0 208 46 Adapted from ( 35, 72, 82)

Table 4. TA loci grouped by gene families as of 2009

ArsR/ Fic/Xre PIN/Xre Bro/Xre Fic/PHD Fic/RHH PHD/PIN Fic/YhfG PIN/RHH RelE/Xre AbrB/Fic Xre/YgiU HipA/Xre AbrB/PIN MazF/Xre MerR/PIN COG3832 PHD/RelE RHH/RelE GNAT/Xre HicA/HicB MazF/PHD AbrB/RelE MazF/RHH AbrB/MazF HEPN/MNT GNAT/RHH DUF397/Xre MazF/XF1863 COG2929/Xre COG3832/Xre COG5654/Xre COG2856/Xre COG2442/PIN COG2886/PIN COG2929/RHH TA Family COG2886/RelE COG5606/RelE # Loci in microbial 69 118 430 49 323 0 58 119 248 64 2 201 5 5 62 250 132 17 24 41 30 182 67 582 267 335 19 189 33 19 42 349 364 611 58 599 765 53 genomes # Loci in bacteria (698 genomes) 69 117 319 46 323 0 58 114 247 36 2 201 5 5 62 250 132 17 24 41 30 179 67 384 235 335 19 188 33 19 42 348 364 513 54 561 765 53 # Loci in archaea (52 genomes) 0111130005128000000000003019832001000109843800 Adapted from (73)

FIGURES

Figure 1. HSP60s from bacteria and archaea. (a) The bacterial GroEL/GroES system. GroEL is composed of 2 7-member rings made of identical subunits, and GroES is made of 1 7-member ring. (b) The archaeal “thermosome” lacks the GroES co-chaperonin; however, the apical domains can adopt a conformation that effectively caps the end of the chaperonin. The “thermosome” is composed of two 8-member rings composed of α, β, and γ subunits. (Figure adapted from (3))

Figure 2. Schematic representation of the organization and mechanism of a Toxin (T)- Antitoxin (A) locus.

Figure 3. 45 of out 60 TA loci in the M. tuberculosis chromosome are from the VapBC family. (http://www1.sdu.dk/Nat/bmb/KGE_research.html)

CHAPTER 2

Role of vapBC Toxin-Antitoxin loci in the thermal stress response of Sulfolobus solfataricus

Charlotte R. Cooper1, Amanda J. Daugherty2, Sabrina Tachdjian1, Paul H. Blum2 and Robert M. Kelly1

1Department of Chemical and Biomolecular Engineering North Carolina State University Raleigh, NC 27695-7905

2Beadle Center for Genetics University of Nebraska-Lincoln Lincoln, NE 68588-0666

Published in Biochemical Society Transactions 37(Pt 1):123-126 (2009)

Abstract

Toxin-antitoxin (TA) loci are ubiquitous in prokaryotic microorganisms, including archaea, yet their physiological function is largely unknown. For example, preliminary reports suggested that TA loci are microbial stress response elements, although it was recently shown that knocking out all known chromosomally encoded TA loci in Escherichia coli did not impact survival under certain types of stress. The hyperthermophilic crenarchaeon

Sulfolobus solfataricus encodes at least 26 vapBC family TA loci in its genome. VapCs are

PilT N-terminus (PIN) domain proteins with putative ribonuclease activity, while VapBs are proteolytically labile proteins, which purportedly function to silence VapCs when associated as a cognate pair. Global transcriptional analysis of S. solfataricus heat shock response dynamics (temperature shift from 80 to 90°C) revealed that several vapBC genes were triggered by the thermal shift, suggesting a role in heat shock response. Indeed, knocking out a specific vapBC locus in S. solfataricus substantially changed the transcriptome and in one case rendered the crenarchaeon heat shock labile. These findings indicate that more work needs to be done to determine the role of VapBCs in S. solfataricus and other thermophilic archaea, especially with respect to post-transcriptional regulation.

Introduction

Toxin-Antitoxin (TA) loci, also known as plasmid addiction or poison/antidote systems, were

first identified as a plasmid maintenance mechanism that activated post-segregational killing

(PSK) in plasmid-free progeny (14). These loci encode a cognate protein pair, consisting of

a proteolytically labile antitoxin (A) and a toxin (T) (16, 36). Available genome sequence

data indicate that TA loci are also chromosomally-encoded and ubiquitous in free-living

prokaryotes (23). The widespread occurrence of chromosomally-encoded TA loci in the

microbial world suggests an important function, although the role of these proteins in either a

specific or general sense is largely unknown.

TA loci are typically arranged in operons with the antitoxin gene preceding the toxin gene

(except for higBA and hipBA where the toxin precedes the antitoxin) (5, 12, 33). The TA

ORFs often overlap making co-expression likely. The functional relationship between toxin- antitoxin proteins appears to be consistent across microbial biology, although there is still only limited experimental evidence along these lines. As long as the antitoxin is present, it interacts with the cognate toxin to presumably neutralize toxic activity, which at least in some cases is ribonucleolytic (2, 3, 7, 10, 19, 40, 41). However, when the antitoxins are lost in plasmid-free progeny or proteolytically degraded in chromosomally-encoded cases, the free toxins are activated. The details of TA function are unique to specific types. For example, in some cases TA operons are apparently regulated by the binding of the N- terminus of the antitoxin to the locus promoter region (20, 21). Toxins may also function as

co-repressors, since their binding to the antitoxin appears to increase the affinity of the latter

for the promoter region (13).

The precise function of chromosomally-encoded TA loci remains controversial. TA loci may

be bacteriocidal (11) or bacteriostatic (24, 25). In fact, hipBA TA loci have been linked to persister cell formation (9). In E. coli K-12, knocking out all five of the known chromosomally encoded TA loci did not significantly impact the survival of pH, nutritional, or antibiotic stress (35). Chromosomally encoded TA loci may act as anti-addiction modules that protect cells from PSK by plasmid-encoded TA loci (28). While TA loci in mesophilic bacteria have been closely examined, this is not the case in the archaea. Many archaeal genomes, thermophilic archaea in particular, encode multiple TA loci. The significance of these TAs under normal or abnormal growth conditions remains to be seen.

vapBC Toxin-Antitoxin Loci and Archaea

The distribution of members of eight TA families in prokaryotic genomes is widespread. Out of 218 prokaryotic genomes surveyed, over 1472 TA loci have been identified with an additional 63 solitary toxins or antitoxins found (22, 23). To date, the vapBC (virulence associated protein) family is the most abundant TA system among prokaryotic genomes,

representing ~40% of all TA loci (23). It is present in high numbers in the archaea,

especially in hyperthermophiles and extreme thermoacidophiles (Figure 1).

Most VapB antitoxins contain a SpoVT/AbrB DNA-binding domain and, as such, belong to

the superfamily of transcriptional regulators of the same name. AbrB, which has been studied

extensively in Bacillus subtilis and Bacillus anthracis, is a transition state regulator (31, 37,

38). SpoVT, an AbrB homologue, was shown to regulate expression of at least 15 genes,

probably via DNA-binding interactions with target promoters (4). VapC toxins are

characterized by a PIN domain (a domain homologous to the N-terminal domain of the pilin biogenesis protein PilT) (1). In eukaryotes, PIN domain proteins are ribonucleases involved in nonsense-mediated mRNA decay and RNAi (8). PIN domains could provide clues to the cellular targets of VapC toxins, but this connection has yet to be made experimentally.

Generally, VapCs are putative ribonucleases, although their precise specificity is not clear.

For example, they were found to have endonuclease activity in mycobacterium and exonuclease activity in the hyperthermophilic crenarchaeon Pyrobaculum aerophilum (2, 3).

Also, a VapC from Haemophilus influenzae was determined to be ribonucleolytic, degrading free RNA in vitro (10).

Heat Shock Response of Hyperthermophilic Archaea

Even though extremely thermophilic archaea thrive at extreme temperatures, they also have thermal limits and display a classic heat shock response when thermally stressed (6, 15, 26,

27, 30, 32, 34, 39). This response involves the thermosome, or rosettasome, a heat-shock responsive HSP60-like molecular chaperone that has been implicated in many cellular roles

(17, 18). Examination of several hyperthermophilic archaea undergoing thermal stress has revealed that heat shock has a profound effect on the transcriptome. Pyrococcus furiosus

response to a temperature shift of 90oC to 105oC, included up-regulation of the thermosome

(PF1974) and a HSP20-like small heat shock protein (PF1883) (30), in addition to several hundred ORFs (Shockley and Kelly, unpublished data). Detailed analysis revealed that a novel heat shock regulator protein, Phr, in P. furiosus prevented synthesis of HSP20, AAA+

ATPase (whose function is unclear), and itself by binding to the promoter regions and blocking the RNA polymerase binding site when under both thermal stress as well as nutrient-limited stationary growth phase (39). Heat shock response of other hyperthermophilic archaea has also been examined. A temperature shift from 85oC to 95oC was lethal for Methanococcus jannaschii; after 20 minutes at 95oC, 76 genes were up- regulated significantly (>2-fold) including a sHSP (MJ0285) and the thermosome (MJ0999)

(6). In Archaeoglobus fulgidus, 10% of the genome was differentially transcribed after only 5 minutes at 89oC (up from the normal growth temperature of 78oC); up-regulated ORFs included 6 of 13 known HS-related genes (AF1296, AF1297, AF1298, AF1451, AF2238, and AF1971). After 1 hour at 89oC, 14% of the A. fulgidus genome displayed changes in mRNA transcription levels (27). Among the hyperthermophilic archaea examined for heat shock response, Sulfolobus solfataricus exhibited the most pronounced change in transcriptome, with approximately one-third of its genome responding to a shift from 80oC to

90oC within 5 minutes of reaching the target temperature; 37% of the up-regulated genes were insertion sequences. Both HSP20 family sHSPs (SSO2427 and SSO2603) were up- regulated, as were many of the vapBC TA loci found in S. solfataricus (32). Some TA loci were constitutively expressed at high levels (e.g., vapBC22), but thermal stress triggered even higher transcription levels (Figure 2). Other TA loci were significantly up-regulated by

thermal stress, such as vapBC6 and vapBC8 of S. solfataricus. Using S. solfataricus

PBL2025, genetic insertions were made to disrupt the function of individual TA genes (29).

When toxin vapC22 was disrupted, no obvious phenotype was noted although approximately

100 ORFS were differentially transcribed 2-fold or more (CR Cooper, AJ Daugherty, S

Tachdjian, PH Blum and RM Kelly, unpublished data). However, disruption of vapB6 (and consequently vapC6) rendered the organism labile to thermal stress (Cooper, AJ Daugherty,

S Tachdjian, PH Blum and RM Kelly, unpublished data). Efforts now underway seek to determine the set of essential TA loci required by S. solfataricus to survive thermal stress.

Concluding Remarks

Because many TA loci are still annotated as “hypothetical proteins”, they are often overlooked as important elements in microbial genomes. Transcriptional data from S. solfataricus heat shock experiments suggested that, while chromosomally encoded TA loci may not play a significant role in mesophilic prokaryotes such as E. coli, they are potentially significant stress response modules in thermophilic archaea. One hypothesis yet to be tested is that TA loci are key components in archaeal RNA management systems. The significance of the large complement of TA loci in thermophilic archaea (as noted there are at 26 vapBC loci in S. solfataricus alone) remains a mystery. Each locus may play a specific role under normal or stressed conditions. Further work is needed to define the targets of specific VapCs in S. solfataricus as this may lead to a clearer picture of TA loci in archaeal biology.

ACKNOWLEDGMENTS

This work was supported in part by the U.S. National Science Foundation. CRC acknowledges support from a US NIH T32 Biotechnology Traineeship.

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FIGURES

Archaeal vap BC Toxin-Antitoxin Loci 40

15 # vapBC TA loci TA vapBC # 10

0 20 40 60 80 100

Topt

Figure 1. The number of chromosomally encoded vapBC TA loci increases with optimal growth temperature for genome-sequenced archaea. Topt data were obtained from The Prokaryotic Growth Temperature Database (http://pgtdb.csie.ncu.edu.tw/).

Before heat shockBefore heat 5 min. after 30 min. after 60 min. after

Figure 2. Sulfolobus solfataricus P2 vapBC TA loci transcriptome before and after heat shock (temperature shift from 80oC to 90oC) (adapted from Tachdjian and Kelly, 2006). Red indicates up-regulation of blue represents down-regulation, relative to the genome-wide average transcription level. Missing vapBs were not annotated at the time the DNA microarray used here was fabricated.

CHAPTER 3

Sulfolobus solfataricus strain PBL2025 during heat shock implicates roles of

transcriptional regulators and VapBC Toxin-Antitoxins Loci

Charlotte R. Cooper1, Yukari Maezato2, Edith Soo2, Karl Dana2,

2 1 Paul H. Blum , and Robert M. Kelly

1Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695-7905

2Beadle Center for Genetics University of Nebraska-Lincoln

To be submitted to: Applied and Environmental Microbiology

ABSTRACT

The heat shock transcriptome of the extremely thermoacidophilic archaeon Sulfolobus solfataricus strain PBL2025 was examined with respect to the role of specific transcriptional regulators and VapBC toxin-antitoxin loci. Temperature shifts from 80 to 90°C were used to trigger dynamic response in S. solfataricus wild-type and mutant strains, which was tracked by transcriptomic analysis. The Sulfolobus heat shock regulator (Shr) (SSO1589) is GntR superfamily transcriptional regulator orthologous to the Phr heat shock regulator (PF1790) previously identified in another hyperthermophile, Pyrococcus furiosus. To further examine the role of this regulator in the heat shock response, the gene encoding a TetR family transcriptional regulator (SSO2506), that was the second highest regulator up-regulated 16- fold in the wild-type upon temperature shift, was disrupted. This resulted in the up- regulation of the Shr over 50-fold upon heat shock of the mutant strain, and indicated a compensatory role in the absence of SSO2506. Under normal and heat shock growth conditions, the most highly transcribed toxin gene was vapC-22 (SSO3078). Disruption of this gene did not result in any noticeable phenotype but led to increased transcription of several antitoxins, including its cognate vapB-22 (SSO11914), vapB-6 (SSO1494), vapB-8

(SSO8620), and vapB-10. This indicates that this ribonucleolytic VapC toxin may target and regulate the levels of cognate and non-cognate antitoxins. VapBC-6, encoded in SSO1493-

1494, was the most heat shock responsive TA loci, increasing over 10-fold within 5 minutes after the temperature shift. Deletion of the entire locus (vapBC-6) rendered the organism thermally labile, while deletion of only vapC-6 left fitness intact and showed increased transcript levels for vapB-6. From the perspective of the transcriptome, these studies show

that the regulation of thermal stress response in S. solfataricus is complex, going beyond recognized heat shock proteins, and involves interplay between transcriptional and post- transcriptional regulation as a coping strategy.

INTRODUCTION

Thermal stress response in bacteria involves the orchestration of specific heat shock proteins (HSPs), universal stress proteins (USPs), chaperones (e.g., DnaK, DnaJ, GroEL,

GroES and GrpE), and proteases (Lon, ClpXP) to minimize cellular disruption arising from protein folding problems and to aid in protein turnover (40). This same phenomenon in the extremely thermophilic archaea, while similar from a functional perspective to bacteria, involves a different set of proteins (e.g., small heat shock proteins, “thermosome” or

“roesttasome”, exosome, proteasome) and regulatory strategies (1, 26, 32, 37, 51, 56).

Functional genomics tools have been used to examine the heat shock transcriptomes for specific extremely thermophilic archaea (7, 43, 46, 49), revealing that a substantial portion of archaeal genomes react to thermal stress. Not only are the expected heat shock elements represented, but also many ORFs encoding hypothetical proteins and annotated genes whose specific role in heat shock is unknown. The hyperthermophile Pyrococcus furiosus, like most archaea, lacks several of the bacterial heat shock response genes, such as the bacterial HSPs

DnaK, DnaJ, and GrpE (32). However, when shifted from 90o Æ 105oC, a targeted DNA microarray revealed that genes encoding the “thermosome” (PF1974) and the HSP20 sHSP

(PF1833) were strongly induced (46). The “thermosome” is a molecular chaperone comprised of α, β, and γ subunits and is the archaeal equivalent of the bacterial GroEL protein refolding system (30, 37, 50). Additionally, two molecular chaperones, VAT-like, were induced (PF1882 and PF0963). The VAT chaperones are homologues of eukaryotic

VCP-like ATPases found in Thermoplasma, and they are predicted to be unfoldases, as seen in Thermoplasma acidophilum (21). A novel heat shock regulator from P. furiosus (Phr) has

been identified using proteomics and transcriptomics (55). Phr (PF1790) has been implicated

in the regulation of a sHSP (hsp20) and an aaa+ ATPase, as well as the phr gene itself (25,

55). Surprisingly, while heat shock from 95Æ103oC dramatically induced the transcript level

of phr (up to 50-fold), cell protein production was only slightly induced, indicating that the

heat shock response is complex and likely involves multiple regulators.

In a similar experiment, hyperthermophilic archaeon Archaeoglobus fulgidus was

heat shocked from 78oÆ89oC (43). A whole-genome transcriptomic analysis revealed that

around 10% of the genome was significantly regulated and that 11 genes were up 5-fold or

more. These genes included two HSP20 sHSPs (AF1296 and AF1971), the thermosome α

and β subunits (AF2238 and AF1451), one known heat shock responsive genes (a cell

division and control protein AF1297), two previously unknown stress responsive genes (a

group II decarboxylase AF1323 and a TBP-interacting protein AF1813), and four

hypothetical proteins (AF1298, AF0172, AF1526, and AF1835). One of the hypothetical

proteins, AF1298, encodes a putative A. fulgidus heat shock regulator (HSR1) that is

orthologus to the Phr. HSR1 was shown to self-regulate, though other genes it potentially

regulates are still unknown.

Methanococcus jannaschii is a hyperthermophilc methanarchaeon that grows

optimally around 85oC (24). Heat shock to 95oC was lethal to a small subset of the cell population, causing up to 10% of the cells to die (7). A whole-genome transcriptomic analysis revealed that during heat shock response, 76 genes were significantly changing, including: ~30 hypothetical proteins, a sHSP (MJ0285), the thermosome (MJ0999), a putative protease regulatory subunit (MJ1494), prefoldin subunit α (MJ0648), and several

CRISPR-associated genes (Boonyaratanakornkit). In the extremely thermoacidophilic

archaeon Sulfolobus Solfataricus, prefoldin is a hetero-hexameric ATP-independent chaperon, predicted to interact with the thermosome and assist in protein folding, although the mechanism is unclear (12). The function of prefoldin is the archaeal homologue to the

GroES systems that interacts with GroEL in bacteria to assist in protein re-folding. CRIPSR- associated (from clustered regularly interspaced short palindromic repeats) proteins are

associated with DNA helicases and other DNA processing proteins, and can protect archaea

and bacteria against mobile genetic elements (23, 52). Almost half of the heat shock responsive genes in M. jannaschii are hypothetical proteins, and unfortunately, this is common observation with all archaea as many genes in these genomes are still not assigned function.

S. solfataricus strain P2, an 80oÆ90oC shift had a profound impact on the

transcriptome, with 1/3 of the genome changing significantly (~1000 genes) within 10

minutes after reaching 90oC (49). As seen with other hyperthermophiles, there was up-

regulation of the sHSPs (SSO2427 and SSO2603), the thermosome (α (SSO0862) and

β (SSO0282) (note that subunit γ (SSO3000) was down-regulated), as well as genes encoding

transcriptional regulators (TetR (SSO2506) and GntR (SSO1589) families) and VapBC

Toxin-Antitoxin (TA) loci (36). The TA genes are of particular interest because many of

them were previously either annotated as hypothetical proteins in S. solfataricus, or not even

identified as ORFs in the genome sequence.

The role of specific TA loci in most microbial genomes has not been defined,

although Type II TA systems have been suggested as stress response elements (11, 20, 28,

29, 49). S. solfataricus has between 17 and 22 TA loci (depending on the strain), all from the

VapBC family ((36); CRC unpublished data). The VapC toxins of the prokaryotic vapBC

family are characterized by a PIN domain (a domain homologous to the N-terminal domain

of the pilin biogenesis protein PilT) (2). In eukaryotes, PIN domain proteins are

ribonucleases involved in nonsense-mediated mRNA decay and RNAi (10). The PIN

domains could provide clues about the cellular targets of VapC toxins. They exhibit

endonuclease activity in mycobacterium and exonuclease activity in the crenarchaeon

Pyrobaculum aerophilum (3, 4). A VapC toxin from Haemophilus influenzae was determined

to be a ribonuclease, degrading free RNA in vitro (13). Ectopic overexpression of VapC

toxins inhibits RNA translation in both Enterobacteria and Mycobacterium smegmatis.

(Winther; Robson) The crystal structure of a VapC from M. tuberculosis was solved in complex with the C-terminal fragment of VapB; ribonuclease activity was confirmed for the toxin in vitro and found to be Mg+2 dependent (35). Most VapB antitoxins contain a

SpoVT_AbrB DNA binding domain. Such proteins belong to the superfamily of

transcriptional regulators of the same name. AbrB, which has been studied extensively in

Bacillus subtilis and B. anthracis, is a transition state regulator (48, 53, 54).

Tachdjian and Kelly (2006) showed that several ORFs encoding VapBC TA loci in S. solfataricus P2 responded to thermal stress (49). VapBC-22 (SSO11914 and SSO3078) was highly transcribed under normal conditions (in the top 5% of all transcripts) and up-regulated by heat shock (in the top 2% of all transcripts). VapBC-6 (SSO1494 and SSO1493), on the other hand, while not highly transcribed during normal growth, was induced by heat shock up to 20-fold above the average transcript level (with vapC-6 in the top 1% of all transcripts

after heat shock). Furthermore, a vapBC-6 disruption mutant in S. solfataricus strain

PBL2025 was thermally labile within 10 minutes after a shift from 80 to 90°C (11). To

determine whether this phenomenon was due to the loss of the toxin or antitoxin, single gene

deletion mutants were constructed (33). Deletion of the gene encoding vapC-6 resulted in a thermally labile phenotype, after longer incubations of 1 hour to 5 hours at 90oC, while deletion of the antitoxin (vapB-6) knocked down both vapB-6 and vapC-6 to very low levels.

The fitness of both mutants was impacted, although the vapC-6 deletion had a larger impact on thermal lability during sustained heat shock.

To further explore the impact of VapBC-6 and VapBC-22 on thermal stress response in S. solfataricus PBL2025, whole-genome transcriptomic analysis was used to examine both wild-type and mutant strains. Also included in this analysis was a mutant deficient in a TetR family transcriptional regulator (SSO2506), since it was highly responsive to heat shock.

Among other things, this study illustrates how genomic elements other than well-studied protein folders and protein degraders play a critical role in thermal stress response.

MATERIALS AND METHODS

S. solfataricus PBL2025 and P2 genome comparison. The genome sequence of S. solfataricus strain PBL2025 (Genbank ID: CP001800.1) was compared to strain P2 (45) using Viroblast BLASTn (15). The genomes were aligned using MAUVE (14) to analyze the regions of synteny (Figure 1).

Mutant strain creation. The gene disruptions were achieved by insertion of a lacS

gene into the target gene sequence in the genome, as described previously (47). Gene

disruption of tetR (SSO2506) created strain PBL2026 and disruption of vapC-22 (SSO3078) created strain PBL2067. The in-frame gene deletions were also made, as described previously (33). Deletion of vapB-6 (SSO1494) created strain PBL2078 and deletion of vapC-6 (SSO1493) created strain PBL2080.

Culture conditions. The S. solfataricus wild-type and mutant strains were routinely

grown at 80°C and on DSMZ 182 medium, pH 4.0; cells were enumerated using

epifluorescence microscopy with acridine orange stain using methods previously reported (9).

Growth and heat shock of S. solfataricus. The thermal stress experiment was

carried out with a modified 3-liter glass fermentor (Applikon, Schiedam, The Netherlands),

as described previously (49) (Figure 2). Briefly, using heated re-circulating water and a

heating mantel, the culture was shifted from 80 to 90°C ( 7 min) at mid-exponential phase

and maintained at 90°C ± 1°C for the duration of the experiment. The optimum growth

temperature for the wild-type strain PBL2025 is 80oC, but slightly lower for the mutant

strains (Figure 3 (33)). Nonetheless, the cultures were all grown at 80oC and heat shocked to

90oC for consistency. Samples were taken 10 min before starting the temperature shift and

then 10 and 30 min after reaching 90°C. Samples (400 ml) were harvested for each time

point and chilled in a dry ice and ethanol bath before harvesting at 4oC for 10 min

(7500RPM). The experiment was biologically repeated under the same conditions.

S. solfataricus PBL2025 transcriptomic analysis. A whole-genome oligonucleotide

microarray for S. solfataricus P2 (DSMZ, Germany) was developed based on the reported

genome sequence (45) and fabricated, as reported previously (Tachdjian paper), but with a

few modifications. Probes were designed for the missing Vap genes in OligoArray 2.0 (44)

and custom synthesized (Integrated DNA Technologies, Coralville, IA). Microarray slides

were printed with four replicates per probe spotted onto each array to fortify statistical analysis. From each sample in the heat shock time course experiment described above, RNA was extracted using the Qiagen RNAqueous kit (Qiagen, Valencia, CA), following the manufacturer’s instructions. Equal amounts of RNA from the biological repeats was pooled and cDNA was synthesized; samples were then hybridized in a three-slide loop design

(Figure 4), as described previously (8), with minor adjustments for long oligonucleotide platforms (49). Microarray slides were scanned with the Axon 400B (Molecular Devices,

Sunnyvale, CA) microarray scanner and raw data were analyzed with GenePix Pro 6.0

(Molecular Devices, Sunnyvale, CA). Data from each experiment were analyzed with JMP

Genomics version 4.0 (SAS, Cary, NC), using a mixed linear analysis of variance model as

described previously (49). A ±2.0-fold change (FC) or higher (where a log2 value of ±1

equals a 2-FC) with significance values at or above the Bonferroni correction, which was 4.8

(equivalent to a P value of 4.0 x 10– 6), defined differential expression for this data set (Table

S1).

RESULTS AND DISCUSSION

S. solfataricus P2 and PBL2025 comparison. Although S. solfataricus strain P2 was

used in initial heat shock studies, here a natural deletion mutant of S. solfataricus 98/2

(PBL2025) was used because it has been established with genetic tools for gene disruption

and deletion (33, 47). The genome sequence of S. solfataricus strain PBL2025 was recently

released (GenBank ID: CP001800.1). The software program Jspecies (41) analyzes the

similarity of organisms based on their genome sequences and showed that S. solfataricus

PBL2025 and P2 are much more similar to each other (scores above 98 out of 100) than to

other Solfolabales, including S. acidocaldarious, S. tokadii, or S. islandicus (scores of 66.5 to

89.6 out of 100). When the S. solfataricus P2 and PBL2025 genomes are aligned with

MAUVE (14), it is readily apparent that the organization of the genomes is conserved, although it does look like the origin of replication in PBL2025 is located downstream of the one in P2 (Figure 1). There are several regions of P2 that have no similarity to PBL2025 and only a few small regions in PBL2025 that are not found in P2, indicating that almost all of the PBL2025 genes have homologues in P2. The genome of PBL2025 is smaller at ~2.6 Kbs compared to P2 at ~3 Kbs. BLASTn analysis showed that approximately 2,000 ORFs are

exactly homologous between strains P2 and PBL2025 (15). Several hundred more ORFs

have 95% or higher homology, and 300-400 P2 ORFs have 70% or less identity with

PBL2025. In terms of the VapBC loci, PBL2025 does not have homologues to VapBC-2,

VapBC-3, VapBC-5, and VapBC-18, but homologues to the remaining Vaps are present

(Figure 1). At this time, it is not clear whether PBL2025 has additional VapBC TA loci not

found in strain P2.

Wild-type S. solfataricus PBL2025 and mutant strains under thermal stress.

Previously, a whole-genome microarray was designed for S. solfataricus P2 (49). After the

identification of the Vap TA loci (36), several probes were added for the missing genes. This

array was used to analyze the heat shock response of PBL2025 and mutant strains (Table 1)

when shifted from 80oÆ90oC. The wild-type strain PBL2025 had a doubling time of 3.5-4 hours (Figure 5) which is comparable to S. solfataricus strain P2 (data not shown). Heat shock revealed no phenotypic differences between PBL2025 and P2. The only phenotypic response seen in the PBL2025 mutants was a slower growth rate (5-9 hrs doubling time, depending on the strain) under normal conditions, and thermal lability of PBL2078 under heat shock conditions. When compared to the S. solfataricus P2 during heat shock, PBL2025 and its derivatives showed equally dramatic transcriptomic responses. In fact, when looking at the initial heat shock response of strains P2 and PBL2025 side-by-side, the magnitude of genes changing is almost identical for the majority of genes; this is a testament to just how similar these strains are (Table 2). In strain PBL2025 and the mutant derivatives, the sHSPs

(SSO2427 and SSO2603) were induced with the universal stress proteins (USPs) (SSO1865,

SSO2778, and SSO3183) except for one USP (SSO0529) which was down-regulated. The proteasome (SSO0738, SSO0278, and SSO0766) remained highly transcribed throughout heat shock, as did the thermosome α and β subunits (SSO0862 and SSO0282), while γ

(SSO3000) was down-regulated (Table 3). Additionally there was a down-regulation of

metabolic elements including the transporters, especially the peptide, oligo/dipeptide, and sugars (like arabinose, glucose, and maltose) (Table 4), as might be expected given the thermal impact on cellular processes.

In the wild-type strain, nearly 600 genes were significantly regulated 2-fold or more, either up or down, within 10 minutes of reaching 90oC, and the number increases to over 700

genes by 30 minutes after heat shock (Figure 6). In addition to the heat shock response, there

were ribosomal proteins down-regulated, as well as RNA polymerase-related proteins and

energy related NADH dehydrogenases (Table S1). This indicates that the cells are regulating

ATP-dependent processes and conserving energy to survive the thermal stress. Furthermore,

the process of translation initiation is down-regulated through the γ subunit of aIF2

(SSO0412). There were two terminal quinol oxidase subunits (SSO0394 and SSO2723) and

a dihydrolipoamide dehydrogenase (SSO2506) that were up-regulated over 8-fold which is

interesting since most energy related processes are down regulated. The dihydrolipoamide

dehydrogenase may be trying to balance the NAD+/NADH ratio but the mechanisms in

archaea are not fully understood (6). Several transcriptional regulators from various structural families were highly differential, with some of the highest fold-changes being tetR

(SSO2506) up 16-fold, gntR (SSO1589) up 16-fold, and putative marR (SSO3242) down 18- fold (Table 4). The Vap genes were also highly differentially transcribed, with toxins VapC-

6 (SSO1493) and VapC-8 (SSO1657) each going up 10-fold (Table 5 and Figure 7). In addition, the four known PIN-domain proteins (SSO1701, SSO1921, SSO2101, and

SSO2783) were induced as high as 15.6-fold within 30 minutes of heat shock, indicating that the Vaps and PINs play a critical role in the thermal stress response of S. solfataricus (Table

5). The genes encoding VapC-22 (SSO3078) and VapC-7 (SSO1651) were the most highly

transcribed toxins under normal growth conditions, and both remained highly transcribed throughout heat shock.

S. solfataricus PBL2025 tetR-like gene mutant under thermal stress. S. solfataricus PBL2025 has 13 transcriptional regulators belonging to three major families

(Figure 8). There are ten MarR/Lsr14 family (SSO0048, SSO0458, SSO1082, SSO1101,

SSO1108, SSO1110, SSO2138, SSO2474, SSO2897, and SSO3242), one TetR family

(SSO2506), two GntR family (SSO1255 and SSO1589) putative transcriptional regulators. In bacteria, MarR-family regulators play many roles such as modulating expression of virulence genes and regulating antiobiotic resistance and oxidative stress resistance (5, 16). Two MarR- family regulators were heat shock responsive in hyperthermophilic bacterium Thermotoga maritima, with one being highly up-regulated and the other only moderately so (38). A

MarR-famliy regulator (BldR) found in S. solfataricus P2 (SSO1352) regulates the detoxification stress response of the organism to toxic aromatic aldehydes (17), indicating that the role of MarR-family regulators in archaea may very greatly from their bacterial counterparts. The tetracycline repressor (TetR) family of transcriptional regulators is well characterized in bacteria (57). They contain a DNA-binding domain and antibiotic tetracycline binding domain, and they have been implicated in everything from antibiotic resistance to biofilm formation (For a comprehensive review see: (57)). Their representation in prokaryotic genomes varies from 151 in Nocardia farcinica IFM 10152 to 1, as is the case in S. solfataricus P2 (39). The role of the TetR-like regulators in archaea is not yet clear,

though there are homologues to the S. solfataricus tetR gene in the other Sulfolobus species,

including S. acidocaldarius, S. tokodaii, and S. islandicus. The GntR-family comes from

the gluconate repressor in Bacillus subtilis (19). The N-terminal domain is highly conserved

while the C-terminal is variable, leading to at least six subfamilies: FadR, HutC, MocR,

YtrA, PlmA, and AraR whose regulation spans a multitude of cellular functions (18, 27, 42).

Under normal growth conditions in S. solfataricus PBL2025, the tetR (SSO2506) and

gntR (SSO1589) transcript levels are average (in the top 50% of all transcripts); however,

upon heat shock the transcript levels are up-regulated, making them two of the most highly

transcribed genes (in the top 1% of all transcripts). The Phr of hyperthermophile Pyrococcus

furiosus belongs to the GntR superfamily, and has a conserved N-terminal winged helix-turn-

helix domain (25, 55). Phr is highly conserved in Pyrococcus species and is extremely heat

shock responsive on the transcript level. Phr has been shown to regulate the transcription of

at least seven heat shock responsive genes, including hsp20 and phr itself, in vitro using cell

free transcription assays and with a combination of runoff transcription of chromosomal

DNA and microarray analysis (25, 31, 55). Interestingly, though the transcript levels of phr

go up to 50-fold higher when P. furiosus is heat shocked from 95oÆ103oC for 90 minutes,

western blot analysis revealed that the protein levels showed only a modest increase,

indicating that Phr may be one of many regulators involved in a complex translational

regulation pathway initiated by thermal shifts. The GntR-like regulator in S. solfataricus is a

putative Phr ortholog for the Sulfolobus strains (Shr). To determine how absence of TetR affects Shr regulation of the heat shock response of S. solfataricus, a disruption mutant was

created with a lacS gene insertion in the middle of tetR (SSO2506), creating strain PBL2026.

It is important to note that due to the nature of the gene disruption it is still possible to detect transcript changes as flanking pieces of the gene are left intact; however, there is no functional protein produced. Under normal growth conditions, the mutant strain showed slower growth than the wild-type PBL2025, with the doubling time varying between 6-9 hours (Figure 9). Heat shock of the mutant from 80oÆ90oC triggered no other phenotypic response. Despite the slower growth of PBL2026, the heat shock transcriptome is remarkably similar to the wild-type strain. By 10 minutes after heat shock, there are almost

650 genes significantly regulated and the number increases to nearly 900 after 30 minutes

(Figure 10). Many of the genes are being regulated by the wild-type and PBL2026 in the same manner but the surprising aspect is the magnitude of regulation. PBL2026 has nearly double the number of genes up 4-fold or more at 30min post heat shock than PBL2025. A dihydrolipoamide dehydrogenase (SSO1565) is again the gene changing the most, up nearly

32-fold 10min after HS and 64-fold 30min after heat shock. Also up-regulated is a hypothetical protein (SSO9092) and an acylaminoacyl peptidase (AAP) (SSO2141). AAP in thermophile Aeropyrum pernix K1 is an exopeptidase, which removes acylated amino acid residues from the N terminus of oligopeptides and has been structurally characterized (22).

Without a functional TetR-like transcriptional regulator, the GntR-like family repressor (SSO1589) is significantly up-regulated, even higher than seen in the wild-type. In fact, it is the highest fold-change in the genome at 52.2 fold. There is a second GntR-like family regulator (SSO1255) that showed a 2.7-fold induction upon heat shock, which is similar to the wild-type. This further emphasizes the idea that the GntR superfamily is diverse and transcriptional regulators play many roles in the regulation of archaea under

stress conditions and otherwise. The proposed Sulfolobus heat shock regulator (Shr) should

be further investigated to identify the gene targets.

The heat shock response of PBL2026 included the differential regulation of many of

the VapBC TA loci and PIN-domain containing proteins. Most notably is the induction of

VapBC-6 (SSO1494 and SSO1493) by almost 26-fold and the induction of VapBC-8

(SSO8620 and SSO1657) by nearly 30-fold for the toxin and 14-fold for the antitoxin (Table

5 and Figure 7). PIN-domain protein SSO1921 followed closely with an 18-fold induction,

and VapC-22 (SSO3078) remained the most highly transcribed toxin under normal and stress

conditions.

S. solfataricus PBL2025 vapC-22 toxin gene mutant under thermal stress. PIN- domain containing VapBC TA loci are one the most abundant families represented in the prokaryotes (34, 36). The VapC toxins have been shown to have ribonuclease activity in mesophilic bacteria Haemophilus influenzae (13) and more recently in extreme thermoacidophile S. solfataricus ((33); Cooper and Kelly, unpublished data). These post- transcriptional regulators are abundant in S. solfataricus PBL2025 with at least 17 pairs, 1 solitary toxin, and 4 uncharacterized PIN-domain containing proteins. VapBC-22

(SSO11914 and SSO3978) are within the top 5% of the most highly transcribed genes in

PBL2025 under normal growth conditions as well as when heat shock occurs. A lacS insertion into vapC-22 (SSO3978) yielded a toxin-deficient mutant strain PBL2067. The vapB-22 (SSO11914) was left intact and transcripts were detectable by qPCR and microarray analysis. As with the tetR mutant strain, the doubling time of the PBl2067 was around 6

hours (Figure 11), and again, heat shock yielded no other phenotypic response. There was, however, a transcriptomic phenotype with almost 700 genes significantly regulated 10 minutes after heat shock and almost 900 genes up or down 2-fold or more after 30 minutes of heat shock (Figure 12). This is ~20% more of the genome responding ±2-fold in the toxin mutant than in the wild-type. The highest genes are a pyruvate dehydrogenase (SSO1565) up

362-fold, a putative translation elongation related protein (SSO0470) up 104-fold, and several hypothetical proteins (SSO9092, SSO1920, and SSO2101) up over 32-fold. The most down-regulated genes are a hypothetical protein (SSO1027) and an ATP-dependent

RNA helicase related protein (SSO1402), which were down 158-fold and 97-fold, respectively. Many ATP-dependent processes are down-regulated to conserve energy and maintain cellular function during thermal stress. Interestingly, the gene encoding the TetR transcriptional regulator goes up 27.7-fold and Shr goes up 24.7-fold (Table 4). Significant up-regulation of the transcriptional regulators and the nature of the VapC toxins as ribonculeolytic post-transcriptional regulators points to a complex heat shock response that involves many levels of regulation. Due to the redundancy of the TA loci in S. solfataricus, it was interesting to see that there is no obvious compensation for the lack of a highly transcribed post-transcriptional regulator, though there is one PIN-domain containing protein

(SSO2101) up 44.1-fold. Many of the Vap loci are significantly differentially expressed, but the obvious difference between the wild-type and PBL2067 is the abundance of several

VapBs. These include VapB-6 (SSO1494), VapB-8 (SSO8620), VapB-10, and VapB-22

(SSO19114) (Table 5 and Figure 7). Each of these VapB antitoxins is significantly up- regulated upon heat shock, but under normal growth conditions their transcript abundance

seems to be lower in the vapC-22 mutant than in the wildtype. These findings indicate that

the toxins may play a role in the abundance of their cognate and non-cognate antitoxins

during normal growth and stress response. The role of VapC22 may be a repressor of

antitoxin transcription to regulate the abundance of silenced and free toxins available in the

cell.

S. solfataricus PBL2025 vapB-6 and vapC-6 gene mutants under thermal stress.

The S. solfataricus vapBC-6 TA locus is unique because it is the only one where there is a small intergenic region between the toxin and antitoxin (all of the others overlap). Also, it is the most responsive toxin to heat shock, going up 10.1-fold after 30 minutes (Table 5 and

Figure 7). Previously, it has been reported that disruption of the vapBC-6 loci in S.

solfataricus PBL2025 led to a thermally labile mutant (11). To investigate whether it is the

loss of the toxin or the antitoxin that leads to such a loss of fitness, two mutants were created

by gene deletion. PBL2078 has the vapB-6 gene deleted, but leaves the vapC-6 gene intact.

