SUPPLEMENTARY TEXT. Systems analysis with the Gaggle mRNA level changes were analyzed simultaneously with / functional associations [operons (Moreno-Hagelsieb and Collado-Vides 2002), phylogenetic profile (Pellegrini et al. 1999), chromosomal proximity (Overbeek et al. 1999)], physical interactions [protein-DNA interactions (Facciotti etal, submitted)], putative functions in the SBEAMS database (http://halo.systemsbiology.net) (Bonneau et al. 2004) along with supporting evidence such as matches in protein databank [PDB (Sussman et al. 1998)], protein families [Pfam (Bateman et al. 2000), COG database (Tatusov et al. 2000)] and metabolic pathways [KEGG (Kanehisa 2002)]. Further, data were extracted into sub- matrices using custom filters, normalized and statistically analyzed using the R statistical package (http://www.r-project.org) and TIGR microarray expression viewer [TMeV (Saeed et al. 2003)]. Given the size and heterogeneity of data and software, we used the Gaggle framework (http://gaggle.systemsbiology.net) (Shannon et al, accepted in BMC Bioinformatics) to facilitate analyses and queries. The Gaggle is an open source Java software framework for seamless desktop integration of diverse databases and software applications. Specifically, all were visualized in Cytoscape (Shannon et al. 2003) as networks of nodes (genes) and edges (functional associations). The genes were organized into various function categories and node color was mapped to mRNA level changes (red for increased and green for decreased mRNA) (Johnson etal, submitted, Shannon etal, submitted); and node size was mapped to statistical significance (λ) (Baliga et al. 2004; Shannon et al. 2003). With this visualization scheme significant changes (genes appearing as large red or green nodes) in various function categories become immediately evident. This also enabled assignment of putative function to genes of previously unknown function by retrieving matching records in SBEAMS, Pfam, PDB, COG, and KEGG. Function assignments were further confirmed with orthogonal sources of information such as expression correlation and functional associations to genes of known function (Bonneau et al. 2004) (ST2).

Physiological reconstruction

Deletion of individual subunits of multi-component ABC transporters does not effect any change in metal sensitivity Multi-subunit ATP-binding cassette (ABC) transporters mediate active transport of sugars, ions, peptides, and oligonucleotides (Schneider and Hunke 1998). At least 50 genes of this function category were differentially regulated in one or more metal (ST2). To evaluate whether these transporters play a role in metal resistance, we selected subunits of four ABC transport systems for further analysis on basis of both their putative functions and their differential regulation. While three of these genes, phoX, appA, and 2- ycdH, encode subunits of transport systems for PO4 , peptides, and Mn(II), respectively, the other two, fepC and VNG2562H, are both putative subunits of the same Fe(II) transport system. Each gene deletion strain was then assayed for altered growth characteristics in varying concentrations of selected metals. Absence of defective growth phenotypes of these strains initially suggested that ABC transporters may not contribute towards metal resistance (ST3). However, given that numerous ABC transport systems were differentially regulated in each metal, we cannot rule out that loss of function of individual subunits in one might be complemented by subunits of other related transport systems.

Responses of metalloenzymes, a ferritin and putative siderophore biosynthesis genes Metalloenzymes. Several metabolic pathways that require metal co-factors were affected during metal stress; for example genes of cobalamin biosynthesis, a pathway that requires Co(II),were differentially regulated in at least four metals Mn(II), Fe(II), Co(II) and Zn(II). The most notable change was Co(II)-specific down regulation of four of seven genes encoding a segment of the pathway that requires this metal ion. Included among these genes is CobN, a putative Co-chelatase which inserts Co into the corrin ring. A second example was Mn(II)- and Fe(II)-specific up regulation of a putative molybdenum sulfurase (VNG1735C) implicated in delivering sulphur to diverse metal-sulphur clusters. Ferritin. Structural analysis of the halobacterial ferritin DpsA has demonstrated that it stores iron in its nontoxic Fe3+ form through a process of matrix-controlled biomineralization (Reindel et al. 2002; Zeth et al. 2004). In conjunction with these reports, increase in dpsA transcript during Fe(II) stress points to a regulatory mechanism that ensures increased abundance of DpsA to minimize Fe(II) toxicity as has been reported for other prokaryotes (Munro and Linder 1978; Nair and Finkel 2004; Theil 1987; Wiedenheft et al. 2005). Siderophore biosynthesis. synthesize low molecular weight compounds termed siderophores for scavenging Fe (Winkelmann 2002). Four of six genes (gabT, bdb, iucA, iucB, hxyA and iucC) in an operon in Halobacterium NRC-1 putatively encode siderophore biosynthesis: two (IucA and IucC ) match a (PF04183) for synthesis of the siderophore aerobactin (de Lorenzo and Neilands 1986); and the other two (Bdb and GabT) putatively act on L-2,4-diaminobutyrate –a precursor of pyovredine siderophore biosynthesis (Vandenende et al. 2004). Although, none of these genes were differentially regulated in presence of Fe(II), addition of Mn(II) resulted in their up regulation (this is also discussed further later). However, we did not detect any growth defects in the ∆iucA strain, with and without Mn(II), upon adding excess Fe(II) or chelating “free” Fe(II) with the Fe(II)-specific chelator 2, 2’-dipyridyl (DIP) (ST3). This suggests that perhaps Fe(II) uptake studies might be better suited to characterize the role of siderophore biosynthesis in Halobacterium NRC-1 Fe(II) response.