PBL2080 has the vapC-6 gene deleted. Previously, when a lacS gene was inserted into vapB-6, the transcripts of vapC-6 were undetectable by qPCR or microarray analysis, leading to a vapBC-6 mutant (11). With the deletion of vapB-6, the transcripts of vapC-6 were greatly reduced to the point where they are not detectable by microarray, but were detectable by qPCR (33). Therefore, for the purpose of this analysis, PBL2078 will be referred to as a vapBC-6 deficient mutant. It should be noted that there did appear to be non-specific binding to the microarray probes for vapB-6 and vapC-6 for PBL2078 and the vapC-6 probe for

PBL2080, however the LSM values (Table 5) show that the transcripts are still much lower than the average level, especially when compared to the wild-type with a functional vapBC-6

locus. The doubling time of the two strains is comparable at 5 hours (Figures 13 and 14), but

upon heat shock the vapBC-6 mutant strain was thermally labile. The vapC-6 mutant displayed no phenotypic response to heat shock in the initial heat shock response (up to 30

minutes after reaching 90oC). However, after longer periods of thermal stress (over 1 hour to

5 hours), the vapC-6 mutant becomes more thermally labile than the vapBC-6 mutant (33).

Both strains had transcriptomic phenotypes, with each having significant differentiation of

nearly 550 genes by 10 minutes post heat shock, and 700-800 genes 30 minutes after

reaching 90oC (Figure 15). After 30min of heat shock for strain PBL2078, some of the highest significantly regulated gene are a dihydrolipoamide dehydrogenase (SSO1565) up

24-fold, two quinol oxidase related proteins (SSO1741 and SSO1742) up 8-fold, and two hypothetical proteins (SSO9092 and SSO2109) up over 8-fold. The most down regulated genes are a MarR family transcriptional regulator (SSO3242) and a putative amino acid transporter (SSO3189), both down more than 8-fold.

In the absence of vapB6 and C-6, vapC-8 is up-regulated almost 15-fold, yet when vapB-6 is present vapC-8 was only up 3.3-fold. The opposite is true for vapB-8, which showed a stronger up-regulation when vapB-6 is present (Table 5 and Figure 7). This indicated that there may be some cross-talk and regulation between the non-cognate TA pairs. As seen with the vapC-22 mutant, there does appear to be a correlation between the transcript levels of vapB-6 and the absence of vapC-6. Even before heat shock, the vapB-6 gene is in the top 10% of the genome-wide gene transcripts, and it moves to the top 5% after heat shock. This was also observed by Maezato et al. who showed that recombinant VapC-6 will degrade vapB-6 transcripts in vitro (33).

VapC-6 has been shown to be a temperature dependent ribonuclease that degrades S.

solfataricus RNA in vitro and is silenced by cognate antitoxin VapB-6 (33). Ribonucleolytic

VapC-6 was also shown to preferentially degrade specific transcripts in vitro that are highly abundant in the absence of the toxin in vivo. The genes tested were an oligopeptide transporter dppB-1 (SSO1274) and transcriptional regulator tetR (SSO2506) because their abundance increased in the vapBC-6 mutant strain, as well as cognate antitoxin vapB-6.

7SRNA (SSO3242) was used as a control since the transcript levels remain constant in the wild-type and mutant strains. VapC-6 did degrade all of the transcripts but the 7SRNA, but because the experiments used such a small subset of genes, it would be interesting to investigate the global regulation of the toxin genes to see if VapC-6 more general role in the thermal stress response.

SUMMARY

The heat shock response of hyperthermophilic archaea is complex and still largely a mystery. Since archaea lack homologues to many of the characterized bacterial heat shock proteins and regulators, transcriptomic analysis of the heat shock response in model extreme thermoacidophile S. solfataricus yields important clues into the novel stress proteins. Several prominent heat shock responsive genes have been identified: two terminal quinol oxidase subunits (SSO0394 and SSO2723), a dihydrolipoamide dehydrogenase (SSO2506), and several hypothetical proteins (SSO9092, SSO1920, and SSO2101). The up-regulation of

quinol oxidases and the dehydrogenase are interesting because most of the energy related proteins are down-regulated under thermal stress.

We have shown that TetR and novel heat shock regulator Shr play a critical role in the transcription regulation of S. solfataricus during thermal stress and may work in a cooperative manner, since there is evidence that Shr compensated for the loss of functional

TetR in strain PBL2026. In direct contrast, a MarR family transcriptional regulator

(SSO3242) is often the most down-regulated gene after thermal stress, indicating that the role transcriptional regulators play in heat shock is complicated and still not well understood.

Also, we have shown the dramatic effects of a single gene deletion or disruption, which

caused everything from slower growth rates to thermal lability. Particularly important is the

better understanding of the interplay between the VapBC toxins and antitoxins and clues

about their cognate and non-cognate interactions. VapBC-6 was demonstrated to be an

“Achilles heel” of thermal stress and the revelation that deletion of one gene pair out of

thousands of genes debilitates cellular fitness is important. It is interesting to note that the

vapBC-6 deletion mutant showed thermal lability for the initial dynamic heat shock, but the vapC-6 mutant was shown to be thermally labile after heat shock persisting over an hour

(33). Further studies should focus on if this is a unique feature of the VapBC-6 locus or if it extends to other loci in S. solfataricus. From this study, it is likely that the Vaps are not redundant, but have specific function(s) in prokaryotes. Efforts are now underway to determine the targets of the VapC toxins and establish cognate and non-cognate binding interactions. It will be interesting to determine the specific genes regulated by the Shr as

well, and to better understand the interplay between transcriptional and post-transcriptional

regulators.

ACKNOWLEDGMENTS

C.R.C. would like to thank Dr. Blumer-Schuette for running the Jspecies analysis. This work was supported in part by the National Science Foundation [grant numbers CBET0730091 and

CBET0617272]. C.R.C. acknowledges support from a National Institutes of Health T32

Biotechnology Traineeship.

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TABLES

Table 1. Summary of PBL2025 mutant strains

Strain Mutation Genes Doubling Phenotypic Transcriptomic response affected time response to 30min post HS (hrs) HS Genes up Genes down PBL2025 None N/A 3.5-4 None 2-fold 363 2-fold 359 4-fold 95 4-fold 67 8-fold 16 8-fold 9 16-fold 2 16-fold 3 PBL2026 Disruption SSO2506 6-9 None 2-fold 471 2-fold 417 4-fold 200 4-fold 95 8-fold 54 8-fold 16 16-fold 16 16-fold 11 32-fold 5 -- -- PBL2067 Disruption SSO3078 6 None 2-fold 488 2-fold 409 4-fold 174 4-fold 129 8-fold 61 8-fold 30 16-fold 17 16-fold 7 32-fold 5 64-fold 2 64-fold 3 128-fold 1 256-fold 1 -- -- PBL2078 Deletion SSO1494 5-6 Thermal 2-fold 381 2-fold 396 SSO1493 lability 4-fold 121 4-fold 68 8-fold 17 8-fold 4 16-fold 4 -- -- PBL2080 Deletion SSO1493 5-6 None 2-fold 402 2-fold 288 4-fold 119 4-fold 40 8-fold 30 8-fold 4 16-fold 7 -- -- 32-fold 3 -- --

Table 2. Fold-change comparison of Sso P2 to Sso PBL2025 after heat shock

ORF name

Category (gene symbol) P2 FC 5-BL P2 FC 30-BL PBL2025 FC 10-BL PBL2025 FC 30-BL DNA polymerase SSO8124 dpo2 4.3 8.4 4.3 5.4 Recombinases (putative) SSO0264 putative recB 1.8 1.4 1.6 1.7 dsDNA break & recombination SSO0777 radA -like 1.1 1.4 1.1 1.2 Partial exonuclease SSO2373 2.1 1.2 1.4 1.4 Putative DNA ligase SSO0189 1.5 2.7 1.8 2.0 SSO2484 ntH-1 1.5 1.2 1.3 1.4 DNA endonuclease III SSO0116 ntH-2 1.3 1.6 1.7 2.7 Tanscription and DNA SSO0269 GTP-binding modification prot. HflX 1.4 1.4 1.1 1.1 Putative mRNA processing factor SSO0386 1.4 1.2 1.1 1.5 SSO0129 cbiG 1.1 1.0 1.0 1.0 SSO2296 cbiE 2.2 1.7 1.8 1.3 Cobalamin SSO3235 cbiB 1.4 1.2 1.1 1.0 SSO0675 mobB 3.8 3.1 4.2 4.0 SSO0676 moeA-1 1.6 2.0 1.3 1.3 MGD SSO0677 mobA 1.2 1.2 1.0 1.3 SSO0485 1.9 4.7 1.9 2.0 Iron transporter SSO0486 3.5 3.1 2.0 3.3 SSO0548 1.8 1.6 1.5 1.7 drug resistance / efflux SSO1162 1.2 1.1 1.7 2.1 TetR family repressor SSO2506 23.5 12.7 15.8 10.9 SSO1255 3.9 3.0 2.7 2.1 GntR family repressors SSO1589 33.1 8.3 15.7 10.9 SSO2427 2.0 3.5 2.1 2.8 sHSP SSO2603 3.3 3.9 2.3 2.4 SSO1859 htpX-1 1.1 1.7 1.3 1.2 SSO2694 1.2 1.2 1.7 2.3 HtpX homolog SSO3231 htpX-2 1.4 1.3 1.2 1.0 USP family SSO0529 2.1 1.4 2.5 2.3 SSO0862 (α) 1.0 1.0 1.6 1.9 SSO0282 (β) 1.0 1.0 1.1 1.5 Thermosome SSO3000 (γ) 3.1 5.2 1.7 2.2 SSO0738 (α) 1.9 1.5 1.9 1.7 SSO0278 (β1) 1.1 1.1 1.3 1.2 Proteasome SSO0766 (β2) 1.7 1.2 1.7 1.7 Exosome SSO0292 csl4 2.6 1.8 2.3 1.9 SSO8620 vapB-8 16.0 6.6 9.1 7.1 SSO1657 vapC-8 7.6 3.4 8.6 10.0 SSO11914 vapB-22 2.2 1.5 1.0 1.2 SSO3078 vapC-22 2.1 1.6 1.4 1.2 SSO12018 vapB-23 1.4 1.0 1.5 1.1 TA loci SSO3128 vapC-23 1.5 1.4 1.4 1.6 SSO2101 PIN-domain 9.7 11.2 8.7 10.9 PIN-domain proteins SSO2783 PIN-domain 5.0 3.2 3.2 4.0

Table 3. Heat shock elements vapB6) BL vapC6) BL vapC22) BL TetR) BL Δ Δ Δ Δ

ORF name

Category (gene symbol) P2 BL SAB P2 05 SAB P2 30 SAB P2 60 SAB FC 5-BL FC 30-BL FC 60-BL BL PBL2025 HS 10 PBL2025 HS 30 PBL2025 FC 10-BL FC 30-BL ( PBL2078 HS 10 PBL2078 HS 30 PBL2078 FC 10-BL FC 30-BL ( PBL2080 HS 10 PBL2080 HS 30 PBL2080 FC 10-BL FC 30-BL ( PBL2067 10 PBL2067 30 PBL2067 FC 10-BL FC 30-BL ( PBL2084 10 PBL2084 30 PBL2084 FC 10-BL FC 30-BL Chaperones, HSPs and chromatin proteins sHSP SSO2427 0.0 1.0 1.8 2.5 2.0 3.5 5.6 0.0 1.1 1.5 2.1 2.8 -0.5 0.3 0.4 1.7 1.8 -0.6 0.0 0.0 1.5 1.6 0.8 1.7 2.0 2.0 2.4 0.0 0.7 1.3 1.7 2.5 SSO2603 1.7 3.4 3.7 3.6 3.3 3.9 3.6 1.6 2.9 2.9 2.3 2.4 2.1 3.1 2.9 2.1 1.8 1.9 3.0 2.9 2.2 2.0 1.5 3.2 3.4 3.2 3.9 2.0 2.9 3.3 1.9 2.4 HtpX homolog SSO1859 htpX-1 -0.9 -1.1 -1.7 -1.8 1.1 1.7 1.8 -2.0 -1.7 -2.3 1.3 1.2 -0.4 -0.7 -1.0 1.2 1.5 -1.6 -1.3 -1.9 1.2 1.2 -1.2 -1.5 -1.9 1.2 1.6 -0.5 -0.7 -0.7 1.1 1.2 SSO2694 -1.7 -1.4 -1.9 -2.0 1.2 1.2 1.2 2.2 1.4 1.0 1.7 2.3 3.3 2.3 2.2 2.1 2.3 2.7 1.9 1.5 1.7 2.2 1.6 1.5 1.4 1.0 1.1 2.8 2.3 2.4 1.5 1.3 SSO3231 htpX-2 0.9 1.4 1.2 1.6 1.4 1.3 1.6 3.5 3.2 3.5 1.2 1.0 3.8 3.3 3.4 1.5 1.4 3.6 3.3 3.1 1.2 1.4 3.5 3.6 3.3 1.1 1.1 4.0 3.5 3.3 1.4 1.6 USP family SSO0529 -0.2 -1.3 -0.6 -0.3 2.1 1.4 1.0 -0.9 -2.2 -2.1 2.5 2.3 1.7 0.4 0.3 2.4 2.6 0.8 -0.7 -1.3 2.7 4.1 -0.1 -0.9 -1.0 1.7 1.9 -0.8 -1.7 -1.8 1.8 2.0 SSO1865 3.5 5.5 4.5 4.3 3.9 2.0 1.7 4.5 4.5 4.8 1.0 1.2 4.3 4.3 4.3 1.0 1.0 3.7 3.9 4.4 1.2 1.7 4.4 4.5 4.6 1.0 1.1 4.4 4.4 4.3 1.0 1.1 SSO2778 -0.9 0.7 0.0 -0.5 3.1 1.8 1.4 -0.9 -0.6 -0.7 1.2 1.2 0.4 1.1 0.7 1.6 1.2 -0.1 0.6 0.8 1.6 1.8 -1.5 -0.1 0.2 2.7 3.4 -0.4 0.0 1.0 1.3 2.6 SSO3183 -2.1 -1.0 -1.6 -1.7 2.0 1.4 1.3 -1.4 -1.2 -1.0 1.1 1.3 -1.6 -1.8 -1.3 1.1 1.2 -1.4 -1.0 -1.3 1.3 1.1 -3.8 -2.4 -1.6 2.7 4.7 -2.0 -1.3 -0.8 1.6 2.2 Thermosome SSO0862 (α) 5.1 5.1 5.0 5.4 1.0 1.0 1.3 3.7 4.3 4.6 1.6 1.9 3.4 4.0 3.8 1.6 1.3 2.8 3.3 3.9 1.4 2.1 4.6 4.6 4.7 1.0 1.0 4.6 4.5 4.4 1.1 1.2 SSO0282 (β) 4.7 4.8 4.7 5.2 1.0 1.0 1.4 4.0 4.1 4.5 1.1 1.5 4.2 4.1 4.2 1.1 1.1 3.4 3.7 4.3 1.2 1.8 4.1 4.2 4.3 1.1 1.2 3.8 3.8 3.7 1.0 1.1 SSO3000 (γ) 2.0 0.4 -0.3 0.0 3.1 5.2 4.0 -0.3 -1.1 -1.5 1.7 2.2 1.4 0.5 0.6 1.9 1.8 0.9 0.0 0.0 1.8 1.9 1.4 0.9 0.8 1.4 1.5 2.1 1.0 1.1 2.1 2.0 Sso7d SSO9180 2.4 4.4 3.7 3.1 3.9 2.5 1.6 4.8 4.6 4.5 1.1 1.3 4.4 4.2 4.3 1.2 1.1 4.0 4.1 4.3 1.1 1.2 2.8 4.2 4.1 2.5 2.4 3.0 3.8 4.5 1.8 2.8 SSO9535 1.6 3.8 3.9 3.2 4.7 4.8 3.0 4.1 3.9 3.5 1.2 1.5 4.4 4.2 4.2 1.1 1.1 3.9 4.1 4.1 1.2 1.1 2.3 3.6 3.5 2.5 2.2 2.7 4.0 4.1 2.6 2.6 SSO10610 1.7 3.9 3.9 3.2 4.5 4.6 2.7 4.5 4.2 4.1 1.2 1.3 4.1 3.9 3.9 1.2 1.1 3.6 3.8 3.7 1.1 1.1 2.9 4.1 3.9 2.4 2.0 3.0 3.9 4.2 1.9 2.4 Protein & mRNA turnover Proteasome SSO0738 (α) 3.7 2.8 3.1 3.5 1.9 1.5 1.2 3.9 3.0 3.2 1.9 1.7 2.4 1.9 1.8 1.4 1.6 2.4 1.8 1.9 1.5 1.4 4.3 4.0 4.1 1.2 1.1 4.3 3.5 3.4 1.7 1.8 SSO0278 (β1) 2.2 2.1 2.0 2.5 1.1 1.1 1.2 4.3 3.9 4.1 1.3 1.2 3.8 3.3 3.4 1.4 1.3 3.3 2.7 2.6 1.4 1.6 3.3 3.3 3.2 1.0 1.1 3.2 2.6 2.5 1.5 1.7 SSO0766 (β2) 2.7 2.0 2.4 3.0 1.7 1.2 1.2 3.1 2.4 2.4 1.7 1.7 3.3 2.6 2.2 1.6 2.1 1.9 1.4 1.8 1.4 1.0 4.1 3.2 3.4 1.8 1.6 3.8 2.6 2.5 2.3 2.5 SSO0271 (PAN) 2.9 0.1 0.0 0.3 7.2 7.5 6.0 0.9 -0.3 -0.1 2.2 1.9 2.1 0.8 0.4 2.4 3.2 1.6 0.7 1.4 1.9 1.2 4.1 1.8 1.6 4.8 5.4 2.1 0.2 0.1 3.6 4.0 Exosome SSO0292 csl4 1.8 0.4 0.9 1.4 2.6 1.8 1.3 2.1 0.9 1.2 2.3 1.9 2.9 1.6 1.3 2.5 3.1 1.9 0.8 0.8 2.1 2.1 1.3 0.3 0.7 2.0 1.6 1.6 0.1 0.1 2.8 2.9 SSO0732 rrp42 3.0 0.3 0.5 1.5 6.7 5.6 2.8 0.7 -0.8 -0.7 2.8 2.5 -0.7 -1.9 -2.1 2.2 2.6 -0.3 -1.5 -1.6 2.3 2.5 1.5 0.1 0.3 2.5 2.2 2.4 0.5 0.1 3.7 4.8 SSO0735 rrp41 3.5 1.4 1.5 2.3 4.2 4.0 2.3 4.7 4.3 4.4 1.4 1.3 4.0 3.2 3.1 1.7 1.8 4.1 4.0 4.1 1.1 1.1 2.9 1.9 1.8 2.1 2.3 2.4 1.3 0.9 2.1 2.7 SSO0736 rrp4 3.9 0.6 1.7 2.4 10.4 4.8 2.9 1.8 0.4 1.0 2.8 1.8 2.7 1.6 0.4 2.2 4.8 1.6 0.6 1.2 2.1 1.3 4.1 2.4 2.7 3.2 2.7 2.9 1.1 1.0 3.5 3.6

Table 4. Metabolic elements vapB6) BL vapC6) BL vapC22) BL BL TetR) Δ Δ Δ Δ

ORF name P2 BL SAB BL P2 P2 05 SAB P2 30 SAB P2 60 SAB FC 5-BL FC 30-BL FC 60-BL BL PBL2025 10 HS PBL2025 FC 10-BL FC 30-BL ( PBL2080 FC 10-BL FC 30-BL FC 10-BL FC 30-BL ( PBL2084 30 PBL2084 FC 10-BL FC 30-BL Category (gene symbol) 30 HS PBL2025 FC 10-BL FC 30-BL ( PBL2078 10 HS PBL2078 30 HS PBL2078 10 HS PBL2080 30 HS PBL2080 ( PBL2067 10 PBL2067 30 PBL2067 10 PBL2084 DNA metabolism SSO1459 dpo2 -0.1 -1.3 -1.4 -2.1 2.3 2.5 4.0 -0.1 -0.1 0.1 1.0 1.2 -0.7 -1.1 -1.6 1.4 1.9 1.3 1.0 1.6 1.2 1.3 1.0 -2.1 -3.1 8.8 16.9 0.1 -2.4 -2.8 5.7 7.4 DNA polymerase SSO8124 dpo2 0.2 -1.9 -2.9 -3.4 4.3 8.4 12.1 1.4 -0.7 -1.1 4.3 5.4 1.6 -0.4 -0.8 3.9 5.1 1.6 -0.1 -0.4 3.4 4.2 1.7 -0.7 -1.6 5.0 9.3 2.1 0.7 -0.1 2.7 4.7 SSO2452 recA -1.4 -0.6 0.4 0.5 1.7 3.5 3.7 -2.4 -1.0 -0.9 2.5 2.7 -2.5 -0.5 -0.9 3.8 3.0 -1.8 -0.1 -0.1 3.3 3.4 1.0 1.0 -3.5 -4.8 -5.5 2.6 3.9 Recombinases (putative) SSO0264 putative recB -1.4 -0.6 -0.9 -0.4 1.8 1.4 2.0 0.0 0.6 0.7 1.6 1.7 -0.2 0.1 0.3 1.2 1.4 0.7 1.4 1.5 1.6 1.7 -1.0 0.3 0.6 2.5 3.0 -0.9 -0.3 -0.6 1.5 1.2 SSO0250 radA 1.9 2.8 3.3 3.1 1.8 2.5 2.3 2.5 3.1 3.3 1.5 1.8 3.3 3.9 3.9 1.6 1.6 2.8 3.0 3.5 1.1 1.6 2.2 2.7 3.2 1.5 2.1 2.5 3.0 2.7 1.4 1.1 dsDNA break & recombination SSO0777 radA -like -2.1 -2.2 -2.6 -2.8 1.1 1.4 1.7 -1.6 -1.7 -1.8 1.1 1.2 -2.2 -2.7 -2.7 1.4 1.5 -1.1 -1.5 -1.8 1.3 1.5 -4.1 -6.1 -6.5 3.8 5.3 -5.0 -4.8 -5.7 1.1 1.6 Partial exonuclease SSO2373 2.1 3.1 2.4 3.1 2.1 1.2 2.1 3.4 3.9 3.9 1.4 1.4 2.8 3.4 3.5 1.5 1.6 2.5 3.3 3.1 1.7 1.5 1.6 3.1 3.1 2.7 2.7 2.3 3.2 3.1 2.0 1.7 Damage-inducible DNA repair polymerase SSO2448 dinP -2.1 -1.7 -0.9 -0.2 1.2 2.3 3.5 -3.7 -2.2 -1.8 2.9 3.7 -3.6 -1.9 -1.7 3.2 3.7 -4.0 -2.3 -2.7 3.2 2.4 -4.1 -1.7 -0.5 5.4 11.7 -3.4 -1.7 -1.2 3.2 4.6 ATP-dependant DNA ligase SSO2734 -1.9 0.4 0.0 0.0 5.1 3.8 3.8 -2.0 -1.4 -0.9 1.6 2.2 -2.0 -0.4 0.0 3.1 4.1 -2.1 -0.2 0.7 3.6 6.7 -0.1 1.5 2.0 2.9 4.3 -1.1 0.8 1.5 3.9 6.1 Putative DNA ligase SSO0189 1.2 1.8 2.7 2.5 1.5 2.7 2.4 0.1 0.9 1.1 1.8 2.0 1.6 2.3 2.3 1.6 1.7 0.1 0.5 1.5 1.3 2.6 2.2 2.7 3.4 1.5 2.4 2.0 2.3 2.9 1.2 1.9 SSO2484 ntH-1 -2.0 -2.6 -2.2 -2.5 1.5 1.2 1.4 -1.7 -1.4 -1.2 1.3 1.4 -1.7 -1.4 -1.3 1.3 1.3 -2.1 -1.6 -1.6 1.4 1.5 -3.7 -3.3 -3.1 1.3 1.5 -3.1 -2.3 -1.7 1.8 2.6 DNA endonuclease III SSO0116 ntH-2 -2.2 -2.6 -2.8 -2.8 1.3 1.6 1.6 -1.1 -1.9 -2.5 1.7 2.7 0.0 -0.7 -0.3 1.6 1.3 -0.6 -1.2 -1.7 1.5 2.2 -2.0 -1.9 -1.6 1.1 1.3 -1.1 -1.9 -1.4 1.7 1.3 MutT-like protein SSO3167 -1.0 -0.1 0.5 0.7 1.8 2.9 3.3 0.9 1.8 2.6 1.9 3.4 0.4 1.4 2.2 2.0 3.6 1.4 2.4 3.3 2.0 3.6 0.7 1.1 1.4 1.3 1.7 0.2 1.7 1.5 2.7 2.5 SSO0265, DNA methylase -0.9 0.6 0.8 1.1 2.9 3.3 4.2 -1.0 -0.7 0.0 1.2 2.0 -0.7 -0.2 0.5 1.3 2.2 -0.4 -0.3 0.4 1.1 1.7 -0.9 -0.1 0.4 1.8 2.6 -2.5 -1.6 -1.2 1.8 2.5 SSO0266 TFE 0.7 2.8 3.3 3.3 4.3 5.9 5.9 1.1 2.1 2.6 2.0 2.8 1.9 2.8 2.7 1.8 1.7 1.7 2.6 3.1 1.9 2.6 2.3 3.3 3.8 2.0 2.7 1.4 2.5 3.2 2.2 3.3 SSO0267 0.1 -0.1 1.7 2.1 1.2 3.0 3.9 -0.1 -0.3 0.1 1.2 1.2 0.1 0.7 0.7 1.4 1.5 -0.5 -0.7 0.4 1.2 1.8 2.1 1.9 3.0 1.2 1.8 0.9 0.9 2.0 1.0 2.1 SSO0269 GTP-binding prot. HflX 1.5 1.1 2.0 2.8 1.4 1.4 2.3 -0.1 0.1 0.0 1.1 1.1 1.5 1.3 1.9 1.2 1.3 0.4 0.4 1.2 1.0 1.8 1.6 2.0 2.8 1.3 2.3 1.9 1.7 2.8 1.1 1.9 Tanscription and DNA SS0270 MBP-like 2.7 3.5 4.2 4.4 1.7 2.7 3.1 3.1 3.3 3.7 1.1 1.5 3.2 3.9 3.8 1.6 1.6 2.8 2.9 3.6 1.0 1.6 3.1 3.6 3.9 1.4 1.7 2.4 3.0 2.9 1.6 1.5 modification mRNA production and processing SSO0225 rpoA1 2.5 -0.1 1.7 2.3 5.8 1.7 1.1 0.4 -0.9 -0.6 2.5 2.0 2.0 0.8 0.6 2.3 2.7 1.6 0.5 0.7 2.2 1.9 3.6 1.7 1.9 3.7 3.3 1.6 0.1 0.0 2.8 3.2 SSO0227 rpoB1 3.9 1.0 2.3 3.3 7.1 2.9 1.5 1.7 1.1 0.8 1.5 1.8 3.1 1.9 1.4 2.3 3.2 1.9 1.1 1.1 1.7 1.8 3.2 1.9 2.2 2.5 2.0 3.7 2.1 1.9 3.1 3.4 SSO3254 rpoB2 3.4 1.1 2.9 3.7 4.8 1.4 1.2 2.7 2.0 1.9 1.6 1.7 2.8 2.0 1.7 1.6 2.1 2.6 1.7 1.8 1.9 1.8 4.4 4.1 4.3 1.3 1.1 4.2 3.3 3.4 1.9 1.8 polymerase SSO0071 rpoD 3.6 -0.1 1.4 2.3 12.6 4.4 2.5 1.7 0.3 0.2 2.8 2.9 0.3 -0.6 -1.1 1.9 2.8 0.4 -1.0 -0.9 2.6 2.3 2.7 1.6 1.8 2.2 1.9 2.3 0.6 0.3 3.1 4.0 RNAPutative mRNA processing facto r SSO0386 -0.6 -1.1 -0.9 -0.2 1.4 1.2 1.3 1.2 1.4 1.8 1.1 1.5 0.5 0.3 0.4 1.1 1.1 0.1 0.1 0.3 1.0 1.2 -0.3 0.6 1.0 1.8 2.4 0.4 0.5 0.7 1.1 1.2 Energy metabolism SSO0324 nuoD 1.6 -1.6 0.9 1.3 9.1 1.6 1.2 2.6 1.5 2.1 2.2 1.4 3.7 2.1 1.6 3.2 4.4 2.8 1.4 1.2 2.5 2.9 4.5 3.6 3.6 2.0 1.9 3.8 2.4 1.9 2.8 3.7 SSO0325 nuoH 1.9 -1.6 0.4 1.4 11.0 2.7 1.4 1.3 0.3 0.8 2.0 1.4 0.7 -1.1 -1.4 3.5 4.4 0.6 -0.6 -0.4 2.3 2.0 1.8 -0.3 0.0 4.1 3.3 1.5 -0.6 -0.5 4.4 4.1 SSO0326 nuoI 2.0 -1.4 1.1 1.6 10.2 1.8 1.3 1.8 0.3 0.8 2.9 2.1 1.9 0.2 0.1 3.3 3.6 1.7 0.0 0.2 3.1 2.8 2.4 -0.1 0.4 5.5 3.9 2.7 0.4 0.9 5.0 3.7 SSO0327 nuoJ 2.7 -0.1 1.4 2.1 7.0 2.4 1.5 2.1 0.6 0.3 2.8 3.5 3.3 1.1 1.1 4.7 4.6 2.9 1.0 1.0 3.7 3.7 4.0 1.7 2.1 5.2 3.7 4.3 2.1 2.5 4.5 3.5 SSO0328 nuoL 1.3 -1.3 -0.3 0.4 6.4 3.2 1.9 2.1 -0.1 -0.7 4.4 6.6 3.1 0.5 0.5 5.9 6.2 2.1 0.1 0.1 4.2 4.0 2.2 -0.6 -0.4 7.3 6.2 2.7 0.4 0.3 5.0 5.4 NADH deh ydrogenase SSO0329 nuoN 1.9 -0.9 0.0 0.5 6.8 3.7 2.6 2.1 1.3 0.8 1.8 2.6 2.3 0.2 -0.2 4.3 5.5 2.0 1.0 1.2 2.0 1.7 2.1 -0.8 -0.9 7.4 7.8 1.0 -0.9 -1.3 3.8 4.9 SSO0210 dfp 2.0 0.4 0.2 0.7 3.2 3.6 2.5 1.5 0.7 0.3 1.7 2.2 2.0 1.1 0.9 1.9 2.3 1.2 0.3 0.4 1.9 1.7 1.7 0.9 1.0 1.7 1.6 0.8 0.1 0.1 1.6 1.6 SSO0584 -1.4 1.1 -0.7 -1.0 5.7 1.7 1.4 0.3 0.6 0.5 1.2 1.1 1.6 1.9 2.1 1.3 1.5 -0.2 1.3 1.1 2.8 2.4 0.4 1.7 1.4 2.4 2.0 0.6 1.6 1.0 2.0 1.3 SSO2348 1.9 0.7 1.3 1.3 2.3 1.6 1.5 2.4 1.5 1.6 1.8 1.7 2.2 1.3 0.6 2.0 3.2 2.1 1.5 1.4 1.6 1.6 4.3 2.9 2.2 2.8 4.4 1.8 0.9 0.0 1.8 3.5 SSO2353 3.8 2.1 2.1 2.5 3.1 3.3 2.4 3.2 2.0 2.1 2.3 2.1 1.9 1.0 0.4 1.8 2.9 1.5 0.7 0.5 1.8 2.1 3.9 3.3 3.0 1.5 1.8 3.8 2.4 2.0 2.6 3.5 SSO2356 sdhA 4.8 3.2 4.6 4.5 2.9 1.1 1.2 1.0 0.5 1.0 1.4 1.0 1.9 1.2 1.0 1.6 1.8 2.1 1.3 1.8 1.8 1.3 4.0 3.5 3.8 1.4 1.2 3.8 3.1 3.3 1.6 1.4 SSO2762 etfA 3.5 4.4 3.7 3.9 2.0 1.1 1.3 2.4 2.7 1.8 1.2 1.5 2.5 2.7 2.1 1.1 1.3 2.6 2.4 2.1 1.1 1.3 3.4 4.0 3.7 1.6 1.2 3.6 3.5 3.6 1.1 1.0 SSO2763 etfB 3.3 4.5 4.3 3.8 2.4 2.1 1.5 1.7 2.4 1.6 1.6 1.1 2.2 2.8 2.2 1.6 1.0 2.4 2.5 2.5 1.1 1.0 4.1 4.3 4.2 1.2 1.1 3.2 3.5 2.9 1.2 1.2 SSO2776 2.7 3.1 3.5 3.7 1.4 1.7 2.0 -1.6 -1.4 -1.1 1.2 1.4 -0.3 -0.3 0.3 1.0 1.5 -0.9 -1.1 -0.2 1.1 1.7 0.9 1.3 2.0 1.3 2.1 0.2 0.4 1.4 1.2 2.2 SSO2817 etfAB/fixAB 3.3 0.8 1.7 2.1 5.4 2.9 2.3 2.3 1.1 1.3 2.3 2.0 2.8 1.1 1.2 3.2 3.0 2.3 1.1 1.3 2.3 2.0 4.6 3.1 2.9 2.9 3.4 3.6 2.2 1.8 2.6 3.4 Flavoproteins SSO2819 3.4 1.8 2.2 2.6 2.9 2.3 1.7 2.2 1.4 1.6 1.7 1.5 2.2 1.4 1.1 1.8 2.2 2.6 1.7 1.9 1.9 1.6 4.4 2.9 2.6 2.9 3.3 3.8 2.1 1.9 3.2 3.7