Transcription regulators selected for further analysis solely on basis of differential regulation and putative functions We also selected transcription regulators for further analysis merely on the basis of their putative functions and differential regulation Specifically, we selected three putative transcription regulators: CspD1, VNG0703H and VNG5176C. CspD1 was selected because its putative function implicated it in stress response (Schindelin et al. 1994) and it was down regulated in Mn(II) and Fe(II) and up regulated in Cu(II) and Zn(II) (ST2). VNG0703H, on the other hand, was selected on basis of its up regulation in Fe(II) and Zn(II) as well as its proximity in the genome to yvgX, which was implicated in mediating resistance to Cu(II). Finally, VNG5176C was selected because it was up regulated in presence of Zn(II) and it was one of four differentially regulated transcription regulators of the ArsR family, which is characterized by the alpha3N metal implicated in binding and mediating responses to Zn(II) (Turner et al. 1996). Therefore, the VNG5176C mutant was predicted to fair poorly under Zn(II) stress. However, the ∆cspD1, ∆VNG0703H and ∆VNG5176C strains did not have any observable growth defects (ST3) ruling out their roles in directly controlling mechanisms that confer resistance to these metals.

SirR may bind up to four different metals with diverse outcomes The putative Mn(II) uptake genes zurA, zurM and ycdH were up regulated by Co(II) and Ni(II) but down regulated by Mn(II) and Fe(II) (Fig 6A). This suggested that SirR, the putative MntR family transcriptional repressor of this uptake system, can functionally bind Mn(II) and Fe(II) to repress these genes. In contrast binding of Co(II) and Ni(II) seems to interfere with normal SirR function. Another possible explanation is that addition of excess Co(II) and Ni(II) changes the balance of the cellular metal ion pool, including relative Mn(II)-availability.

Supplementary References

Baliga, N.S., S.J. Bjork, R. Bonneau, M. Pan, C. Iloanusi, M.C.H. Kottemann, L. Hood, and J. DiRuggiero. 2004. Systems Level Insights Into the Stress Response to UV Radiation in the Halophilic Archaeon Halobacterium NRC-1. Genome Res. 14: 1025-1035. Bateman, A., E. Birney, R. Durbin, S.R. Eddy, K.L. Howe, and E.L. Sonnhammer. 2000. The Pfam protein families database. Nucleic Acids Res 28: 263-266. Bonneau, R., N.S. Baliga, E.W. Deutsch, P. Shannon, and L. Hood. 2004. Comprehensive de novo structure prediction in a systems-biology context for the Halobacterium sp. NRC-1. Genome Biol 5: R52. de Lorenzo, V. and J.B. Neilands. 1986. Characterization of iucA and iucC genes of the aerobactin system of plasmid ColV-K30 in Escherichia coli. J Bacteriol 167: 350- 355. Kanehisa, M. 2002. The KEGG database. Novartis Found Symp 247: 91-101; discussion 101-103, 119-128, 244-152. Moreno-Hagelsieb, G. and J. Collado-Vides. 2002. A powerful non- method for the prediction of operons in prokaryotes. Bioinformatics 18 Suppl 1: S329- 336. Munro, H.N. and M.C. Linder. 1978. Ferritin: structure, biosynthesis, and role in iron metabolism. Physiol Rev 58: 317-396. Nair, S. and S.E. Finkel. 2004. Dps protects cells against multiple stresses during stationary phase. J Bacteriol 186: 4192-4198. Overbeek, R., M. Fonstein, M. D'Souza, G.D. Pusch, and N. Maltsev. 1999. The use of gene clusters to infer functional coupling. Proc Natl Acad Sci U S A 96: 2896- 2901. Pellegrini, M., E.M. Marcotte, M.J. Thompson, D. Eisenberg, and T.O. Yeates. 1999. Assigning protein functions by comparative genome analysis: protein phylogenetic profiles. Proc Natl Acad Sci U S A 96: 4285-4288. Reindel, S., S. Anemuller, A. Sawaryn, and B.F. Matzanke. 2002. The DpsA-homologue of the archaeon Halobacterium salinarum is a ferritin. Biochim Biophys Acta 1598: 140-146. Saeed, A.I., V. Sharov, J. White, J. Li, W. Liang, N. Bhagabati, J. Braisted, M. Klapa, T. Currier, M. Thiagarajan, A. Sturn, M. Snuffin, A. Rezantsev, D. Popov, A. Ryltsov, E. Kostukovich, I. Borisovsky, Z. Liu, A. Vinsavich, V. Trush, and J. Quackenbush. 2003. TM4: a free, open-source system for microarray data management and analysis. Biotechniques 34: 374-378. Schindelin, H., W. Jiang, M. Inouye, and U. Heinemann. 1994. Crystal structure of CspA, the major cold shock protein of Escherichia coli. Proc Natl Acad Sci U S A 91: 5119-5123. Schneider, E. and S. Hunke. 1998. ATP-binding-cassette (ABC) transport systems: functional and structural aspects of the ATP-hydrolyzing subunits/domains. FEMS Microbiol Rev 22: 1-20. Shannon, P., A. Markiel, O. Ozier, N.S. Baliga, J.T. Wang, D. Ramage, N. Amin, B. Schwikowski, and T. Ideker. 2003. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13: 2498- 2504. Sussman, J.L., D. Lin, J. Jiang, N.O. Manning, J. Prilusky, O. Ritter, and E.E. Abola. 1998. (PDB): database of three-dimensional structural information of biological macromolecules. Acta Crystallogr D Biol Crystallogr 54: 1078-1084. Tatusov, R.L., M.Y. Galperin, D.A. Natale, and E.V. Koonin. 2000. The COG database: a tool for genome-scale analysis of protein functions and . Nucleic Acids Res 28: 33-36. Theil, E.C. 1987. Ferritin: structure, gene regulation, and cellular function in animals, plants, and microorganisms. Annu Rev Biochem 56: 289-315. Turner, J.S., P.D. Glands, A.C. Samson, and N.J. Robinson. 1996. Zn2+-sensing by the cyanobacterial metallothionein repressor SmtB: different motifs mediate metal- induced protein-DNA dissociation. Nucleic Acids Res 24: 3714-3721. Vandenende, C.S., M. Vlasschaert, and S.Y. Seah. 2004. Functional characterization of an aminotransferase required for pyoverdine siderophore biosynthesis in Pseudomonas aeruginosa PAO1. J Bacteriol 186: 5596-5602. Wiedenheft, B., J. Mosolf, D. Willits, M. Yeager, K.A. Dryden, M. Young, and T. Douglas. 2005. An archaeal antioxidant: characterization of a Dps-like protein from Sulfolobus solfataricus. Proc Natl Acad Sci U S A 102: 10551-10556. Winkelmann, G. 2002. Microbial siderophore-mediated transport. Biochem Soc Trans 30: 691-696. Zeth, K., S. Offermann, L.O. Essen, and D. Oesterhelt. 2004. Iron-oxo clusters biomineralizing on protein surfaces: structural analysis of Halobacterium salinarum DpsA in its low- and high-iron states. Proc Natl Acad Sci U S A 101: 13780-13785.

SUPPLEMENTARY FIGURE LEGENDS

Supplementary Figure 1. Viability of Halobacterium NRC-1 cells after prolonged exposure to six transition metals. A mid-log phase culture of Halobacterium NRC-1 was exposed to a growth-inhibitory concentration of each metal over 27 hours at 37oC. Culture aliquots were serially diluted (effectively reducing metal ion concentration by over 10,000-fold) and 5 ul of 10-4, 10-5 and 10-6 dilutions were plated on to CM agar plates. survival was evaluated in seven days by counting colonies The five panels show no significant change in cell viability under constant metal stress up until 27 hours suggesting the metals caused growth inhibition and not killing.

Supplementary Figure 2. mRNA level changes for the P1 ATPase (YvgX) and its putative transcription regulator VNG1179C in ~240 different experimental conditions including gene expression changes described in this study.

Supplementary Figure 3. Growth phenotypes of Halobacterium NRC-1 ∆ura3 and Halobacterium NRC-1 ∆ura3 ∆zntA in increasing concentrations of CoSO4 (A), NiSO4 (B), CuSO4 (C), and ZnSO4 (D).

Supplementary Figure 4. Investigation of mechanistic basis of SirR function. A. Inferelator prediction of influence of putative regulators of a bicluster containing Mn(II) uptake genes. Red arrows indicate “activate” and green arrows indicate “repress” influences and the numbers by the influences indicate their relative contribution in predicting mRNA levels of genes in that bicluster. The blue lines indicate that the transcription regulators combine through “AND”/”OR” gates to exert influence on the genes. The black lines indicate that those regulators were included as part of the bicluster. B. Properties of the bicluster containing Mn(II) uptake genes. The genes in this bicluster have correlated mRNA level change in ~150 (to the left of the red vertical line) out of ~256 conditions that were analyzed. SirR was not a part of this bicluster but is included for purpose of illustration. Three motifs were detected de novo by cMonkey and may be responsible for mediating some of the predicted regulatory influences. The locations of the three motifs in the upstream regions of the various genes is indicated with red, green and blue boxes for motifs 1, 2 and 3 respectively. C. Differences in normalized (mean=0, variance=1) mRNA level changes in Halobacterium NRC-1 ∆ura3 and Halobacterium NRC-1 ∆ura3 ∆sirR were determined using the SAM algorithm by Tusher et al 2001. Among the two groups are those that appear to be under negative control of SirR (Group I) and those that are under positive control of SirR (Group II). The inset plot shows mRNA profiles for three genes (zurA, zurM and ycdH) of a putative Mn-uptake ABC transport system in the wt, ∆ura3 and ∆ura3 ∆sirR in presence of Mn and Fe.

Supplementary Figure 5. Investigation of mechanistic basis of VNG1179C function. A. Differences in normalized (mean=0, variance=1) mRNA level changes in Halobacterium NRC-1 ∆ura3 and Halobacterium NRC-1 ∆ura3 ∆VNG1179C were determined using the SAM algorithm by Tusher et al 2001. B. Significant differences among the two sets were hierarchically clustered to reveal two distinct groups. C. Among the two groups are those that appear to be under negative control of VNG1179C (Group I) and those that are under positive control of VNG1179C (Group II). The inset

plot shows mRNA profile for YvgX a Cu-efflux P1 ATPase in ∆ura3 and ∆ura3 ∆VNG1179C backgrounds in presence of Cu.

Supplementary Figure 6. mRNA level changes for the putative Mn-uptake ABC transporter system operon zurA, zurM and ycdH and its putative transcription regulator SirR in ~250 different experimental conditions including gene expression changes described in this study.

Supplementary Figure 7. Correspondence analysis (contd. from Fig. 5 in main text). CoA plots with Axes 1 and 3 (A) , and Axes 2 and 3 (2), demonstrate relationships among responses to different metals. Panel C shows mRNA level changes for 23 genes at a higher concentration of Co(II) appear to be similar to mRNA level changes at 0.05mM Zn(II). Each dot in this graph represents a particular experiment, for example Co (0.5) refers to mRNA level changes in 0.5mM Co(II). Eigen values are plottted for each of the first two dimensions, indicated as Axes 1 and 2, which represent inertia values of 26.34 and 19.45, respectively.

Supplementary Figure 8. Similarity dendogram of transcript level changes for transcription regulators. log10 ratios of the 48 transcription regulators were normalized (variance=0, mean=1) and hierarchically biclustered (genes and conditions; Euclidean distance, Average Linkage). Regulators containing any of the four different metal-binding protein family (Pfam) signatures are indicated along with GTFs, genes that were succesfully deleted, and genes that we were unable to delete.