Table 4. Continued SSO0044 doxB 3.8 1.6 2.3 3.0 4.8 3.0 1.8 4.8 4.3 4.7 1.3 1.0 4.2 3.5 3.5 1.7 1.6 4.4 3.3 3.2 2.0 2.2 4.5 3.6 4.1 1.8 1.4 4.6 3.8 3.7 1.7 1.8 SSO0045 doxC 3.3 1.2 1.7 2.3 4.4 3.2 2.0 3.8 3.1 3.9 1.6 1.1 4.1 3.3 3.3 1.8 1.7 3.4 2.3 2.2 2.2 2.3 4.0 3.1 3.5 1.9 1.4 4.0 2.9 3.0 2.1 2.0 SSO1741 doxA -0.9 0.2 0.8 0.9 2.1 3.3 3.3 -2.3 2.0 2.4 20.3 25.9 -2.0 2.0 2.1 16.3 17.9 -1.2 1.6 1.3 6.8 5.5 -0.6 3.3 3.9 14.4 22.0 0.7 3.5 4.2 6.9 11.2 SSO1742 doxD -0.6 0.9 1.2 1.1 2.7 3.3 3.2 1.0 4.2 4.3 9.2 10.1 0.2 2.9 3.6 6.4 10.7 0.6 3.4 3.7 7.1 8.8 0.1 3.6 4.0 11.1 15.2 1.1 3.8 4.0 6.6 7.6 SSO1881 doxD -like -1.8 -1.6 -2.0 -2.3 1.2 1.2 1.4 -0.6 0.7 1.1 2.5 3.3 -0.8 -0.1 0.3 1.6 2.1 -1.3 0.5 0.3 3.6 2.9 -1.8 0.3 0.3 4.4 4.2 0.2 2.1 2.0 3.5 3.5 SSO1882 doxA -like -2.2 -1.8 -2.1 -2.0 1.2 1.1 1.1 -1.9 -0.1 -0.2 3.5 3.4 -3.2 -1.2 -1.4 4.1 3.5 -4.0 -1.8 -2.0 4.4 3.9 -4.5 -0.9 -0.8 12.2 13.3 -2.3 -1.1 -0.9 2.2 2.6 Terminal oxidase SSO5098 doxE 1.4 0.1 0.5 0.7 2.5 1.8 1.6 4.1 3.3 4.1 1.7 1.0 3.7 2.5 2.8 2.2 1.8 3.6 2.7 2.6 2.0 2.1 2.8 2.4 2.6 1.3 1.1 4.5 3.0 3.4 2.9 2.3 SSO0368 trxA-1 -0.9 -0.1 -0.2 0.5 1.8 1.6 2.7 1.1 1.4 1.7 1.2 1.5 2.1 2.2 2.5 1.0 1.3 0.6 1.1 1.4 1.5 1.8 1.1 1.4 2.1 1.2 2.0 1.0 1.5 2.5 1.4 2.8 SSO2232 trxA-2 1.0 1.9 2.7 2.4 2.0 3.3 2.6 0.2 0.8 1.2 1.6 2.0 0.3 1.3 1.8 1.9 2.7 1.1 1.9 2.5 1.7 2.6 2.8 3.8 4.1 1.9 2.4 1.4 2.5 3.1 2.1 3.2 Misc. redox proteins SSO2765 trxB-2 0.9 1.6 2.3 2.6 1.7 2.7 3.2 4.7 4.4 4.6 1.3 1.1 4.3 4.0 4.1 1.2 1.1 4.1 4.0 4.1 1.1 1.0 2.9 2.9 2.6 1.0 1.2 2.3 3.2 3.0 1.9 1.7 Cofactor biosynthesis SSO0129 cbiG -1.8 -1.9 -1.8 -1.8 1.1 1.0 1.0 0.0 -0.1 0.0 1.0 1.0 0.1 0.1 0.0 1.0 1.0 -0.7 -0.2 -0.4 1.4 1.2 -2.2 -1.2 -0.8 2.0 2.6 -1.8 -1.5 -1.0 1.3 1.7 SSO2296 cbiE 1.8 0.6 1.0 1.2 2.2 1.7 1.5 0.8 -0.1 0.4 1.8 1.3 0.6 0.0 0.2 1.6 1.3 1.3 0.6 1.2 1.6 1.0 3.1 2.0 2.2 2.2 1.8 2.1 1.4 1.8 1.7 1.3 SSO2297 cbiG- like 3.1 1.4 1.6 2.4 3.2 2.9 1.6 0.6 0.3 0.4 1.3 1.1 0.0 -0.2 0.0 1.1 1.0 -0.6 -0.8 -0.2 1.2 1.2 1.0 0.5 1.0 1.4 1.0 0.5 -0.1 0.4 1.5 1.0 SSO2299 cbiF 3.0 1.0 1.9 2.4 3.8 2.1 1.5 -0.1 -0.8 -0.6 1.7 1.5 0.8 0.5 0.7 1.3 1.1 0.9 0.4 1.2 1.4 1.3 2.9 2.3 2.7 1.5 1.1 2.2 1.5 1.9 1.6 1.2 SSO2301 cbiL 3.2 1.3 2.5 2.7 3.8 1.6 1.4 -0.1 -1.1 -0.6 1.9 1.4 -0.4 -0.8 -0.5 1.3 1.1 -0.4 -1.2 -0.1 1.7 1.2 2.2 1.5 2.1 1.6 1.0 1.7 1.0 1.6 1.7 1.1 SSO2303 cbiT 4.4 2.5 3.7 3.8 3.7 1.6 1.5 1.5 1.0 1.2 1.4 1.3 1.1 1.2 1.5 1.1 1.3 1.1 1.0 1.9 1.0 1.7 3.9 3.2 3.6 1.6 1.2 3.0 2.0 2.5 2.0 1.4 SSO2305 cbiD 3.6 1.3 2.4 3.0 5.1 2.4 1.6 1.6 0.7 1.1 1.9 1.5 1.1 0.2 0.5 1.9 1.6 1.3 0.5 0.9 1.8 1.3 2.6 1.7 2.2 1.9 1.4 2.9 2.0 2.3 1.9 1.6 SSO2306 cbiH 3.7 2.0 3.2 3.5 3.2 1.4 1.1 1.7 1.1 1.2 1.5 1.4 1.9 1.4 1.6 1.4 1.3 1.8 1.2 1.5 1.5 1.3 2.4 2.0 2.4 1.3 1.0 2.4 2.1 2.8 1.3 1.3 Cobalamin SSO3235 cbiB -2.7 -3.1 -3.0 -3.1 1.4 1.2 1.3 -0.5 -0.6 -0.5 1.1 1.0 -1.4 -1.7 -1.6 1.2 1.1 0.0 -0.2 -0.7 1.2 1.7 -3.7 -4.0 -4.8 1.2 2.2 -3.0 -2.8 -3.3 1.1 1.2 SSO0675 mobB -2.0 -0.1 -0.4 -0.7 3.8 3.1 2.6 -1.2 0.9 0.9 4.2 4.0 -0.3 1.2 1.1 2.8 2.6 -1.1 0.4 0.3 2.9 2.6 -1.9 -0.2 0.6 3.2 5.7 -0.9 0.4 0.7 2.4 2.9 SSO0676 moeA-1 -0.3 0.3 0.6 0.7 1.6 2.0 2.0 -1.1 -0.8 -0.7 1.3 1.3 -1.2 -0.8 -0.6 1.3 1.5 -0.9 -0.5 -0.1 1.3 1.8 -0.8 -0.5 0.1 1.2 1.9 -1.9 -1.0 -1.0 1.9 2.0 MGD SSO0677 mobA -2.7 -2.9 -2.9 -2.7 1.2 1.2 1.0 -1.8 -1.8 -1.4 1.0 1.3 -2.4 -2.4 -2.0 1.0 1.3 -3.1 -2.7 -2.2 1.3 1.8 -1.1 -2.9 -3.8 3.7 6.8 -1.7 -1.5 -1.5 1.1 1.2 Lipids SSO0534 acaB -1 2.3 -1.3 -1.9 -0.4 12.2 17.5 6.5 1.2 -0.2 -0.2 2.6 2.6 0.3 -1.1 -1.4 2.5 3.1 0.3 -0.9 -0.5 2.4 1.7 1.1 -0.2 0.5 2.4 1.5 1.4 0.7 1.4 1.7 1.0 SSO2062 acaB -3 3.5 2.1 2.9 3.2 2.5 1.5 1.2 -0.2 -0.7 -0.8 1.4 1.5 1.5 0.8 0.6 1.7 1.9 -0.1 -0.8 -0.6 1.6 1.4 3.5 2.5 2.6 2.0 1.9 3.5 2.5 2.5 2.1 2.0 SSO2377 acaB -4 1.9 1.8 3.4 3.8 1.1 2.7 3.7 0.1 -0.1 1.1 1.1 2.1 0.6 0.4 1.1 1.1 1.4 0.5 0.2 1.1 1.3 1.5 1.9 1.9 2.9 1.1 2.0 1.4 1.2 1.7 1.2 1.2 SSO2496 acaB -5 0.5 1.4 2.0 2.3 1.8 2.7 3.4 0.0 1.2 1.8 2.2 3.5 -0.2 -0.4 0.5 1.1 1.7 -0.7 -0.8 0.1 1.1 1.7 -2.5 -2.6 -1.4 1.1 2.1 -0.9 -0.4 -0.5 1.4 1.3 SSO2508 acaB -6 -1.0 -0.1 -0.1 0.1 1.9 1.8 2.1 -0.6 0.1 1.0 1.6 3.0 -0.2 -0.2 0.6 1.0 1.8 -0.4 0.7 1.3 2.2 3.4 0.5 1.9 2.2 2.6 3.3 1.0 2.3 1.7 2.4 1.6 SSO2697 acaB -8 2.9 1.2 1.3 1.2 3.1 3.0 3.2 1.7 0.2 -0.3 2.9 4.0 1.3 0.4 -0.6 1.8 3.6 1.9 0.8 0.5 2.1 2.7 3.3 1.6 0.7 3.3 6.2 2.7 1.3 0.6 2.7 4.4 Acetyl-CoA C-acetyltransferase SSO3113 acaB -10 -0.1 -1.7 -1.8 -2.2 3.1 3.2 4.2 1.1 -0.3 -0.6 2.6 3.2 0.4 -1.6 -2.1 3.8 5.7 0.7 -1.1 -1.7 3.5 5.2 1.0 -1.2 -2.0 4.5 8.4 1.0 -0.2 -0.9 2.3 3.7 SSO1030 fabG -2 -0.4 -1.7 -2.1 -2.0 2.5 3.2 3.0 -1.1 -1.6 -1.5 1.4 1.3 -1.7 -2.2 -1.9 1.4 1.1 -1.8 -2.2 -2.1 1.3 1.2 -0.8 -2.4 -3.1 3.1 4.9 -0.7 -2.0 -1.9 2.6 2.4 SSO1542 fabG -3 0.3 2.5 2.5 2.0 4.3 4.4 3.2 -2.4 -1.2 -0.7 2.3 3.1 -2.3 -1.0 -0.8 2.5 2.8 -1.9 -0.2 0.3 3.2 4.6 0.3 2.2 2.3 3.8 3.9 0.4 2.3 2.2 3.7 3.5 SSO2205 fabG -4 1.4 0.6 0.2 0.2 1.8 2.3 2.3 -0.4 -0.9 -1.5 1.4 2.1 0.5 0.0 -0.7 1.4 2.2 0.1 -0.6 -0.6 1.7 1.7 1.7 1.1 0.9 1.5 1.8 1.8 0.7 0.8 2.3 2.1 SSO2276 fabG -5 2.2 1.1 1.7 2.2 2.3 1.5 1.0 1.8 0.9 1.2 1.9 1.6 2.0 1.2 0.9 1.7 2.2 2.5 1.5 1.3 2.1 2.3 3.5 2.3 2.0 2.3 2.7 3.0 2.2 1.6 1.8 2.7 SSO2289 fabG 0.5 0.5 0.6 0.8 1.0 1.1 1.3 -0.3 -0.3 -0.4 1.0 1.1 -0.1 -0.3 -0.2 1.2 1.1 0.3 0.1 0.2 1.1 1.0 -0.8 -0.6 -0.1 1.1 1.5 -0.6 -0.1 0.4 1.5 2.0 SSO2500 fabG -7 -0.7 0.8 0.4 0.2 2.8 2.1 1.8 -1.9 -0.4 -1.0 2.7 1.8 -1.9 -0.5 -0.6 2.5 2.3 -1.5 -0.4 -0.4 2.2 2.0 -5.9 -1.5 -0.6 20.5 38.2 -2.5 -1.5 -1.8 2.0 1.7 SSO2664 fabG-8 -1.3 -0.5 -0.6 -0.5 1.7 1.6 1.8 -0.5 0.2 0.1 1.6 1.5 -1.7 -0.9 -0.7 1.8 2.0 -0.2 0.2 0.6 1.4 1.7 -0.9 -0.4 -0.2 1.4 1.6 -1.4 -0.9 -0.5 1.4 1.9 Fatty acid biosynthesis SSO3114 fabG -10 1.5 0.1 -0.1 0.0 2.6 3.0 3.0 1.1 -0.2 -0.3 2.4 2.6 0.6 -0.7 -1.0 2.4 2.9 0.2 -1.3 -1.3 2.8 2.9 1.8 0.2 -0.2 3.1 4.2 1.2 -0.1 -0.3 2.5 2.9 Transporters SSO0485 1.9 1.0 -0.3 -0.3 1.9 4.7 4.4 -0.3 -1.3 -1.3 1.9 2.0 1.6 0.5 0.5 2.1 2.1 -0.9 -1.8 -1.5 1.9 1.5 -3.1 -3.5 -3.0 1.3 1.1 -1.1 -1.6 -2.3 1.3 2.3 Iron SSO0486 -0.3 -2.1 -1.9 -2.5 3.5 3.1 4.6 -1.7 -2.7 -3.4 2.0 3.3 -0.8 -2.1 -2.9 2.4 4.1 -1.8 -2.5 -2.2 1.7 1.3 -9.2 -6.3 -2.0 7.2 147.2* -4.0 -4.9 -5.4 1.8 2.5 SSO1892 0.3 -1.5 -1.6 -2.0 3.5 3.7 5.0 1.4 0.2 -0.1 2.2 2.7 0.9 -0.4 -0.7 2.6 3.1 0.6 -0.3 -0.8 1.9 2.7 1.2 0.0 -0.6 2.2 3.4 1.9 0.8 0.2 2.2 3.4 SSO1893 1.0 -0.7 -1.0 -1.3 3.2 4.0 5.0 1.0 0.6 0.0 1.4 2.0 0.2 -0.8 -1.3 2.0 2.7 -0.2 -1.2 -1.6 2.0 2.6 0.7 -0.7 -1.5 2.6 4.7 0.3 -0.4 -0.7 1.6 2.0 Cobalt SSO1894 1.7 0.8 0.1 -0.5 1.8 3.0 4.5 0.2 0.6 0.2 1.3 1.0 -0.2 -0.4 -1.0 1.1 1.8 0.2 -0.2 -0.2 1.3 1.3 1.0 0.1 -0.6 1.8 3.0 0.1 0.0 -0.6 1.0 1.5 Sulfate SSO2469 0.7 -1.3 -0.8 -0.5 3.9 2.8 2.3 1.8 0.3 -0.1 2.7 3.6 0.9 -0.2 -0.4 2.1 2.6 0.9 -0.4 -0.7 2.6 3.1 -0.4 -1.9 -2.7 2.8 5.2 0.2 -0.9 -1.4 2.2 3.0

Table 4. Continued SSO1009 -1.7 -2.8 -2.7 -2.5 2.2 2.0 1.8 0.6 -1.3 -1.9 3.5 5.4 0.9 -0.5 -0.6 2.6 2.7 -0.1 -0.9 -1.2 1.8 2.2 0.7 -1.4 -1.6 4.3 5.1 0.2 -1.0 -0.7 2.3 2.0 SSO1069 1.9 -0.8 0.4 0.3 6.5 2.8 3.0 -1.1 -0.6 -0.4 1.5 1.6 -1.4 -0.7 -0.5 1.6 1.9 -1.3 -0.6 -0.5 1.6 1.8 -3.0 -3.3 -4.3 1.2 2.5 -3.3 -3.0 -1.8 1.2 2.9 SSO1173 0.3 -1.5 -1.3 -1.3 3.4 3.1 3.0 0.9 -0.3 -0.9 2.3 3.4 0.6 -0.3 -1.2 1.8 3.4 -0.7 -1.5 -1.1 1.7 1.3 1.7 0.0 -0.3 3.4 4.3 1.5 -0.2 -0.8 3.1 4.9 SSO1463 -1.9 -2.1 -2.1 -2.3 1.2 1.2 1.3 -1.7 -1.9 -2.3 1.1 1.5 -1.5 -2.3 -1.9 1.8 1.3 -2.3 -1.9 -2.5 1.3 1.2 -1.7 -2.2 -1.3 1.4 1.3 -1.1 -1.6 -2.5 1.4 2.7 SSO1693 -2.2 -1.7 -2.1 -2.2 1.4 1.1 1.0 -1.9 -0.5 -0.2 2.7 3.4 -2.6 -1.0 -1.4 3.0 2.4 -1.9 -0.4 -0.3 2.8 3.1 -1.5 -1.9 -3.4 1.3 3.6 -2.1 -0.9 -1.3 2.3 1.8 SSO1906 4.4 5.0 4.7 4.2 1.5 1.2 1.1 0.3 1.1 1.0 1.8 1.6 3.0 3.2 3.2 1.1 1.2 1.6 2.2 2.7 1.5 2.1 3.2 3.7 3.7 1.4 1.4 3.0 3.1 3.4 1.1 1.3 SSO2043 -2.0 -2.3 -2.2 -2.6 1.2 1.2 1.6 -4.1 -3.6 -3.6 1.3 1.4 -3.9 -3.3 -3.5 1.5 1.3 -4.0 -3.8 -3.4 1.1 1.5 -8.9 -5.6 -5.9 9.8 7.9 -6.3 -5.2 -6.9 2.1 1.5 SSO2292 putative 1.0 -1.8 -2.1 -2.1 7.1 8.6 8.3 1.6 -0.3 -1.1 3.8 6.2 2.5 0.5 -0.4 3.8 7.4 2.1 0.4 0.0 3.2 4.2 1.9 -0.1 -1.2 3.9 8.4 3.1 1.0 0.3 4.2 7.1 SSO2549 putative -2.6 -2.3 -2.1 -2.3 1.2 1.4 1.2 -2.6 -2.4 -1.7 1.2 1.9 -2.3 -2.8 -2.3 1.4 1.0 -2.7 -1.7 -2.0 2.1 1.7 -6.6 -3.9 -2.0 6.5 24.2* -2.9 -1.9 -2.6 2.0 1.2 SSO2728 0.4 -1.2 -0.9 -1.2 3.1 2.5 3.0 -0.7 -1.5 -1.8 1.8 2.2 -1.0 -1.1 -0.8 1.1 1.1 -1.2 -1.5 -0.9 1.2 1.2 0.3 -0.3 -0.2 1.5 1.4 0.9 -0.2 0.3 2.1 1.5 SSO3189 putative 2.1 -0.8 -1.4 -1.6 7.6 11.6 13.3 3.1 0.5 0.2 6.3 7.7 3.0 0.0 -0.4 8.0 10.7 2.9 0.4 -0.2 5.6 8.6 3.2 0.8 0.0 5.6 9.8 3.7 1.4 1.1 4.9 6.2 Amino Acids SSO3224 -0.1 -0.5 -0.2 0.2 1.3 1.1 1.2 2.4 1.9 1.9 1.4 1.4 2.2 1.7 1.3 1.4 1.9 2.1 1.7 1.9 1.3 1.1 3.3 2.6 2.3 1.7 2.0 3.3 2.0 1.8 2.5 2.9 SSO2615 dppF-3 2.5 0.2 1.3 1.8 4.9 2.2 1.6 3.6 2.5 2.7 2.1 1.8 3.3 2.4 2.2 1.8 2.1 3.4 2.2 2.7 2.2 1.6 1.9 1.2 0.6 1.7 2.5 1.7 1.7 1.0 1.1 1.7 SSO2616 dppD-3 2.7 -0.1 2.4 2.2 7.1 1.3 1.4 2.1 0.2 0.8 3.8 2.5 2.0 0.3 0.0 3.3 4.1 2.5 0.6 0.9 3.7 3.0 4.1 1.8 1.7 4.7 5.2 3.5 1.0 1.0 5.4 5.5 SSO2617 dppC-3 2.6 -0.5 1.5 1.9 8.5 2.1 1.6 1.2 -0.1 -0.2 2.4 2.7 3.0 1.1 0.5 3.9 5.8 2.0 0.2 0.3 3.6 3.2 3.2 1.0 0.9 4.6 4.8 3.3 0.9 1.2 5.4 4.5 SSO2618 dppB-3 2.0 0.0 1.2 1.4 4.1 1.8 1.6 3.4 1.9 2.5 2.9 2.0 2.8 1.0 1.0 3.3 3.4 2.0 0.5 0.4 2.8 3.1 3.9 1.9 1.7 3.9 4.7 3.8 1.8 1.7 4.0 4.4 Peptides SSO2619 dppA 4.8 4.9 4.4 4.7 1.1 1.3 1.1 4.8 3.8 4.1 2.0 1.6 4.1 3.5 3.4 1.5 1.6 4.0 3.6 2.9 1.3 2.1 2.9 2.9 3.0 1.0 1.1 4.3 3.8 3.4 1.4 1.9 SSO1274 dppB-1 3.0 -1.0 0.1 0.5 15.9 7.3 5.9 2.0 0.2 0.4 3.3 3.0 1.8 -0.5 -0.7 4.8 5.5 1.5 -0.4 -0.7 3.7 4.6 2.4 0.2 0.4 4.6 3.9 3.2 1.1 0.5 4.1 6.4 SSO1275 dppC-1 2.3 -1.6 -0.5 -0.5 15.3 7.1 7.4 -0.4 -1.2 -1.2 1.8 1.8 0.6 -0.3 -0.6 1.8 2.2 -0.2 -1.3 -0.7 2.0 1.4 3.0 0.5 0.5 5.7 5.5 1.0 -1.0 -1.0 4.0 4.0 SSO1276 dppD-1 2.9 0.0 0.4 0.3 7.3 5.7 5.9 2.8 1.6 1.6 2.3 2.3 1.5 0.7 1.2 1.8 1.3 1.7 0.7 1.2 2.0 1.4 1.9 0.0 0.0 3.8 3.7 2.3 1.1 0.7 2.2 3.0 SSO1277 dppF-1 1.0 -2.0 -2.1 -1.9 8.0 8.5 7.9 -0.6 -1.1 -0.9 1.4 1.2 0.7 0.2 0.4 1.5 1.2 0.1 -0.7 -0.4 1.7 1.3 2.1 0.3 0.6 3.4 2.7 0.6 -0.3 -0.4 1.9 1.9 SSO1281 dppF-2 -0.1 -1.3 -0.7 -0.7 2.4 1.5 1.5 0.4 -0.2 -0.1 1.5 1.4 -0.2 -0.5 -0.1 1.2 1.0 -0.2 -1.2 -0.6 2.0 1.3 1.3 -0.5 -0.3 3.4 3.0 0.2 -0.8 -1.0 2.0 2.3 SSO1282 dppD-2 1.6 -0.8 0.8 0.2 5.1 1.7 2.7 1.5 1.0 1.0 1.4 1.4 -0.2 -0.6 -0.6 1.3 1.3 -0.1 -1.1 -0.4 2.1 1.3 3.5 1.4 1.5 4.2 4.2 1.6 0.2 0.4 2.7 2.3 Oligo/dipeptides SSO1283 dppC-2 2.7 0.0 1.0 1.1 6.3 3.3 3.0 3.0 2.5 2.1 1.4 1.8 2.8 1.8 1.1 2.0 3.3 1.6 0.8 0.8 1.7 1.7 3.2 1.7 1.6 2.7 3.1 1.8 1.0 0.9 1.7 1.8 SSO3066 0.3 -0.8 -2.0 -2.2 2.2 4.9 5.6 4.6 2.2 1.0 5.5 12.6 4.1 2.6 1.8 2.8 4.7 2.9 1.5 0.5 2.6 5.4 3.5 2.0 1.0 2.7 5.7 4.0 2.1 1.4 3.6 5.8 SSO3067 -1.6 -3.2 -3.1 -3.4 3.1 2.8 3.6 1.4 -1.7 -2.3 9.0 13.4 1.0 -1.7 -2.3 6.4 10.0 0.1 -2.1 -2.4 4.5 5.5 1.6 -1.8 -2.2 10.4 13.4 0.9 -1.3 -2.3 4.5 9.2 SSO3068 0.5 -0.1 -0.1 -0.3 1.5 1.5 1.8 2.3 0.4 -0.1 3.7 5.3 2.6 0.7 0.4 3.6 4.7 2.0 0.6 0.5 2.6 2.7 1.5 -1.3 -1.9 6.7 10.5 1.1 -0.4 -1.0 2.8 4.3 Arabinose SSO3069 -1.7 -1.5 -2.5 -2.7 1.1 1.7 2.0 -0.3 -0.6 -1.7 1.2 2.7 0.3 0.4 -0.2 1.0 1.4 -0.5 -0.4 -0.6 1.1 1.0 -2.1 -1.7 -1.5 1.4 1.6 -1.4 -1.1 -0.7 1.2 1.7 SSO2848 2.0 0.1 0.4 0.0 3.6 3.0 3.8 3.3 2.0 1.8 2.5 3.0 2.9 1.7 1.6 2.3 2.4 2.5 1.8 2.1 1.7 1.3 -0.2 -0.9 -0.9 1.6 1.7 1.5 0.8 0.3 1.6 2.3 SSO2849 1.6 -0.5 0.0 0.1 4.3 2.9 2.8 3.3 2.0 1.7 2.6 3.0 3.0 1.5 1.1 2.7 3.6 3.2 1.7 1.2 2.9 4.0 1.6 0.4 0.0 2.4 3.2 3.3 2.2 1.3 2.1 4.1 Glucose SSO2850 0.4 -1.1 -1.2 -1.0 2.7 3.0 2.6 -2.2 -2.6 -2.7 1.3 1.4 -2.8 -3.5 -3.7 1.7 1.9 -1.9 -2.8 -2.5 1.8 1.5 -8.1 -6.7 -6.1 2.6 4.0 -5.5 -4.3 -7.0 2.4 2.8 SSO1000 3.2 1.3 3.2 3.6 3.6 1.0 1.4 2.6 1.8 1.8 1.7 1.7 1.0 0.0 0.5 1.9 1.5 1.3 0.4 0.5 1.8 1.7 1.7 0.6 1.5 2.2 1.2 2.1 1.4 1.7 1.7 1.4 SSO1170 1.4 -0.4 0.2 0.0 3.5 2.3 2.6 -1.0 -1.2 -1.2 1.1 1.1 -1.4 -1.6 -1.6 1.1 1.1 -1.6 -1.9 -1.6 1.2 1.0 -3.2 -3.6 -3.4 1.3 1.1 -1.1 -1.5 -1.9 1.4 1.8 SSO3053 -1.3 -2.1 -2.6 -2.6 1.7 2.4 2.5 3.0 0.9 0.5 4.3 5.6 3.2 1.0 0.9 4.6 4.8 0.9 0.2 0.1 1.6 1.7 0.3 -0.1 -0.4 1.3 1.6 1.5 1.0 0.3 1.4 2.3 Maltose SSO3058 -0.7 -0.3 0.5 0.8 1.4 2.3 2.9 2.4 2.5 2.4 1.1 1.0 1.6 1.7 1.8 1.0 1.1 1.3 1.7 1.7 1.3 1.3 1.2 1.9 2.0 1.7 1.8 1.2 1.4 1.6 1.2 1.3 SSO0548 -2.0 -2.9 -2.6 -2.8 1.8 1.6 1.8 -0.4 -1.0 -1.2 1.5 1.7 -1.4 -1.9 -2.5 1.4 2.1 -1.4 -2.0 -2.2 1.5 1.8 -1.0 -2.6 -3.7 3.0 6.6 -1.6 -2.2 -2.6 1.5 2.0 SSO1162 -2.2 -1.9 -2.0 -2.3 1.2 1.1 1.1 -2.9 -2.1 -1.8 1.7 2.1 -2.4 -2.2 -2.1 1.2 1.3 -1.6 -0.8 -0.4 1.7 2.3 -2.0 -1.8 -2.0 1.1 1.1 -2.0 -1.7 -1.3 1.3 1.7 SSO1773 -2.1 -2.3 -2.1 -2.1 1.2 1.0 1.0 0.5 1.8 2.3 2.4 3.4 -0.2 0.4 -0.1 1.4 1.1 -0.2 0.9 0.1 2.0 1.2 -0.5 0.4 0.1 1.9 1.5 -0.2 0.2 -0.7 1.3 1.4 SSO1863 -1.2 -1.4 -1.5 -1.7 1.1 1.2 1.5 -0.7 -1.0 -1.7 1.3 2.0 -0.3 -1.2 -1.6 1.9 2.5 -0.8 -0.8 -1.6 1.0 1.7 -0.3 -0.3 -0.9 1.0 1.5 0.9 1.1 0.3 1.1 1.5 SSO2035 -1.2 -0.8 0.0 -0.2 1.4 2.4 2.0 -2.5 -1.8 -2.1 1.7 1.4 -2.5 -1.7 -1.5 1.7 2.1 -2.9 -1.9 -1.7 2.0 2.3 -5.4 -4.2 -2.9 2.3 5.4 -3.0 -2.4 -2.7 1.5 1.2 SSO2135 0.4 2.2 2.4 2.6 3.5 4.0 4.5 -1.9 0.7 0.2 5.7 4.2 -1.1 0.1 0.6 2.3 3.3 -1.3 0.2 0.4 2.9 3.4 -1.9 1.0 1.9 7.3 13.7 -0.5 2.2 2.7 6.4 9.1 SSO2137 3.4 4.6 5.0 5.0 2.3 3.1 3.0 3.3 3.4 3.2 1.1 1.1 2.5 2.8 3.1 1.2 1.5 3.4 3.7 3.5 1.2 1.1 1.5 3.6 3.7 4.1 4.5 1.8 3.4 3.6 3.0 3.5 SSO2228 -1.5 0.9 0.0 0.3 5.4 2.9 3.7 -0.2 1.9 2.5 4.2 6.3 -0.3 1.2 2.1 2.9 5.5 -1.5 1.0 0.9 5.4 5.0 -1.6 0.6 1.1 4.6 6.3 -0.7 1.2 1.4 3.5 4.2 SSO2704 -0.4 1.1 1.8 1.1 2.8 4.5 2.9 -0.2 -0.1 -0.1 1.1 1.1 0.4 -0.1 0.0 1.4 1.3 0.9 0.6 0.8 1.3 1.1 2.8 2.3 2.1 1.4 1.6 2.5 2.4 2.1 1.0 1.3 drug resistance / efflux SSO2716 -1.4 -1.5 -1.8 -2.0 1.1 1.4 1.6 0.7 0.2 0.0 1.4 1.6 0.3 -0.6 -0.5 1.8 1.8 -0.3 0.2 -0.6 1.4 1.2 -2.0 -1.6 -1.8 1.3 1.1 0.6 0.2 -0.8 1.4 2.6 SSO1319 -0.4 -0.3 -0.4 -0.3 1.1 1.0 1.0 2.2 1.3 1.1 1.9 2.2 0.7 0.2 0.1 1.4 1.5 2.3 1.7 1.2 1.5 2.0 -4.6 -4.3 -3.9 1.3 1.6 -3.4 -4.4 -3.4 1.9 1.0 antibiotic SSO1934 -0.7 -0.2 -0.2 -0.7 1.5 1.4 1.0 -0.5 -0.2 -0.3 1.3 1.2 -1.6 -0.5 -0.9 2.2 1.7 -1.0 -0.4 -0.1 1.6 1.9 0.0 0.2 0.4 1.1 1.3 -1.2 -0.4 0.2 1.7 2.5

Table 4. Continued Regulators TetR family repressor SSO2506 -0.6 3.9 3.0 3.1 23.5 12.7 13.4 0.6 4.6 4.0 15.8 10.9 0.8 4.3 3.7 11.2 7.5 0.9 3.7 4.0 7.1 8.6 -0.7 3.8 4.1 22.8 27.7 -3.9 -2.7 -1.5 2.4 5.3 SSO1255 -1.5 0.4 0.1 -0.1 3.9 3.0 2.6 -0.2 1.3 0.9 2.7 2.1 -1.4 0.0 -0.1 2.6 2.4 -1.8 -0.6 -0.5 2.3 2.5 -2.6 -0.5 -0.6 4.4 4.0 -2.2 -0.2 -0.6 3.9 2.9 GntR family repressors SSO1589 0.4 5.5 3.5 2.8 33.1 8.3 5.0 1.0 5.0 4.5 15.7 10.9 0.9 4.3 4.1 9.9 9.0 1.3 4.4 4.2 8.3 7.5 -1.4 2.9 3.2 19.2 24.7 -1.6 3.6 4.1 37.4 52.2 SSO0048 2.4 4.1 3.4 2.9 3.2 2.0 1.4 4.5 4.5 4.5 1.0 1.0 3.8 4.0 4.3 1.2 1.4 3.5 3.8 4.1 1.2 1.5 2.8 4.0 4.2 2.2 2.5 3.1 3.8 4.0 1.7 1.9 SSO0458 -0.1 1.9 1.0 0.6 4.1 2.3 1.7 2.7 4.1 3.6 2.5 1.9 1.2 3.0 2.5 3.5 2.5 2.5 3.5 3.1 2.1 1.5 0.9 2.9 2.8 4.0 3.7 0.8 2.7 2.9 3.7 4.3 SSO1082 -0.1 2.2 1.6 1.5 4.9 3.2 2.9 1.8 2.9 2.6 2.2 1.7 0.5 2.2 2.1 3.2 3.1 1.0 2.5 2.7 2.8 3.3 0.3 2.2 2.5 3.7 4.5 0.0 2.1 2.4 4.2 5.2 SSO1101 5.1 4.8 4.9 5.3 1.3 1.2 1.1 4.4 4.7 5.0 1.2 1.5 4.3 4.3 4.3 1.0 1.0 3.6 3.9 4.6 1.3 2.0 4.6 4.6 4.7 1.0 1.1 4.1 4.7 4.6 1.5 1.3 SSO1108 1.5 3.4 2.0 1.3 4.0 1.4 1.1 3.9 4.3 4.2 1.3 1.2 3.7 4.2 4.3 1.4 1.5 3.2 3.7 4.2 1.4 2.0 3.3 4.3 4.1 2.1 1.8 3.2 4.4 3.7 2.4 1.5 SSO1110 -0.9 1.0 0.0 -0.2 3.7 1.9 1.7 0.5 1.7 1.8 2.4 2.5 -0.1 1.4 1.8 2.8 3.9 -0.1 1.5 1.6 3.1 3.3 -1.6 0.1 0.7 3.1 4.8 -1.3 0.3 1.1 3.2 5.3 SSO2138 3.1 4.7 5.2 4.5 3.0 4.1 2.7 -1.1 2.2 1.5 9.5 5.8 -0.4 2.2 2.0 6.0 5.2 -0.8 1.7 2.4 5.9 9.3 1.4 3.8 4.0 5.2 6.1 0.8 3.2 3.6 5.0 6.7 SSO2474 0.5 0.7 0.1 0.0 1.1 1.3 1.4 -0.7 -0.8 -1.1 1.1 1.3 -1.1 -1.4 -1.4 1.3 1.2 -0.3 -0.8 -0.8 1.4 1.4 -2.8 -3.8 -3.8 2.0 1.9 -1.9 -2.0 -2.3 1.1 1.4 SSO2897 -0.1 2.5 2.9 1.8 6.1 8.0 3.8 0.8 3.2 3.3 5.3 5.6 0.5 3.3 3.2 6.6 6.5 0.4 2.9 3.2 5.5 7.0 1.2 3.7 3.9 5.6 6.2 1.1 3.4 3.8 5.1 6.8 MarR/Lrs14 family repressors SSO3242 2.5 1.4 -0.5 -0.9 2.1 8.1 10.3 3.5 0.3 -0.6 9.8 18.3 3.8 1.4 0.4 5.0 10.5 3.5 1.2 0.5 4.7 7.8 3.0 0.6 -0.2 5.2 9.3 3.2 1.4 0.6 3.4 5.8 SSO0200 -1.0 -2.0 -2.5 -2.4 2.0 2.9 2.7 -0.5 -1.7 -1.6 2.2 2.1 -1.0 -1.9 -1.8 1.8 1.7 -1.4 -2.2 -2.1 1.7 1.6 -3.2 -3.4 -2.9 1.1 1.2 -2.0 -2.6 -2.8 1.6 1.7 SSO0239 2.3 0.6 0.7 1.2 3.3 3.1 2.1 0.4 -0.2 -0.5 1.5 1.9 1.7 0.8 0.6 1.9 2.2 0.5 -0.5 -0.4 1.9 1.9 1.5 0.8 0.8 1.6 1.6 1.3 -0.1 0.0 2.6 2.5 SSO0618 -1.6 0.5 1.0 1.6 4.2 6.0 8.9 -2.3 -0.4 0.8 3.6 8.4 -1.2 0.4 1.6 3.2 7.0 -4.2 -1.0 0.8 8.7 31.6 -0.6 2.1 3.3 6.8 15.1 -2.4 0.1 1.1 5.7 11.5 SSO0669 0.7 1.2 2.0 1.8 1.5 2.5 2.2 -1.4 -0.9 -0.4 1.4 2.0 0.2 1.1 1.0 1.8 1.7 -0.1 0.1 0.9 1.1 2.1 2.6 2.9 3.5 1.3 1.9 0.8 1.5 2.0 1.5 2.2 SSO5522 1.2 2.8 2.5 2.4 3.2 2.5 2.3 4.2 4.3 4.4 1.0 1.1 4.4 4.4 4.4 1.0 1.0 3.9 4.0 4.0 1.1 1.1 4.4 4.6 4.6 1.1 1.2 4.0 4.4 4.6 1.3 1.5 SSO2131 -1.0 0.2 -0.9 -0.8 2.4 1.1 1.2 -1.8 0.5 0.5 5.1 4.9 -1.6 0.0 0.6 3.0 4.6 -2.5 -0.3 -1.0 4.6 2.9 -4.8 -2.0 -0.8 6.9 16.2 -3.6 -1.0 -0.2 6.2 10.9 SSO2827 0.5 2.6 1.9 1.4 4.1 2.6 1.9 2.2 3.4 3.3 2.2 2.0 2.2 3.3 3.3 2.1 2.0 1.8 3.4 3.6 3.0 3.4 1.6 3.2 3.4 3.1 3.5 0.6 2.6 3.7 3.9 8.4 Misc regulators SSO10340 -0.4 1.2 0.3 0.2 2.9 1.6 1.4 1.5 2.1 1.7 1.5 1.2 3.2 3.8 3.7 1.5 1.4 2.3 3.4 3.3 2.1 2.0 0.4 1.9 2.0 2.8 3.1 1.3 2.6 3.4 2.5 4.5

Table 5. Toxin-Antitoxin Loci and PIN-domain proteins L vapB6) BL vapC22) B vapC6) BL BL TetR) Δ Δ Δ Δ

ORF name PBL2025 HS 30 HS PBL2025 PBL2025 BL PBL2025 10 HS PBL2025 10-BL FC 30-BL FC ( PBL2078 10 HS PBL2078 30 HS PBL2078 10 HS PBL2080 30 HS PBL2080 ( PBL2067 10 PBL2084 10-BL FC Cate gory (gene s ymbol ) P2 BL SAB P2 05 SAB P2 30 SAB P2 60 SAB 5-BL FC 30-BL FC 60-BL FC 10-BL FC 30-BL FC ( PBL2080 10-BL FC 30-BL FC 10 PBL2067 30 PBL2067 10-BL FC 30-BL FC ( PBL2084 30 PBL2084 30-BL FC

TA loci vapB-1 2.8 2.2 2.2 1.6 1.6 2.2 1.2 0.7 2.0 2.8 3.7 2.8 3.1 1.8 1.4 2.5 0.6 0.3 3.7 4.4 1.9 0.4 -0.2 3.0 4.5 SSO0414 va pC-1 -0.1 -2.9 -2.1 -1.1 6.9 4.1 2.0 1.1 -0.5 -0.8 3.2 3.8 1.7 -0.1 -0.1 3.4 3.5 0.6 -1.0 -1.2 3.0 3.5 0.7 -0.6 -0.4 2.6 2.1 1.5 -0.5 -0.6 3.8 4.2 vapB-4 0.1 1.2 1.2 2.2 2.1 0.5 2.1 2.0 3.0 2.9 1.7 2.7 3.0 2.1 2.6 -1.3 -0.1 0.6 2.4 3.7 -0.3 1.4 2.4 3.2 6.9 SSO1243 va pC-4 -2.0 -1.3 -1.6 -1.8 1.6 1.3 1.1 -0.3 0.5 0.4 1.8 1.6 -0.1 0.3 0.8 1.4 1.8 -0.5 0.6 1.0 2.1 2.8 -1.3 -0.9 -0.5 1.3 1.7 -0.9 -0.1 1.6 1.7 5.5 SSO1494 va pB-6 0.2 4.0 3.0 2.6 14.0 7.0 5.3 -0.3 1.7 2.0 4.0 4.9 -3.3 -1.9 -1.7 2.7 3.1 3.2 3.7 3.9 1.4 1.7 -0.5 3.3 3.3 13.5 13.5 -1.0 3.2 3.7 19.4 25.9 SSO1493 va pC-6 0.7 5.0 3.7 3.1 20.0 8.1 5.2 -2.1 1.2 1.1 10.1 9.3 -4.1 -2.4 -1.5 3.3 6.1 -4.4 -2.3 -1.9 4.1 5.5 0.2 3.7 3.8 10.7 11.9 -1.0 3.2 3.5 18.4 22.5 vapB-7 -1.6 0.6 0.1 4.8 3.4 -0.4 2.0 2.4 5.4 6.9 -0.2 1.6 1.6 3.3 3.6 -1.5 0.8 1.3 5.2 6.9 -0.1 2.3 3.3 5.3 10.4 SSO1651 va pC-7 3.2 3.2 2.5 2.6 1.0 1.7 1.6 4.8 4.3 4.4 1.4 1.3 4.2 3.8 3.9 1.3 1.2 4.2 4.0 4.1 1.1 1.1 3.0 2.3 1.8 1.5 2.2 3.0 2.9 2.4 1.0 1.5 SSO8620 va pB-8 -1.0 3.0 1.7 1.3 16.0 6.6 5.1 1.0 4.2 3.8 9.1 7.1 1.1 3.9 4.0 7.2 7.6 0.2 3.7 4.0 10.8 13.6 -0.8 3.1 3.9 14.8 25.7 -0.7 3.6 4.2 20.0 29.3 SSO1657 va pC-8 -0.6 2.3 1.2 1.0 7.6 3.4 3.1 -2.5 0.6 0.9 8.6 10.0 -0.5 2.6 3.4 8.4 14.9 -0.1 1.6 0.8 3.3 1.9 -0.8 1.9 2.9 6.6 12.8 -0.3 2.7 3.5 7.9 13.9 vapB-9 -1.4 -0.8 -1.2 1.5 1.2 -0.9 -0.6 -0.7 1.2 1.1 -0.5 -0.1 -0.3 1.3 1.2 -1.5 -1.9 -2.0 1.4 1.4 -1.5 -1.3 -1.5 1.1 1.0 SSO8813 va pC-9 -3.0 -2.9 -3.2 -3.3 1.0 1.2 1.2 -1.3 -1.5 -1.5 1.2 1.1 -1.8 -2.1 -2.0 1.2 1.1 -1.9 -2.0 -2.3 1.1 1.3 -1.9 -2.7 -3.0 1.7 2.2 -2.3 -2.0 -2.0 1.2 1.2 vapB-10 2.1 3.3 3.1 2.3 2.0 0.8 2.3 3.1 2.9 5.0 3.1 3.8 3.9 1.5 1.7 -1.8 1.1 1.9 7.2 12.6 -0.2 2.1 3.5 5.0 12.6 SSO1746 va pC-10 1.6 3.1 2.5 2.2 2.9 1.9 1.5 1.8 3.0 3.0 2.4 2.2 1.4 3.2 3.7 3.4 4.9 3.3 4.0 3.1 1.6 1.1 0.6 3.1 3.5 5.5 7.3 0.2 3.1 3.5 7.8 9.7 vapB-11 -0.7 2.4 2.3 8.1 7.6 0.7 3.0 3.2 4.9 5.6 1.3 2.8 3.4 2.8 4.3 0.4 2.9 3.4 5.7 7.9 0.1 2.9 3.7 7.1 12.8 SSO1786 va pC-11 -0.3 1.7 0.9 1.1 4.0 2.4 2.6 1.4 3.3 3.8 3.8 5.4 1.8 3.4 3.9 2.9 4.4 0.3 2.6 3.1 4.9 7.1 0.3 2.7 3.8 5.1 11.4 1.1 3.0 3.8 3.6 6.4 SSO1867 va pB-12 0.3 1.3 0.8 0.2 2.1 1.4 1.1 -0.2 0.7 0.8 1.8 2.0 -0.8 0.4 0.9 2.2 3.2 -0.5 0.3 1.2 1.8 3.4 0.8 1.9 2.5 2.1 3.2 0.6 2.1 3.2 2.9 6.1 SSO1868 va pC-12 -1.5 0.1 -0.7 -0.9 3.0 1.7 1.5 -1.1 -0.5 0.3 1.5 2.5 -1.7 -0.9 0.0 1.7 3.2 -0.7 -0.1 0.2 1.6 1.9 -2.2 -0.7 -0.3 2.9 3.8 -2.4 -1.2 -0.2 2.4 4.7 vapB-13 0.9 3.6 3.1 6.6 4.6 1.9 3.8 4.0 3.6 4.2 1.7 3.8 3.9 4.4 4.6 0.2 3.1 3.2 7.4 8.2 1.0 2.9 3.5 3.8 5.9 SSO1914 va pC-13 -0.8 2.2 1.4 0.5 7.9 4.6 2.5 -1.9 0.0 0.1 3.6 4.0 -0.9 1.2 1.9 4.4 6.8 -1.0 1.1 1.3 4.2 4.9 -0.2 2.5 2.9 6.6 8.5 -2.4 0.0 0.7 5.4 8.7 vapB-14 0.5 0.5 0.3 1.0 1.1 -0.8 0.2 0.0 2.0 1.7 2.0 1.8 1.6 1.1 1.3 -2.8 -2.2 -1.7 1.5 2.2 -1.0 -0.4 -0.5 1.5 1.4 SSO1922 va pC-14 -1.9 -0.9 -2.0 -2.0 2.0 1.0 1.1 -1.9 -0.1 -0.9 3.4 2.0 -1.5 -0.1 0.0 2.8 2.8 -4.1 -1.6 -1.6 5.8 5.8 -5.5 -3.2 -1.4 4.9 17.6 -2.1 -0.9 -1.1 2.4 2.1 SSO9378 va pB-15 -0.7 2.1 0.7 0.2 7.3 2.8 1.9 2.1 4.0 3.9 3.8 3.6 2.0 3.7 3.6 3.2 2.9 1.8 3.9 3.8 4.3 3.9 1.8 3.5 4.0 3.3 4.7 1.2 3.6 4.1 5.3 7.6 SSO1968 va pC-15 -0.5 0.2 -0.4 -0.6 1.6 1.1 1.1 2.1 2.0 2.7 1.1 1.5 1.1 1.3 2.2 1.1 2.1 0.1 1.0 1.4 1.8 2.5 0.8 1.3 1.7 1.4 1.9 0.1 1.3 2.3 2.3 4.6 vapB-16 -1.4 0.7 0.3 4.4 3.3 -0.2 1.5 1.6 3.4 3.7 -0.2 1.2 1.5 2.7 3.4 -1.5 0.1 0.6 3.2 4.5 -1.2 1.0 1.8 4.6 7.9 SSO1969 va pC-16 -1.2 -0.9 -1.5 -1.6 1.2 1.2 1.3 -0.6 0.0 0.1 1.6 1.7 0.8 1.2 1.7 1.3 1.8 -1.8 -0.7 -0.3 2.1 2.8 -1.7 -1.3 -0.5 1.4 2.4 -1.1 -0.5 1.1 1.6 4.6 vapB-17 2.6 2.7 2.5 1.0 1.1 1.6 2.2 2.7 1.5 2.1 3.7 3.6 3.6 1.1 1.0 -0.8 0.0 0.5 1.8 2.5 0.3 1.4 2.6 2.1 4.7 SSO1970 va pC-17 -1.2 -0.7 -1.3 -1.5 1.5 1.1 1.2 2.4 1.8 2.0 1.6 1.3 2.2 1.6 2.1 1.5 1.1 2.6 2.3 1.8 1.3 1.8 1.4 1.4 1.4 1.0 1.0 1.2 1.9 1.9 1.7 1.6 vapB-19 0.0 2.1 2.1 4.2 4.4 1.4 3.2 3.5 3.4 4.4 1.3 2.7 3.2 2.7 3.8 0.6 2.7 3.6 4.4 7.7 0.6 3.1 3.7 5.7 8.8 SSO2096 va pC-19 0.7 2.9 3.5 3.4 4.6 7.3 6.9 1.2 2.8 3.2 3.0 3.8 2.0 3.6 4.0 2.9 3.9 0.4 2.4 3.5 4.1 8.8 -0.1 2.2 3.3 4.9 10.8 0.6 2.6 3.8 4.0 9.0 vapB-20 0.0 0.2 0.2 1.2 1.2 0.9 1.0 1.1 1.1 1.2 0.9 0.9 0.9 1.0 1.0 0.4 0.6 0.8 1.1 1.3 2.1 2.3 1.9 1.1 1.1 SSO2218 va pC-20 0.3 1.1 0.7 1.0 1.7 1.3 1.6 2.3 2.4 2.6 1.1 1.3 1.9 2.1 2.5 1.1 1.5 1.5 2.1 2.2 1.5 1.6 1.9 2.5 2.7 1.5 1.7 2.4 3.0 3.0 1.5 1.5 SSO2579 va pC-21 -1.4 0.0 -0.4 0.2 2.5 1.9 2.9 -3.0 -1.7 -1.7 2.5 2.4 -2.2 -1.2 -1.0 2.0 2.4 -3.6 -1.6 -1.2 4.0 5.2 -2.6 -0.3 -0.1 5.1 5.8 -1.6 0.0 0.4 2.9 4.0 SSO11914 va pB-22 4.6 5.7 5.2 5.2 2.2 1.5 1.5 4.1 4.2 4.4 1.0 1.2 4.1 4.0 4.1 1.1 1.0 3.0 3.5 4.2 1.4 2.2 2.3 3.6 4.2 2.5 3.8 2.7 3.3 3.9 1.5 2.2 SSO3078 va pC-22 3.7 4.8 4.4 4.1 2.1 1.6 1.3 3.8 4.2 4.1 1.4 1.2 3.0 3.8 3.8 1.7 1.7 2.8 3.5 3.9 1.6 2.1 0.3 1.3 2.0 1.9 3.2 4.0 4.0 4.0 1.0 1.0 SSO12018 va pB-23 0.0 0.4 0.0 -0.4 1.4 1.0 1.3 0.4 0.9 0.2 1.5 1.1 0.6 0.9 0.5 1.3 1.1 1.0 1.5 1.8 1.4 1.8 0.0 0.0 0.2 1.1 1.2 -0.6 0.3 1.4 1.8 3.9 SSO3128 vapC-23 -2.3 -1.7 -1.9 -2.4 1.5 1.4 1.1 -1.5 -1.9 -2.2 1.4 1.6 -1.5 -1.3 -1.1 1.1 1.3 -2.2 -1.5 -0.6 1.7 3.0 -3.7 -2.4 -1.1 2.5 6.2 -2.5 -2.1 -1.4 1.3 2.1 PIN-domain SSO1701 PIN-domain 0.1 4.5 4.0 3.6 20.2 14.6 11.3 1.9 3.8 3.9 3.8 4.0 2.5 3.9 4.3 2.7 3.4 1.6 3.9 4.2 5.0 6.1 1.2 4.0 4.2 7.1 8.2 2.0 3.9 3.8 3.8 3.5 proteins SSO1921 PIN-domain -0.8 2.9 1.7 1.3 12.3 5.3 4.0 -1.2 2.4 2.7 12.2 15.6 -2.6 1.1 2.1 12.5 25.9 -3.3 0.9 1.7 18.0 32.3 -4.7 0.5 1.8 38.8 91.9 -2.6 1.5 2.6 17.8 36.3 SSO2101 PIN-domain -1.4 1.9 2.1 1.9 9.7 11.2 10.0 -1.8 1.3 1.6 8.7 10.9 -2.5 0.8 0.9 9.5 10.6 -4.3 -0.2 1.0 16.5 37.9 -3.5 0.9 2.0 19.9 44.1 -2.5 0.5 1.2 8.1 13.2 SSO2783 PIN-domain -0.8 1.5 0.9 1.0 5.0 3.2 3.5 0.6 2.3 2.6 3.2 4.0 1.5 2.6 3.5 2.2 3.9 0.4 2.2 2.8 3.4 5.2 0.2 2.0 2.6 3.5 5.4 1.2 2.5 3.6 2.4 5.2 X indicates probe was missing from original microarray. Light yellow indicated the p-value falls bellows the acceptable cut-off. Dark yellow indicated the highest significant fold- change for the gene in each microarray loop. LSM values are shown in columns preceeding the fold-changes.