30 minutes 90 minutes 3 hours 6 hours 27 hours Dilution 10-4 10-5 10-6 10-4 10-5 10-6 10-4 10-5 10-6 10-4 10-5 10-6 10-4 10-5 10-6

CM CM + 9.8mM FeSO4 CM +2 mM MnSO4 CM + 1.25 mM CuSO4 CM + 0.5mM CoSO4 CM + 0.05mM ZnSO4 CM + 2.5mM NiSO4 Supplementary Figure 1. Viability of Halobacterium NRC-1 cells after prolonged exposure to six transition metals. A mid-log phase culture of Halobac- terium NRC-1 was exposed to a growth-inhibitory concentration of each metal over 27 hours at 37oC. Culture aliquots were serially diluted (effectively reducing metal ion concentration by over 10,000-fold) and 5 ul of 10-4, 10-5 and 10-6 dilutions were plated on to CM agar plates. Cell survival was evalu- ated in seven days by counting colonies The five panels show no significant change in cell viability under constant metal stress up until 27 hours suggesting the metals caused growth inhibition and not killing.

0.5 Zn Zn

0.4 Cu 0.3 ) s o i t a

r 0.2 0 1 g o l

( 0.1 s e v e l 0 A N R -0.1 m

-0.2

-0.3 VNG0700G (yvgX) VNG1179C -0.4 1 11 21 31 41 51 61 71 81 91 101 111 121 131 141 151 161 171 181 191 201 211 221 231 241 Experiments

Supplementary Figure 2. mRNA level changes for the P1-type ATPase (YvgX) and its putative transcription regulator VNG1179C in ~240 different experimental conditions including gene expression changes described in this study.

Growth rate assay

∆yvgX ∆VNG1179C i 1.4 ii ∆yvgX (0.6mM Cu) 1.4 ∆VNG1179C (0.6mM Cu) 1.2 ∆ura3 ∆ura3 ∆ura3 (0.6mM Cu) 1.2 ) ) ∆ura3 (0.6mM Cu) m 1 m n n 1 0 0 0 0 6 6 ( 0.8 ( 0.8 . . D D . . O 0.6 O 0.6

0.4 0.4

0.2 0.2

0 0 14h 17h 20h 23h 26h 29h 32h 35h 14h 17h 20h 23h 26h 29h 32h 35h Time (hours) Time (hours) ∆zntA ∆sirR iii 1.2 ∆zntA (0.005mM Zn) iv 1.2 ∆sirR (0.4mM Mn) ∆ura3 ∆ura3 1 ∆ura3 (0.005mM Zn) 1 ∆ura3 (0.4mM Mn) ) ) m m n n 0 0.8 0 0.8 0 0 6 6 ( ( . . D 0.6 D 0.6 . . O O 0.4 0.4

0.2 0.2

0 0 14h 17h 20h 23h 26h 29h 32h 35h 14h 17h 20h 23h 26h 29h 32h 35h Time (hours) Time (hours) Supplementary Figure 3. Growth phenotypes of Halobacterium NRC-1 ∆ura3 and Halobacterium NRC-1 ∆ura3 ∆zntA in increasing concentrations of CoSO4 (A), NiSO4 (B), CuSO4 (C), and ZnSO4 (D). A B

VNG1405C

-0.14 VNG2476C Prp1 AND 0.15 2- Mn(II), PO4 and Co(II) KaiC 0.12 transport PhoU

0.12

SirR

C II

I

SAM analysis on 352 genes with significant (λ>15.0) change in at least two microarray experiments. I Genes directly or indirectly repressed by SirR II Genes directly or indirectly activated by SirR VNG0037H VNG0037H VNG0101G cspD1 Cold shock protein (putative regulator) VNG0293H VNG0293H putative transcription regulator, Rosetta prediction VNG0146H VNG0146H VNG0452G pstB2 Phosphate transport ATP-binding VNG0285C VNG0285C transposase VNG0453G pstA2 Phosphate ABC transporter permease VNG0394C VNG0394C VNG0457G phoX Phosphate ABC transporter periplasmic PO4-binding VNG0426G rpoM DNA-directed RNA-polymerase subunit M VNG0962G flaB3 Flagellin B3 precursor VNG0491G dnaK Chaperone protein dnaK (Hsp70) VNG0974G cheY CHEY and CHEB genes (Chemotaxis protein) VNG0659H VNG0659H VNG0997G acs2 Acetyl-CoA synthetase VNG0702H VNG0702H heavy metal transport protein VNG1047H VNG1047H VNG0765H VNG0765H VNG1121G aspC2 Aspartate aminotransferase VNG0863H VNG0863H VNG1295H VNG1295H VNG1007H VNG1007H VNG1332G sod2 Superoxide dismutase [Mn] 2 VNG1132G rps13p 30S ribosomal protein S13P/S18E (HS13) VNG1380H VNG1380H VNG1289H VNG1289H VNG1529G mmdA Methylmalonyl-CoA decarboxylase, subunit alpha VNG1320G cbp Calcium-binding protein homology VNG1794C VNG1794C Staphylococcal nuclease homolog VNG1433G rps17e 30S ribosomal protein S17e VNG1801G hsp1 Small heat shock protein VNG1541G sucC Succinyl-CoA synthetase beta chain VNG1806H VNG1806H VNG1542G sucD Succinyl-CoA synthetase alpha chain VNG1912G trpD2 Phosphoribosyl VNG1591H VNG1591H VNG1973H VNG1973H VNG1624G mdh Malate dehydrogenase VNG2109H VNG2109H VNG1692G rpl2p 50S ribosomal protein L2P VNG2246H VNG2246H VNG1695G rpl22p 50S ribosomal protein L22P (HHAL22) VNG2410G gbp3 GTP-binding protein homolog VNG1698G rpl29p 50S ribosomal protein L29P (HHAL29) VNG2431C VNG2431C VNG1699C VNG1699C putative RNAse P encoded in ribosomal operon VNG2433H VNG2433H VNG1727G cmk Cytidylate kinase VNG2436G argH Argininosuccinate VNG1830G pyrG CTP synthase/GATase VNG2437G argG Argininosuccinate synthetase VNG2273H VNG2273H VNG2473G radA1 DNA repair and recombination protein radA VNG2467G rpl31e 50S ribosomal protein L31e change shared with ∆sirR VNG2482G pstB1 Phosphate ABC transporter ATP-binding VNG2469G rpl39e 50S ribosomal protein L39e change inverse to ∆sirR VNG2484G pstC1 Phosphate transporter permease VNG2643H VNG2643H VNG2486G yqgG Phosphate ABC transporter binding Protease/nuclease/chaperone VNG2657G rps7p 30S ribosomal protein S7P VNG2508C VNG2508C Dehydrogenase//redoxins VNG2661G nusA NusA protein homolog VNG2624G ribC Riboflavin synthase alpha subunit Putative transcription regulators VNG6332H VNG6332H VNG6162H VNG6162H VNG0287H VNG0287H VNG6241G gvpA2 Gas vesicle structural protein 2 (GVP) (C-VAC) VNG2664G rpoA DNA-directed RNA polymerase subunit A zurM ycdH zurA VNG5008H VNG5008H VNG6262G zurM ABC transporter, permease protein 2 )

o 1.5 i

VNG6264G zurA ABC transporter, ATP-binding protein t VNG5019G gvpM1 GvpM protein, cluster A

a 1 r

0 0.5 VNG6265G ycdH Adhesion protein 1 VNG5071C VNG5071C sugar (and other) transporter g 0 o l ( -0.5

VNG1406G rhl putative DNA helicase A VNG1653H VNG1653H transposase

N -1 R -1.5 VNG5028G gvpE1 GvpE protein, cluster A m VNG2059H VNG2059H -2 -2.5 VNG5029G gvpD1 GvpD protein, cluster A 0 0 VNG2118G pyrE2 Orotate phosphoribosyltransferase VNG5032G gvpC1 GvpC protein, cluster A Fe Mn Mn Mn VNG2634H VNG2634H wt wt ∆ura3 ∆ura3∆sirR VNG5033G gvpN1 GvpN protein, cluster A VNG6181H VNG6181H VNG5183G arsC VNG6182H VNG6182H IS200-like transposase VNG6255C VNG6255C VNG6256G lipB Lipoate protein VNG6305C VNG6305C radical SAM superfamily protein VNG5044H VNG5044H transposase Supplementary Figure 4. Investigation of mechanistic basis of SirR function. A. Inferelator prediction of influence of putative regulators of a bicluster containing Mn(II) uptake genes. Red arrows indicate “activate” and green arrows indicate “repress” influences and the numbers by the influences indicate their relative contribution in predicting mRNA levels of genes in that bicluster. The blue lines indicate that the transcription regulators combine through “AND”/”OR” gates to exert influence on the genes. The black lines indicate that those regulators were included as part of the bicluster. B. Properties of the bicluster containing Mn(II) uptake genes. The genes in this bicluster have correlated mRNA level change in ~150 (to the left of the red vertical line) out of ~256 conditions that were analyzed. SirR was not a part of this bicluster but is included for purpose of illustration. Three motifs were detected de novo by cMonkey and may be responsible for mediating some of the predicted regulatory influences. The locations of the three motifs in the upstream regions of the various genes is indicated with red, green and blue boxes for motifs 1, 2 and 3 respectively. C. Differences in normalized (mean=0, variance=1) mRNA level changes in Halobacterium NRC-1 ∆ura3 and Halobacterium NRC-1 ∆ura3 ∆sirR were determined using the SAM algorithm by Tusher et al 2001. Among the two groups are those that appear to be under negative control of SirR (Group I) and those that are under positive control of SirR (Group II). The inset plot shows mRNA profiles for three genes (zurA, zurM and ycdH) of a putative Mn-uptake ABC transport system in the wt, ∆ura3 and ∆ura3 ∆sirR in presence of Mn and Fe. A B 1179C ura3 Cu Cu II

I

I

II

SAM analysis on 352 genes with significant (λ>15.0) change in at least two microarray experiments.

Hierarchical clustering (Euclidean distance/Average Linkage) on I Genes directly or indirectly repressed by VNG1179C normalized data (variance = 1; mean = 0) for the 139/352 genes VNG0154G merA Mercury(II) reductase with significant differences between ∆sirR/∆ura3 and ∆ura3 VNG0166G psmA2 Proteasome alpha subunit VNG0261H VNG0261H VNG0291H VNG0291H VNG0293H VNG0293H putative transcription regulator VNG0457G phoX Phosphate ABC transporter periplasmic phosphate-binding VNG0466C VNG0466C VNG0467G yafB Aldehyde reductase VNG0518H VNG0518H II Genes directly or indirectly activated by VNG1179C VNG0520H VNG0520H VNG0013C VNG0013C VNG0557H VNG0557H putative protease VNG0049H VNG0049H Protein-L-Isoaspartate O-Methyltransferase VNG0679G acd4 Acyl-CoA dehydrogenase VNG0749G prk Protein kinase VNG0101G cspD1 Cold shock protein (putative regulator) VNG0814C VNG0814C VNG0146H VNG0146H VNG0815G yfmJ Quinone oxidoreductase VNG0285C VNG0285C transposase VNG0930G yvbT Alkanal monooxygenase homolog VNG0374G nusG Transcription termination-antitermination factor VNG0933G yqjM NADH-dependent flavin oxidoreductase VNG0375G secE Preprotein secE subunit VNG0934H VNG0934H VNG0935G noxC NADH oxidase VNG0410G rfbU2 LPS biosynthesis