FIGURES

\ Sso PBL2025

Sso P2 40 vapBC-2 vapBC-3 vapBC-5 vapBC-18

b. 30

25 20

5 0nt 1nt 90nt 186nt or more Difference between Sso P2 Toxin and Antitoxin Genes found in Sso PBL2025 (# of nt) 0

Figure 1. Comparison of S. solfataricus P2 to PBL2025. (a) Genome alignment using MAUVE (43). Missing Vaps are indicated on genome map. (b) The Vap homologues in S. solfataricus P2 and PBL2025 are compared on the nt level.

Figure 2. Fermentor set-up. A 3-L Applikon fermentor was adapted for heat shock experiments. A heating jacket and recirculating hot water were used to quickly heat the culture. Samples could be harvested quickly through the sampling line using a vacuum pump and side-arm flasks.

T (oC)

0.08 90 85 80 75 70

0.07 ) -1 0.06 k (hr

0.05

0.04 2.75 2.80 2.85 2.90 o 1000/T ( K)

Figure 3. Optimum growth temperatures for PBL2025 and mutant strains. Closed circles (●) represent wild-type PBL2025 and open circles (○) represent vapB6::lacS disruption strain PBL2066. Due to the mutations, the optimum growth temperature is shifted slightly (112).

Baseline—10 min. before HS

30 min. after 10 min. after HS HS

Figure 4. Experimental design for microarray hybridization. Samples were taken 10 min before starting the temperature shift (80Æ90oC) and then 5 and 10 min. after reaching 90°C. cDNA samples were then hybridized in a three-slide loop design. Dots (●) and arrowheads (◄) represent cyanine 3- and cyanine 5-labeled samples, respectively.

PBL2025 HS Curve 1.00E+09

1.00E+08 Heat shock

growth curve Cells/mlCells/ml HS #1 1.00E+07 HS #2

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 1.00E+06 Hours

Figure 5. Wild-type growth curves. Strain PBL2025 growth curves for heat shock experiments. HS is indicated with arrows for the biological repeats.

Figure 6. Wild-type Venn diagram of total genes significantly regulated. Gene totals are indicated in parentheses for each timepoint.

100

Figure 7. Heat plots of TA loci and PIN-domain proteins. X indicates the probe was missing from the array at the time of the experiment. Red indicated the transcript levels are above average and blue indicates the transcript levels are below average. Gray represents average transcript levels.

101

Figure 8. Heat plot of Sso transcriptional regulators. Differential transcription of the 13 transcriptional regulators during heat shock.

102

PBL2026 HS Curve 1.00E+09

1.00E+08

Heat shock

Cells/ml Cells/ml growth curve HS #1 1.00E+07 HS #2

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 1.00E+06 Hours

Figure 9. ΔTetR mutant growth curves. Strain PBL2026 growth curves for heat shock experiments. HS is indicated with arrows for the biological repeats.

103

Figure 10. ΔTetR mutant (strain PBL2026) Venn diagram of total genes significantly regulated. Gene totals are indicated in parentheses for each timepoint.

104

PBL2067 HS Curve

1.00E+09

1.00E+08

Heat shock

Cells/mlCells/ml growth curve HS #1 1.00E+07 HS #2

1.00E+06 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Hours

Figure 11. ΔVapC-22 mutant growth curves. Strain PBL2067 growth curves for heat shock experiments. HS is indicated with arrows for the biological repeats.

105

Figure 12. ΔVapC22 mutant (strain PBL2026) Venn diagram of total genes significantly regulated. Gene totals are indicated in parentheses for each timepoint.

106

PBL2078 HS Curve

1.00E+09

1.00E+08 Heat shock

Cells/mlCells/ml growth curve HS #1 HS #2 1.00E+07

1.00E+06 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Hours

Figure 13. ΔVapBC-6 mutant growth curves. Strain PBL2078 growth curves for heat shock experiments. HS is indicated with arrows for the biological repeats.

107

PBL2080 HS Curve 1.00E+09

1.00E+08 Heat shock

Cells/mlCells/ml growth curve HS #1 1.00E+07 HS #2

1.00E+06 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Hours

Figure 14. ΔVapC-6 mutant growth curves. Strain PBL2078 growth curves for heat shock experiments. HS is indicated with arrows for the biological repeat.

108

PBL2078 PBL2080

Figure 15. ΔVapBC-6 mutant (strain PBL2078) and ΔVapC-6 mutant (strain PBL2080) Venn diagrams of total genes significantly regulated. Gene totals are indicated in parentheses for each timepoint.

109

APPENDICIES

110

APPENDIX 3.A

Table S1: Log2 values of all genes significantly regulated 2-fold or more (log2≥1) PBL2025 PBL2078 PBL2080 PBL2067 PBL2026

Name Annotation (10)-(BL) (30)-(BL) (10)-(30) (10)-(BL) (30)-(BL) (10)-(30) (10)-(BL) (30)-(BL) (10)-(30) (10)-(BL) (30)-(BL) (10)-(30) (10)-(BL) (30)-(BL) (10)-(30) SSO0006 Metallo-beta-lactamase superfamily, putative -1.4 -1.3 -1.0 -1.4 -1.1 -1.4 -1.2 -1.2 -1.3 SSO0008 hypothetical protein 2.6 2.2 2.3 2.1 2.4 2.3 2.5 2.2 1.9 1.8 SSO0010 proline dipeptidase, putative -1.0 SSO0012 conserved hypothetical transmembrane protein, putative 1.0 SSO0014 conserved hypothetical protein -1.3 -1.6 -1.3 -1.1 SSO0015 Domain of unknown function, putative -1.4 -1.0 -1.6 -1.0 -1.7 -1.4 SSO0016 exsB protein, putative -1.2 SSO0018 4Fe-4S binding domain protein -1.9 -1.8 -1.8 -2.3 -1.7 -1.7 -1.8 -2.0 -1.9 -2.2 SSO0019 molybdenum cofactor biosynthesis protein B, putative -1.2 SSO0021 hypothetical protein -1.0 -1.4 -1.0 SSO0023 hypothetical protein 1.1 1.3 SSO0024 conserved hypothetical protein -1.2 -1.2 -1.2 SSO0026 phosphoribosylglycinamide formyltransferase 2 -1.4 -1.2 -1.1 -1.8 -1.2 -1.8 SSO0031 Carbon-nitrogen hydrolase, putative 1.1 1.1 1.0 SSO0032 hypothetical protein -1.0 SSO0034 ParA family protein, putative -2.2 -2.8 -1.8 -2.6 -2.6 -2.4 -2.1 -2.6 -2.5 -2.4 SSO0035 conserved hypothetical protein -1.0 -2.1 -2.2 -2.1 -1.9 SSO0036 hypothetical protein 1.0 SSO0037 hypothetical protein -1.6 -1.8 -1.1 -1.1 -1.2 -1.4 -1.3 SSO0038 conserved hypothetical protein 1.2 1.5 1.6 2.1 1.6 2.2 1.3 1.9 2.0 2.6 SSO0040 conserved hypothetical protein 1.3 1.2 1.4 1.6 1.0 1.4 SSO0041 conserved hypothetical protein 1.9 2.1 1.3 2.0 2.0 1.9 2.0 2.2 SSO0043 conserved hypothetical protein 1.0 SSO0044 subunit I-homologue of cytochrome oxidase, putative -1.0 -1.2 SSO0045 subunit of the terminal oxidase with unknown homologue -1.1 -1.2 -1.1 -1.0 SSO0046 hydrolase, putative 1.3 2.0 1.6 2.3 1.9 2.9 -1.1 2.5 1.2 2.6 -1.4 SSO0048 transcriptional regulator Lrs14 1.2 1.3 SSO0049 30S ribosomal protein S14 homolog 1.8 1.7 1.9 2.1 2.1 3.2 -1.1 1.8 2.6 2.0 2.8 Annotations are from TIGR/JCVI Database.

111

Table S1 Continued SSO0051 conserved hypothetical protein -1.1 -1.6 1.1 SSO0057 conserved hypothetical protein 1.01.1 1.01.0 1.61.8 SSO0060 UDP-N-acetylglucosamine-dolichyl-P N-AcGluNH-phosphotransferase -1.3 -2.0 SSO0061 geranylgeranyl pyrophosphate synthetase -1.0 -1.5 -1.8 -1.4 -1.7 SSO0064 conserved hypothetical protein -1.8 -1.6 SSO0065 conserved hypothetical protein -1.8 -1.9 -1.7 -2.0 -1.4 -1.2 -2.1 -1.6 -1.8 -1.7 SSO0066 Protein of unknown function superfamily -1.8 -1.8 -1.4 -1.7 -1.2 -1.2 -1.2 -1.4 SSO0067 ribosomal protein S2 -1.4 -1.3 -1.0 -1.3 -1.6 SSO0071 DNA-directed RNA polymerase subunit D -1.5 -1.5 -1.5 -1.4 -1.2 -1.1 -1.6 -2.0 SSO0072 ribosomal protein S11 -1.4 -1.7 -1.4 -1.9 -1.2 -1.4 -1.0 -1.4 -2.0 SSO0073 ribosomal protein S4 -1.0 -1.1 -1.1 -1.8 -1.2 -1.3 -1.3 -1.5 SSO0074 30S ribosomal protein S13p -1.4 -1.4 -1.2 -1.4 -1.4 -1.6 SSO0078 tyrosyl-tRNA synthetase 1.0 1.7 -1.0 SSO0079 Toprim domain protein 1.6 1.4 2.3 1.1 SSO0082 Acetyltransferase (GNAT) family family 2.1 1.6 1.6 1.7 1.9 1.8 2.1 2.5 2.1 2.5 SSO0083 cell division protein pelota 1.3 1.0 1.2 1.4 SSO0084 ribosomal protein S15 -1.2 SSO0085 conserved hypothetical protein -1.1 -1.2 SSO0087 Sterol-regulatory element binding protein (SREBP) site 2 protease fami -1.3 -1.1 SSO0091 30S ribosomal protein hs6-like -1.6 -2.2 -1.0 SSO0097 conserved hypothetical protein -1.2 -1.1 SSO0098 methionine aminopeptidase, type II -1.4 -1.2 -1.0 -1.1 -1.6 -1.9 1.1 SSO0099 conserved hypothetical protein -1.4 -1.2 -1.3 -1.1 SSO0100 probable phenylalanyl-tRNA synthetase -1.3 -1.5 -1.9 -1.2 -1.7 -1.8 -1.6 -2.1 SSO0101 phenylalanyl-tRNA synthetase, beta subunit -1.8 -1.8 -1.1 -1.5 -1.6 -1.4 -1.8 -1.5 -2.0 -1.7 SSO0106 conserved hypothetical protein -1.1 -1.2 SSO0107 Helix-turn-helix domain protein -1.5 -2.0 -1.0 SSO0108 conserved hypothetical protein -2.9 -3.4 -1.9 -2.1 -2.2 -2.8 -2.4 -2.7 -2.6 -2.9 SSO0111 1.7 SSO0112 ATP-dependent helicase -1.3 SSO0116 endonuclease III -1.4 -1.2 SSO0117 conserved hypothetical protein -2.5 -3.3 -2.2 -2.8 -1.6 -2.3 -1.9 -2.8 -2.6 -3.0 SSO0118 c04004 -1.5 -2.0 -1.7 -2.2 1.4 -1.7 1.2 -1.7 SSO0119 Integral membrane protein superfamily -1.4 -1.4 -1.0 -1.5 -1.1 -1.4 -1.9 -1.2 -1.4 SSO0120 virB homolog -1.6 -2.1 -1.6 -2.2 -3.4 1.2 -2.2 -2.5

112

Table S1 Continued SSO0121 conserved hypothetical protein -1.6 -1.8 -1.2 -1.2 -1.2 -1.8 -1.2 -1.8 SSO0123 hypothetical protein 2.3 2.8 SSO0124 C-terminal part of protein without known homologue -1.2 -1.2 -1.1 -1.2 -1.1 SSO0125 4-hydroxybenzoate octaprenyltransferase, putative -1.2 -1.1 -1.6 -1.7 -1.1 -1.2 -1.3 -2.2 SSO0127 2-isopropylmalate synthase/homocitrate synthase family protein -1.5 -2.0 -1.1 -1.4 -1.4 -1.7 -2.0 -1.6 -1.8 SSO0131 hypothetical protein -1.2 1.0 SSO0133 hypothetical protein 1.1 1.3 1.2 1.6 1.3 1.6 SSO0134 hypothetical protein 1.5 1.3 1.2 1.3 1.5 1.0 1.2 1.5 SSO0141 conserved hypothetical protein 1.5 1.2 1.8 1.5 1.1 1.4 1.3 1.9 SSO0144 transporter, NadC/P/Pho87 family, putative -1.3 -1.1 SSO0149 hypothetical protein -1.1 SSO0150 hypothetical protein -1.0 SSO0152 hypothetical protein -1.1 -1.1 -1.3 -1.3 -1.5 -1.5 -1.4 -1.3 SSO0159 ribosomal protein S6 modification protein 1.0 1.2 1.4 1.1 SSO0160 acetylornithine aminotransferase 1.0 SSO0162 Peptidase family M20/M25/M40 superfamily 1.3 1.5 2.0 1.6 2.6 1.0 1.8 SSO0164 ribosomal protein S8.e -1.4 -1.1 SSO0165 SRP19 protein -1.0 SSO0168 hypothetical protein -1.2 -1.2 -1.3 -1.0 SSO0169 hypothetical protein -1.0 SSO0173 aspartyl-tRNA synthetase -1.1 -1.7 -1.8 SSO0174 Uncharacterized protein family UPF0024 superfamily -1.0 -1.1 -2.5 SSO0175 conserved hypothetical protein -1.8 SSO0176 transitionAL endoplasmic reticulum ATPase -1.1 1.9 -1.5 SSO0179 FEN-1 2.3 -1.8 SSO0182 glutamate-1-semialdehyde-2,1-aminomutase -1.1 -1.0 SSO0183 porphobilinogen deaminase, putative 1.2 1.4 1.9 1.1 1.9 1.6 1.9 SSO0184 uroporphyrinogen III synthase, putative 1.4 -1.2 1.4 1.6 -1.2 1.3 -1.5 SSO0185 conserved hypothetical protein -1.1 -1.2 SSO0189 DNA ligase 1.0 1.4 -1.0 1.3 SSO0190 deoxycytidine triphosphate deaminase, putative -1.8 -1.8 -1.5 -1.5 -1.7 -1.4 -1.9 -1.9 -1.7 -1.6 SSO0195 conserved hypothetical protein 1.2 1.9 1.5 2.3 1.2 1.4 2.0 2.7 1.7 2.1 SSO0197 RIO1/ZK632.3/MJ0444 family family 1.1 1.2 SSO0198 conserved hypothetical protein 1.1 1.5 1.5 1.4 1.8 1.1 1.8 1.4 SSO0199 Protein of unknown function superfamily -1.7 -2.5 -1.4 -1.7 -1.5 -2.0 -1.7 -1.9 -2.1 -1.6

113

Table S1 Continued SSO0200 transcriptional regulator, ArsR family domain protein -1.2 SSO0202 hexulose-6-phosphate synthase SgbH, putative -1.1 -1.5 SSO0206 conserved hypothetical protein -1.1 -1.2 -1.1 -1.6 -1.5 -1.4 -1.5 -1.2 -1.4 -1.3 SSO0207 phosphoglucomutase/phosphomannomutase family protein, putative -1.1 -1.0 -1.1 -1.4 -1.0 -1.0 -1.1 -1.0 SSO0210 dfp protein -1.1 -1.2 SSO0214 conserved hypothetical protein -1.2 SSO0215 ribosomal protein S10 -1.7 -1.8 -1.5 -2.0 -1.3 -1.7 -1.4 -1.4 -1.9 -2.5 SSO0216 translation elongation factor EF-1, subunit alpha -1.6 -1.3 SSO0217 ribosomal protein S7 -1.4 -1.5 -1.1 -1.4 -1.2 -1.3 -1.2 -1.1 -1.1 -1.7 SSO0218 conserved hypothetical protein 1.0 1.3 1.0 SSO0220 N utilization substance protein A, putative -1.0 SSO0225 DNA-directed RNA polymerase subunit a -1.3 -1.0 -1.2 -1.4 -1.2 -1.9 -1.7 -1.5 -1.7 SSO0227 DNA-directed RNA polymerase subunit b -1.2 -1.7 -1.3 -1.0 -1.6 -1.8 SSO0228 translation initiation factor aIF-2 1.0 SSO0230 nucleoside diphosphate kinase I -1.5 SSO0231 uracil phosphoribosyltransferase -1.7 -1.4 SSO0232 glutamyl-tRNA(Gln) amidotransferase, B subunit -1.3 -1.2 -2.0 -1.6 SSO0233 Phosphoribosyl transferase domain protein -1.4 -1.2 -1.3 -1.5 -1.1 -1.6 -1.1 -2.2 -1.9 SSO0234 hypothetical protein -1.2 -1.1 -1.1 -1.6 -1.4 SSO0235 moaA/nifB/pqqE family protein, putative -1.3 -1.5 -1.1 -1.1 -1.3 SSO0239 conserved hypothetical protein -1.1 -1.4 -1.3 SSO0240 adenylosuccinate lyase -1.2 -1.4 -1.2 -1.6 -1.6 -1.6 -1.4 -1.4 -1.4 -1.7 SSO0246 chorismate mutase/prephenate dehydratase, putative -1.0 SSO0250 DNA repair protein rada 1.1 SSO0251 conserved hypothetical protein -1.0 -1.0 SSO0254 Protein of unknown function superfamily 1.1 1.2 1.0 SSO0256 Protein of unknown function superfamily -1.3 -1.6 -1.9 -2.4 -2.4 -2.1 SSO0257 ATPase, AAA family domain protein -1.2 -1.6 -3.2 -4.2 -3.0 -2.2 SSO0261 conserved hypothetical protein -1.1 -1.4 SSO0262 conserved hypothetical protein 1.1 1.2 SSO0264 invalid gene, putative 1.3 1.6 SSO0265 5-methylcytosine methyltransferase 1.0 1.2 1.4 1.3 SSO0266 conserved hypothetical protein 1.0 1.5 1.4 1.0 1.4 1.1 1.7 SSO0267 Protein of unknown function superfamily -1.1 -1.1 1.1 -1.1 SSO0269 GTP-binding protein HflX 1.2 -1.1

114

Table S1 Continued SSO0270 conserved hypothetical protein SSO0271 26S protease regulatory subunit 4 -1.3 -1.7 -2.3 -2.4 -1.8 -2.0 SSO0273 conserved hypothetical protein -1.0 SSO0280 conserved hypothetical protein -1.0 -1.1 -1.6 -1.3 SSO0281 tRNA intron endonuclease, putative -1.0 SSO0283 Domain of unknown function domain protein -1.1 -1.1 -2.1 -1.3 SSO0284 hypothetical protein -1.3 -1.1 SSO0287 RNase L inhibitor 1.1 1.1 1.7 1.5 SSO0290 inorganic phosphate transporter, putative -1.5 -1.3 -1.2 -1.4 -1.8 -1.7 1.0 SSO0292 conserved hypothetical protein -1.2 -1.3 -1.6 -1.1 -1.1 -1.0 -1.5 -1.5 SSO0293 diphthamide biosynthesis protein DPH2-related protein -1.7 -1.7 -1.3 -1.7 -1.5 -1.2 -1.3 -1.1 -1.6 -2.1 SSO0294 ribosomal protein L10.e -1.4 SSO0295 hypothetical protein -1.1 -1.3 SSO0296 hypothetical protein -1.2 -1.8 -2.0 -2.7 -2.1 1.2 SSO0297 transketolase, putative -1.0 -1.0 SSO0298 thiF protein, putative -1.1 -1.1 -1.1 -1.4 SSO0299 transketolase C-terminal section -1.0 SSO0302 chorismate mutase/prephenate dehydrogenase, putative -1.0 -1.0 -1.0 -1.1 -1.4 SSO0305 3-dehydroquinate synthase -1.0 SSO0306 shikimate 5-dehydrogenase -1.2 -1.3 -1.1 SSO0313 DNA REPAIR HELICASE RAD3, putative -1.0 SSO0314 conserved hypothetical protein 1.0 SSO0316 superoxide dismutase (EC 1.15.1.1) (Fe/Mn) [validated] -1.5 -1.3 -1.3 SSO0318 hypothetical protein -1.1 -1.1 -1.0 -1.2 SSO0320 hypothetical protein -1.4 -1.5 -1.1 -1.1 -1.1 -1.3 SSO0321 Thymidylate synthase complementing protein family -1.4 -1.3 -1.0 -1.3 -1.3 SSO0324 NADH dehydrogenase I chain D -1.1 -1.7 -2.1 -1.3 -1.5 -1.0 -1.5 -1.9 SSO0325 F420H2:quinone oxidoreductase, 41.2 kDa subunit, putative, putative -1.0 -1.8 -2.1 -1.2 -1.0 -2.0 -1.7 -2.1 -2.0 SSO0326 NADH dehydrogenase I chain i -1.5 -1.1 -1.7 -1.8 -1.7 -1.5 -2.5 -1.9 -2.3 -1.9 SSO0327 NADH dehydrogenase I chain J, putative -1.5 -1.8 -2.2 -2.2 -1.9 -1.9 -2.4 -1.9 -2.2 -1.8 SSO0328 hypothetical NADH dehydrogenase (ubiquinone) chain 13 -2.1 -2.7 -2.6 -2.6 -2.1 -2.0 -2.9 -2.6 -2.3 -2.4 SSO0329 NADH dehydrogenase subunit 2, putative -1.4 -2.1 -2.5 -1.0 -2.9 -3.0 -1.9 -2.3 SSO0330 1L-myo-inositol-1-phosphate synthase -1.1 -1.1 -1.5 -1.9 -1.1 -1.3 SSO0333 thiazole biosynthesis protein ThiI 1.1 1.1 1.2 1.5 1.6 1.2 1.1 SSO0337 conserved hypothetical protein 2.3 1.4 1.6 1.9 1.0 1.3

115

Table S1 Continued SSO0340 conserved hypothetical protein 1.1 -1.0 -1.4 SSO0341 alanyl-tRNA synthetase -1.0 SSO0344 ribosomal protein L10 -1.5 -1.7 -1.0 -1.4 -1.1 -1.2 SSO0345 L1P family of ribosomal proteins -1.5 -1.7 -1.3 -1.2 SSO0348 signal recognition particle-docking protein FtsY -1.2 -1.1 -1.1 -1.3 SSO0349 conserved hypothetical protein -1.3 -1.1 SSO0351 translation initiation factor eIF-6, putative -1.2 1.3 -1.1 SSO0353 Ribosomal protein S19e -1.1 SSO0354 nicotinate phosphoribosyltransferase, putative -1.1 SSO0359 conserved hypothetical protein 1.3 SSO0363 proline dipeptidase, putative -1.0 SSO0365 conserved hypothetical protein 1.1 1.4 1.2 SSO0366 glutamine synthetase, type I -1.8 -2.4 -1.1 -1.8 -1.5 -1.6 -2.0 -2.5 -1.5 -2.0 SSO0367 anion permease 1.6 1.5 1.2 1.5 -1.0 SSO0368 thioredoxin 1 1.0 1.5 -1.0 SSO0369 long-chain-fatty-acid--CoA ligase, putative -1.1 SSO0370 kinase, GHMP family, group 1 1.1 -1.0 SSO0372 carbonic anhydrase, family 3 -1.0 -1.3 -1.0 -1.2 -1.0 SSO0373 heme biosynthesis protein-related -1.3 -1.3 -1.1 -1.3 -1.6 SSO0375 integrase/recombinase XerD -1.6 -1.8 -1.5 -1.3 -1.3 -1.4 -1.6 -1.8 -1.7 -1.7 SSO0376 Sodium/hydrogen exchanger family family 1.2 2.0 2.7 2.1 3.0 3.3 4.0 SSO0379 hypothetical serine-threonine rich protein, putative -1.9 -2.6 -1.9 -2.1 -1.0 -1.8 -2.8 1.0 SSO0381 TDP-D-Glucose synthase, putative -1.0 -1.0 -1.1 -1.0 -1.0 SSO0384 threonyl-tRNA synthetase 1.4 SSO0386 mRNA 3-end processing factor, putative 1.3 SSO0387 Helix-turn-helix domain protein -1.1 SSO0388 LPS biosynthesis protein, putative -1.2 -1.5 -1.6 -2.2 SSO0390 hypothetical protein -1.0 SSO0394 large helicase-related protein 1.4 -1.3 1.7 SSO0395 hypothetical protein -1.2 -1.6 -1.2 SSO0396 hypothetical protein -1.4 -1.7 -1.1 -2.1 -1.3 -1.6 -1.8 -2.1 -1.5 -1.8 SSO0397 proliferating cell nuclear antigen homolog (pcna), putative -1.2 -1.4 -1.4 -1.1 -1.2 -1.8 -1.6 SSO0398 Chlorohydrolase, putative -1.1 SSO0402 3,4-dihydroxy-2-butanone 4-phosphate synthase 1.2 -1.0 SSO0403 conserved hypothetical protein -1.0

116

Table S1 Continued SSO0406 5-methyltetrahydropteroyltriglutamate--homocysteine methyltransferase -1.0 -1.1 SSO0407 5-methyltetrahydropteroyltriglutamate--homocysteine methyltransferase -1.0 -1.2 -1.1 -1.4 -1.1 -1.2 SSO0411 SSU ribosomal protein S6E -1.3 -1.6 -1.1 -1.4 -1.1 -1.1 -1.3 -1.2 -1.7 -1.6 SSO0412 translation elongation factor Tu -2.3 -2.4 -2.1 -2.5 -2.1 -2.5 -1.8 -2.1 -1.8 -2.4 SSO0414 vapC-1 -1.7 -1.9 -1.8 -1.8 -1.6 -1.8 -1.4 -1.1 -1.9 -2.1 SSO0415 DNA-directed RNA polymerase -1.1 -1.1 SSO0420 reverse gyrase -1.4 -1.2 -2.0 -1.9 -1.5 -1.6 SSO0421 transitionAL endoplasmic reticulum ATPase -1.1 SSO0422 Carbon-nitrogen hydrolase superfamily -1.5 -1.5 SSO0427 protein-L-isoaspartate methyltransferase homolog, putative -1.2 -1.3 -1.1 -1.1 -1.3 SSO0428 methyl-accepting chemotaxis protein, putative -1.1 SSO0429 ATP synthase (C/AC39) subunit -1.4 -1.2 -1.5 -1.5 -1.2 -1.2 -1.0 SSO0435 SSU ribosomal protein S24E, putative -1.5 -1.3 SSO0436 thiazole biosynthesis enzyme -1.1 -1.1 SSO0437 3-octaprenyl-4-hydroxybenzoate carboxy-lyase 1.1 1.4 SSO0438 conserved hypothetical protein 1.1 1.1 SSO0439 tRNA intron endonuclease -1.0 -1.0 -1.3 -1.1 -1.0 -1.0 SSO0440 conserved hypothetical protein -1.1 -1.0 -1.3 SSO0444 glycyl-tRNA synthetase -1.0 SSO0445 agmatinase, putative -1.3 1.0 SSO0446 transcription initiation factor iib homolog 1.0 SSO0447 conserved hypothetical protein -1.0 SSO0451 conserved hypothetical protein -3.2 -4.7 -2.0 -1.7 -3.3 1.6 -2.5 -3.6 1.1 -2.4 -3.1 SSO0452 tryptophanyl-tRNA synthetase -1.5 -1.9 -1.1 -1.9 -1.2 -1.9 -1.3 -1.9 -1.1 -2.1 1.0 SSO0454 Helix-turn-helix domain, fis-type protein 1.1 1.1 1.3 SSO0455 putative integral membrane protein, putative -1.0 SSO0458 transcriptional regulator Lrs14, putative 1.3 1.8 1.3 1.0 2.0 1.9 1.9 2.1 SSO0461 conserved hypothetical protein -1.1 -1.1 -1.3 -1.1 -1.5 4.2 -2.5 SSO0465 hypothetical protein 1.1 1.0 SSO0467 hypothetical protein -1.0 SSO0470 translation factor EF-1 alpha, putative-related 4.4 6.7 SSO0472 alcohol dehydrogenase class III 1.6 1.2 1.6 SSO0480 histone acetyltransferase, ELP3 family subfamily 1.1 1.2 1.2 2.2 -1.1 1.2 SSO0482 mttA/Hcf106 family family 1.1 1.3 1.4 SSO0485 Periplasmic binding protein, putative -1.0 -1.1 -1.2

117

Table S1 Continued SSO0486 hemin permease (hemU), putative -1.7 -1.3 -2.0 SSO0488 phosphate ABC transporter, ATP-binding protein -1.2 1.4 -2.6 SSO0496 hypothetical protein -1.6 -2.3 -2.1 -3.3 -4.7 -2.7 -3.3 SSO0497 hypothetical protein -1.7 -2.1 -2.1 -2.8 -1.4 -1.4 -2.3 -2.8 -2.1 -3.0 SSO0499 hypothetical protein -1.2 -1.5 -1.1 -1.4 -1.5 -1.9 SSO0502 conserved hypothetical protein 1.1 SSO0503 conserved hypothetical protein 1.4 SSO0504 leucyl-tRNA synthetase 1.1 -1.1 SSO0505 probable nucleoside hydrolase, putative -1.3 -1.2 SSO0506 hypothetical protein 1.5 2.1 1.9 2.0 SSO0507 hypothetical protein 1.41.8 1.61.9 1.21.8 1.41.5 SSO0509 conserved hypothetical protein 1.1 1.5 1.7 2.5 1.0 1.3 1.7 2.3 1.4 2.3 SSO0510 conserved hypothetical protein 1.0 SSO0511 conserved hypothetical protein 1.1 1.2 SSO0514 putative invertase/transposase 1.9 2.0 1.7 2.0 1.9 2.1 1.7 2.0 1.2 1.3 SSO0518 conserved hypothetical protein 2.7 2.7 1.8 2.0 2.0 2.4 1.7 1.9 1.4 1.6 SSO0519 conserved hypothetical protein -1.1 SSO0520 -1.0 SSO0522 conserved hypothetical protein -1.1 -1.0 SSO0523 conserved hypothetical protein 1.7 SSO0525 putative transposase 1.6 2.4 1.5 2.5 -1.0 1.6 2.1 1.1 1.5 1.5 2.0 SSO0527 phosphoglycerate kinas -1.1 -1.0 SSO0528 glyceraldehyde 3-phosphate dehydrogenase (gap) -1.2 -1.1 -1.3 -1.3 SSO0529 universal stress protein family, putative -1.3 -1.2 -1.3 -1.4 -1.5 -2.0 SSO0530 serine hydroxymethyltransferase, putative -1.1 -1.1 -1.2 -1.5 SSO0531 3-hydroxy-3-methylglutaryl Coenzyme A reductase -1.5 SSO0532 Domain of unknown function -1.1 -1.0 -1.2 -1.7 -1.2 SSO0534 nonspecific lipid-transfer protein, putative -1.4 -1.4 -1.3 -1.6 -1.3 -1.3 SSO0535 condensing enzyme, putative, FabH-related -1.9 -1.7 -1.8 -1.2 SSO0536 Uncharacterized ACR, COG1586 superfamily -1.4 -1.1 -1.6 -1.1 SSO0537 conserved hypothetical protein -1.7 -2.1 -1.3 -1.3 -2.0 SSO0539 conserved hypothetical protein 1.4 1.4 1.2 1.2 1.9 1.8 SSO0540 conserved hypothetical protein 1.7 -1.2 1.4 1.8 SSO0544 exsB protein -1.5 1.2 SSO0546 coenzyme pqq synthesis protein, putative -1.2 -1.0

118

Table S1 Continued SSO0548 multidrug-efflux transporter, putative -1.0 -1.6 -2.7 -1.0 SSO0549 conserved hypothetical protein 1.0 1.8 1.3 1.6 1.4 2.1 1.1 1.4 1.4 1.6 SSO0551 rnase P RNA component 1.0 1.5 1.2 1.5 1.1 1.0 1.9 2.3 SSO0553 conserved hypothetical protein -1.1 -1.5 -1.3 -1.0 -1.5 -1.4 SSO0554 conserved hypothetical protein -1.4 -1.7 -1.2 -1.7 -1.4 -1.4 -1.5 -1.5 -1.2 -1.4 SSO0555 conserved hypothetical protein -1.5 -2.2 -1.4 -2.3 -1.2 -1.8 -1.4 -2.0 -1.3 -1.6 SSO0557 conserved hypothetical protein -1.4 -1.3 -1.2 SSO0558 methionyl-tRNA synthetase -1.0 SSO0559 V-type ATPase 116kDa subunit family superfamily -2.3 -2.2 -2.2 -2.7 -1.1 -2.0 -2.4 -2.4 -3.1 SSO0566 V-type ATPase, subunit D -1.8 -1.6 -1.3 -1.5 -1.6 -1.5 -1.3 -1.3 -1.2 SSO0569 prolyl-tRNA synthetase -1.6 -1.8 -1.1 -1.8 -1.5 -1.2 -2.1 -1.8 SSO0570 pyridoxine biosynthesis protein -1.0 SSO0575 Holliday junction resolvase 1.0 SSO0576 ketol-acid reductoisomerase 1.1 1.4 1.5 SSO0577 conserved hypothetical protein -1.0 SSO0579 acetolactate synthase, large subunit, biosynthetic type -1.2 SSO0582 conserved hypothetical protein -1.2 SSO0583 4-hydroxybenzoate octaprenyltransferase, putative -1.6 1.2 SSO0584 conserved hypothetical protein 1.5 1.3 1.3 1.0 SSO0588 conserved hypothetical protein subfamily 1.6 1.8 1.1 1.2 1.8 2.1 1.6 2.3 1.8 SSO0592 histidinol-phosphate aminotransferase 1.2 SSO0594 Phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase (hisA) -1.1 -1.0 SSO0596 imidazoleglycerol-phosphate dehydratase -1.2 -1.4 -1.1 -1.5 -1.5 -1.1 -1.0 -1.0 SSO0597 imidazoleglycerol phosphate synthase, cyclase subunit -1.2 SSO0601 Domain of unknown function domain protein 1.71.7 1.31.5 1.21.4 SSO0602 seryl-tRNA synthetase 1.1 SSO0607 conserved hypothetical protein -1.2 -1.3 SSO0610 dihydroorotate dehydrogenas -1.0 -1.7 -1.4 SSO0611 dihydroorotas -1.8 -1.4 -1.5 -1.2 -1.0 -1.5 -1.3 -1.0 SSO0612 conserved hypothetical protein -1.0 SSO0617 conserved hypothetical protein -1.3 SSO0618 phosphate regulatory protein, putative, putative 1.8 3.1 -1.2 1.7 2.8 2.8 3.9 -1.2 2.5 3.5 SSO0619 conserved hypothetical protein -1.7 -1.4 -1.1 -1.1 SSO0621 conserved hypothetical protein 1.1 SSO0622 Uncharacterized ACR, COG1590 superfamily 1.0 1.1

119

Table S1 Continued SSO0626 phosphoribosylaminoimidazole-succinocarboxamide synthase 1.3 -1.6 -1.0 SSO0627 methionyl-tRNA synthetase C-terminal region, beta subunit -1.1 -1.4 -1.0 SSO0628 phosphoribosylformylglycinamidine synthas, putative -1.2 SSO0629 phosphoribosylformylglycinamidine synthase II, putative -1.1 -1.1 -1.0 SSO0632 amidophosphoribosyltransferase -1.2 -1.3 -1.4 -1.3 -1.2 -1.5 SSO0633 amidophosphoribosyltransferase -1.1 -1.0 -1.0 SSO0635 phosphoribosylamine--glycine ligase -1.0 SSO0637 conserved hypothetical protein -1.1 -1.3 -1.2 -1.0 SSO0641 carbamoyl-phosphate synthase large chain -1.0 -1.0 -1.0 SSO0645 ribosomal protein S6 modification protein, putative 1.21.7 1.52.0 1.31.8 1.11.7 SSO0647 3-hydroxyacyl-CoA dehydrogenase family protein 1.6 1.2 1.3 1.2 SSO0648 gentisate 1,2-dioxygenase, putative 1.2 -1.0 1.3 -1.4 SSO0650 conserved hypothetical protein -1.2 -1.1 -1.1 SSO0651 conserved hypothetical protein 1.0 1.2 1.0 SSO0652 conserved hypothetical protein -1.3 -1.5 -1.5 -1.5 -1.9 -3.7 1.9 SSO0653 conserved hypothetical protein 1.3 1.1 SSO0655 conserved hypothetical protein 1.3 1.3 1.5 1.7 1.2 SSO0656 cytochrome C oxidase assembly factor 1.1 SSO0659 Helix-turn-helix protein, copG family domain protein 1.0 1.3 1.6 1.1 1.6 1.3 1.7 1.5 1.9 SSO0664 Sua5/YciO/YrdC/YwlC family protein, putative -1.0 -1.0 -1.2 -1.0 -1.3 SSO0669 SloR, putative 1.0 1.0 1.2 SSO0670 conserved hypothetical protein 1.6 1.6 1.3 1.3 1.0 1.2 1.3 1.6 1.3 SSO0675 molybdopterin-guanine dinucleotide biosynthesis protein MobB 2.1 2.0 1.5 1.4 1.5 1.4 1.7 2.5 1.3 1.5 SSO0678 conserved hypothetical protein 1.2 1.2 1.7 1.2 1.9 1.2 2.3 -1.1 SSO0679 atrazine chlorohydrolas, putative 1.3 1.3 1.2 1.7 SSO0680 Glycosyl transferases domain protein 2.2 -1.5 1.3 SSO0683 Domain of unknown function domain protein -1.2 -1.2 -1.0 SSO0684 glutamate synthase (NADH) precursor -1.3 -1.5 -2.1 -1.4 SSO0686 ATP-binding cassette transporter-like protein -1.2 SSO0687 conserved hypothetical protein -2.0 -2.0 -2.2 -2.5 -1.8 -2.1 -1.9 -2.1 -1.9 -2.3 SSO0688 ABC transporter, ATP-binding protein -1.2 -1.3 SSO0690 Protein of unknown function superfamily 2.4 SSO0691 conserved hypothetical protein -1.2 -1.9 -1.1 -1.3 -1.4 -1.7 SSO0692 cytidylate kinase -1.1 -1.5 -1.2 -1.6 -1.4 SSO0693 conserved hypothetical protein -1.6 -1.3 -1.0 -1.4 -1.8

120

Table S1 Continued SSO0694 adenylate kinas -1.2 -1.2 -1.5 -1.3 -1.9 -1.5 SSO0695 preprotein translocase, SecY subunit -1.2 -1.7 -1.9 -1.2 -1.4 -1.5 -1.5 -1.3 -1.9 SSO0696 ribosomal protein L27a -1.0 SSO0697 50S ribosomal protein L30, putative -1.0 -1.1 -1.7 -1.0 -1.1 -1.7 -1.8 -2.0 -2.0 SSO0698 ribosomal protein S5 -1.2 -1.2 -1.1 -1.3 -1.4 -1.3 SSO0699 50S ribosomal protein L18 -1.0 -1.3 -1.0 -1.0 -1.0 -1.1 SSO0703 SSU ribosomal protein S8P -1.1 SSO0704 50S ribosomal protein L5 -1.3 -1.0 -1.4 -1.4 SSO0705 30S ribosomal protein S4, putative -1.0 -1.1 -1.0 -1.0 -1.1 -1.1 -1.5 SSO0707 ribosomal protein L24 -1.2 -1.0 -1.0 -1.2 SSO0708 ribosomal protein L14 -1.1 -1.3 SSO0712 ribosomal protein S3 -1.2 -1.2 SSO0713 ribosomal protein L22 -1.0 SSO0715 ribosomal protein S19 -1.1 -1.1 -1.1 SSO0716 ribosomal protein L2 -1.1 -1.2 -1.5 -1.0 -1.5 -1.4 SSO0718 ribosomal protein L4/L1 family -1.0 -1.2 -1.3 -1.2 -1.9 SSO0719 50s ribosomal protei -1.2 -1.3 -1.1 SSO0722 isoleucyl-tRNA synthetase, putative -1.1 -1.7 -1.1 -2.0 -1.9 -1.7 -1.7 SSO0725 conserved hypothetical protein 1.1 SSO0726 phosphoglycolate phosphatase, putative, putative -1.4 -1.3 -1.1 SSO0728 translation elongation factor aEF-2 -1.8 -1.8 -1.7 -2.1 -1.2 -1.5 -1.5 -1.9 -2.1 SSO0730 KE2 family protein superfamily -1.2 -1.4 -1.0 -1.0 SSO0732 3 exoribonuclease family -1.5 -1.3 -1.2 -1.4 -1.2 -1.3 -1.3 -1.2 -1.9 -2.3 SSO0735 polyribonucleotide nucleotidyltransferase, putative -1.0 -1.2 -1.1 -1.5 SSO0736 KH domain protein -1.1 -1.7 -1.4 -1.8 -1.9 SSO0737 conserved hypothetical protein -1.2 -1.1 -1.1 -1.1 -1.3 -1.5 SSO0743 GTP-binding protein, YchF family 1.1 SSO0746 30S ribosomal protein S3AE -1.0 -1.2 -1.1 -1.2 -1.3 -1.0 SSO0747 RNA methyltransferase, TrmH family, group 1 -1.0 -1.2 -1.2 SSO0751 conserved hypothetical protein -1.2 -1.0 SSO0753 conserved hypothetical protein -1.6 SSO0754 s-adenosylhomocysteine hydrolase 1.7 1.1 2.1 -1.0 1.9 3.8 -2.0 SSO0755 adenosylhomocysteinase -1.2 -1.0 -1.3 -1.1 -1.1 -1.2 SSO0757 spermidine synthase -2.1 -1.9 -2.1 -2.3 -1.5 -2.2 -1.3 -1.7 -1.9 -2.5 SSO0758 fkbp-type peptidyl-prolyl cis-trans isomerase -1.6 -1.7 -1.7 -2.0 -1.1 -1.8 -1.3 -1.0 -1.7

121

Table S1 Continued SSO0760 glycerol 1-phosphate dehydrogenase -1.0 SSO0761 mRNA 3-end polyadenylation factor 1.4 SSO0763 conserved hypothetical protein SSO0765 glutamyl-tRNA(Gln) amidotransferase, subunit A -1.1 -1.0 -1.3 SSO0766 proteasome, subunit beta -1.1 -1.2 -1.3 SSO0769 replication factor C, large subunit -1.0 SSO0771 cell division control 6 (cdc6) protein, putative -1.1 SSO0772 conserved hypothetical protein 1.3 SSO0774 minichromosome maintenance (MCM) protein 1.0 1.2 1.1 1.1 1.7 1.4 SSO0775 conserved hypothetical protein -1.6 -1.1 -1.7 -1.0 -1.0 -1.2 SSO0776 Putative integral membrane protein superfamily -1.1 -1.1 SSO0778 conserved hypothetical protein 1.4 1.3 SSO0858 hypothetical protein -2.1 -2.5 -1.6 -2.0 -1.1 -2.3 1.2 -1.5 -2.4 -1.4 -1.4 SSO0860 conserved hypothetical protein 1.3 1.4 SSO0862 thermosome subunit 1.1 SSO0863 hypothetical protein 1.1 1.1 1.2 SSO0867 conserved hypothetical protein -1.0 -1.1 SSO0870 hypothetical protein 1.3 1.2 1.2 1.8 SSO0873 PHP domain N-terminal region family -1.2 SSO0876 aspartokinase III, lysine-sensitive, putative -1.1 -1.0 SSO0878 threonine synthase -1.2 -1.2 -1.0 SSO0879 conserved hypothetical protein 1.0 SSO0881 conserved hypothetical protein -2.7 -4.1 1.3 -1.8 -2.6 -1.7 -3.3 1.6 -1.1 -1.8 SSO0883 phosphoenolpyruvate synthase -1.1 -1.0 -1.2 -1.0 -1.0 -1.0 -1.3 SSO0886 1L-myo-inositol-1-phosphate synthase -1.2 -1.1 -1.2 SSO0887 Acylphosphatase 1.2 1.2 1.3 1.4 SSO0888 tryptophan synthase beta chain -1.0 -1.0 SSO0889 tryptophan synthase, alpha subunit -1.1 -1.7 -1.7 -1.7 -1.4 SSO0890 anthranilate phosphoribosyltransferase -1.2 -1.4 -1.2 -1.2 -1.2 -1.4 -1.3 -1.3 SSO0892 N-(5phosphoribosyl)anthranilate isomerase -1.5 -1.6 -1.2 -2.1 -1.6 -1.7 -1.2 SSO0893 anthranilate synthase component I -1.0 SSO0897 aspartate aminotransferase -1.1 -1.2 -1.2 -1.0 -1.4 SSO0898 conserved hypothetical protein -1.1 SSO0899 valyl-tRNA synthetase -1.1 -1.1 -1.5 -1.1 SSO0904 conserved hypothetical protein -1.1 -1.1

122

Table S1 Continued SSO0905 D-3-phosphoglycerate dehydrogenase, putative -1.1 -1.0 -1.2 -1.4 SSO0906 aminotransferase, class V, putative -1.3 -1.6 -1.2 -1.2 SSO0907 DNA topoisomerase I 1.1 SSO0909 cell division control protein 48, AAA family -1.5 -1.5 -1.0 -1.0 -1.2 SSO0910 conserved hypothetical protein -1.5 -1.5 -1.2 SSO0911 conserved hypothetical protein -1.4 -1.0 -1.1 SSO0913 enolase -1.0 SSO0914 hypothetical protein 1.1 SSO0917 glycine dehydrogenase (decarboxylating) subunit 2 -1.0 SSO0919 glycine cleavage system T protein -1.0 SSO0920 glycine cleavage system H protein -1.1 -1.0 -1.6 SSO0921 Protein of unknown function family -1.3 SSO0927 ABC transporter membrane protein 1.2 1.1 1.1 SSO0928 Uncharacterized protein family (UPF0051) family 1.1 SSO0929 ribonucleoside-diphosphate reductase, alpha subunit, putative -1.5 -2.0 -1.1 -1.7 -1.0 -1.0 -1.1 -1.4 SSO0931 conserved hypothetical protein 1.3 1.5 SSO0936 aspartyl-tRNA(Asn) amidotransferase, B subunit, putative -1.2 SSO0939 NOP56 homolog -1.3 -1.5 -1.0 -1.0 -1.2 SSO0940 fibrillarin -1.3 -1.4 -1.3 SSO0941 Protein of unknown function superfamily -1.1 -1.3 -1.6 SSO0944 conserved hypothetical protein 1.3 1.3 1.5 1.3 1.1 1.9 1.1 2.0 SSO0946 transcription initiation factor IIB -2.7 -2.6 -2.0 -2.1 -2.1 -2.0 -1.9 -2.1 -2.5 -2.7 SSO0947 conserved hypothetical protein -1.0 SSO0955 Domain of unknown function, putative -1.4 -1.3 -1.4 -1.9 -1.4 -1.5 -1.6 -1.3 -1.5 -1.7 SSO0956 moaA / nifB / pqqE family family -1.2 -1.1 -1.1 SSO0963 reverse gyrase -1.3 -1.6 -1.1 SSO0965 DEAD/DEAH box helicase domain protein -1.3 -1.1 SSO0966 Uncharacterized protein family UPF0027 superfamily -1.0 SSO0967 deoxyhypusine synthase, putative -1.2 -1.1 -1.1 -1.0 -1.3 -1.0 -1.1 -1.4 SSO0968 DNA topoisomerase VI, B subunit -1.4 -1.4 -1.4 -1.3 SSO0971 fifty-four homologue of SRP54 -1.0 -1.2 SSO0972 conserved hypothetical protein -1.2 -1.2 -1.4 -1.1 -1.5 -1.5 SSO0976 uridylate kinase, putative 1.4 1.0 1.4 SSO0977 2-isopropylmalate synthase 1.2 SSO0978 ribose 5-phosphate isomerase -1.3 -1.1

123

Table S1 Continued SSO0980 conserved hypothetical protein 1.0 1.3 SSO0981 pyruvate kinase 1.1 1.2 1.2 SSO0983 hypothetical protein 1.6 1.2 1.7 SSO0986 aerobically inducible protein, putative -1.2 -1.3 -1.0 -1.9 -1.4 SSO0987 glycogen synthase, putative -1.2 -1.2 -1.4 -1.8 -1.2 SSO0990 glucoamylase TGA, putative -1.0 -1.1 SSO0991 glycogen debranching enzyme-related protein, putative -1.1 -1.0 SSO0994 30S ribosomal protein S14 homolog 1.1 1.2 1.5 1.9 SSO0997 L-aspartate oxidase 1.0 1.0 1.7 -1.1 1.6 SSO0998 quinolinate synthetase complex, subunit A 1.3 1.2 2.8 4.0 SSO1000 inner membrane protein MalF, putative -1.1 SSO1004 D-lactate dehydrogenase, putative -1.1 -1.1 -1.5 -1.4 SSO1005 conserved hypothetical protein -1.6 -1.5 -1.1 -1.1 -2.0 -1.9 -1.6 -1.3 SSO10051 1.0 1.4 1.6 SSO1007 acetylornithine deacetylase, putative -1.1 -1.2 SSO1008 conserved hypothetical protein 1.3 1.5 1.1 1.5 1.3 2.0 SSO1009 pheP phenylalanine-specific permease, putative -1.8 -2.4 -1.4 -1.4 -1.2 -2.1 -2.4 -1.2 SSO1010 NADH oxidase, putative 1.6 SSO1012 1.3 1.4 1.5 SSO1019 cysA2 -1.2 -1.4 SSO1022 NADH-ubiquinone oxidoreductase, 20 Kd subunit -1.2 -1.1 -1.4 -1.5 -1.3 -2.2 -2.3 -1.3 -1.1 SSO1023 formate hydrogenlyase subunit 5 precursor, putative -1.5 -1.9 -1.2 -1.8 -2.1 SSO1024 NADH dehydrogenase, putative -1.4 -1.3 -1.4 -1.1 SSO1025 hypothetical protein -1.0 -1.2 -1.5 SSO10258 1.2 1.1 -1.8 1.1 SSO1026 formate hydrogenlyase subunit 4, putative 1.4 SSO1027 hypothetical protein -1.6 -1.8 -2.1 -2.6 -1.7 -1.1 -5.0 -7.3 2.3 -3.1 -3.8 SSO1028 hydrolase, putative -1.1 SSO1029 formate dehydrogenase alpha subunit -1.0 -1.2 -1.5 SSO1030 mocC protein -1.7 -2.3 -1.4 -1.2 SSO1031 conserved hypothetical protein -1.1 SSO1032 sulfate ABC transporter, permease protein, putative -1.1 -1.2 -2.9 SSO1033 hypothetical protein -1.6 -2.1 -1.1 -1.3 -1.8 -1.5 -1.9 SSO1034 ABC transporter, ATP-binding protein, putative 1.3 1.0 1.1 SSO10340 transcription regulator, putative 1.1 1.5 1.6 1.3 2.2

124

Table S1 Continued SSO10342 hypothetical protein -1.1 -1.6 -1.3 -1.2 -1.5 -2.0 -1.0 -1.2 SSO10348 hypothetical protein -1.4 -1.7 -1.2 -1.6 -1.4 -1.7 -1.2 -1.5 -1.1 -1.5 SSO1035 hypothetical protein 1.5 1.2 1.0 1.0 SSO1036 hypothetical protein 1.1 1.4 1.6 1.9 1.5 1.7 SSO1038 conserved hypothetical protein 1.1 SSO1041 conserved hypothetical protein -1.4 -1.4 -1.4 -1.2 -1.5 -1.4 -1.3 -1.0 SSO1042 Protein of unknown function superfamily -1.3 -1.3 -1.1 -1.0 -1.5 -1.4 -1.3 -1.5 SSO1043 AtsA/ElaC family protein, putative 1.62.2 1.21.6 1.0 SSO10449 hypothetical protein 1.7 2.0 SSO1045 Pk-RPPK -1.4 -1.5 SSO1046 conserved hypothetical protein 1.0 1.0 SSO1047 proliferating cell nuclear antigen (pcna) -1.0 -1.2 -1.3 -1.2 -1.2 -1.1 -1.3 SSO1052 CG1518 gene product, putative -1.2 -1.4 -1.4 -1.1 -1.5 -1.0 -1.6 -2.1 SSO1053 hypothetical protein -1.1 SSO1054 ammonium transporter -1.4 -1.6 -1.3 -1.8 -1.1 -1.4 -1.6 -2.2 SSO1057 hypothetical protein 1.5 1.8 1.4 2.3 1.2 1.0 2.2 2.4 1.4 1.0 SSO1059 hypothetical protein 2.3 1.7 -1.6 SSO1060 lipoate protein ligase A related -1.4 -1.9 -1.3 SSO10604 Partial transposase ISC1058 1.7 1.2 1.0 1.3 SSO10610 7 KD DNA-binding protein (SSO7D) (SSH7A) (ssh7A /Sso7d-1) 1.3 1.0 1.2 SSO1062 hypothetical protein -1.0 -1.1 -1.7 1.1 SSO1064 phosphoribosylaminoimidazole carboxylase, catalytic subunit -2.7 -1.9 -2.3 -1.6 -1.9 -2.0 -2.5 -1.6 -2.0 SSO1065 phosphoribosylaminoimidazole carboxylase, ATPase subunit -1.2 -1.2 -1.8 -1.7 SSO1070 Transposase IS116/IS110/IS902 family domain protein 2.3 2.5 1.8 2.3 1.6 1.5 1.4 1.7 SSO10704 hypothetical protein -1.4 -2.5 -2.9 SSO1073 IS1537, resolvase, putative 1.2 1.0 SSO1075 inosine-5-monophosphate dehydrogenase related 1.1 SSO1076 hypothetical protein 1.4 1.1 1.1 1.2 1.6 2.6 SSO1077 fumarate hydratase class ii -1.2 -1.5 SSO1078 ABC transporter, ATP-binding protein 2.4 SSO1079 conserved hypothetical protein -1.3 -1.1 -1.5 SSO10802 Carbon monoxide dehydrogenase, small chain. Carboxy-end fragment (cutC-2) -1.2 SSO1082 transcriptional regulator, MarR family, putative 1.1 1.7 1.7 1.5 1.7 1.9 2.2 2.1 2.4 SSO10828 Quinol oxidase (SoxABC), cytochrome B subunit, C-terminal part (soxC) -1.4 -1.8 -1.5 -1.1 -1.4 -1.7 -2.0 SSO1083 probable CoA transferase, subunit A, putative 1.1 1.4 1.3 1.2 1.5 1.1 1.8 1.5

125

Table S1 Continued SSO1085 probable CoA transferase, subunit B, putative 1.21.1 1.31.7 1.21.3 SSO1086 conserved hypothetical protein 1.4 1.0 1.1 1.1 1.2 1.1 1.6 1.4 SSO1087 hypothetical carbamoylphosphate synthetase, putative 1.1 1.1 SSO1088 heterocyst to vegetative cell connection protein -1.4 -2.2 -1.3 -1.9 -1.2 -1.8 -1.4 -2.2 -2.1 SSO1090 protein-serine/threonine phosphatase -1.6 -2.2 -1.4 -2.1 -1.3 -1.7 -1.8 -2.6 -1.2 -2.3 1.1 SSO1091 von Willebrand factor type A domain protein -1.1 -1.1 -1.3 -1.0 SSO1092 hypothetical protein 1.0 1.4 1.7 1.6 1.8 SSO1093 conserved hypothetical protein 1.3 -1.3 1.0 -1.1 SSO1095 aconitate hydratase (ec 4.2.1.3) (citrate hydro-lyase) (aconitase) -1.1 SSO10975 Second ORF in transposon ISC1225 1.5 1.5 1.6 1.2 1.8 SSO10982 Partial transposase ISC1225 2.4 2.2 2.2 2.9 1.8 2.6 1.8 1.9 2.6 3.2 SSO1100 hypothetical protein 1.5 SSO1101 transcriptional regulatory protein, AsnC family, putative 1.0 SSO1102 hypothetical protein 1.6 1.6 2.1 2.4 2.6 4.3 -1.6 1.3 1.0 SSO11020 hypothetical protein -1.0 SSO1104 glycoprotein B, putative 1.2 1.4 SSO1105 glycine cleavage system H protein, putative 1.1 1.2 1.6 2.2 SSO1106 hypothetical protein 1.4 1.0 1.5 1.9 1.1 1.3 SSO1107 hypothetical protein 1.3 1.8 1.0 1.3 SSO11071 Pyruvate synthase delta chain (Pyruvic-ferredoxin oxidoreductase delta chain) (porD-2) -1.1 -1.0 SSO1108 transcriptional regulator Lrs14 1.3 SSO1109 hypothetical protein 1.0 1.7 -1.1 SSO1110 transcriptional regulator Lrs14 1.3 1.3 1.5 2.0 1.6 1.7 1.6 2.3 1.7 2.4 SSO11114 Ferredoxin like protein (zfx-like1) 1.2 1.1 SSO11133 hypothetical protein 1.92.2 1.82.0 2.43.1 2.23.4-1.2 SSO11138 Second ORF in transposon ISC1225 1.5 1.9 1.6 2.0 1.8 2.2 1.9 2.2 2.0 2.2 SSO11139 hypothetical protein 1.3 1.5 1.4 1.7 SSO1114 conserved hypothetical protein -1.1 -1.1 -1.2 -1.1 -1.2 -1.4 SSO1115 conserved hypothetical protein -1.0 -1.3 -1.3 -1.4 SSO1118 hypothetical protein 1.1 1.2 1.1 1.7 2.0 SSO1119 hypothetical protein 1.0 1.4 1.7 1.1 SSO1120 glutaredoxin-related protein 1.1 1.1 SSO1121 hypothetical protein -1.1 1.1 SSO1123 pyruvate dehydrogenase, E3 component, lipoamide dehydrogenase, putative 1.4 2.5 SSO11231 Ferredoxin like protein (zfx-like2) -1.3 -1.3 -1.1 -1.1 -1.7 -1.5 -2.0 -1.5 -2.1

126

Table S1 Continued SSO1125 conserved hypothetical protein 2.0 2.3 1.2 1.8 1.0 2.0 1.1 2.2 -1.0 1.4 2.8 -1.5 SSO1126 conserved hypothetical protein 1.2 1.1 SSO11277 Second ORF in transposon ISC1190 1.0 1.1 1.1 SSO11281 Last part of transposase in ISC1250 1.2 -1.2 SSO1129 conserved hypothetical protein 1.5 1.4 -1.1 2.2 -1.5 SSO1131 heterodisulfide reductase 1.6 1.3 1.9 -1.2 1.1 1.9 -1.2 SSO1133 conserved hypothetical protein -1.1 SSO1134 heterodisulfide reductase, subunit C 1.6 -1.1 SSO1135 heterodisulfide reductase subunit B, putative 1.0 SSO11362 First ORF in transposon ISC1225 1.0 1.1 SSO1137 hypothetical protein 1.4 1.7 1.7 -1.0 1.7 -1.2 1.5 SSO11387 hypothetical protein 1.1 SSO11389 First ORF in transposon ISC1225 1.1 1.6 2.0 2.4 1.5 2.2 1.7 2.3 1.6 2.2 SSO1140 conserved hypothetical protein -1.0 -1.1 -1.2 -1.2 -1.9 -1.0 SSO1141 hypothetical protein -1.1 -1.2 SSO11412 Copper binding protein 2.3 2.1 2.3 2.2 1.3 1.8 1.5 1.6 2.3 2.7 SSO1142 oxalate/formate antiporter, putative -1.4 -2.1 -1.6 -2.0 -1.7 -1.2 -2.2 1.0 SSO1143 conserved hypothetical protein -1.0 SSO1145 tryptophan synthase, beta subunit homolog -1.3 -1.1 SSO1147 hypothetical protein -1.1 SSO1148 streptococcal aci, putative 1.7 2.6 1.3 1.4 1.1 1.5 SSO1151 tldD protein, putative -1.1 SSO1152 Putative modulator of DNA gyrase family -1.2 -1.5 -1.6 SSO1154 FkbR2, putative -1.4 SSO11553 ORF in partial transposon ISC774 1.8 SSO11575 hypothetical protein 2.0 2.4 2.2 2.2 2.4 2.9 SSO1159 haloacetate dehalogenase H-2 1.1 SSO1160 hypothetical protein 1.4 1.5 1.8 1.3 1.4 1.3 1.4 SSO1161 conserved hypothetical protein 1.2 1.1 1.2 1.3 SSO11614 hypothetical protein -1.4 -1.4 SSO1162 drug transporter, putative 1.1 1.2 SSO11637 glucoamylase TGA, putative 1.2 1.1 1.0 SSO1167 hypothetical protein -2.8 SSO1168 sugar ABC transporter, ATP-binding protein 1.2 1.3 1.1 SSO1169 cymG protein, putative -1.3

127

Table S1 Continued SSO1171 maltose ABC transporter, periplasmic maltose-binding protein, putative 1.41.5 1.92.0 SSO1172 Uncharacterized ACR, COG1543 family -1.0 -1.3 -1.9 SSO1173 pheP phenylalanine-specific permease, putative -1.2 -1.8 -1.8 -1.7 -2.1 -1.7 -2.3 SSO1175 hypothetical protein -1.1 -1.4 -1.1 SSO1176 Holliday junction resolvase, putative -1.4 SSO1180 conserved hypothetical protein 1.7 2.4 1.4 2.3 1.6 2.2 1.2 1.6 1.0 SSO1181 IS1537, resolvase 1.4 1.2 1.7 1.3 1.8 SSO1184 Isochorismatase 1.0 SSO1186 hypothetical protein 1.5 1.9 1.7 SSO11867 Alpha-fucosidase N-terminal fragment (fucA1) 1.2 SSO1188 Phytoene dehydrogenase related protein -1.0 -1.2 SSO1190 hypothetical protein 1.4 1.1 1.3 1.3 SSO11914 vapB-22 1.1 1.3 1.9 1.1 SSO11915 hypothetical protein 1.0 1.4 1.4 SSO11917 Partial ORF in transposon ISC1212 1.0 1.1 1.1 SSO11934 hypothetical protein -2.1 -2.6 -2.0 -2.5 SSO11939 hypothetical protein -1.8 -1.8 1.0 -1.2 -1.6 -2.4 -1.6 -1.9 SSO1197 conserved hypothetical protein 1.1 1.2 1.1 SSO11972 hypothetical protein 1.1 1.1 1.1 SSO1198 putative DNA-invertase -1.0 SSO1200 conserved hypothetical protein 1.2 1.2 1.1 1.1 SSO12018 vapB-23 2.0 -1.1 SSO1202 putative transposase 1.4 1.1 SSO1203 conserved hypothetical protein 1.1 SSO1206 PorB subunit of pyruvate:flavodoxin oxidoreductase 1.1 1.1 1.2 1.7 SSO12063 Third ORF in partial transposon ISC1160 1.6 1.7 -1.6 1.1 1.2 1.8 1.5 2.1 SSO12067 Second ORF in partial transposon ISC1160 1.5 1.5 2.0 2.8 1.6 2.0 2.1 2.2 2.4 2.6 SSO12068 First ORF in partial transposon ISC1160 1.2 1.2 1.9 2.6 2.1 2.3 3.2 4.3 -1.1 SSO1207 2-ketovalerate ferredoxin oxidoreductase alpha-2 1.4 1.1 1.3 1.6 1.5 1.7 SSO1208 2-oxoisovalerate oxidoreductase, gamma subunit, putative 1.4 1.3 SSO12083 hypothetical protein 2.7 2.3 2.2 2.8 2.7 2.5 2.5 2.0 1.8 SSO1209 oxidoreductase 1.2 1.0 1.1 1.3 1.4 1.2 1.8 SSO1210 hypothetical protein 1.31.5 1.31.6 2.12.3 SSO1212 putative transposase 1.5 1.1 SSO12252 hypothetical protein -1.4 -1.7 -1.3 -1.9 -1.2 -1.2 -2.0 -1.2 -1.8

128

Table S1 Continued SSO12256 hypothetical protein -1.8 -1.4 -1.5 -1.4 -2.2 -2.8 SSO1250 IS element ISC1217 1.0 1.4 2.5 2.2 SSO1255 transcriptional regulator, GntR family, putative 1.5 1.0 1.4 1.3 1.2 1.3 SSO1260 altronate hydrolase 1.8 1.6 1.9 2.5 1.1 1.2 1.5 2.0 2.2 2.7 SSO1262 hypothetical protein -1.1 -1.5 SSO1263 conserved hypothetical protein 2.0 2.1 1.2 2.2 -1.0 1.4 1.5 2.0 2.2 1.9 SSO1264 conserved hypothetical protein 1.4 1.7 1.0 1.3 SSO1265 hypothetical protein -1.1 -1.3 -1.5 -1.1 SSO1266 ABC transporter, ATP-binding protein, putative 1.5 SSO1268 glucosamine--fructose-6-phosphate aminotransferase (isomerizing) -1.0 -1.0 SSO1269 hypothetical protein -1.0 SSO1273 conserved hypothetical protein -1.1 -1.0 SSO1274 peptide ABC transporter, permease protein, putative -1.7 -1.6 -2.3 -2.5 -1.9 -2.2 -2.2 -2.0 -2.0 -2.7 SSO1275 peptide ABC transporter, permease protein, putative -1.0 -2.5 -2.4 -2.0 -2.0 SSO1276 Oligo/dipeptide transport, ATP binding protein (dppD-1) -1.2 -1.2 -1.0 -1.9 -1.9 -1.2 -1.6 SSO1277 oligopeptide ABC transporter, ATP-binding protein -1.8 -1.5 SSO1278 hypothetical protein -1.2 SSO1279 dipeptide ABC transporter, ATP-binding protein -1.0 -1.9 -1.7 -1.6 SSO1280 conserved hypothetical protein 1.3 2.2 1.3 1.3 1.8 1.4 1.6 1.9 SSO1281 hypothetical protein -1.0 -1.8 -1.6 -1.2 SSO1282 oligopeptide ABC transporter, ATP-binding protein -1.0 -2.1 -2.1 -1.4 -1.2 SSO1283 hypothetical oligopeptide transport system permease protein, putative -1.0 -1.7 -1.5 -1.6 SSO1284 oligopeptide ABC transporter, permease protein, putative -1.1 -1.1 -1.2 SSO1285 hypothetical protein 1.2 1.4 1.5 SSO1288 unknown protein, putative 1.0 SSO1289 conserved hypothetical protein 1.5 1.9 1.8 2.0 SSO1292 CoxF protein 1.1 1.2 SSO1294 hypothetical protein 2.6 2.9 2.2 2.7 2.2 2.8 2.1 2.9 2.2 3.5 -1.3 SSO1298 hypothetical protein 1.0 SSO1299 n-acetylglucosaminyltransferase, putative -2.4 -1.1 SSO1302 conserved hypothetical protein 1.1 1.1 -1.1 1.1 SSO1387 ISA1214-6, putative transposase -1.1 SSO1388 hypothetical protein 1.5 -1.0 SSO1391 conserved hypothetical protein 1.3 1.2 SSO1392 Domain of unknown function domain protein 1.0 1.3 2.0 2.2 2.4

129

Table S1 Continued SSO1393 hypothetical protein 1.2 1.3 1.2 1.3 1.3 1.3 1.0 1.1 SSO1395 hypothetical protein 1.2 2.6 -1.6 SSO1397 conserved hypothetical protein 1.0 SSO1399 Protein of unknown function superfamily -1.2 SSO1400 conserved hypothetical protein -1.2 SSO1401 hypothetical protein -1.1 -1.0 -1.2 SSO1402 conserved hypothetical protein -4.3 -6.6 SSO1403 hypothetical protein -2.6 -3.0 SSO1404 Uncharacterized ACR, COG1343 superfamily 2.0 2.6 SSO1405 Protein of unknown function superfamily 1.3 1.1 1.1 1.1 1.5 1.5 SSO1406 hypothetical protein 1.7 1.4 1.5 1.4 2.9 2.8 2.1 SSO1456 DNA helicase 1.9 1.9 1.3 2.4 -1.1 2.7 3.6 4.5 SSO1457 nADP-specific glutamate dehydrogenase , fragment -1.3 1.5 1.1 -1.4 SSO1458 hypothetical protein -1.2 -1.8 SSO1459 DNA polymerase ii -3.1 -4.1 -2.5 -2.9 SSO1460 penicillin G acylase, putative -1.2 -1.8 1.1 -1.1 1.0 SSO1463 amino acid permease, putative -1.4 SSO1465 pyrrolidone-carboxylate peptidase 1.2 1.2 1.3 SSO1466 putative transposase 1.9 1.2 1.6 1.4 2.2 1.1 1.5 1.7 2.2 SSO1468 conserved hypothetical protein 1.3 1.6 2.0 SSO1469 coiled-coil protein, putative 1.4 1.3 1.1 1.2 1.3 1.4 1.6 1.5 1.8 2.0 SSO1472 conserved hypothetical protein 1.1 1.1 1.2 1.5 1.2 1.1 1.3 1.0 1.2 SSO1473 putative transposase 1.2 1.3 SSO1474 IS1004 transposase 1.3 1.3 1.0 1.0 1.6 2.0 1.3 1.3 2.2 2.5 SSO1475 putative neutral protease 1.1 1.3 1.3 1.4 1.1 1.6 2.0 SSO1478 hypothetical protein 1.4 1.4 1.1 1.1 1.4 1.4 1.9 2.0 SSO1479 hypothetical protein 1.3 1.1 1.3 1.1 1.3 1.6 2.0 SSO1484 putative invertase/transposase 1.7 1.4 1.5 1.8 1.8 2.0 1.5 1.7 SSO1486 hypothetical protein 1.1 1.5 1.8 2.2 1.8 1.7 1.8 1.8 1.8 2.2 SSO1487 putative transposase 1.4 1.0 SSO1491 hypothetical protein 1.1 1.1 1.2 1.5 2.2 2.2 2.6 1.0 1.5 SSO1493 vapC-6 3.3 3.2 2.0 2.5 3.4 3.6 4.2 4.5 SSO1494 vapB-6 2.0 2.3 1.4 1.6 3.8 3.8 4.3 4.7 SSO1495 hypothetical protein 1.6 1.7 -1.1 2.1 3.7 1.9 2.5 SSO1496 hypothetical protein -1.5

130

Table S1 Continued SSO1497 IS1537, resolvase, putative 1.8 2.2 SSO1498 putative resolvase/transposase 1.4 1.3 SSO1500 hypothetical protein -1.1 -1.4 -1.1 -1.4 -1.2 -1.3 -1.6 -1.6 SSO1504 Probable transposase family 1.0 SSO1507 hypothetical protein 1.2 1.2 SSO1510 hypothetical protein -1.7 -1.4 -1.3 -1.8 -1.3 -1.2 -1.4 -1.4 -2.4 -2.0 SSO1512 hypothetical protein -1.3 -1.1 -1.7 -1.8 -1.7 -1.6 -1.0 -1.1 -1.3 -1.6 SSO1513 hypothetical protein -1.7 -1.1 -1.3 -1.2 SSO1516 conserved hypothetical protein 1.5 SSO1522 conserved hypothetical protein 1.7 1.3 1.5 1.4 1.4 1.5 1.6 1.8 1.7 2.5 SSO1523 conserved hypothetical protein 1.0 1.3 1.0 1.0 1.0 1.5 1.8 1.4 SSO1524 2-oxo acid dehydrogenase, E3 component, lipoamide dehydrogenase, putative 1.1 1.3 1.2 1.4 1.1 2.4 2.5 1.1 1.2 SSO1525 probable dehydrogenase E1 component 1.2 SSO1529 2-oxoglutarate dehydrogenase, E2 component 1.1 1.0 1.1 SSO1530 Dihydrolipoamide S-acetyltransferase, carboxy-end (pdhC) 1.1 SSO1531 alkyldihydroxyacetonephosphate synthase, putative -1.2 SSO1533 oxidoreductase, putative 1.9 1.5 1.7 1.3 2.3 2.3 1.9 2.0 SSO1537 putative metallohydrolase, putative 1.3 1.8 2.0 1.3 2.7 2.1 1.4 1.2 SSO1538 acetylornithine deacetylase, putative 1.4 2.3 2.8 2.6 3.1 SSO1539 probable MFS transporter, putative 1.1 SSO1540 conserved hypothetical protein 1.1 1.4 1.4 1.0 2.0 1.9 2.1 2.0 SSO1542 3-oxoacyl-[acyl-carrier protein] reductase precursor 1.31.5 1.72.2 1.92.0 1.91.8 SSO1545 conserved hypothetical protein 1.6 SSO1546 conserved hypothetical protein 1.2 1.0 1.0 1.0 SSO1548 putative invertase/transposase 2.0 2.0 1.7 2.2 1.7 1.8 2.3 2.5 1.5 1.9 SSO1552 astB/chuR-related protein 1.0 1.0 1.2 1.5 SSO1553 hypothetical protein 3.2 3.0 3.0 3.4 3.6 4.7 -1.2 3.5 4.3 3.2 4.1 SSO1555 hypothetical protein 2.1 2.1 2.0 1.8 1.6 2.0 1.2 1.4 SSO1556 probable transposase, putative 2.0 1.9 2.1 2.4 1.6 1.9 1.6 1.5 1.9 1.9 SSO1560 3-hydroxyisobutyrate dehydrogenase, putative -1.1 SSO1563 conserved hypothetical protein 1.3 SSO1564 IS1537, resolvase, putative 2.9 2.8 2.2 2.0 2.3 2.0 3.0 3.4 2.9 3.4 SSO1565 pyruvate dehydrogenase, E3 component, lipoamide dehydrogenase, putative 3.3 3.9 3.5 4.6 -1.1 3.9 4.0 7.2 8.5 -1.3 4.7 6.0 -1.3 SSO1568 hypothetical protein 3.3 3.7 3.0 2.9 4.0 4.2 3.3 3.5 3.1 3.1 SSO1569 hypothetical protein 2.0 2.5 1.1 1.8 2.9 3.3 2.2 1.8

131

Table S1 Continued SSO1570 conserved hypothetical protein 1.1 1.2 1.8 1.3 SSO1571 nitric oxide reductase, putative -1.1 1.6 -1.2 SSO1572 conserved hypothetical protein 1.5 1.1 1.6 1.2 1.7 SSO1574 reductase, assembly protein 1.5 2.1 2.4 1.4 2.9 -1.5 SSO1577 4Fe-4S binding domain protein 1.5 1.7 1.4 SSO1578 hypothetical protein 1.3 -1.0 -1.5 SSO1579 formate dehydrogenase, iron-sulfur subunit 1.4 SSO1588 Transcription regulator -1.1 SSO1589 Bacterial regulatory proteins, gntR family, putative 4.0 3.4 3.3 3.2 3.1 2.9 4.3 4.6 5.2 5.7 SSO1590 conserved hypothetical protein 3.2 2.4 2.9 1.9 3.7 3.0 1.3 1.8 SSO1602 conserved hypothetical protein 2.0 1.2 1.1 SSO1606 coiled-coil protein, putative 1.3 SSO1607 pyrrolidone-carboxylate peptidase 1.3 1.1 SSO1608 putative integral membrane protein 1.7 1.3 1.3 1.3 2.1 SSO1610 hypothetical protein -1.5 SSO1625 hypothetical protein -1.1 -1.1 SSO1631 coenzyme PQQ synthesis protein -1.8 -2.0 -1.3 SSO1632 coenzyme pqq synthesis protein, putative 2.0 -1.1 2.1 -1.3 2.6 -1.6 SSO1634 conserved hypothetical protein 1.9 2.3 SSO1638 penicillin G acylase, putative -3.1 SSO1639 putative invertase/transposase 1.5 SSO1640 Nucleotidyltransferase domain protein -1.3 SSO1642 conserved hypothetical protein 1.3 2.3 -1.0 1.2 1.7 1.4 2.0 1.1 1.6 1.7 2.2 SSO1643 conserved hypothetical protein -1.1 SSO1646 alcohol dehydrogenase 1.5 1.6 1.4 1.7 1.7 1.5 1.7 1.8 2.0 1.8 SSO1647 acetolactate synthase III, large subunit, putative 1.1 SSO1648 unnamed protein product, putative 1.0 1.2 1.4 1.4 1.3 1.6 SSO1650 hypothetical protein 1.72.1 1.81.8 1.81.8 2.12.3 SSO1651 vapC-7 -1.2 SSO1655 helicase, SNF2/RAD54 family 1.4 2.3 SSO1656 conserved hypothetical protein -1.1 -1.4 SSO1657 vapC-8 3.1 3.3 3.1 3.9 2.7 3.7 3.0 3.8 SSO1658 putative resolvase 3.0 2.8 2.3 2.1 2.3 1.9 2.8 3.4 2.8 3.3 SSO1659 putative resolvase/transposase 1.3 1.3 1.0 1.4 1.0 1.7 1.8 2.6 1.8 2.4 SSO1662 AgaG protein, putative -2.0 -2.6 -1.1