VNG0997G acs2 Acetyl-CoA synthetase ) VNG0426G rpoM DNA-directed RNA-polymerase subunit M o i VNG0998G yajO2 Probable oxidoreductase t VNG0473G porB Pyruvate ferredoxin oxidoreductase, subunit beta

a 0.4 VNG1012H VNG1012H glutaredoxin r VNG0620G edp Proteinase IV homolog 0

VNG1021C VNG1021C 1 VNG0628G gdhA1 Glutamate dehydrogenase g

VNG1052H VNG1052H o l VNG0659H VNG0659H VNG1190G sod1 Superoxide dismutase [Mn] 1 ( 0.0 VNG1295H VNG1295H A VNG0700G yvgX Molybdenum-binding protein N -0.2 VNG0702H VNG0702H heavy metal transport protein VNG1301G cysK Cysteine synthase R VNG1339C VNG1339C m Cu Cu VNG0782H VNG0782H HEAT repeat-containing protein VNG1364G ocd2 Ornithine cyclodeaminase ura3 1179C VNG0863H VNG0863H VNG1438H VNG1438H VNG0864G purL Phosphoribosylformylglycinamidine synthase II VNG1473H VNG1473H VNG1104G rpl10p Acidic ribosomal protein P0 homolog (L10E) VNG1529G mmdA Methylmalonyl-CoA decarboxylase, subunit alpha VNG1801G hsp1 Small heat shock protein VNG1105G rpl1p 50S ribosomal protein L1P (HL8) VNG1802H VNG1802H VNG1117C VNG1117C VNG1806H VNG1806H VNG1137G rpl18e 50S ribosomal protein L18e (HeL18) VNG1821G adh4 Alcohol dehydrogenase VNG1141G rpoK DNA-directed RNA polymerase subunit K VNG2081H VNG2081H VNG1143G rps2p 30S ribosomal protein S2P VNG2091H VNG2091H putative phosphatase VNG2094G trh4 Transcription regulator VNG1173G eef1b Elongation factor 1-beta (EF-1-beta) (aEF-1beta) VNG2115H VNG2115H glutaredoxin VNG1263C VNG1263C PTS IIC components, Glu.Ma/N-AcGln specific VNG2226G cctA Thermosome alpha subunit VNG1289H VNG1289H VNG2310H VNG2310H VNG1318H VNG1318H VNG2311H VNG2311H VNG1414G glyA Serine hydroxymethyltransferase VNG2423G serB Phosphoserine phosphatase VNG1493G purF Amidophosphoribosyl-pyrophosphate amidotransferase VNG2430G thrC1 Threonine synthase VNG2431C VNG2431C VNG1494G rpl37e 50S ribosomal protein L37e VNG2437G argG Argininosuccinate synthetase VNG1496G snp snRNP homolog VNG2443G dpsA Starvation induced DNA binding protein VNG1564H VNG1564H VNG2482G pstB1 Phosphate ABC transporter ATP-binding VNG1591H VNG1591H VNG2484G pstC1 Phosphate transporter permease VNG1653H VNG1653H transposase. VNG2486G yqgG Phosphate ABC transporter binding VNG1688C VNG1688C VNG2513G aldY1 Aldehyde dehydrogenase (Retinol) VNG2537G entB Isochorismatase VNG1690G rpl4e 50S ribosomal protein L4E VNG2600G trxA2 Thioredoxin VNG1692G rpl2p 50S ribosomal protein L2P VNG2616G cxp Probable carboxypeptidase VNG1695G rpl22p 50S ribosomal protein L22P (HHAL22) VNG2619H VNG2619H VNG1698G rpl29p 50S ribosomal protein L29P (HHAL29) VNG6133H VNG6133H VNG1727G cmk Cytidylate kinase VNG6201G hsp5 Heat shock protease protein VNG1830G pyrG CTP synthase/GATase VNG6206H VNG6206H VNG6262G zurM ABC transporter, permease protein VNG1912G trpD2 Phosphoribosyl transferase VNG6264G zurA ABC transporter, ATP-binding protein VNG2076G rpl40e 50S ribosomal protein L40E VNG6265G ycdH Adhesion protein VNG2118G pyrE2 Orotate phosphoribosyltransferase VNG6277G ugpB Glycerol-3-phosphate-binding protein precursor VNG2150G etfB Electron transfer flavoprotein subunit beta VNG6281G ugpC Sn-glycerol-3-phosphate transport system ATP-binding VNG2467G rpl31e 50S ribosomal protein L31e VNG6294G perA Peroxidase/catalase VNG6301G aph Alkaline phosphatase VNG2469G rpl39e 50S ribosomal protein L39e VNG6313G nhaC3 Na+/H+ antiporter VNG2555C VNG2555C putative ferredoxin VNG6315G arcB Ornithine carbamoyltransferase VNG2599H VNG2599H Sec61beta subunit VNG6316G arcC carbamate kinase VNG2643H VNG2643H VNG6317G arcA Arginine DESAMINASE (Arginine deiminase) VNG6255C VNG6255C VNG1349C VNG1349C Predicted acyl dehydratase VNG6256G lipB Lipoate protein ligase VNG5032G gvpC1 GvpC protein, cluster A VNG5033G gvpN1 GvpN protein, cluster A VNG0287H VNG0287H VNG5068G boa3 bacterio-opsin activator-like protein VNG1561C cbiX Putative cobaltochelatase with ferredoxin VNG5144H VNG5144H Transcriptional regulator PadR-like family VNG5008H VNG5008H VNG5145H VNG5145H likely involved in protein transport change shared with ∆sirR VNG5175H VNG5175H VNG5176C VNG5176C transcriptional regulator, arsR family, change inverse to ∆sirR VNG5177C VNG5177C VNG5178H VNG5178H Protease/nuclease/chaperone VNG5180G arsA2 Dehydrogenase/oxidoreductase/redoxins VNG5181G arsD VNG5183G arsC Putative transcription regulators Supplementary Figure 5. Investigation of mechanistic basis of VNG1179C function. A. Differences in normalized (mean=0, variance=1) mRNA level changes in Halobacterium NRC-1 ∆ura3 and Halobacterium NRC-1 ∆ura3 ∆VNG1179C were determined using the SAM algorithm by Tusher et al 2001. B. Significant differences among the two sets were hierarchically clustered to reveal two distinct groups. C. Among the two groups are those that appear to be under negative control of VNG1179C (Group I) and those that are under positive control of VNG1179C (Group II). The inset plot shows mRNA profile for YvgX a Cu-efflux P1-type ATPase in ∆ura3 and ∆ura3 ∆VNG1179C backgrounds in presence of Cu. 0.4