132

Table S1 Continued SSO1663 N-methylhydantoinase -2.1 -2.7 SSO1664 conserved hypothetical protein 2.3 2.5 1.9 2.4 2.4 2.8 1.7 2.0 2.8 3.5 SSO1665 cytosine permease, putative -1.2 -1.6 -1.7 -1.2 -1.2 -2.1 -2.7 -1.4 SSO1666 hypothetical protein -1.0 -1.6 -2.0 -1.4 -1.4 -2.1 -2.5 -1.3 -1.8 SSO1667 unknown conserved protein in others -1.2 -1.5 -1.5 -1.9 -2.1 -2.5 -1.5 -1.2 SSO1668 hydantoinase -1.2 -1.9 -2.3 SSO1673 Nucleotidyltransferase domain, putative 1.5 1.1 1.4 1.0 1.7 SSO1674 conserved hypothetical protein 1.4 1.8 1.6 SSO1676 hypothetical protein -1.0 SSO1679 hypothetical protein 1.0 1.0 1.0 SSO1680 conserved hypothetical protein 1.7 2.1 1.7 1.8 1.3 1.7 1.4 2.4 2.5 SSO1681 conserved hypothetical protein 1.1 1.5 1.1 1.9 2.0 3.1 2.4 SSO1682 Partial transposase in ISC1316 1.2 SSO1683 conserved hypothetical protein 1.3 SSO1684 putative invertase/transposase 2.5 SSO1686 conserved hypothetical protein 1.2 1.2 1.1 1.3 1.1 1.6 1.4 SSO1689 hypothetical protein 1.0 1.2 1.4 1.1 1.1 1.0 1.7 2.0 SSO1731 putative invertase/transposase 1.3 1.3 1.3 1.7 1.6 1.8 1.2 1.9 SSO1732 IS element ISC1217 1.1 1.5 SSO1736 hypothetical protein -1.9 -2.4 -1.3 -1.5 -1.1 -1.3 -1.1 SSO1739 conserved hypothetical protein 1.0 1.4 1.5 1.8 1.1 1.4 SSO1740 chromosome assembly protein, putative 1.6 1.6 1.7 2.6 SSO1741 subunit II of the terminal oxidase 4.3 4.7 4.0 4.2 3.8 4.5 2.8 3.5 SSO1742 subunit of the terminal oxidase with unknown homologue 3.2 3.3 2.7 3.4 2.8 3.1 3.5 3.9 2.7 2.9 SSO1744 putative transposase 1.5 1.1 SSO1745 putative transposase 1.6 1.4 1.1 1.2 1.5 1.6 SSO1746 conserved hypothetical protein 1.8 2.3 2.5 2.9 3.0 3.3 SSO1747 conserved hypothetical protein 1.2 SSO1748 ISA1083-2, ISORF2, putative 1.3 1.5 1.3 1.0 1.5 SSO1749 conserved hypothetical protein 1.9 1.8 1.9 2.5 2.1 2.2 2.4 2.6 1.2 1.7 SSO1750 conserved hypothetical protein 1.7 1.4 1.4 1.5 1.4 1.4 1.1 SSO1752 coiled-coil protein, putative 1.1 SSO1755 conserved hypothetical protein -1.2 -1.0 -1.3 SSO1757 putative membrane protein 1.6 1.6 SSO1758 hypothetical protein 1.1 1.3 1.7 1.2 1.0

133

Table S1 Continued SSO1760 IS1537, resolvase, putative 1.6 1.5 1.1 1.3 1.9 2.6 2.2 2.7 SSO1762 hypothetical protein 1.3 SSO1764 vapC-10 1.4 1.4 2.9 2.9 3.0 SSO1765 vapC-11 1.2 1.3 SSO1766 hypothetical protein 1.0 1.3 1.3 1.4 SSO1767 hypothetical protein 1.6 1.6 SSO1769 transposase, putative 1.5 1.5 1.2 1.6 1.2 1.7 SSO1772 transposase, putative 1.9 1.8 1.9 1.8 1.6 1.7 1.6 1.9 1.9 SSO1773 conserved hypothetical protein 1.3 1.8 1.0 SSO1775 conserved hypothetical protein 1.0 1.0 1.1 1.2 SSO1781 dTDP-glucose 4,6-dehydratase -1.8 -2.4 -1.2 SSO1782 glucose-1-phosphate thymidyltransferase -1.2 -1.7 -1.4 -1.0 -1.6 -1.8 -1.0 -1.3 SSO1783 dTDP-4-dehydrorhamnose reductase -1.1 -1.3 -1.1 SSO1784 hypothetical protein 1.3 1.5 1.5 1.6 1.6 1.8 2.4 2.0 2.3 SSO1786 hypothetical protein 1.9 2.4 1.6 2.1 2.3 2.8 2.4 3.5 -1.2 1.9 2.7 SSO1787 conserved hypothetical protein 1.1 SSO1788 conserved hypothetical protein 1.0 1.0 1.3 2.1 SSO1789 hypothetical protein 1.7 2.6 SSO1790 hypothetical protein 1.5 1.6 2.2 2.0 SSO1791 putative invertase/transposase 1.1 1.2 1.7 1.5 1.9 1.3 1.4 SSO1793 transposase related protein, putative 1.1 1.2 1.2 SSO1795 conserved hypothetical protein 2.2 2.1 2.2 2.6 2.1 2.4 1.8 1.9 1.7 2.0 SSO1799 hypothetical protein 2.8 3.3 2.0 2.6 2.3 2.9 1.8 2.2 1.8 2.3 SSO1800 hypothetical protein 1.0 1.3 1.2 1.4 1.8 SSO1801 conserved hypothetical protein 1.2 SSO1802 hypothetical protein 2.4 1.7 1.3 1.2 1.3 1.4 1.6 SSO1803 hypothetical protein 1.8 2.2 1.2 1.8 1.1 1.6 SSO1804 hypothetical protein -1.1 -1.0 SSO1806 acetyl-CoA synthetase 1.1 1.8 2.5 SSO1807 conserved hypothetical protein 2.3 3.5 1.3 2.3 2.5 2.1 2.4 3.4 2.3 2.4 SSO1808 probable menaquinone biosynthesis methlytransferase, putative 1.3 1.5 SSO1809 hypothetical protein 3.1 3.8 1.6 2.7 -1.1 2.5 3.3 2.4 3.4 -1.0 2.1 2.8 SSO1810 Predicted permease family 1.1 1.4 SSO1812 hypothetical protein 1.2 3.1 SSO1813 hypothetical protein 1.3 2.1 1.4 1.5 1.4 1.1 1.9

134

Table S1 Continued SSO1815 hypothetical protein 1.1 SSO1816 4Fe-4S binding domain protein 2.3 SSO1817 thiosulfate sulfurtransferase SseA, putative 2.0 1.8 2.0 1.2 1.5 1.2 2.1 1.7 1.3 SSO1818 chromosome assembly protein homolog, putative 1.1 1.3 1.4 1.7 2.1 SSO1820 hypothetical protein -1.0 SSO1823 hypothetical protein -2.1 -1.1 -2.1 1.0 -1.3 1.1 SSO1825 hypothetical protein 1.4 1.5 1.1 1.4 1.2 1.6 SSO1826 hypothetical protein 1.2 1.7 SSO1827 hypothetical protein -1.2 -1.6 SSO1831 hypothetical protein 2.2 2.2 2.0 1.8 1.7 1.9 1.4 1.7 1.6 2.0 SSO1833 conserved hypothetical protein 1.2 SSO1834 conserved hypothetical protein 1.2 1.3 1.1 1.2 1.1 1.5 1.8 SSO1835 Nucleotidyltransferase domain protein 1.0 1.1 1.4 1.4 1.5 1.6 1.5 SSO1837 putative transposase 1.6 1.2 1.1 1.3 SSO1838 conserved hypothetical protein 3.4 2.9 2.7 2.5 2.7 3.4 1.9 2.0 2.7 2.1 SSO1839 heme biosynthesis protein 2.2 2.4 1.6 2.6 1.6 3.5 -1.9 1.9 3.0 -1.1 1.8 2.7 -1.0 SSO1840 coenzyme PQQ synthesis protein 1.2 1.6 1.4 1.8 1.5 2.8 -1.2 1.4 2.5 -1.1 SSO1842 glyceraldehyde-3-phosphate dehydrogenase, NADP-dependent 1.0 2.1 SSO1850 conserved hypothetical protein 1.0 1.2 1.3 1.2 1.3 SSO1851 conserved hypothetical protein 1.0 1.0 1.2 1.2 SSO1857 glutamine amidotransferase class-I 1.2 1.3 1.7 1.8 1.3 1.8 2.1 2.0 1.8 SSO1861 conserved hypothetical protein 1.1 1.1 1.0 2.0 2.1 1.6 1.9 SSO1863 conserved hypothetical protein -1.3 SSO1864 dipeptidase 1.4 1.9 SSO1867 vapB-12 1.0 1.2 1.7 1.8 1.1 1.7 1.6 2.6 -1.0 SSO1868 Helix-turn-helix domain, fis-type protein 1.3 1.7 1.5 1.9 1.3 2.2 SSO1869 conserved hypothetical protein 2.9 SSO1870 rusticyanin 1.4 1.9 1.1 1.7 1.2 1.4 1.0 SSO1875 hypothetical protein 1.4 1.4 1.7 2.0 3.0 2.5 3.0 2.0 SSO1876 transcriptional regulatory protein, AsnC family, putative 2.3 2.6 2.5 3.0 2.5 3.4 2.6 3.1 2.6 3.4 SSO1877 hypothetical protein 1.4 1.0 1.8 1.6 1.1 2.8 SSO1881 subunit of the terminal oxidase with unknown homologue 1.3 1.7 1.8 1.6 2.1 2.1 1.8 1.8 SSO1882 subunit II of the terminal oxidase, putative 1.8 1.8 2.0 1.8 2.1 3.6 3.7 1.1 1.4 SSO1885 hypothetical protein 1.3 2.1 1.7 1.3 SSO1888 putative transposase 1.6 1.1 1.0

135

Table S1 Continued SSO1889 ATP-dependent RNA helicase DeaD -1.1 1.1 -1.3 1.4 -1.3 SSO1890 conserved hypothetical protein SSO1892 Cobalt transport protein family -1.1 -1.4 -1.4 -1.6 -1.4 -1.7 -1.1 -1.8 SSO1893 ABC transporter, ATP-binding protein, putative -1.4 -1.0 -1.4 -1.4 -2.2 -1.0 SSO1894 hypothetical protein -1.6 SSO1897 conserved hypothetical protein 2.1 2.2 1.6 2.5 2.1 2.3 2.3 2.6 1.2 2.1 SSO1902 hypothetical protein 1.4 SSO1904 conserved hypothetical protein 1.6 2.5 1.3 2.3 1.0 1.2 1.2 1.7 1.6 1.9 SSO1905 probable transporter, putative -1.1 -1.1 SSO1906 amino acid permease, putative -1.2 SSO1908 conserved hypothetical protein 1.1 1.0 1.1 1.2 1.1 SSO1910 conserved hypothetical protein 1.0 1.3 1.4 1.4 1.8 1.8 1.9 2.4 SSO1912 hypothetical protein 1.9 1.7 SSO1913 Protein of unknown function family 1.0 SSO1914 vapC-13 1.8 2.0 2.1 2.8 2.1 2.3 2.7 3.1 2.4 3.1 SSO1918 conserved hypothetical protein 1.2 1.7 1.6 1.1 1.1 4.2 SSO1920 hypothetical protein 2.0 3.4 3.4 5.5 -2.1 3.0 4.8 -1.7 SSO1921 PIN-domain protein 3.6 4.0 3.6 4.7 -1.1 4.2 5.0 4.2 5.2 -1.0 SSO1922 vapC-14 1.8 1.0 1.5 1.5 1.3 SSO1923 dTDP-4-dehydrorhamnose 3,5-epimerase 1.1 1.4 SSO1924 putative invertase/transposase 1.2 1.0 SSO1926 putative invertase/transposase 1.5 1.3 1.5 1.7 1.7 1.8 1.9 1.9 1.2 1.9 SSO1927 IS1537, resolvase, putative 1.5 1.5 1.6 1.2 1.3 1.2 SSO1928 conserved hypothetical protein 1.8 1.9 1.7 1.5 1.3 1.3 1.8 2.5 2.1 2.6 SSO1929 conserved hypothetical protein 1.3 SSO1930 nADP-specific glutamate dehydrogenase , fragment 1.4 1.1 -1.3 -2.3 1.1 1.0 SSO1931 hypothetical protein 3.1 2.8 3.0 2.9 2.9 3.4 1.6 1.6 2.2 2.4 SSO1932 hypothetical protein 3.4 3.7 2.9 3.0 2.9 3.8 2.1 2.2 2.2 2.1 SSO1933 hypothetical protein 1.7 1.1 1.3 1.4 1.4 1.3 1.2 1.2 1.3 1.3 SSO1934 antibiotic ABC transporter, ATP-binding protein, putative 1.1 1.3 SSO1939 conserved hypothetical protein 1.7 2.2 1.9 2.4 1.4 2.5 -1.1 2.0 3.2 -1.3 1.5 2.9 -1.4 SSO1941 thiamine biosynthesis protein ThiC 1.1 1.9 1.5 -1.0 1.7 1.0 2.1 -1.1 1.5 2.1 SSO1942 ketol-acid reductoisomerase 1.3 SSO1947 conserved hypothetical protein 1.4 1.7 SSO1948 hypothetical protein -1.1

136

Table S1 Continued SSO1949 endoglucanase A precursor 1.9 2.2 SSO1951 putative invertase/transposase 1.5 1.4 1.6 1.7 1.8 1.9 1.3 1.4 1.9 SSO1952 carboxypeptidase (cpsA) 1.0 SSO1954 dolichyl-phosphate mannose synthase related protein -1.2 -1.0 -1.6 SSO1956 hypothetical protein 1.0 SSO1958 TRANSPORT PROTEIN, permease, putative 1.3 SSO1959 hypothetical protein 1.6 1.4 2.1 2.1 1.6 1.4 1.9 1.4 SSO1960 hypothetical protein 1.1 SSO1961 hypothetical protein 1.2 1.0 1.4 SSO1963 conserved hypothetical protein 1.5 1.5 SSO1964 IS1537, resolvase, putative 1.0 1.3 SSO1966 putative invertase/transposase 1.7 1.4 1.3 1.5 1.4 1.4 1.9 SSO1967 conserved hypothetical protein -1.1 SSO1968 vapC-15 1.0 1.3 1.2 2.2 SSO1969 vapC-16 1.5 1.3 2.2 -1.6 SSO2060 acyl-CoA dehydrogenase 1.3 1.5 1.3 1.6 1.3 1.2 1.8 1.3 2.3 -1.0 SSO2062 nonspecific lipid-transfer protein, putative -1.0 -1.1 SSO2063 Domain of unknown function domain protein -1.2 -1.0 -1.3 -1.2 -1.2 SSO2064 Domain of unknown function, putative -1.0 -1.4 -1.0 SSO2067 indolepyruvate ferredoxin oxidoreductase alpha subunit 1.1 1.2 SSO2070 acetyl-CoA synthase, putative 2.4 1.7 -1.6 SSO2071 bacterioferritin comigratory protein 1.3 1.5 1.3 1.6 2.0 2.4 1.3 1.6 1.6 2.0 SSO2072 Metallo-beta-lactamase superfamily domain protein 1.9 2.1 1.7 1.6 2.1 1.5 2.7 -1.2 1.7 2.9 SSO2073 Protein of unknown function superfamily 2.2 2.3 3.1 3.8 2.8 3.2 3.1 3.7 SSO2081 conserved hypothetical protein 1.11.1 1.31.3 1.21.4 1.0 SSO2083 conserved hypothetical protein 1.3 1.1 1.3 1.3 1.3 SSO2085 conserved hypothetical protein 1.2 1.1 1.3 1.0 1.3 1.1 2.2 2.0 SSO2086 putative transposase 1.3 2.2 1.6 2.3 1.2 2.0 1.2 1.5 1.5 2.0 SSO2088 conserved hypothetical protein 1.2 1.2 1.8 SSO2090 conserved hypothetical protein 1.9 1.8 1.5 2.3 2.1 2.1 2.1 2.0 2.2 2.5 SSO2095 alpha-amylase -1.4 -1.3 -1.1 SSO2096 vapC-19 1.6 1.9 1.5 2.0 2.0 3.1 -1.1 2.3 3.4 2.0 3.2 -1.2 SSO2097 putative riboflavin biosynthesis enzyme 1.1 1.0 SSO2099 conserved hypothetical protein 1.2 SSO2100 conserved hypothetical protein 1.9 2.6 1.0 1.8 1.5 1.9 3.1 -1.2

137

Table S1 Continued SSO2101 conserved hypothetical protein 3.1 3.4 3.2 3.4 4.0 5.2 4.3 5.5 -1.2 3.0 3.7 SSO2102 conserved hypothetical protein 1.4 1.0 1.1 1.9 2.0 2.1 2.5 SSO2103 conserved hypothetical protein 1.5 1.4 1.7 1.1 1.6 1.6 1.9 1.4 2.0 SSO2104 conserved hypothetical protein -1.1 -1.4 SSO2107 Nucleotidyltransferase domain, putative 1.1 SSO2109 conserved hypothetical protein 2.4 2.7 3.2 3.7 5.5 2.9 4.0 -1.1 SSO2110 conserved hypothetical protein 1.2 1.4 1.0 1.0 1.0 1.0 SSO2111 conserved hypothetical protein -1.0 SSO2118 conserved hypothetical protein 1.2 1.0 2.2 3.4 SSO2120 conserved hypothetical protein 1.5 -1.0 1.5 1.5 1.8 SSO2121 Methanobacterium orfk homolog 1.1 1.1 1.2 1.2 SSO2123 Probable transposase superfamily 1.1 SSO2125 conserved hypothetical protein 1.3 1.2 1.9 1.8 2.5 SSO2126 L-lactate permease -1.5 SSO2128 4Fe-4S binding domain protein 2.4 1.8 2.9 2.6 1.6 1.9 2.5 2.8 2.3 2.8 SSO2129 pyruvate-ferredoxin oxidoreductase alpha-2 1.0 1.7 1.6 1.7 2.0 SSO2130 pyruvate-ferredoxin oxidoreductase beta-2 1.0 1.6 2.3 1.7 SSO2131 AsnC family family 2.3 2.3 1.6 2.2 2.2 1.5 2.8 4.0 2.6 3.4 SSO2132 conserved hypothetical protein 2.1 2.3 1.5 2.5 -1.0 2.1 2.2 2.3 2.7 1.0 2.0 SSO2133 glycerol kinase 1.4 1.3 1.6 1.5 2.0 1.9 SSO2134 sugar transporter 1.0 1.2 1.4 2.0 1.7 2.0 1.0 SSO2135 ABC transporter efflux protein, DrrB family, putative 2.5 2.1 1.2 1.7 1.5 1.8 2.9 3.8 2.7 3.2 SSO2137 daunorubicin resistance ATP-binding protein DrrA 2.0 2.2 1.6 1.8 SSO2138 conserved hypothetical protein 3.3 2.5 2.6 2.4 2.6 3.2 2.4 2.6 2.3 2.8 SSO2139 conserved hypothetical protein 1.6 2.0 2.1 1.3 1.3 4.1 4.7 SSO2140 uncharacterized domain 1, putative 2.1 1.9 1.7 2.0 2.6 3.2 2.5 2.7 SSO2141 acylamino-acid-releasing enzyme 2.2 2.7 3.2 4.6 5.3 SSO2142 conserved hypothetical protein 1.3 1.3 1.9 1.9 SSO2144 conserved hypothetical protein 1.1 SSO2145 conserved hypothetical protein 1.4 2.3 1.6 2.5 1.3 1.9 1.3 1.6 2.0 SSO2146 conserved hypothetical protein 1.9 1.8 1.6 2.1 1.9 2.1 1.8 2.4 1.6 1.9 SSO2148 conserved hypothetical protein 1.0 1.8 1.1 1.6 1.3 2.3 SSO2152 conserved hypothetical protein -1.1 SSO2154 aminopeptidase -1.0 -1.0 -1.8 SSO2155 conserved hypothetical protein 1.2 1.4 2.0

138

Table S1 Continued SSO2159 haloacid dehalogenase-like hydrolase superfamily -1.0 1.1 SSO2162 FAD binding domain protein 1.1 -1.1 SSO2164 conserved hypothetical protein 1.3 1.0 1.0 SSO2165 conserved hypothetical protein 1.4 1.4 1.3 1.1 1.1 SSO2166 hypothetical protein 1.6 1.1 1.5 1.6 1.2 1.3 1.3 SSO2167 MutT/nudix family protein 1.0 1.1 1.3 1.3 1.5 2.2 SSO2178 aspartate-semialdehyde dehydrogenase 1.4 1.1 SSO2181 conserved hypothetical protein -1.0 -1.1 -1.2 -1.4 -1.6 -1.1 SSO2183 hypothetical protein 1.1 SSO2184 conserved hypothetical protein -1.2 SSO2186 hypothetical protein -1.0 -1.2 SSO2187 pterin-4-alpha-carbinolamine dehydratase, putative 1.1 1.0 1.3 1.3 SSO2190 hypothetical protein -1.0 -1.2 SSO2199 hypothetical protein -1.2 -1.2 -1.0 SSO2200 Domain of unknown function domain protein -2.0 -2.0 -1.8 -2.0 -2.3 -2.2 SSO2202 hypothetical protein -1.6 -1.1 -1.0 -1.0 -1.1 SSO2203 hypothetical protein -1.6 -2.1 -1.7 -2.1 -1.6 -1.9 -2.0 -3.4 1.4 -1.8 -2.3 SSO2205 3-oxoacyl-(acyl carrier protein) reductase, putative -1.1 -1.1 -1.2 -1.0 SSO2206 hypothetical transcriptional activator TENA, putative -1.4 -1.7 -1.5 -2.1 -1.4 -1.3 -2.0 -1.4 -2.3 SSO2207 maturase-like rf3 protein, putative -1.4 -1.7 -2.0 -1.5 -1.3 -2.2 SSO2208 Protein of unknown function superfamily 1.2 SSO2209 ceramide glucosyltransferase, putative -2.1 -2.6 -2.0 -2.4 -1.9 -2.4 -2.5 -3.6 1.1 -1.5 -2.6 SSO2210 hypothetical protein -1.0 SSO2213 conserved hypothetical protein 1.1 SSO2214 hypothetical protein 1.0 1.0 1.3 SSO2215 aminotransferase, class V, putative 1.4 2.8 SSO2221 hypothetical protein 1.2 1.5 1.8 2.5 1.3 2.4 -1.1 SSO2224 conserved hypothetical protein 1.5 1.9 1.5 1.3 1.4 2.1 SSO2225 conserved hypothetical protein 1.4 1.7 1.6 1.6 1.8 2.5 1.5 2.0 SSO2227 hypothetical protein 1.1 SSO2228 conserved hypothetical integral membrane protein, putative 2.1 2.7 1.5 2.5 2.4 2.3 2.2 2.7 1.8 2.1 SSO2231 hypothetical protein 1.3 1.5 1.1 1.6 1.1 SSO2232 thioredoxin (trxA) 1.0 1.4 1.4 1.3 1.1 1.7 SSO2234 conserved hypothetical protein -1.1 -1.1 -1.2 -1.6 SSO2236 phosphoglycerate mutase 1.5 1.5

139

Table S1 Continued SSO2241 chromosome assembly protein homolog, putative 1.7 -1.3 SSO2244 ferric uptake regulation protein, putative 1.2 1.3 1.7 1.9 1.5 1.6 1.7 1.9 1.1 1.5 SSO2245 hypothetical protein 1.0 SSO2248 conserved hypothetical protein 1.0 1.1 1.0 2.1 -1.0 1.8 -1.7 SSO2249 purine NTPase, putative 1.6 1.4 1.8 2.0 -1.1 1.2 2.0 2.1 SSO2254 Uncharacterized protein family UPF0031 family -1.3 -1.2 SSO2255 antioxidant, AhpC/Tsa family -1.4 -1.1 SSO2259 conserved hypothetical protein 1.1 1.0 1.2 1.4 1.3 SSO2261 sulfide-quinone reductase, putative 1.1 1.2 1.4 1.3 SSO2265 LAO/AO transport system ATPase 1.4 1.0 2.1 -1.1 1.4 3.0 -1.6 1.9 3.1 -1.3 1.0 3.0 -2.0 SSO2266 methylmalonyl Coenzyme A mutase 1.6 1.2 2.3 -1.1 1.3 2.1 1.7 2.7 -1.0 1.9 3.2 -1.3 SSO2269 hypothetical protein -1.1 -1.3 -1.5 1.0 -1.2 -1.5 SSO2272 membrane protein 1.4 1.4 1.0 SSO2273 ABC transporter, permease protein, putative 1.4 1.4 1.1 1.6 1.1 SSO2274 dihydrodipicolinate synthase -1.1 -1.4 -1.1 -1.5 SSO2276 3-ketoacyl-acyl carrier protein reductase, putative -1.1 -1.0 -1.2 -1.2 -1.4 -1.4 SSO2277 hypothetical protein -1.3 -1.0 -1.5 -2.0 -1.7 SSO2279 hypothetical protein -1.5 -1.3 -1.4 -1.1 SSO2280 cysteinyl-tRNA synthetase -1.1 SSO2284 hypothetical protein -1.3 SSO2285 Domain of unknown function domain protein -1.1 1.0 SSO2288 hypothetical protein -1.9 -1.5 -1.5 -2.3 -3.0 -1.8 -2.4 SSO2292 hypothetical protein -1.9 -2.6 -1.7 -2.1 -2.0 -3.1 1.1 -2.1 -2.8 SSO2296 CbiG protein, putative -1.2 SSO2301 cobalamin biosynthesis precorrin-3 methylase (cbiF) -1.1 SSO2303 cobalamin biosynthesis precorrin-8W decarboxylase, putative -1.0 SSO2309 coenzyme pqq synthesis protein, putative 1.6 1.7 1.7 1.5 1.5 1.6 1.6 1.7 1.5 2.3 SSO2311 hypothetical protein -1.0 SSO2312 putative invertase/transposase 1.7 1.4 1.4 1.8 1.6 1.7 1.5 1.8 1.8 SSO2314 hypothetical protein 1.1 1.4 1.7 2.1 1.5 1.5 1.8 2.0 1.8 SSO2316 type II secretion system protein, putative -1.3 -1.4 -1.1 -1.9 -1.8 -1.9 -1.3 SSO2318 flagella-related protein H, putative -1.7 -1.6 -1.5 -1.6 -1.2 -1.9 -2.1 -2.3 -1.9 SSO2319 hypothetical protein -2.2 -2.5 -1.8 -1.7 -2.0 -2.4 -3.0 -2.5 SSO2320 conserved hypothetical protein 1.2 1.0 SSO2322 hypothetical protein -2.1 -2.3 -1.5 -2.0 -1.5 -1.8 -1.9 -2.6 -2.2 -2.6

140

Table S1 Continued SSO2323 flagellin, putative -1.5 -1.5 -1.5 -1.1 SSO2324 hypothetical protein -1.3 -1.5 -1.5 -1.8 -1.1 SSO2325 hypothetical protein -1.4 -1.6 -1.6 -1.9 SSO2326 hypothetical protein 1.5 1.4 1.1 1.0 1.6 1.4 1.4 1.6 1.4 SSO2327 hypothetical protein -1.1 -1.2 -1.3 -1.3 SSO2331 hypothetical protein -1.4 -1.3 -1.1 -1.3 SSO2336 conserved hypothetical protein -1.1 SSO2337 hypothetical protein 1.8 1.5 2.0 1.9 3.6 2.0 2.2 1.6 2.0 SSO2338 conserved hypothetical protein -1.7 -1.9 -1.0 -1.9 -2.8 -1.6 -2.3 SSO2339 peptide chain release factor eRF/aRF, subunit 1 -1.1 SSO2342 purine phosphoribosyltransferase -1.3 -1.3 -1.3 -1.0 SSO2344 hypothetical protein -1.3 -1.1 -2.0 -1.1 -1.9 -1.2 -2.1 -1.6 1.0 SSO2345 polyprenyl synthetase, putative -1.0 -1.4 -1.0 SSO2347 transcriptional regulator AsnC -1.0 -1.2 -1.4 -1.5 SSO2348 conserved hypothetical protein -1.7 -1.5 -2.1 -1.8 SSO2349 hypothetical protein -1.3 -1.3 -1.9 -1.2 -1.3 -1.7 -1.3 -1.5 SSO2350 asparaginase, putative -1.2 -1.0 -1.2 SSO2351 hypothetical protein -1.1 SSO2353 geranylgeranyl hydrogenase, putative -1.2 -1.1 -1.5 -1.1 -1.4 -1.8 SSO2354 hypothetical protein -1.2 -1.6 -1.0 -1.4 SSO2357 fumarate reductase, iron-sulfur protein -1.0 -1.1 -1.2 -1.4 -1.3 -1.2 SSO2358 heterodisulfide reductase subunit B -1.2 -1.1 -1.2 -1.2 -1.1 -1.3 SSO2359 succinate dehydrogenase subunit D -1.3 SSO2367 homoserine kinase -1.7 -1.6 -1.4 -1.9 -1.6 -1.5 -2.1 -2.2 -1.7 -1.6 SSO2368 cystathionine gamma-synthase -1.6 -1.6 SSO2369 hypothetical protein -2.3 -2.6 -2.2 -2.4 -2.2 -2.4 -2.3 -2.9 -1.8 -1.9 SSO2370 hypothetical protein -2.9 -3.0 -2.3 -3.2 -2.6 -2.7 -2.4 -3.3 -2.9 -3.1 SSO2371 conserved hypothetical protein -1.1 -1.3 SSO2373 conserved hypothetical protein 1.4 1.5 SSO2377 acetyl-CoA acetyltransferase 1.1 -1.2 1.0 SSO2383 conserved hypothetical protein -1.1 -1.4 SSO2384 ribonuclease HII 1.6 -1.3 -1.1 SSO2386 Integral membrane protein superfamily -1.3 -1.0 -1.0 -1.0 SSO2387 type II secretion system protein -1.1 -1.0 -1.2 -1.3 SSO2389 putative bi-functional transferase/deacetylase-related -1.3 -2.0 -1.4 -1.9 -1.0

141

Table S1 Continued SSO2390 inorganic pyrophosphatase -1.2 SSO2395 hypothetical protein -2.0 -2.2 -1.4 -1.8 -1.3 -1.6 -2.2 -3.2 SSO2397 Protein of unknown function superfamily -1.4 -1.3 1.1 SSO2399 Protein of unknown function family -1.2 SSO2405 P50 adhesin 1.4 2.0 1.6 2.0 1.4 1.8 1.2 1.7 SSO2406 conserved hypothetical protein -1.3 -1.1 SSO2407 2-isopropylmalate synthase -1.2 -1.1 -1.2 -1.3 -1.2 -1.1 -1.1 -1.2 -1.8 -1.4 SSO2408 Protein of unknown function superfamily -1.0 -1.0 -1.1 -1.1 SSO2409 hypothetical protein -1.5 -1.0 -1.3 -1.3 -1.6 SSO2410 hypothetical protein -1.5 -2.2 -1.4 -2.5 1.1 -1.6 -2.1 -1.2 -1.8 -1.3 SSO2422 Phytoene dehydrogenase related protein -1.5 -2.2 -1.3 -2.3 -1.4 -1.6 -1.2 -2.0 -1.9 SSO2423 conserved hypothetical protein -1.5 -2.2 -1.3 -2.2 -1.1 -2.0 -1.0 -2.2 1.2 -1.8 SSO2424 purine phosphoribosyltransferase -1.5 -2.1 -1.4 -2.2 -1.2 -2.1 -1.2 -2.3 1.1 -1.0 -1.9 SSO2426 Lactoylglutathione lyase (glyoxalase I), putative 1.2 1.2 1.1 1.3 1.4 2.1 SSO2427 small heat shock protein 1.1 1.5 1.3 1.3 SSO2436 hypothetical protein 1.2 1.9 1.6 1.5 1.6 1.2 1.7 2.0 SSO2438 chlorohydrolase, putative -1.4 -1.3 -1.4 -1.1 -1.7 SSO2440 glutamine synthetase, type I, putative -1.1 -1.2 SSO2442 ribosomal protein L13 (breast basic conserved protein 1) 1.8 1.5 2.5 2.0 2.9 2.3 2.1 1.5 2.4 2.1 SSO2446 Probable RNA-3 terminal phosphate cyclase -1.1 -1.1 -1.0 -1.3 -1.1 -1.0 -1.4 SSO2448 DNA-damage-inducible protein P, putative 1.5 1.9 1.7 1.9 2.4 3.5 1.7 2.2 SSO2451 GMP synthetase, C-terminal domain 1.5 SSO2452 recombinase related 1.3 1.4 1.9 1.6 1.7 1.8 SSO2453 conserved hypothetical protein 1.3 1.1 1.4 SSO2455 pyrazinamidase/nicotinamidase -1.2 -1.1 SSO2462 helicase 1.1 SSO2466 acetyl-coenzyme A carboxylase, biotin carboxylase -1.0 -1.1 SSO2468 ABC transporter nitrate permease, putative -1.0 -1.1 SSO2469 sulfate ABC transporter, ATP-binding protein -1.4 -1.8 -1.1 -1.4 -1.4 -1.6 -1.5 -2.4 -1.1 -1.6 SSO2473 glucoamylase TGA, putative -2.6 SSO2481 hypothetical protein -1.1 -3.3 SSO2487 methylated-DNA--protein-cysteine methyltransferase 1.2 SSO2490 iron-containing alcohol dehydrogenase family protein RfbM, putative 1.5 1.1 1.9 1.8 2.2 2.6 SSO2493 lipase/esterase -1.5 SSO2494 alcohol dehydrogenase 1.1

142

Table S1 Continued SSO2496 3-ketoacyl-CoA thiolase 1.1 1.8 SSO2498 ribonucleoside-diphosphate reductase, beta subunit, putative 1.3 1.7 1.4 2.1 1.3 2.4 -1.0 SSO2500 3-oxoacyl-(acyl carrier protein) reductase (fabG-7) 1.5 1.3 1.2 1.1 1.0 SSO2502 hypothetical protein -1.1 1.5 -1.7 SSO2506 transcriptional regulator, tetR family domain protein 4.0 3.4 3.5 2.9 2.8 3.1 4.5 4.8 2.4 SSO2507 conserved hypothetical protein 1.9 SSO2508 Thiolase, putative 1.6 1.1 1.8 1.4 1.7 1.2 SSO2509 Domain of unknown function, putative 1.2 1.1 1.6 1.9 SSO2510 medium-chain acyl-CoA ligase 1.0 1.0 1.1 1.7 1.7 SSO2511 acyl-CoA dehydrogenase, putative 1.3 SSO2519 dehydrase, putative 1.2 SSO2520 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase (hpcE-1) 1.8 1.4 2.3 2.9 1.5 SSO2521 lipase/esterase 1.1 1.0 1.1 1.1 SSO2522 parathion hydrolase 2.5 1.7 3.0 2.8 2.8 2.7 3.4 3.7 2.4 2.9 SSO2523 long-chain-fatty-acid--CoA ligase, putative 1.6 1.2 1.0 1.1 SSO2528 probable MFS transporter, putative 1.1 -1.1 -1.7 SSO2530 conserved hypothetical protein 1.2 1.1 1.3 SSO2532 conserved hypothetical protein 1.3 1.6 1.1 1.7 1.2 1.6 1.1 SSO2534 endoglucanase, putative 1.0 1.4 1.3 SSO2536 nad-dependent alcohol dehydrogenase 1.1 1.3 SSO2537 phosphoenolpyruvate carboxykinase, putative 1.7 SSO2538 glycogen phosphorylase family protein, putative -1.2 -1.3 -1.8 SSO2540 conserved hypothetical protein 1.5 2.4 1.3 2.1 1.0 1.4 1.4 1.6 1.9 SSO2542 putative invertase/transposase 1.4 1.4 1.3 1.7 1.6 1.7 1.3 1.9 SSO2549 hypothetical protein 1.1 SSO2550 hypothetical protein 2.7 2.9 2.5 3.2 3.3 3.7 3.0 2.9 3.2 3.5 SSO2551 conserved hypothetical protein -1.1 -1.7 -1.1 -1.8 -1.1 -1.4 -1.0 -1.7 -1.0 -1.6 SSO2555 hypothetical protein 1.1 1.3 1.4 1.3 1.7 1.4 1.6 1.5 1.9 SSO2556 hypothetical protein 1.4 1.1 1.3 SSO2558 probable MFS transporter, putative -1.7 -2.9 1.2 -1.9 -2.9 1.0 -1.7 -2.7 -2.0 -3.1 1.1 -2.1 -3.0 SSO2560 Blub protein 4.6 1.4 2.3 SSO2566 conserved hypothetical protein 1.2 SSO2568 conserved hypothetical protein, putative 1.1 -1.6 1.5 -1.1 1.2 2.5 -1.4 2.4 -1.4 SSO2569 hypothetical protein -1.4 -1.2 -1.0 -1.5 -1.0 -1.0 SSO2572 hypothetical protein 1.0 1.2 1.7 1.4

143

Table S1 Continued SSO2573 hypothetical protein 1.5 1.3 1.2 1.2 1.8 SSO2575 PDZ domain (Also known as DHR or GLGF). protein -1.2 SSO2577 putative transposase 1.4 1.0 SSO2579 vapC-21 1.2 2.0 2.4 2.4 2.5 1.6 2.0 SSO2581 transcriptional regulatory protein, AsnC family, putative 1.1 1.1 1.4 1.5 1.2 1.5 1.1 1.2 1.5 1.6 SSO2582 hypothetical protein 1.1 1.0 SSO2585 L-lactate dehydrogenase (sqdB) 1.2 SSO2586 conserved hypothetical protein -1.3 -1.4 -1.2 -1.0 -1.4 -1.9 -1.4 -1.7 SSO2588 inosine-5-monophosphate dehydrogenase related 1.0 1.0 SSO2590 hypothetical protein 1.0 1.1 1.8 2.2 1.2 1.3 SSO2592 triosephosphate isomerase -1.5 -1.5 -1.0 -1.5 -1.2 SSO2595 Protein of unknown function family 1.5 SSO2597 aminotransferase, class V, putative -1.5 -1.6 -1.1 -1.2 -1.3 -1.6 -2.0 -1.4 -1.7 SSO2599 protein-L-isoaspartate O-methyltransferase 1.9 1.7 2.5 3.0 1.0 1.9 SSO2602 hypothetical protein 1.1 1.3 1.1 1.7 1.0 1.3 1.0 SSO2603 small heat shock protein 1.2 1.3 1.1 1.1 1.0 1.7 2.0 1.3 SSO2605 ubiquinone biosynthesis protein AarF, putative 1.2 SSO2608 hypothetical protein -1.1 -1.1 -1.6 -1.3 -1.6 SSO2609 Glycosyl transferases group 1 domain protein -1.0 SSO2611 conserved hypothetical protein 1.4 1.2 SSO2612 protease DegS, putative 1.1 1.4 SSO2615 oligopeptide ABC transporter, ATP-binding protein -1.1 -1.1 -1.1 -1.3 SSO2616 oligopeptide ABC transporter, ATP-binding protein -1.9 -1.3 -1.7 -2.0 -1.9 -1.6 -2.2 -2.4 -2.4 -2.5 SSO2617 peptide ABC transporter, permease protein, putative -2.0 -2.5 -1.8 -1.7 -2.2 -2.3 -2.4 -2.2 SSO2618 dipeptide ABC transporter, permease protein (dppB) -1.5 -1.0 -1.7 -1.8 -1.5 -1.6 -1.9 -2.2 -2.0 -2.1 SSO2619 conserved hypothetical protein -1.0 -1.1 SSO2620 Conserved hypothetical protein 1.7 -1.3 1.3 1.1 1.8 -1.1 1.5 -1.2 SSO2621 hypothetical protein 1.9 -1.2 SSO2624 3-hydroxyacyl-CoA dehydrogenase 1.1 SSO2627 Phenylacetyl-CoA-Ligase 1.3 2.2 SSO2628 conserved hypothetical protein 1.3 SSO2632 hypothetical protein 1.1 1.1 1.8 SSO2634 conserved hypothetical protein -1.1 -1.3 1.1 -1.6 1.2 -1.2 -1.0 SSO2635 hypothetical protein -1.0 -1.1 1.1 SSO2639 oxidoreductase -1.2 -1.0 -1.1 -1.2

144

Table S1 Continued SSO2640 hypothetical protein 1.9 SSO2641 hypothetical protein 1.0 SSO2643 anaerobic glycerol-3-phosphate dehydrogenase, subunit C, putative -1.4 -1.2 -1.8 -1.2 -1.3 SSO2644 hypothetical protein -1.1 -1.1 -1.0 -2.1 -1.3 SSO2645 hypothetical protein 1.3 1.7 1.9 1.9 SSO2648 conserved hypothetical protein 1.1 1.5 1.4 SSO2651 cation-transporting ATPase, E1-E2 family 1.2 SSO2653 bacitracin transport permease protein bcrc, putative -1.0 -1.6 -1.5 -1.5 -1.2 -1.0 SSO2656 Quinol oxidase (SoxABC), cytochrome B subunit (soxC) -1.4 -1.1 -1.4 -1.4 -1.1 SSO2657 quinol oxidase polypeptide I -1.3 -1.3 -1.6 -1.8 -1.3 -1.9 -1.2 SSO2659 conserved hypothetical protein -1.0 SSO2661 hypothetical protein -1.2 -1.4 SSO2662 hypothetical protein -1.2 -1.2 SSO2663 hypothetical protein -1.0 -1.1 -1.0 -1.0 SSO2664 glucose 1-dehydrogenase, putative 1.0 SSO2667 hypothetical protein -1.5 SSO2668 peptide ABC transporter, permease protein, putative -1.0 SSO2669 Bacterial extracellular solute-binding proteins, family 5, putative 1.4 SSO2670 peptide ABC transporter, ATP-binding protein 1.0 1.4 1.3 SSO2671 peptide ABC transporter, permease protein, putative -1.1 -1.1 -1.2 SSO2675 aminopeptidase N, putative -1.7 -1.8 -1.2 -1.7 -1.9 -2.8 -1.6 SSO2683 hypothetical protein 1.1 1.4 SSO2687 conserved hypothetical protein 1.1 SSO2689 mercuric reductase 1.7 -1.6 1.7 -1.7 2.3 -2.0 3.0 -2.2 2.9 -2.4 SSO2690 conserved hypothetical protein 1.2 2.2 1.6 1.0 1.7 SSO2691 CBS domain protein protein 1.2 1.8 SSO2693 hypothetical acylamino-acid-releasing enzyme -1.2 -1.0 SSO2694 heat shock protein, putative -1.2 -1.1 -1.2 -1.1 SSO2697 nonspecific lipid-transfer protein, putative -1.5 -2.0 -1.9 -1.1 -1.4 -1.7 -2.6 -1.4 -2.1 SSO2699 conserved hypothetical protein 1.1 SSO2703 hypothetical protein -1.2 -1.6 -1.2 -1.4 -2.0 -1.7 -2.6 -1.9 SSO2708 AIR synthase related protein, putative -1.2 -1.0 -1.3 -1.5 SSO2712 hypothetical protein -1.0 -1.1 -1.1 -1.4 -2.3 SSO2713 maltose/maltodextrin ABC transporter, ATP-binding protein -1.1 -1.6 SSO2714 Binding-protein-dependent transport systems inner membrane component D -1.2

145

Table S1 Continued SSO2716 drug transporter, putative -1.4 SSO2717 starvation sensing protein rspb, putative -1.2 -1.8 -1.2 -1.7 -1.4 -1.8 -1.2 -1.4 SSO2718 putative transporter protein, putative -1.0 -1.7 -1.5 -2.1 -1.8 -3.5 1.6 -1.1 -2.2 1.1 SSO2719 N-carbamoyl-beta-alanine amidohydrolase 1.8 2.8 -1.0 1.3 2.9 -1.6 3.2 2.6 1.2 1.3 2.7 1.8 SSO2721 hypothetical protein 1.01.1 1.71.9 SSO2722 putative invertase/transposase 2.1 2.2 1.6 2.0 1.8 1.9 2.2 2.5 1.4 1.6 SSO2723 conserved hypothetical protein 2.6 2.7 2.0 2.5 1.8 1.9 2.0 2.1 1.5 2.1 SSO2725 conserved hypothetical protein -1.1 -1.2 SSO2726 hypothetical protein -1.2 -1.0 -1.4 SSO2728 Amino acid permease superfamily -1.1 -1.1 SSO2729 conserved hypothetical protein 1.3 SSO2730 conserved hypothetical protein 1.3 1.2 1.1 1.1 SSO2732 agmatinase -1.1 1.0 SSO2733 phage SPO1 DNA polymerase-related protein 1.1 1.3 1.3 SSO2734 hypothetical protein 1.7 2.0 2.7 1.5 2.1 1.9 2.6 SSO2736 hypothetical protein -1.0 SSO2740 inosine-5-monophosphate dehydrogenase related protein II, putative 1.0 1.2 1.5 1.6 1.3 1.4 1.4 1.5 SSO2744 Domain of unknown function, putative -1.1 -1.2 -1.7 -1.0 -1.5 -1.8 -1.0 -1.8 SSO2745 conserved hypothetical protein 1.0 1.7 SSO2746 conserved hypothetical protein -1.7 -1.5 -2.2 -1.2 -2.0 -1.8 -3.0 1.2 -1.6 SSO2748 hypothetical protein -1.1 SSO2750 hypothetical protein -1.6 -1.9 -1.0 -1.1 -1.0 -1.1 -1.5 -1.2 SSO2751 PepK protein -1.4 -1.1 -1.6 -1.5 -1.7 -2.9 -3.1 SSO2752 hypothetical protein -1.1 SSO2753 hypothetical protein -1.1 SSO2754 conserved hypothetical protein -1.5 -1.2 -1.1 -1.0 -1.7 -1.5 SSO2755 Glycosyltransferase, putative 1.1 1.5 1.3 1.2 1.8 1.8 1.0 1.3 SSO2756 PorB subunit of pyruvate:flavodoxin oxidoreductase -1.3 -1.2 -1.1 -1.2 -1.0 SSO2757 2-ketovalerate ferredoxin oxidoreductase alpha-2 -1.0 -1.3 -1.3 -1.4 -1.6 SSO2758 2-oxoisovalerate oxidoreductase, gamma subunit, putative -1.0 -1.1 SSO2759 anaerobic dimethyl sulfoxide reductase chain b -1.0 -1.1 SSO2760 oxidoreductase -1.0 -1.1 -1.4 -1.0 SSO2762 electron transfer flavoprotein, subunit alpha SSO2764 4Fe-4S binding domain protein -1.2 -1.3 SSO2768 conserved hypothetical protein 1.1 1.2

146

Table S1 Continued SSO2770 cytosine deaminase -1.0 SSO2772 hypothetical protein -1.1 -1.2 SSO2774 acetolactate synthase, large subunit, putative -1.2 -1.2 -1.2 -1.0 -1.2 -1.7 -1.4 SSO2775 LPS biosynthesis RFBU related protein, putative 1.3 -1.1 SSO2776 fixc protein, putative 1.1 1.2 SSO2777 hypothetical protein 1.3 1.8 1.7 1.8 1.4 1.7 1.4 2.0 1.6 2.1 SSO2778 universal stress protein family, putative 1.4 1.8 SSO2783 PIN-domain protein 1.7 2.0 1.1 2.0 1.7 2.4 2.4 1.3 2.4 SSO2784 conserved hypothetical protein -1.5 -1.6 1.1 SSO2785 Probable transposase family 1.0 1.3 1.5 1.8 1.1 1.1 1.0 SSO2787 DedA family protein 1.2 1.4 1.0 1.5 1.1 SSO2788 hypothetical protein 1.4 SSO2789 phospholipase, putative -1.2 -1.1 -1.2 -2.1 -1.4 SSO2793 hypothetical protein -1.1 -1.6 -1.1 SSO2794 nitrate reductase, beta subunit -1.3 SSO2796 surface-located membrane protein 1, putative 1.1 1.6 1.2 1.2 1.5 1.0 1.3 SSO2797 hypothetical protein -1.2 SSO2798 hypothetical protein 1.2 1.6 1.2 1.7 2.2 1.6 -1.1 SSO2799 hypothetical protein 1.1 -1.0 SSO2802 cytochrome b558/566, subunit B -1.6 -1.4 -1.5 -1.6 -1.6 -2.7 -1.1 SSO2803 Rieske-I iron sulfur protein 3.2 3.2 SSO2805 cytochrome b -1.1 SSO2807 hypothetical protein 1.2 1.2 1.2 SSO2813 heterocyst differentiation protein, putative 1.8 1.8 1.4 1.6 1.9 1.9 2.1 2.1 1.7 1.9 SSO2814 conserved hypothetical protein 1.2 1.1 2.0 1.9 1.9 2.2 2.1 2.9 SSO2817 electron transfer flavoprotein alpha-subunit -1.2 -1.0 -1.7 -1.6 -1.2 -1.0 -1.5 -1.8 -1.4 -1.8 SSO2819 FIXC protein homolog (fixC) -1.1 -1.5 -1.7 -1.7 -1.9 SSO2825 hypothetical protein 1.1 SSO2827 conserved hypothetical protein 1.1 1.0 1.6 1.8 1.6 1.8 2.0 3.1 -1.1 SSO2830 hypothetical protein -1.0 -1.2 -1.2 SSO2834 conserved hypothetical protein 1.6 2.4 1.4 2.3 1.0 1.2 1.2 1.8 1.5 1.8 SSO2835 hypothetical branched-chain amino acid binding protein, putative -1.1 -1.7 -1.6 -1.2 -2.0 -1.8 1.3 SSO2836 branched chain amino acid ABC transporter, ATP-binding protein -1.4 -1.3 SSO2845 conserved hypothetical protein 1.0 1.5 1.2 1.7 SSO2847 conserved hypothetical protein -1.0 -1.5 -1.6

147

Table S1 Continued SSO2848 ABC transporter, putative -1.3 -1.6 -1.2 -1.3 -1.2 SSO2849 ABC transporter, putative -1.4 -1.6 -1.4 -1.9 -1.5 -2.0 -1.2 -1.7 -1.1 -2.0 SSO2852 sugar transporter family protein, putative -2.0 -2.3 -1.8 -1.9 -1.7 -2.4 -1.2 -1.3 -2.8 SSO2855 conserved hypothetical protein 1.1 1.1 1.0 1.2 1.0 1.1 1.6 SSO2856 conserved hypothetical protein -1.6 SSO2857 hypothetical protein 1.2 1.5 1.5 1.5 1.3 1.4 1.3 SSO2858 hypothetical protein 1.3 SSO2861 uncharacterized domain 1, putative 1.8 1.5 1.8 SSO2864 GPR1/FUN34/yaaH family superfamily -1.4 -1.4 -1.8 -1.1 -2.2 1.1 -1.5 -2.2 1.4 SSO2866 hypothetical protein -1.1 SSO2867 conserved hypothetical protein -1.4 SSO2868 Integral membrane protein, putative -1.4 -1.3 -1.6 -4.6 -2.0 1.6 SSO2872 Domain of unknown function domain protein -1.0 -1.4 -1.2 -1.4 -1.1 -1.0 SSO2875 4-coumarate:CoA ligase, putative 1.5 -1.1 1.0 1.3 SSO2878 threonine dehydrogenase, putative 1.7 1.3 2.1 1.9 2.0 2.3 2.7 3.2 1.7 2.3 SSO2879 Uncharacterized BCR, COG1975 superfamily 1.1 1.2 1.3 2.5 2.5 2.3 2.3 SSO2880 conserved hypothetical protein -1.9 -2.2 -1.4 -1.7 -1.2 -1.6 -1.6 -2.4 -1.1 -1.6 SSO2884 3-oxoadipate enol-lactone hydrolase/4-carboxymuconolactone decarboxylase 1.0 SSO2885 oxidoreductase, aldo/keto reductase family -1.2 SSO2887 hypothetical protein -1.2 -1.3 -1.1 -1.2 SSO2889 conserved hypothetical protein -1.0 -1.3 -1.1 SSO2890 metallo-beta-lactamase-related protein -1.1 -1.2 SSO2892 flavoprotein reductase, putative 1.6 2.2 1.1 1.7 1.1 1.3 1.4 1.6 1.1 1.3 SSO2893 putative transposase 1.1 SSO2894 putative invertase/transposase 1.1 1.6 1.1 1.8 2.1 1.2 2.0 SSO2897 Protein of unknown function, putative 2.4 2.5 2.7 2.7 2.5 2.8 2.5 2.6 2.3 2.8 SSO2899 Domain of unknown function domain protein 1.7 1.6 1.7 1.7 2.0 2.4 1.8 1.8 SSO2900 LPS biosynthesis protein, putative -1.2 SSO2901 Uncharacterized LmbE-like protein, COG2120 superfamily 1.0 1.1 SSO2905 phytoene synthase, putative 1.0 SSO2906 beta-carotene hydroxylase -1.0 -2.1 1.1 -1.4 SSO2907 phytoene dehydrogenase, putative -2.2 -3.7 SSO2908 uroporphyrin-III C-methyltransferase 1.0 SSO2909 sulfite reductase (NADPH) hemoprotein beta-component, putative -2.4 1.6 -1.3 -1.6 -1.2 -2.2 -1.7 -2.3 SSO2911 phosphoadenosine phosphosulfate reductase -1.5 2.2 -1.3 -1.5 1.3 -1.2

148

Table S1 Continued SSO2912 sulfate adenylyltransferase -2.0 1.7 -1.1 -1.5 -1.3 -2.4 1.1 -1.2 -2.3 1.0 SSO2913 conserved hypothetical protein -1.1 -2.5 1.9 -1.5 -2.0 -1.1 -2.1 -1.3 -2.0 SSO2914 hypothetical protein -1.6 -1.7 -2.3 SSO2916 putative transposase 1.0 1.2 SSO2917 hypothetical protein 1.2 1.3 1.2 1.1 SSO2918 Insertion sequence IS1126-like gene 1.1 1.0 1.4 1.2 1.8 SSO2919 putative transposase 1.1 1.0 SSO2922 conserved hypothetical protein -1.3 SSO2925 transposase related protein, putative 1.0 1.3 1.3 1.2 SSO2928 conserved hypothetical protein 1.4 1.0 1.1 1.3 1.3 SSO2930 probable transposase, putative 1.2 SSO2931 transposase related protein, putative 1.1 1.0 1.2 SSO2933 conserved hypothetical protein 1.5 1.1 1.9 2.4 1.6 SSO2936 N-methylhydantoinase -1.1 -1.1 SSO2938 osmoprotectant uptake system A -1.1 1.0 SSO2939 hypothetical arylmalonate decarboxylase 1.7 1.2 1.9 1.4 1.5 1.8 1.0 1.9 1.4 SSO2940 Metallo-beta-lactamase superfamily domain protein 1.1 SSO2941 2-hydroxyhepta-2 ,4-diene-1,7-dioate isomerase (hpcE-3) 1.4 SSO2943 putative resolvase 1.5 1.9 1.7 SSO2946 hypothetical tropomyosin, putative -1.3 -1.3 -1.5 -2.1 -1.8 1.1 SSO2947 transposase related protein, putative 1.0 1.1 SSO2949 probable transposase, putative 1.2 1.1 1.1 SSO2951 putative invertase/transposase 1.9 1.8 1.8 1.9 2.1 2.3 1.7 1.9 1.6 2.0 SSO2953 hypothetical protein 1.0 1.3 1.5 SSO2954 deoxycytidine triphosphate deaminase, putative -1.0 -1.0 -1.0 SSO2956 hypothetical protein 1.2 1.0 1.1 1.2 1.5 1.7 SSO2957 Helix-turn-helix domain, fis-type protein 2.2 2.2 3.1 2.1 1.7 2.8 3.4 SSO2959 Pfmdr2 protein -1.3 2.4 1.0 1.3 SSO2960 NADH dehydrogenase, putative -1.4 SSO2965 Sodium/hydrogen exchanger family family -1.1 SSO2968 conserved hypothetical protein -1.3 -1.8 -1.9 -1.1 SSO2970 cytochrome b, putative -1.1 -1.4 -1.6 -1.6 -2.3 SSO2971 Rieske-I iron sulfur protein -1.5 -1.2 -1.3 -1.0 -1.5 -1.3 SSO2973 cytochrome c oxidase polypeptide I, putative -1.2 -1.3 -1.9 -1.0 -2.0 SSO2975 hypothetical protein -1.3 -1.5 -1.5 -1.7 -1.3 -1.4 -1.0

149

Table S1 Continued SSO2976 hypothetical protein 1.0 1.3 1.1 1.1 1.6 SSO2977 conserved hypothetical protein -1.2 SSO2979 conserved hypothetical protein -1.1 -1.0 SSO2980 hypothetical protein 1.1 1.0 SSO2982 hypothetical protein -1.2 1.0 SSO2985 conserved hypothetical protein 1.2 2.1 1.7 2.6 1.1 1.9 1.3 1.6 1.5 2.2 SSO2986 conserved hypothetical protein 1.3 2.0 1.5 1.5 1.2 1.7 SSO2989 mevalonate diphosphate decarboxylase, putative -1.1 -1.4 -1.2 SSO2991 hypothetical protein -1.0 SSO2992 conserved hypothetical protein 1.4 1.0 SSO2993 conserved hypothetical protein 2.0 1.9 1.4 1.9 2.1 2.2 1.9 1.7 1.8 2.2 SSO2994 hypothetical protein 1.1 SSO3052 hypothetical protein -1.3 SSO3053 Bacterial extracellular solute-binding proteins, family 5, putative -2.1 -2.5 -2.2 -2.3 -1.2 SSO3055 ABC transporter, ATP-binding protein 1.5 SSO3056 putative transposase 1.3 1.0 1.2 SSO3059 peptide ABC transporter, permease protein, putative 1.0 SSO3060 alpha-L-fucosidase -1.0 SSO3061 conserved hypothetical protein 1.1 1.2 SSO3066 hypothetical protein -2.5 -3.7 -1.5 -2.2 -1.4 -2.4 1.0 -1.5 -2.5 1.0 -1.9 -2.5 SSO3067 ABC transporter, putative -3.2 -3.7 -2.7 -3.3 -2.2 -2.5 -3.4 -2.2 -3.2 SSO3068 ABC transporter, putative -1.9 -2.4 -1.9 -2.2 -1.4 -1.5 -2.7 -3.4 -1.5 -2.1 SSO3069 iron(III) ABC transporter, ATP-binding protein -1.4 1.1 SSO3071 conserved hypothetical protein -1.5 -2.1 -1.0 SSO3073 conserved hypothetical protein -1.3 -1.1 SSO3075 putative DNA-invertase -1.0 1.3 SSO3078 vapC-22 1.1 1.7 SSO3079 probable MFS transporter, putative -1.0 -1.5 -1.5 -1.7 -1.3 -2.2 -3.2 1.0 -1.8 -2.5 SSO3080 hypothetical protein 1.31.3 1.21.5 SSO3081 hypothetical protein -1.1 SSO3082 hypothetical protein -1.1 -1.5 -1.2 -1.6 -1.1 -1.6 -1.3 -2.0 -1.6 1.0 SSO3083 ABC transporter, ATP-binding protein, putative -1.3 -1.0 SSO3084 conserved hypothetical protein -1.1 -1.1 -1.3 -1.5 -1.1 -2.0 -2.5 -1.7 -1.9 SSO3085 conserved hypothetical protein -1.9 -2.6 -2.1 -2.8 -1.7 -1.5 -2.3 -3.2 -2.2 -3.0 SSO3086 hypothetical protein -1.1 -1.3

150

Table S1 Continued SSO3087 hypothetical protein -1.0 -1.3 SSO3089 hypothetical protein -1.1 -1.4 -1.0 -1.5 -1.7 -1.0 -1.9 -1.1 -2.0 SSO3090 hypothetical protein -1.6 -2.1 -1.7 -2.5 -1.6 -2.3 -1.7 -2.5 -1.2 -2.8 1.6 SSO3091 conserved hypothetical protein -1.2 -1.6 -1.5 -2.1 -1.4 -2.0 SSO3092 hypothetical protein -1.1 SSO3093 ABC transporter, ATP-binding protein, putative 1.2 SSO3095 hypothetical protein -1.0 -1.0 -1.2 SSO3097 conserved hypothetical protein 1.4 1.5 1.0 1.2 SSO3098 conserved hypothetical protein -1.3 -1.7 -1.4 -1.4 -1.3 -1.6 -1.8 -2.1 -1.3 SSO3099 hypothetical protein -1.0 SSO3105 thermostable carboxypeptidase 1, putative -1.2 -1.7 SSO3107 dihydroxy-acid dehydratase -1.5 -2.3 -1.4 -1.9 1.1 -1.6 -1.2 -2.2 1.0 SSO3111 conserved hypothetical protein -1.3 SSO3112 Domain of unknown function, putative -1.5 -1.6 -2.3 -2.7 -1.1 -1.5 SSO3113 Thiolase, putative -1.4 -1.7 -1.9 -2.5 -1.8 -2.4 -2.2 -3.1 -1.9 SSO3114 3-oxoacyl-(acyl-carrier-protein) reductase -1.3 -1.4 -1.2 -1.5 -1.5 -1.5 -1.6 -2.1 -1.3 -1.6 SSO3115 proline iminopeptidase -1.1 SSO3117 aldehyde dehydrogenase, thermostable -1.5 -1.8 -1.3 -1.0 -1.1 -1.8 SSO3118 hypothetical protein -1.9 -2.5 -1.1 -1.3 -1.3 -1.5 -1.6 SSO3120 sugar transporter, putative -1.6 -2.0 -1.4 -1.6 -1.1 -1.0 -2.1 -2.5 -1.5 -2.0 SSO3121 probable MFS transporter 1.3 3.1 SSO3124 Mandelate racemase / muconate lactonizing enzyme family, putative -1.3 -1.5 SSO3127 seed imbibition protein -1.5 -1.3 -1.5 -1.6 SSO3129 multidrug transporter, putative -1.4 -1.1 -1.7 SSO3130 iron-sulfur cluster binding protein -1.1 -1.1 -1.4 SSO3131 conserved hypothetical protein -1.0 -1.1 SSO3132 metallo-beta-lactamase superfamily protein 1.1 1.1 SSO3136 FkbR2 -1.1 SSO3139 hypothetical protein -1.1 -1.3 -1.0 SSO3144 fatty acid-CoA racemase, putative -1.3 -1.2 -1.5 -1.4 -1.6 SSO3145 probable acyl-CoA dehydrogenase -1.1 SSO3146 conserved hypothetical protein -1.0 -2.6 -4.1 1.6 SSO3147 probable MFS transporter, putative -1.0 -1.5 -1.4 SSO3148 NADH oxidase, putative 2.2 2.1 1.6 2.0 2.0 1.7 2.4 2.4 1.8 1.9 SSO3150 conserved hypothetical protein 1.3 1.1 1.2

151

Table S1 Continued SSO3151 conserved hypothetical protein 1.4 1.4 1.6 1.4 1.8 SSO3153 putative resolvase/transposase 1.1 1.0 SSO3155 Trp repressor-binding protein -1.3 1.0 SSO3159 lipoate-protein ligase A, putative -1.0 -1.0 SSO3163 glycolate oxidase, subunit GlcD -1.3 -1.5 -1.5 -1.6 -1.5 -1.3 -1.5 -1.6 -1.4 -1.1 SSO3164 hypothetical protein 1.0 SSO3165 D-lactate dehydrogenase, putative -1.2 -1.6 -1.2 -1.1 -1.9 SSO3167 mutator MutT protein (mutT) 1.8 1.0 1.8 1.0 1.8 SSO3168 conserved hypothetical protein -1.2 -1.1 SSO3170 conserved hypothetical protein 1.3 1.4 SSO3171 IS1537, resolvase, putative 1.2 1.3 SSO3172 putative resolvase/transposase -1.3 SSO3174 inosine-5-monophosphate dehydrogenase related 1.0 1.5 1.4 1.2 1.2 1.6 1.5 SSO3175 hypothetical protein -1.8 -1.9 -1.7 -1.8 -1.4 -1.4 -1.0 -1.6 SSO3178 hypothetical protein 2.3 1.8 2.0 1.4 2.0 1.8 2.7 2.5 SSO3179 hypothetical protein 1.1 SSO3180 phosphate permease -1.9 -2.3 -1.4 -1.2 -1.2 -1.1 SSO3182 Eukaryotic protein kinase domain protein 1.2 SSO3184 pheP phenylalanine-specific permease, putative -1.0 -1.7 -1.2 -1.8 -1.7 -1.8 1.0 -1.5 1.3 SSO3188 hypothetical protein 1.2 1.2 SSO3189 hypothetical protein -2.6 -2.9 -3.0 -3.4 -2.5 -3.1 -2.5 -3.3 -2.3 -2.6 SSO3192 Sodium/calcium exchanger protein family 1.8 2.0 1.7 1.9 2.1 3.1 2.5 2.6 SSO3197 N-acetylneuraminate lyase, putative -1.0 -1.0 SSO3200 Domain of unknown function domain protein -1.4 -1.0 SSO3201 molybdopterin oxidoreductase, putative -1.0 SSO3207 Eukaryotic protein kinase domain protein -1.4 -2.4 -2.6 -2.8 -2.6 SSO3210 acetolactate synthase large subunit, putative -1.1 SSO3214 Acetyltransferase (GNAT) family -1.0 -1.2 SSO3220 putative transposase 1.6 1.2 1.0 1.0 1.1 1.3 SSO3224 pheP phenylalanine-specific permease, putative -1.0 -1.3 -1.5 SSO3225 conserved hypothetical protein -2.1 1.3 -1.3 -2.5 1.2 -1.2 -2.6 1.4 -2.1 1.3 -3.0 1.7 SSO3226 Deoxyribose-phosphate aldolase superfamily -1.0 -1.0 SSO3227 hypothetical protein -1.1 -1.0 -1.2 SSO3228 hypothetical protein -1.0 SSO3230 inosine-5-monophosphate dehydrogenase related 1.3 1.1 1.5 1.2 1.0 1.8 2.4

152

Table S1 Continued SSO3233 cobyric acid synthase CobQ -1.3 -1.2 SSO3234 cobalamin (5-phosphate) synthase -1.1 -1.4 -2.1 SSO3237 threonine 3-dehydrogenase, putative -1.1 -1.3 SSO3239 oxidoreductase -1.1 -1.1 SSO3240 uncharacterized domain 2 protein -1.0 SSO3241 apolipoprotein n-acyltransferase Lnt/dolichol-phosphate-mannosyl transferase -1.2 -1.0 -1.0 -1.2 -1.6 -1.4 SSO3242 transcriptional regulator, DeoR family domain protein -3.3 -4.2 -2.3 -3.4 1.1 -2.2 -3.0 -2.4 -3.2 -1.8 -2.5 SSO3243 mu-crystallin homolog, putative -1.4 -1.9 -1.1 -1.4 SSO3246 hypothetical protein -1.7 -2.5 -1.8 -2.1 -1.0 -1.8 -1.4 -2.1 -1.4 -1.5 SSO3248 conserved hypothetical protein 1.0 1.2 SSO3251 conserved hypothetical protein -1.1 -1.1 SSO3254 DNA-directed RNA polymerase subunit B -1.1 SSO3259 conserved hypothetical protein -1.0 SSO5027 hypothetical protein 1.7 1.2 1.5 1.3 1.9 SSO5098 small hydrophobic subunit of the terminal oxidase with unknown homologue -1.1 -1.0 -1.5 -1.2 SSO5140 DNA-directed RNA polymerase subunit n-related protein -1.1 SSO5209 conserved hypothetical protein -2.0 -3.0 -1.9 -2.7 -1.5 -2.4 -1.9 -2.7 -1.6 -1.9 SSO5336 Ribosomal L40e family 1.3 SSO5343 conserved hypothetical protein -1.0 -1.6 -1.1 -1.7 -1.4 SSO5345 translation elongation factor aEF-1 beta -1.1 -1.1 -1.3 -1.0 -1.5 SSO5410 snRNP, putative -1.4 -1.5 -1.3 -1.1 -1.5 -1.3 SSO5478 Ribosomal protein L24e -1.3 -2.0 -1.7 -1.7 -1.5 SSO5559 hypothetical protein -1.1 -1.1 SSO5576 DNA-directed RNA polymerase subunit M -1.1 SSO5577 RNA polymerases L / 13 to 16 kDa subunit -1.4 -1.2 -1.2 -1.3 -1.2 -1.1 -1.0 -1.5 -1.5 SSO5668 50S ribosomal protein Lx -1.3 -1.0 SSO5671 Ribosomal protein L31e 1.5 SSO5761 tRNA pseudouridine synthase subunit A (truB) -1.0 -1.0 -1.1 SSO5763 Ribosomal protein L14 -1.2 -1.2 SSO5826 conserved hypothetical protein -1.8 -1.6 -1.5 -1.6 -1.3 -1.5 -1.3 -1.5 SSO5865 DNA-directed RNA polymerase, putative 1.0 1.0 SSO5866 ribosomal protein SUI1 1.2 1.0 1.0 1.0 1.4 SSO5909 conserved hypothetical protein 1.6 1.1 1.2 SSO6008 hypothetical protein 1.7 2.3 1.5 2.1 1.7 2.2 1.5 1.8 1.4 2.0 SSO6024 SSV1 virus orf B-129 fragment-like protein 1.0 1.5 1.7

153

Table S1 Continued SSO6175 membrane-associated ATPase epsilon chain-related protein -1.8 -1.9 -1.5 -2.0 -1.9 -2.3 -1.2 -1.3 -1.2 SSO6264 conserved hypothetical protein -1.2 SSO6374 Ribosomal protein L34e -1.1 -1.5 -1.2 -1.4 -1.0 -1.6 -1.4 SSO6391 ribosomal protein S14 -1.4 -1.4 SSO6401 ribosomal protein L23 -1.1 -1.3 -1.1 -1.6 -1.1 -1.2 -1.1 -1.1 -1.7 SSO6418 Ribosomal L37ae protein family -1.5 SSO6454 snRNP, putative -1.2 -1.0 -1.1 -1.3 SSO6468 conserved hypothetical protein -1.2 -1.6 SSO6469 hypothetical protein 1.2 SSO6570 Fourth ORF in transposon ISC1225 1.0 SSO6687 hypothetical protein -1.6 -2.5 -1.6 -1.1 -1.9 -1.2 -2.1 -1.5 SSO6716 hypothetical protein 1.31.2 2.22.7 1.41.9 SSO6768 DNA-directed RNA polymerase subunit K -1.1 -1.0 -1.3 -1.3 SSO6778 hypothetical protein -1.3 -1.9 -2.0 -1.7 -1.2 -1.7 -2.6 -1.4 -2.1 SSO6817 ribosomal S30/ubiquitin fusion 1.3 1.7 SSO6877 conserved hypothetical protein -1.0 SSO6901 hypothetical ribonuclease p3-related protein 1.4 1.1 1.5 1.4 2.1 1.9 2.2 2.6 2.3 2.9 SSO6904 conserved hypothetical protein 1.01.8 1.32.0 2.3-1.8 SSO7111 Ribosomal protein L44 -1.3 -1.1 SSO7114 Ribosomal protein S27 -1.2 -1.1 SSO7115 conserved hypothetical protein -1.3 SSO7348 sugar carrier protein 1.1 1.1 2.3 2.0 SSO7351 conserved hypothetical protein 1.5 1.6 1.1 1.7 1.6 1.9 SSO7412 pyuvate ferredoxin oxidoreductase 1.4 SSO7578 hypothetical protein 1.4 1.3 1.2 1.1 1.6 1.0 1.5 1.5 SSO7607 First ORF in partial transposon ISC1160 1.3 1.4 2.4 1.4 1.6 2.3 2.8 2.6 3.2 SSO7610 Second ORF in partial transposon ISC1160 2.0 2.3 2.6 3.1 2.6 3.7 -1.1 2.5 3.0 2.0 2.8 SSO7621 First ORF in transposon ISC1160 1.7 1.7 2.5 1.8 2.5 2.8 3.9 -1.1 1.8 2.7 SSO7627 Second ORF in transposon ISC1160 1.5 -1.1 SSO7635 conserved hypothetical protein 2.0 -1.2 1.2 1.6 1.4 1.8 SSO7754 Third ORF in transposon ISC1160 1.2 1.7 SSO7756 Second ORF in transposon ISC1160 1.7 1.3 1.6 2.5 3.6 2.4 2.8 SSO7758 First ORF in transposon ISC1160 1.5 1.8 1.8 2.5 1.6 2.4 2.5 3.4 2.3 3.1 SSO7893 putative DNA-invertase 1.3 1.2 1.4 1.2 1.1 1.2 1.1 1.8 SSO8090 hypothetical protein 1.1 1.1

154

Table S1 Continued SSO8124 DNA polymerase II (DNA polymerase B2) amino-end (dpo2) -2.1 -2.4 -2.0 -2.4 -1.8 -2.1 -2.3 -3.2 -1.5 -2.2 SSO8189 hypothetical protein 1.0 SSO8195 Third part of one of two inversely orientated ORFs in ISC1043 -1.8 SSO8278 hypothetical protein 1.1 1.1 1.1 1.1 SSO8288 putative resolvase/transposase -1.4 -1.1 -1.2 SSO8351 hypothetical protein 1.8 2.4 2.2 2.9 SSO8380 First ORF in transposon ISC1160 1.1 1.1 1.8 -1.0 1.2 2.2 2.6 SSO8382 Second ORF in transposon ISC1160 1.82.5 1.52.1 1.92.4 2.53.3 SSO8386 Third ORF in transposon ISC1160 1.0 1.6 -1.0 SSO8426 hypothetical protein 1.0 SSO8472 conserved hypothetical protein 1.2 1.1 SSO8481 hypothetical protein 1.1 1.0 3.7 3.7 SSO8549 hypothetical protein -1.1 SSO8568 conserved hypothetical protein 1.1 1.0 SSO8620 vapB-8 3.2 2.8 2.8 2.9 3.4 3.8 3.9 4.7 4.3 4.9 SSO8684 vapC-12 1.0 1.2 1.2 1.3 SSO8685 conserved hypothetical protein 1.0 1.1 SSO8687 conserved hypothetical protein -1.3 SSO8875 hypothetical protein 1.1 1.0 1.3 1.2 1.1 1.6 SSO8892 hypothetical protein 1.0 SSO8906 hypothetical protein 1.1 1.7 SSO8910 hypothetical protein 2.7 2.3 2.5 2.9 3.3 2.8 3.4 3.3 3.5 SSO8936 probable arginyl-tRNA synthetase (argrs) 1.0 1.2 1.3 1.1 1.3 1.6 SSO8938 hypothetical protein 1.9 2.4 2.2 2.9 2.6 2.8 4.2 SSO8948 DNA-binding protein-related protein 2.02.3 3.03.3 4.5 SSO8998 hypothetical protein 1.6 1.4 2.1 1.2 2.0 1.9 2.2 2.1 1.5 1.7 SSO9043 transposase 1.9 2.3 2.9 3.0 2.7 4.0 3.8 SSO9092 conserved hypothetical protein 2.2 2.8 3.6 4.7 -1.1 2.0 5.4 6.0 4.3 5.6 -1.4 SSO9115 conserved hypothetical protein 1.41.4 1.21.8 1.01.3 1.42.5-1.1 SSO9134 Site-specific recombinases, putative 2.3 2.4 3.5 SSO9135 Site-specific recombinases, putative 2.9 2.9 3.3 3.9 3.4 4.2 4.2 4.5 SSO9136 conserved hypothetical protein 1.0 1.1 SSO9180 dna-binding protein 7 1.3 1.2 1.5 SSO9268 conserved hypothetical protein 1.2 1.1 4.1 3.4 SSO9378 vapB-15 1.9 1.9 1.7 1.5 2.1 2.0 1.7 2.2 2.4 2.9

155

Table S1 Continued SSO9761 First ORF in transposon ISC1225 1.8 1.7 1.8 2.2 1.9 2.3 1.5 2.1 SSO9763 1.7 1.8 1.8 2.2 1.6 2.0 1.7 2.2 2.0 SSO9953 transcription regulatory protein 1.1 1.0 1.4 1.5 1.4 1.8 1.4 2.1 vapB-1 vapB-1 -1.0 -1.5 -1.9 -2.1 -1.6 -2.2 vapB-10 vapB-10 1.2 1.5 2.3 2.8 3.7 3.7 vapB-11 vapB-11 3.0 2.9 2.3 2.5 2.5 3.0 2.8 3.7 vapB-13 vapB-13 2.7 2.2 1.9 2.1 2.1 2.2 2.9 3.0 1.9 2.6 vapB-16 vapB-16 2.1 1.7 1.8 1.9 1.7 2.2 2.2 3.0 vapB-17 vapB-17 1.1 1.3 1.1 2.2 -1.1 vapB-19 vapB-19 2.1 2.1 1.8 2.1 1.4 1.9 2.1 2.9 2.5 3.1 vapB-4 vapB-4 1.2 1.1 1.6 1.5 1.1 1.4 1.3 1.9 1.7 2.8 vapB-7 vapB-7 2.2 1.8 2.4 2.8 1.7 1.8 2.4 2.8 2.4 3.4

156

CHAPTER 4

VapB Antitoxin is associated with the aIF2-γ subunit in archaeal genomes

Charlotte R. Cooper1, Sabrina Tachdjian1, Paul H. Blum2, and Robert M. Kelly1

1Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695-7905

2Beadle Center for Genetics University of Nebraska-Lincoln

To be submitted to: Nucleic Acids Research

157

ABSTRACT

Archaea lack many of the RNA management tools found in Eukaryotes and Bacteria,

such as RNA interference (RNAi), small interfering RNAs (siRNAs), bacterial Rho

transcription termination factor, and bacterial degradasomes. What little is known about the

translation machinery in Archaea indicates that a heterotrimeric translation initiation factor,

aIF2, is responsible for bringing initiator tRNA to the ribosomal complex. VapBC toxin-

antitoxin loci are common in many prokaryotic genomes, although their functional roles have

not been determined. As such, it was interesting to note that in most archaeal genomes

sequenced to date, the gene encoding a putative VapC toxin often overlaps an upstream gene,

which encodes aIF2 subunit γ. This curious genomic locus has implications as a potential

archaeal RNA management strategy. In the extremely thermoacidophilic archaeon Sulfolobus

solfataricus, VapB-1 is contained within SSO0412, which encodes aIF2γ. In fact, the crystal structure of recombinant Sso aIF2γ (PDB:3cw2) indicates VapB-1 is represented in the

second and third domains. aIF2 would not assemble into the heterotrimeric complex if the

VapB-1 (here referred to at VapBγ) component was removed from the aIF2γ subunit,

indicating that VapBγ remains as part of the aIF2γ structure. Furthermore, VapC-1

(SSO0414) overlaps aIF2γ and was co-transcribed under several different growth conditions

(here referred to as VapCγ). Recombinant Sso VapCγ was determined to be a monomeric

ribonuclease, which did not associate with recombinant Sso aIF2γ, heterotrimeric Sso aIF2,

or VapBγ in vitro. By contrast, the recombinant versions of a typical TA pair in S.

solfaraticus, VapBC18 (unannotated and SSO1975, respectively), readily formed a multimer,

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consisting of three or four dimeric pairs of VapB18 and VapC18. While there was no

evidence that VapBγ associated with VapCγ in S. solfataricus, the deletion of the gene

encoding VapCγ resulted in a non-viable phenotype. The interesting localization of Sso

VapBγ, and importance of S. solfataricus VapCγ for cell viability, suggests a significant, and

perhaps unique, functional role of this atypical TA pair in S. solfataricus and orthologues in

other archaea, potentially related to RNA management strategies.

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INTRODUCTION

For messenger RNA to be translated into functional proteins, translation must be initiated by a multimeric complex that brings the initiator methionyl-tRNA (tRNAi) to the correct site of the ribosome (11). This process varies greatly in prokaryotes and eukaryotes, while archaea seem to have a juxtaposition of the two, with a few unique quirks all their own.