0.2

0 s l o e i t v a e r l -0.2 0 A 1 N g R o l m -0.4

VNG0536G (sirR) -0.6 VNG6262G (zurM) VNG6264G (zurA) VNG6265G (ycdH) -0.8 1 11 21 31 41 51 61 71 81 91 101 111 121 131 141 151 161 171 181 191 201 211 221 231 241 251 Experiments Supplementary Figure 6. mRNA level changes for the putative Mn-uptake ABC transporter system operon zurA, zurM and ycdH and its putative tran- scription regulator SirR in ~250 different experimental conditions including gene expression changes described in this study.

A B Axes 1 and 3 Axes 2 and 3

0.221 0.221 Ribosomal Gas Vesicle biogenesis RadA1 0.177 0.177 Ferredoxin/quinone Fe (2.0) Fe (2.0) oxidoreductase Fe (4.0) Cu (0.7) Fe (4.0) Cu (0.7) 0.133 0.133 Sod2 Fe (6.0) Fe (6.0) Cu (0.85) Co (0.2) Cu (1.0) Cu (0.85) Fe (7.0) Fe (7.0) 0.089 0.089Co (0.2) Cu (1.0) Co (0.3) Co (0.3)

0.044 Ni (0.75) 0.044 Ni (0.5) Ni (0.5) Ni (0.75)

-0.304 -0.243 -0.182 -0.122 -0.061 0.061 0.122 0.182 0.243 0.304 -0.304 -0.243 -0.182 -0.122 -0.061 0.061 0.122 0.182 0.243 0.304

-0.044 -0.044 Co (0.5) Co (0.5) Zn (0.005) Ni (1.5) Ni (1.5) -0.089 -0.089 Zn (0.005)

ZntA,YvgX, CpX -0.133 Arsenic resistance genes -0.133 Siderophore biosynthesis Fbr (putative fibrillarin) Zn (0.01) Zn (0.01) Iron transport plastocyanin Zn (0.02) Zn (0.02) -0.177 Aldehyde reductase (Zn-binding) -0.177 Mn (1.0) Zn-binding proteases Mn (1.0) Mn (1.5) lom protease, proteasome Mn (1.5) -0.221 -0.221 Mn (0.8) Mn (0.8)

C Supplementary Figure 7. Correspondence analysis (contd. from Fig. 5 in main text). CoA plots with Axes 1 and 3 (A) , and Axes 2 and 3 (2), demonstrate relationships among re- sponses to different metals. Panel C shows mRNA level changes for 23 genes at a higher concentration of Co2+ appear to be similar to mRNA level changes at 0.05mM Zn2+. Each dot in this graph represents a particular experiment, for example Co (0.5) refers to mRNA level changes in 0.5mM Co2+. Eigen values are plottted for each of the first three dimen- sions, indicated as Axes 1, 2 and 3, which represent inertia values of 26.34, 19.45 and 16.41, respectively. Supplementary Table 1. Numbers of genes that changed (�� > 15) in at least one concentration of each of the six metals Metal mM up down Mn 0.8 76 186 1 111 124 1.5 182 167 Fe 2 43 54 4 75 31 6 61 26 7 11 17 Co 0.2 17 15 0.3 56 13 0.5 59 15 Ni 0.5 38 30 0.75 40 36 1.5 21 30 Cu 0.7 57 23 0.85 11 7 1 34 4 Zn 0.005 40 17 0.01 134 40 0.02 106 53 The values for Fe do not include changes in the time series experiment Supplementary Table 2. I. Genes of known function (including genes in operon) A. Transporters Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG0002G yvrO Amino acid ABC transporter, ATP-binding protein VNG0123G trp1 ABC transport protein VNG0124C VNG0124C creatinine , Creatinine (EC:3.5.2.10), or creatininase, catalyses the hydrolysis of creatinine to . VNG0149G zntA Broad specificity P1-ATPase for Co, Ni, Cu and Zn VNG0316C VNG0316C FhuD is an ATP-binding cassette-type (ABC-type) binding protein involved in the uptake of hydroxamate-type siderophores VNG0365G arsA1 Arsenical pump-driving ATPase VNG0452G pstB2 Phosphate transport ATP-binding VNG0453G pstA2 Phosphate ABC transporter permease VNG0455G pstC2 Phosphate ABC transporter permease VNG0457G phoX Phosphate ABC transporter periplasmic phosphate-binding VNG0465G nosF2 Copper transport ATP-binding protein VNG0524G yurY ABC transporter, ATP-binding protein VNG0525C VNG0525C Predicted component of ABC transporter system (COG0719) VNG0527C VNG0527C Predicted component of ABC transporter system (COG0719) VNG0700G yvgX Cu-transporting P1-ATPase VNG0702H VNG0702H heavy metal transport protein VNG0727C VNG0727C Multi Antimicrobial Extrusion (drug/sodium antiporter) VNG0897G rbsC1 Ribose ABC transporter permease VNG0898G rbsC2 Ribose ABC transporter permease VNG0901G rbsA Ribose ABC transporter ATP-binding VNG0903C VNG0903C Basic membrane protein VNG0921G potA1 Spermidine/putrescine ABC transporter ATP-binding VNG0923G sfuB Iron transporter-like protein VNG0924G ibp Iron-binding protein VNG0938G gufA GufA protein, putative divalent cation transporter (PFAM, COG matches). VNG1367G srp19 Signal recognition particle 19 kDa protein (SRP19) VNG1369G hemV1 Iron (III) ABC transporter ATP-binding VNG1370G hemU Iron (III) ABC transporter permease VNG1371G hemV2 Iron (III) ABC transporter ATP-binding VNG1631G cbiO2 Cobalt transport ATP-binding protein VNG1632G cbiQ Cobalt transport protein VNG2358G appA Oligopeptide binding protein VNG2359G appB Oligopeptide ABC permease VNG2361G appC Oligopeptide transport permease protein VNG2363G oppD1 Oligopeptide ABC transporter ATP-binding VNG2365G appF Oligopeptide ABC transporter ATP-binding VNG2482G pstB1 Phosphate ABC transporter ATP-binding VNG2483G pstA1 Phosphate ABC transporter permease VNG2484G pstC1 Phosphate transporter permease VNG2486G yqgG Phosphate ABC transporter binding VNG2527G dppD Dipeptide ABC transporter ATP-binding VNG2529G dppB2 Dipeptide ABC transporter permease VNG2531G dppC1 Dipeptide ABC transporter permease VNG2532H VNG2532H VNG2558G fepC Ferric enterobactin transport protein VNG2560G yfmD2 Ferrichrome ABC transporter permease protein VNG2562H VNG2562H periplasmic binding protein, probably involved in iron transport VNG2581H VNG2581H heavy metal transport protein VNG2582H VNG2582H VNG5071C VNG5071C sugar (and other) transporter VNG5180G arsA2 Anion transporting ATPase VNG5181G arsD VNG5183G arsC Arsenate reductase VNG6262G zurM ABC transporter, permease protein VNG6264G zurA ABC transporter, ATP-binding protein VNG6265G ycdH Adhesion protein VNG6277G ugpB Glycerol-3-phosphate-binding protein precursor VNG6279G ugpA Sn-glycerol-3-phosphate transport system permease VNG6281G ugpC Sn-glycerol-3-phosphate transport system ATP-binding