There are several protein factors involved in translation initiation, but initiation factor 2 (IF2) is a key component because it is responsible for binding and delivering the Met-tRNAiMet to

the mRNA start site (13).

The form and function of IF2 in bacteria is still not completely understood, but based

on information derived from a few crystal structures, bacterial IF2 is a large six-domain

monomeric protein (5, 6, 16). In contrast, eukaryotes have a heterotrimeric initiation factor

(eIF2) consisting of α, β, and γ subunits. Archaea have a system homologous to eukaryotes

(aIF2), although the β subunit is typically smaller (24). The crystal structures for the α−β−γ

complex with and without GTP have been determined for the extreme thermoacidophile

Sulfolobus solfataricus (25), and the interaction of the subunits has been studied extensively

(17, 20-22, 25). The α subunit contains three domains: flexible domains 1 and 2 and rigid domain 3, which complexes with γ’s domain II. α is predicted to play a significant role in tRNAi and/or rRNA binding. In fact, by having just domain 3 of α pre-bound to γ, the tRNAi binding affinity is 2 orders of magnitude higher than γ alone (26). By contrast, β shows no significant role in tRNAi binding. The smallest of the three subunits, it interacts

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with γ at domain I (also known at the G-domain) and only interacts with α through γ (19).

Gamma is the largest subunit, with three distinct domains (I, II, and III). In domain I, there is

Met a GTP binding site and a binding pocket is formed for Met-tRNAi in domains I and II.

The methionine group on the tRNA is key for binding, as it was demonstrated that non-

initiator tRNAs aminoacylated with methionine can bind γ and are protected from

deacylation (26).

There are two modes of translation initiation in archaea. One involves a Shine-

Delgarno (SD) motif that, similar to bacteria, pairs with the anti-SD on the 16S ribosomal

subunit (6, 10). The second mechanism involves what is known as “leaderless mRNAs” which completely lack a 5’ untranslated region. Because archaea lack homologues to the

eukaryotic cap complex that binds mRNA and scans for the start codon (namely eIF4F, eIF3,

and the GDP-GTP exchange factor for eIF2 (7)), it has been shown that the ribosome must

have pre-bound tRNAi to interact in a codon-anticodon fashion at the start site of leaderless

transcripts (1).

In an intriguing revelation, it was shown that in S. solfataricus the translation

initiation factor aIF2 and subunit γ alone can bind mRNA at the triphosphorylated 5’ end and

protect it from 5’Æ3’ mRNA decay (9). Because mRNA binding and Met-tRNAiMet binding occurs at the γ-subunit, and there may be a complex mechanism of competition for the site

(8), it lends increased significance for this subunit in the role of translation regulation. It has been shown that free γ prefers mRNA, while ribosome associated γ prefers tRNAi (9). It has also been noted that there is no clear mechanism for dissociating the bound γ from mRNA and that bound mRNA is untranslatable.

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Archaea lack many of the eukaryotic and bacterial mechanisms for RNA and protein management, such as RNA interference and the Clp and Lon proteases, but Type II Toxin-

Antitoxin loci have been proposed as a compensatory mechanism (2). Several families of toxins (T) including the VapC (from virulence associate protein), which have a characteristic

PIN-domain that is homologous to the PilT protein N-terminus, have been shown to have ribonuclease activity and may play a significant role in stress response (2, 3, 12, 15, 23), though their exact mechanism is still mostly a mystery.

While the vast majority of TA loci are independent, free-standing open reading frames (ORFs), the predicted vapB-1 gene in S. solfataricus overlaps with the C-terminus of

SSO0412, encoding the γ-subunit of translation initiation factor aIF2 (Figure 1) (18). Both

Sso aIF2γ and vapB-1 (from here denoted vapBγ) are encoded on the same DNA strand, implying that VapBγ is contained within the structure of aIF2γ as the C-terminal domain of the subunit (domains II and III). Sso VapC-1 (denoted VapCγ), a suspected ribonuclease, is located directly downstream of predicted VapBγ and overlaps 36 nucleotides so that co- transcription is likely. Since VapCγ is thought to bind VapBγ, and the latter almost certainly remains the undissociated C-terminal domain of aIF2γ, VapCγ might play several intriguing roles: 1) blocking aIF2 complex formation by binding the γ subunit, 2) mRNA degradation of transcripts bound to the γ subunit to free the subunit for α−β−γ initiation complex formation,

3) regulation of mismethylated tRNAs bound to the aIF2 complex.

Here, we present evidence that aIF2γ/vapBγ co-location is highly conserved among the Archaea. Furthermore, we show that VapCγ from S. solfataricus is a traditional toxin

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displaying ribonuclease activity, and examine its association with the cognate antitoxin, either VapBγ free from aIF2γ, the entire γ subunit, or heterotrimeric complex α−β−γ. We also determine if the VapBγ portion of aIF2γ is necessary for complex formation and if

VapCγ will bind γ and inhibit complex formation. Finally, we discuss the possible roles that aIF2γ/vapBγ collocation followed by a PIN-domain protein may play in RNA management in

Archaea.

MATERIALS AND METHODS

Comparative Genomic Analysis. To determine if the co-location of vapBγ with aIF2γ followed by a predicted PIN-domain containing ribonuclease is conserved, a comparative analysis of 70 sequenced archaeal genomes was performed using

MicrobesOnline (4). The genomes with conserved aIF2γ homologues (COG-GCD11) followed by vapCγ homologues (COG1412) are listed in Table 1. Whenever possible, the vapCγ and vapBγ were checked against the predictions by Pandey and Gerdes (18) (Table

S1).

Cloning and Expression of Sso aIF2, VapBγ, VapCγ, and VapBC18. As previously noted for S. solfataricus (19), recombinant aIF2 subunits expressed with an N- terminal hexahisitdine (N-His) tag does not interfere with protein production or complex formation. Therefore, SSO1050 (α), SSO2381 (β), SSO0412 (γ), and a truncated version of

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γ without the vapBγ (denoted γ-Bγ) were cloned into pET28b+ (Novagen). All of the primers

used for cloning are provided in Table 2. SSO0414 (VapCγ) and SSO1975 (VapC18) were

cloned into pET21b+ (Novagen) with a C-terminal hexahistidine (C-His) tag, and VapCγ was

also cloned with no tag. VapBγ was cloned into both pET28b+ and pET21b+ to yield N-His,

C-His, and untagged proteins. VapB18 was cloned into pET21b+ with no His-tag added. All of the genes were expressed in Rosetta (DE3) (Novagen). As a negative control, pET21b+ with no insert was also transformed and expressed in exactly the same manner as the recombinant genes. For expression, 50ml overnight cultures of each subunit were grown in

LB with chloramphenicol and kanamycin or ampicillin. Larger 1L cultures were inoculated with overnight cultures to an OD600~0.1 and grown to OD600~0.5 where they were induced with 1mM IPTG (Inalco). Expression was allowed to proceed for 4 hours at which point the cultures were harvested at 10,000xg. The pellets were frozen overnight. The pellets were then thawed and re-suspended in ~10ml of “Buffer A” (50mM Na-Phos, pH 8 and 300mM

NaCl) with 100μg/ml Lysozyme (Sigma) and sonicated for 10 minutes at 50% Amplitude

(10s on, 10s off). Alternatively, the pellets were re-suspended in 10ml of “Reaction Buffer”

(50mM Tris, pH6, 150mM NaCl, 250mM KCl, and 10mM MgCl2) and French Pressed at

approximately 15,500 psi. The cell debris was pelleted at 20,000xg and the soluble fraction

was sterile filtered with a 0.22 μm filter. The presence of untagged VapB18 and VapCγ were confirmed by mass spec analysis.

Protein Purification. Soluble protein from recombinant expression trials was concentrated and purified further by FPLC with an IMAC column (BioRad). The protein

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was loaded in “Buffer A” and eluted with “Buffer B” (Buffer A + 500mM Imidazole). The

purified proteins were concentrated and dialyzed into the “Reaction Buffer”. To ensure that

downstream results of the RNase assays were not the consequence of E. coli contamination,

pET21b+ was also purified with the IMAC column. Although E. coli naturally has some protein that will bind the column, only the fractions identical to those used for VapCγ C-His and VapC18 C-His were collected and processed. This “mock extract” ensured that the same residual E. coli RNase activity was quantified correctly. All of the cultures yielded soluble protein, except for the vapBγ constructs. The VapBγ protein was purified from inclusion

bodies and refolded with a Protein Refolding Kit (Novagen). The refolded protein was

soluble in 20mM Tris-HCl, pH 8.5 (the Dialysis buffer from the kit), but precipitated if left in

“Buffer A” for Nickel column purification, dialyzed into “Reaction Buffer”, or subjected to

65oC heat treatment.

Sso aIF2γ-Bγ was produced in soluble form, but in much smaller quantities than the

other aIF2 subunits. Upon concentration, it was apparent that there were several

contaminating proteins from E. coli. As such, γ-Bγ protein was further purified on a HighQ

Anion exchange column (BioRad). The protein was first dialyzed into “Buffer A” (20mM

Tris-HCl, pH 8) and loaded onto the column. It was eluted with a linear gradient (0-40%) of

“Buffer B” (20mM Tris-HCl, pH 8, 1M NaCl). The resulting protein showed fewer

contaminating bands after concentration and dialysis back into the “Reaction Buffer”.

Assembling the aIF2 complex. To assemble the complex, the α, β, and γ or γ-Bγ proteins were combined in varying amounts and incubated in the “Reaction Buffer” at 65oC

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for 15 minutes as previously described (19). To visualize the complexes, the reaction

products were electrophoresed on non-denaturing polyacrylamide gels. Due to the pI of the

proteins (mostly 8 or above), acidic native gels were used and prepared as described

previously (26). Briefly, a 15% acrylamide gel was prepared in potassium acetate buffer

(1.5M Acetate-KOH, pH 4.3 stock solution). The stacking gel was 4% acrylamide (0.25M

Acetate-KOH, pH 6.8 stock solution), while the running buffer was 350 mM β-Alanine and

140mM acetic acid, pH 4.3. To determine the effects of temperature on complex assembly,

o the samples were incubated at 65, 70, 75, 80, and 85 C for 15 min. To assess whether VapCγ interfered with complex formation, VapCγ was added with γ and α−β−γ and incubated at 65,

70, and 75oC for 15min. The assembly products were run on a native gel.

VapCγ ribonuclease activity assay. To determine the ribonuclease activity of purified recombinant VapCγ, the RNaseAlert Lab Test Kit (Ambion) was used, as previously

described for Haemophilus influenzae (3). The RNaseAlert substrate, which is labeled with

fluorophores and quenchers, was re-suspended in 5μl of 10X RNaseAlert Buffer. 45μl of

~0.6 mg/ml total protein VapCγ sample was added to one tube, as was ~0.3 mg/mg total protein VapC18 sample for comparison. As a positive control, 5μl RNase A was added to another tube. For negative controls, the pET21b+ mock extract was added to a separate tube, as was the reaction buffer. The tubes were wrapped in aluminum foil to shield them from light and placed at 37oC for 30min. After that time, the tubes were viewed on a UV- transilluminator. The fluorescence of the VapCγ and VapC18 tubes were compared to the

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RNase A control. The tubes were incubated at 37oC for an additional 30min. and reassessed.

After 1hr, RNase A was added to the two negative control tubes to show that labeled RNA

was present.

VapC “fishing” experiments. To establish an interaction between a typical cognate

TA pair in S. solfataricus, VapBC18 were utilized. VapB18 with no His-tag was produced in

soluble form in E. coli cell extract, and VapC18 C-His was produced in soluble form in a

separate culture. VapC18 C-His and VapB18 were incubated separately at 95oC for 10 minutes then combined and incubated together for 2 hours at 80oC, as described for

recombinant VapBC-6 previously (12). The sample was centrifuged at 20,000xg to remove

insoluble protein and sterile filtered with a 0.22μm filter. The protein was applied to an

IMAC column loaded in “Reaction Buffer” and eluted with a linear gradient (0-100%) of

“Reaction Buffer + 500mM Imidazole”. The experiment was repeated for VapB18 alone to

confirm that it did not bind to the column without VapC18 C-His (data not shown). For

fishing with the α−β−γ complex and γ alone, the temperature was lowered to 65oC. To

o determine if VapCγ interacted with γ, the γ subunit was incubated at 65 C for 10 minutes

before VapCγ with no His-tag was added, and the incubation was extended for 2 hours at

65oC. To test the entire complex, α, β, and γ were incubated at 65oC for 15 minutes together

before VapCγ with no His-tag was added, and then incubated for 2 additional hours. The samples were treated in the same manner as VapBC18 and applied to the IMAC column as

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described. The same experiments were run with VapCγ C-His in place of the VapCγ untagged protein.

Determination of VapB, VapC, and VapBC molecular assembly. The VaBC18 complex that eluted from the IMAC column separated into two clean bands on an SDS

PAGE gel. Densiotometry reveled there was a 1:1 ratio of VapC18 C-His to VapB18 protein

(Carestream Molecular Imaging Software SE v. 5.0.2.30, Carestream Health, Woodbridge,

CT). The complex was separated on a native page gel and run with the Native Mark ladder

(both from Invitrogen). The gels were run in the dark blue cathode buffer, per the manufacturer’s instructions. The gel was de-stained using the quick de-stain protocol, per the manufacturer’s instructions. The complex was run on a Sephacryl 200 sizing column

(GE Healthcare) in the “Reaction Buffer”. The VapB18 protein and VapC18 C-His protein were run individually on a Sephacryl 75 sizing column (GE Healthcare) in the “Reaction

Buffer”. VapCγ C-His was also run on the S75 sizing column.

RESULTS AND DISCUSSION

aIF2γ/vapBγ co-location is conserved in archaea. Of the 70 sequenced archaeal genomes examined, 46 of the 70 showed aIF2γ homologues (COG-GCD11), followed by predicted vapCγ homologues (COG1412) (Table 1). The aIF2γ/VapCγ gene arrangement falls into one of three categories (2): (1) the genes overlap, (2) there are a few nucleotides between aIF2γ and vapCγ, or (3) an ORF has been inserted between the two genes. The third

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category only applies for two organisms, Pyrococcus abyssi and Pyrococcus horikoshii

shinkaj, and in both cases, the vapBγ was predicted to be part of the intervening gene by

Pandey and Gerdes (18). For the other organisms with TA systems predicted (10 of the

remaining 44), VapCγ’s cognate antitoxin was contained within aIF2γ. Missing from the

conserved co-location were all sequenced Nanoarchaeota and Korarchaeota, as well as

certain Euryarchaeota (Methanococcus and Methanocaldococcus species), and Crenarchaeota

(Pryobaculum species).

Role of VapC in S. solfataricus. Recombinant VapCγ (C-His) and VapC18 (C-His)

were determined to be ribonucleolytic (Figure 3). Efforts to silence the VapC toxins were

unsuccessful, as recombinant VapB18 and γ/αβγ exhibited ribonuclease activity (data not

shown) even after several attempts to remove this background activity through various heat

treatment and purification steps. Note that recombinant VapB-6 was able to silence

recombinant VapC-6 in vitro (12).

Assembly of S. solfataricus aIF-2 subunits α−β−γ. To ensure the recombinant α, β,

and γ proteins were properly folded, the complex was assembled at 65, 70, 75, 80, and 85oC

and viewed on an acidic native gel (Figures 4 and 5). Note that the γ-subunit becomes

o unstable at 75 C. When the VapBγ sequence was removed from recombinant γ, leaving γ-Bγ, the complex would not assemble (Figure 6), suggesting that the γ subunit is not likely proteolytically cleaved in vivo to release VapBγ. Efforts to produce VapBγ by itself were

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futile, as the recombinant protein was insoluble and unstable when re-folded; this indicates

that VapBγ is encoded in the γ-subunit and not produced as a separate gene product from γ.

When α, β, and γ were incubated in the presence of VapCγ, the α−β−γ complex still formed

(Figure 7), indicating that under the conditions of the reaction assay VapCγ did not compete

for γ and does not interfere with the binding of α and β to γ.

Cognate TA interactions. To establish whether the cognate toxins and putative

antitoxins interact, the recombinant tagged proteins were used to “fish” for their recombinant

binging partner expressed in soluble form in E. coli cell extracts. VapC18 C-His was able to

specifically bind and co-elute from an IMAC column with VapB18 (Figure 8). It is

important to note that there was no visible non-specific binding of VapC18 C-His to E. coli

native proteins and that VapBC18 elutes later in the Immidazole gradient than VapC18 C-His

alone. For VapCγ, the untagged protein did not associate with γ (Figure 9) or αβγ (Figure

10), as it did not co-elute from the column. When the experiment was repeated with VapCγ

C-His, the protein bound the column, but eluted before the complex in the same manner as it does when the complex or γ is not present (Figures 11, 12, and 13).

Molecular assembly of toxins, antitoxins, and complexes. Because the elution of

VapBC18 was shifted higher in the Immidazole gradient, it was suspected that the toxin and antitoxin were forming a complex. Running the protein that eluted from the IMAC column on a native gel revealed the presence of one or more higher-molecular weight conformations

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(Figure 14). To determine the composition of the complex, the individual proteins were run

on an S75 sizing column (Figures 15 and 16). VapC18 C-His is most likely a dimer, as is

VapB18. From the S200 elution profile, the VapBC18 complex is predicted to be either 3:3

or 4:4 VapB18:VapC18 (Figure 17). There may be a mixture of multimers, as this has been

reported for other TA families (14). In contrast, VapCγ formed a monomer (Figure 18).

Given that γ which includes VapBγ is part of the heterotrimeric aIF-2 (αβγ), it would seem

likely that VapCγ would be monomeric if it interacts in any way with the γ-associated VapBγ.

However, all attempts to show an interaction between VapCγ and VapBγ in its various forms

were unsuccessful.

SUMMARY

VapCγ is ribonucleolytic toxin whose co-location with the γ subunit of the archaeal translation initiation factor is highly conserved in the Crenarchaeota and Euryarchaeota. It is a unique monomeric toxin compared to others in S. solfataricus like dimeric VapC18. The cognate antitoxin for VapBγ is predicted to be part of aIF2γ (18), and we have shown that

VapBγ is not cleaved from aIF2γ as this inhibits the aIF2 complex formation. It is also

unlikely that VapBγ is produced as a separate protein as efforts to produce it recombinantly were futile. As the C-terminal domains II and III of the aIF2 subunit γ, there was no

evidence that the protein bound to VapCγ (neither by itself nor complexed with α and β), as

was seen with VapBC18 complex formation. However, due to the size, shape, and function

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of the aIF2 complex, the binding mechanism with VapCγ is likely different, or there may not

be binding at all depending on the specificity of the ribonuclease. It is important to note that

the presence of VapCγ does not inhibit the formation of the α−β−γ complex, so it is not likely that VapCγ competes for interaction with γ. Efforts to make a VapCγ deficient mutant of S.

solfataricus PBL2025 have proven lethal thus far, further indicating the importance of this

specific toxin (P. Blum, unpublished data).

The co-location of the crucial subunit of the translation initiation factor with a post-

transcriptional regulator could serve as a novel RNA management system in Archaea. It has been shown that the initiation complex can bind non-initiator tRNAs that have been esterfied by methionine, but there is no known mechanism to remove the errant tRNA so that initiator can bind (25). VapCγ could degrade the errant tRNA and release the α−β−γ complex (Figure

19a). γ and α−β−γ not bound to the 30S ribosome have also been shown to bind to the 5’

end of mRNA to protect them from 5’Æ3’ degradation (9). Once the mRNA is bound, as

with the errant tRNAs, there is no known mechanism to remove the transcript and the

translation initiation process stalls. VapCγ could degrade mRNA transcripts as needed to

release the aIF2 complex and mediate the rate of translation initiation (Figure 19b). Further

studies are needed to characterize this unique toxin further and establish the extent of the interaction with the aIF2 subunit γ.

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ACKNOWLEDGMENTS

This work was supported in part by the National Science Foundation [grant numbers

CBET0730091 and CBET0617272]. C.R.C. acknowledges support from a National Institutes of Health T32 Biotechnology Traineeship. Helpful discussions with J. Brown (Department of

Microbiology) are also acknowledged.

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TABLES

Table 1. Co-location and arrangement of eIF2γ and VapC in the Archaea

COG-GCD11 COG1412 (aIF2γ (VapC Phylum Organism homologue) homologue) Overlap* Crenarchaeota Aeropyrum pernix K1 APE2366 APE_2365.1 A Desulfurococcus kamchatkensis 1221n DKAM_1347 DKAM_1346 A Hyperthermus butylicus DSM 5456 Hbut_0441 Hbut_0440 A Metallosphaera sedula DSM 5348 Msed_2243 Msed_2242 A Nitrosopumilus maritimus SCM1 Nmar_0072 Nmar_0073 A Staphylothermus marinus F1 Smar_0831 Smar_0832 B Sulfolobus acidocaldarius DSM 639 Saci_0832 Saci_0833 A Sulfolobus islandicus L.S.2.15 LS215_1820 LS215_1819 A Sulfolobus islandicus M.14.25 M1425_1692 M1425_1691 A Sulfolobus islandicus M.16.27 M1627_1808 M1627_1807 A Sulfolobus islandicus M.16.4 M164_1739 M164_1738 A Sulfolobus islandicus Y.G.57.14 YG5714_1808 YG5714_1807 A Sulfolobus islandicus Y.N.15.51 YN1551_1118 YN1551_1119 A Sulfolobus solfataricus P2 SSO0412 SSO0414 A Euryarchaeota Sulfolobus tokodaii str. 7 ST0381 ST0380 A Archaeoglobus fulgidus DSM 4304 AF_0592 AF_0591 A Candidatus Methanoregula boonei 6A8 Mboo_0247 Mboo_0246 B Candidatus Methanosphaerula palustris E1-9c Mpal_0605 Mpal_0604 A Haloarcula marismortui ATCC 43049 rrnAC2827 rrnAC2828 B Halobacterium salinarum R1 OE3876R OE3875R B Halobacterium sp. NRC-1 VNG2056G VNG2054H B Halomicrobium mukohataei DSM 12286 Hmuk_0730 Hmuk_0729 A Haloquadratum walsbyi DSM 16790 HQ1335A HQ1336A B Halorhabdus utahensis DSM 12940 Huta_2209 Huta_2208 A Halorubrum lacusprofundi ATCC 49239 Hlac_2398 Hlac_2397 B Methanobrevibacter smithii ATCC 35061 Msm_0200 Msm_0199 B Methanobrevibacter smithii DSM 2375 METSMIALI_00179 METSMIALI_00180 B Methanococcoides burtonii DSM 6242 Mbur_2278 Mbur_2279 A Methanoculleus marisnigri JR1 Memar_2240 Memar_2241 A Methanopyrus kandleri AV19 MK1447 MK1448 B Methanosaeta thermophila PT Mthe_1319 Mthe_1320 B Methanosarcina acetivorans C2A MA3690 MA3691 A Methanosarcina barkeri str. fusaro Mbar_A0287 Mbar_A0286 A Methanosarcina mazei Goe1 MM0594 MM0595 A Methanosphaera stadtmanae DSM 3091 Msp_0627 Msp_0626 B Methanospirillum hungatei JF-1 Mhun_2861 Mhun_2862 A Methanothermobacter thermautotrophicus str. Delta H MTH261 MTH262 B Natronomonas pharaonis DSM 2160 NP5084A NP5082A B Pyrococcus abyssi GE5 PAB2040 PAB2042 C Pyrococcus furiosus DSM 3638 PF1717 PF1716 B Pyrococcus horikoshii shinkaj OT3 PH1706 PH1710 C Thermococcus gammatolerans EJ3 TGAM_1286 TGAM_1285 B Thermococcus kodakaraensis KOD1 TK1946 TK1945 B Thermococcus onnurineus NA1 TON_1944 TON_1943 B Thermococcus sibiricus MM 739 TSIB_0720 TSIB_0721 A

*Overlap refers to Figure 2

178

Table 2. Primers used for cloning eIF2 and vapBC genes

Gene Primers PlasmidTag F: catATGATTTACAGTAGAAGCAAACTACCCTC SSO1050 (aIF2α) pET28b+ N-His R: ctcgagTCATTTCTTAACCACACTTATATCTAC F: gctagcaTGAGTTCAGAAAAAGAATACGTAG SSO2381 (aIF2β) pET28b+ N-His R: ctcgagTCATAGTGGTTTCACTGGTGTTTGAG F: gct agc aTG GCGTGGCCTAAAGTTCAAC SSO0412 (aIF2γ) pET28b+ N-His R: ctcgagTTAGATCTCTACTAAACCCCATCCTATC F: gctagcATGGCGTGGCCTAAAGTTCAAC aIF2γ-VapB (γ-B ) pET28b+ N-His γ γ R: ctcgagTTAAAGCATAACAGGTTTTTGTGAGAG F: catATGATAAGAAGTTTTGACGTTAATAAGCCCGGT pET21b+ No tag R: ctcgagTTAGATCTCTACTAAACCCCATCCT VapB γ F: catATGATAAGAAGTTTTGACGTTAATAAGCCCGGT pET21b+ C-His R: ctcgagGATCTCTACTAAACCCCATCC F: catATGGAGAATGATAGGATGGGGTTTAGT pET21b+ No tag R: ctcgagTTAGATAGGATATAAGGATTTTATAAT TTTACT SSO0414 (VapC ) γ F: catATGGAGAATGATAGGATGGGGTTTAGT pET21b+ C-His R: ctcgagGATAGGATATAAGGATTTTATAATTTTACT F: catATGTACACATCATATATGAAAAC VapB18 pET21b+ No tag R: ctcgagTTAATAATTGCTGAGCTTTC F: catATGAAAGTATTAATAGAAAGCTCAGCA SSO1975 (VapC18) pET21b+ C-His R: ctcgagAGTAATTAGAGCGACTTTCAGTTCA “F” refers to the forward primer and “R” refers to the reverse primer. The restriction site is indicated in red.

179

FIGURES

Domain I

Domain II

Domain III

Figure 1. Structure of the archaeal initiation factor aIF2g from S. solfataricus (PDB:

2PLF (26)). Within the gamma subunit, the C-terminal domain corresponding to the VapBg amino-acid sequence is highlighted in yellow, covering domains II and III.

180

A vapBγ vapCγ

B vapBγ vapCγ

C aIF2γ vapBγ vapCγ

Figure 2. Co-location of of aIF2γ with vapCγ. (A) The genes overlap by several

nucleotides, (B) there are zero or several nucleotides between aIF2γ and vapCγ, or (C) there

is a gene between aIF2γ and vapCγ. For (A) and (B), vapBγ is predicted to be part of aIF2γ.

For the cases where (C) has occurred, the vapBγ is predicted to be part of the intervening gene.

181

a. 1 2 3 4 5

b. 1 2 3 4 5

c. 1 2 3 4 5

Figure 3. RNase Alert ribonuclease activity assay. Tubes: 1. RNase A control; 2.

“reaction buffer” control; 3. pET21b+ mock extract control; 4. VapCγ; 5. VapC18. (a.) Reactions incubated at 37oC for 30min. (b). Reactions incubated at 37oC for 60min. (c) RNaseA added to negative control tubes 2 and 3.

182

αγ + αβγ

γ βγ α

Figure 4. Assemby of the aIF2 complex. The reactions were incubated at 65oC for 15min prior to running the acidic native gel. Lane 1: α; lane 2: β; lane 3: γ; lane 4: α and β; lane 5: α and γ; lane 6: β and γ; lane 7: α, β, and γ. α and β do not interact, but α−γ, β−γ, and α−β−γ complexes are formed.

183

αβγ

γ α

β -

65oC 70oC 75oC 80oC 85oC -

Figure 5. Temperature dependence of α−β−γ complex formation. The individual subunits were incubated at 65oC (lanes 1-3) and the complex was incubated at 65, 70, 75, 80, and 85oC (lanes 4-8). The γ subunit is stable up to 75oC, while α and β are stable at 85oC.

184

βγ αγ αβγ +

γ α γ−

Figure 6. The effect of VapBγ on complex formation. The subunits were incubated at 65oC for 15min. Lane 1: α; lane 2: β; lane 3: γ; lane 4: g-; lane 5: α and β; lane 6: α and γ; land 7: α and γ−; lane 8: β and γ; lane9: β and γ−; lane 10: α, β, and γ; lane 11: α, β, and γ−. The truncated version of γ was insufficient for complex formation.

185

+ αβγ

γ α

β - C1 C-His 65oC 70oC 75oC -

Figure 7. Effect of VapCγ on α−β−γ complex formation. The individual subunits were o o incubated at 65 C. VapCγ+γ and VapCγ+α−β−γ were incubated at 65, 70, and 75 C. The prescence of VapCγ does not affect the formation of the α−β−γ complex, nor does it appear to associate with the complex.

186

116kDa

36.5kDa C18 C-His 14.4kDa B18 6kDa S P Fractions 1-16

116kDa

36.5kDa

14.4kDa C18 C-His B18 6kDa Fractions 17-35

Figure 8. Co-elution of VapB18 and VapC18-C. Top Gel lane 1: Mark 12 protein ladder; lane 2: empty; lane 3: soluble protein from VapBC18 “fishing”; lane 4: insoluble protein; lanes 5-20: Fractions 1-16 from Nickel column purification. Bottom gel 2 lane 1: Mark 12 protein ladder; lanes 2-20: Fractions 17-35 from Nickel column purification. Note that the molecular weight of VapC18 C-His is ~15kDa but runs slightly lower. VapB18 is ~10kDa. VapC18 C-His binds VapB18 in a specific manner and co-elutes from the column. The elution profile is shifted from that of VapC18 C-His alone (data not shown).

187

γ 116kDa

55.4kDa

14.4kDa 6kDa Cγ S Fractions 2-19

γ 116kDa

55.4kDa

14.4kDa 6kDa

Fractions 20-38

Figure 9. VapCγ association with aIF2γ. Top gel lane 1: Mark 12 protein ladder; lane 2:

soluble protein from VapCγ and aIF2γ “fishing”; lanes 3-20: Fractions 2-19 from Nickel column purification. Bottom gel lane 1: Mark 12 protein ladder; lanes 2-20: Fractions 20-38 from Nickel column purification. The molecular weight of VapCγ is 16.5kDa and γ N-His is

49kDa. VapCγ is not co-eluted with aIF2γ N-His. VapCγ is eluted in the flow through, but aIF2γ binds the column and elutes in the immidazole gradient.

188

γ 116kDa α 55.4kDa β

14.4kDa 6kDa Cγ S Fractions 2-19

γ 116kDa α

55.4kDa β

14.4kDa 6kDa

Fractions 20-38

Figure 10. VapCγ association with α−β−γ. Top gel lane 1: Mark 12 protein ladder; lane 2: soluble protein from VapCγ and aIF2 α−β−γ “fishing”; lanes 3-20: Fractions 2-19 from Nickel column purification. Bottom gel lane 1: Mark 12 protein ladder; lanes 2-20:

Fractions 20-38 from Nickel column purification. The molecular weight of VapCγ is

16.5kDa, α N-His is 32kDa, β N-His is 18kDa, and γ N-His is 49kDa. VapCγ does not elute with the α−β−γ complex. The unincorporated β subunits elute from the column first followed by the assembled α−β−γ complex.

189

116kDa 55.4kDa

14.4kDa 6kDa Cγ C-His

S Fractions 2-19

116kDa 55.4kDa

14.4kDa 6kDa

Fractions 20-38

Figure 11. VapCγ C-His purification. Top gel lane 1: Mark 12 protein ladder; lane 2: soluble protein from VapCγ C-His; lanes 3-20: Fractions 2-19 from Nickel column purification. Bottom gel lane 1: Mark 12 protein ladder; lanes 2-20: Fractions 20-38 from

Nickel column purification. The molecular weight of VapCγ is 18kDa. VapCγ C-His binds the Nickel column and elutes as a pure protein.

190

γ 116kDa

55.4kDa

14.4kDa 6kDa Cγ C-His S Fractions 2-19

116kDa γ

55.4kDa

Cγ C-His

14.4kDa 6kDa

Fractions 20-38 Figure 12. VapCγ C-His association with aIF2γ. Top gel lane 1: Mark 12 protein ladder; lane 2: soluble protein from VapCγ C-His and aIF2γ “fishing”; lanes 3-20: Fractions 2-19 from Nickel column purification. Bottom gel lane 1: Mark 12 protein ladder; lanes 2-20:

Fractions 20-38 from Nickel column purification. VapCγ C-His elutes from the Nickel column in the same fractions as aIF2γ, but the elution profiles are identical to the non-mixed samples. This indicates that the VapCγ and aIF2γ are eluting independently rather than as an associated complex.

191

γ 116kDa α 55.4kDa β

14.4kDa 6kDa Cγ C-His S Fractions 2-19

γ 116kDa α

55.4kDa β

Cγ C-His

14.4kDa 6kDa

Fractions 20-38

Figure 13. VapCγ C-His association with α−β−γ. Top gel lane 1: Mark 12 protein ladder; lane 2: soluble protein from VapCγ C-His and aIF2 α−β−γ “fishing”; lanes 3-20: Fractions 2- 19 from Nickel column purification. Bottom gel lane 1: Mark 12 protein ladder; lanes 2-20:

Fractions 20-38 from Nickel column purification. VapCγ C-His binds and eluted the Nickel column before the assembled α−β−γ complex, indicating there is no association.

192

1048kDa 1048kDa

720kDa 720kDa 480kDa 480kDa 242kDa 242kDa 146kDa 146kDa 66kDa 66kDa

20kDa 20kDa 1 2 3 4 5

Figure 14. Native gel of VapBC18 complex. Lane 1: heat treated VapB18; lane 2: VapC18 C-His heat treated; lane 3: B18 purified; lane 4: Purified VapBC18 complex formation; lane 5: Crude VapBC18 complex formation. Native gel analysis indicates the formation of multimers of VapBC18.

193

VapC18 C-His on S75

0.5 UV 0.45 59.5 Conductivity 0.4 58.5 0.35

0.3 57.5

0.25 AU 56.5 0.2

0.15 55.5

0.1 54.5 0.05

0 53.5 0 5 10 15 20 25 30 35 40 45 50 Time (min)

116kDa

55.4kDa

14.4kDa

6kDa

Figure 15. VapC18 C-His size estimation. Sizing analysis with a Sephacryl 75 column indicated VapC18 C-His forms a dimer.

194

VapB-18 on S75

2 UV 54.9 1.8 Conductivity 1.6 54.7

1.4 54.5 1.2 54.3 1 AU

0.8 54.1

0.6 53.9 0.4 53.7 0.2

0 53.5 0 5 10 15 20 25 30 35 40 45 50 Time (min)

116kDa

55.4kDa

14.4kDa 6kDa VapB18

Figure 16. VapB18 size estimation. Sizing analysis with a Sephacryl 75 column indicated VapB18 forms a dimer.

195

VapBC18 on S200

0.05 UV 73.5 0.045 Conductivity 0.04

0.035 68.5

0.03

0.025 AU 63.5 0.02

0.015 58.5 0.01

0.005

0 53.5 0 5 10 15 20 25 30 35 40 45 50 Time (min)

116kDa

55.4kDa

14.4kDa

6kDa

Figure 17. VapBC18 C-His size estimation. Sizing analysis with a Sephacryl 200 column indicated VapBC18 C-His forms a heterohexamer (3:3) or heterooctamer (4:4). The SDS PAGE gel was silver stained to visualize protein.

196

VapC1γ C-His on S75

0.5 58 UV 0.45 Conductivity 57.5

0.4 57 0.35 56.5 0.3 56 0.25 AU 55.5 0.2 55 0.15

0.1 54.5

0.05 54

0 53.5 0 5 10 15 20 25 30 35 40 45 50 Time (min)

Figure 18. VapCγ size estimation. Sizing analysis with a Sephacryl 75 column indicated

VapCγ C-His is a monomer.

197

Figure 19. Putative mechanisms for VapCγ/aIF2γ interaction and function. Schematic representation of co-location of the aIF2 translation initiation complex and the

ribonucleolytic toxin VapCγ. (a) VapCγ may degrade non-initiator tRNAs that have been

esterfied by methionine. (b) VapCγ may also play a role in degrading mRNAs bound to the intitation complex by the 5’ end. The complex can protect the mRNAs from degradation but there is no know mechanism to release the aIF2 complex to be recycled.

198

APPENDICIES

199

APPENDIX 4.A

Table S1. Detailed information on archaeal VapCγ/aIF2γ co-location

COG-GCD11 COG1412 Pandey and Gerdes Phylum Organism (aIF2γ homologue) (VapC homologue) Strand Overlap Annotation Crenarchaeota Aeropyrum pernix K1 APE2366 APE_2365.1 - -33 vapBC-6 Desulfurococcus kamchatkensis 1221n DKAM_1347 DKAM_1346 - -13 Hyperthermus butylicus DSM 5456 Hbut_0441 Hbut_0440 - -21 Nitrosopumilus maritimus SCM1 Nmar_0072 Nmar_0073 + -7 Staphylothermus marinus F1 Smar_0831 Smar_0832 + 20 Sulfolobus acidocaldarius DSM 639 Saci_0832 Saci_0833 + -33 Sulfolobus islandicus L.S.2.15 LS215_1820 LS215_1819 - -21 Sulfolobus islandicus M.14.25 M1425_1692 M1425_1691 - -21 Sulfolobus islandicus M.16.27 M1627_1808 M1627_1807 - -21 Sulfolobus islandicus M.16.4 M164_1739 M164_1738 - -21 Sulfolobus islandicus Y.G.57.14 YG5714_1808 YG5714_1807 - -21 Sulfolobus islandicus Y.N.15.51 YN1551_1118 YN1551_1119 + -21 Sulfolobus solfataricus P2 SSO0412 SSO0414 + -36 vapBC-1 Sulfolobus tokodaii str. 7 ST0381 ST0380 - -36 vapBC-4 Euryarchaeota Archaeoglobus fulgidus DSM 4304 AF_0592 AF_0591 - -33 vapBC-9 Candidatus Methanoregula boonei 6A8 Mboo_0247 Mboo_0246 - 27 Candidatus Methanosphaerula palustris E1-9c Mpal_0605 Mpal_0604 - -3 Haloarcula marismortui ATCC 43049 rrnAC2827 rrnAC2828 + 0 Halobacterium salinarum R1 OE3876R OE3875R - 5 Halobacterium sp. NRC-1 VNG2056G VNG2054H - 5 vapBC-3 Halomicrobium mukohataei DSM 12286 Hmuk_0730 Hmuk_0729 - -3 Haloquadratum walsbyi DSM 16790 HQ1335A HQ1336A + 176 Halorhabdus utahensis DSM 12940 Huta_2209 Huta_2208 - -3 Halorubrum lacusprofundi ATCC 49239 Hlac_2398 Hlac_2397 - 2 Metallosphaera sedula DSM 5348 Msed_2243 Msed_2242 - -21 Methanobrevibacter smithii ATCC 35061 Msm_0200 Msm_0199 - 10 Methanobrevibacter smithii DSM 2375 METSMIALI_00179 METSMIALI_00180 + 10 Methanococcoides burtonii DSM 6242 Mbur_2278 Mbur_2279 + -3 Methanoculleus marisnigri JR1 Memar_2240 Memar_2241 + -3 Methanopyrus kandleri AV19 MK1447 MK1448 + 7 vapBC-2 Methanosaeta thermophila PT Mthe_1319 Mthe_1320 + 14 Methanosarcina acetivorans C2A MA3690 MA3691 + -3 vapBC-4 Methanosarcina barkeri str. fusaro Mbar_A0287 Mbar_A0286 - -3 Methanosarcina mazei Goe1 MM0594 MM0595 + -3 vapBC-1 Methanosphaera stadtmanae DSM 3091 Msp_0627 Msp_0626 - 31 Methanospirillum hungatei JF-1 Mhun_2861 Mhun_2862 + -3 Methanothermobacter thermautotrophicus str. Delta H MTH261 MTH262 + 25 vapBC-1 Natronomonas pharaonis DSM 2160 NP5084A NP5082A - 0 Pyrococcus abyssi GE5 PAB2040 PAB2042 - 1219 part of PAB2041 is predicted as VapB-3 Pyrococcus furiosus DSM 3638 PF1717 PF1716 - 6 vapBC-15 Pyrococcus horikoshii shinkaj OT3 PH1706 PH1710 + 1241 part of PH1708 predicted as VapB-10 Thermococcus gammatolerans EJ3 TGAM_1286 TGAM_1285 - 23 Thermococcus kodakaraensis KOD1 TK1946 TK1945 - 43 Thermococcus onnurineus NA1 TON_1944 TON_1943 - 29 Thermococcus sibiricus MM 739 TSIB_0720 TSIB_0721 + -3

200