B. Oxidative stress Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG0154G merA Mercury(II) reductase VNG0281G soxB . Sarcosine oxidase VNG0439C VNG0439C Pyridine nucleotide-disulfide oxidoreductase VNG0467G yafB Aldehyde reductase VNG0474G porA Pyruvate ferredoxin oxidoreductase, subunit alpha VNG0523G inb Oxidoreductase homolog VNG0563G ndhG2 NADH dehydrogenase/oxidoreductase VNG0628G gdhA1 Glutamate dehydrogenase VNG0635G nolB NADH dehydrogenase/oxidoreductase-like protein VNG0637G ndhG5 NADH dehydrogenase/oxidoreductase VNG0639G ndhG4 NADH dehydrogenase/oxidoreductase VNG0640G nolD NADH dehydrogenase/oxidoreductase-like protein VNG0641C VNG0641C NADH-ubiquinone/plastoquinone oxidoreductase chain 6 VNG0642C VNG0642C NADH-ubiquinone oxidoreductase VNG0643G nolC NADH dehydrogenase/oxidoreductase-like protein VNG0646G nuoL F420H2:quinone oxidoreductase chain L VNG0647G nuoM F420H2:quinone oxidoreductase chain M VNG0677H VNG0677H VNG0678G acaB1 3-ketoacyl-CoA thiolase VNG0679G acd4 Acyl-CoA dehydrogenase VNG0771G aldY2 Aldehyde dehydrogenase (Retinol) VNG0815G yfmJ Quinone oxidoreductase VNG0891G yjlD NADH dehydrogenase VNG0930G yvbT Alkanal monooxygenase homolog VNG0931G acaB2 3-ketoacyl-CoA thiolase VNG0933G yqjM NADH-dependent flavin oxidoreductase VNG0935G noxC NADH oxidase VNG0937G gap Glyceraldehyde-3-phosphate dehydrogenase VNG0998G yajO2 Probable oxidoreductase VNG1011C VNG1011C Uncharacterized conserved protein VNG1012H VNG1012H glutaredoxin VNG1018G adh3 Alcohol dehydrogenase VNG1070G gpdA1 FAD-dependent oxidoreductase VNG1128G korA Putative 2-ketoglutarate ferredoxin oxidoreductase (Alpha) VNG1190G sod1 Superoxide dismutase [Mn] 1 Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG1259G trxB2 Thioredoxin reductase VNG1306G sdhA Succinate dehydrogenase subunit A VNG1308G sdhB Succinate dehydrogenase subunit B VNG1309G sdhD Membrane anchor VNG1310G sdhC Succinate dehydrogenase hydrophobic membrane anchor protein

VNG1332G sod2 Superoxide dismutase [Mn] 2 VNG1644G nrdB2 Ribonucleoside reductase large chain VNG1774G hemA Glutamyl-tRNA reductase VNG1775C VNG1775C Nad-Dependent Dehydrogenase and Ferrochelatase Involved In Siroheme Synthesis. VNG1821G adh4 Alcohol dehydrogenase VNG2023G gsp General stress protein 69 VNG2106G sdh Succinate dehydrogenase subunit VNG2115H VNG2115H glutaredoxin VNG2171G adh1 Alcohol dehydrogenase VNG2218G pdhB Pyruvate dehydrogenase beta subunit VNG2219G dsa Dihydrolipoamide S-acetyltransferase VNG2220G lpdA Dihydrolipoamide dehydrogenase VNG2299H VNG2299H VNG2301G txrB3 Thioredoxin reductase VNG2420G metA Probable homoserine O-acetyltransferase VNG2513G aldY1 Aldehyde dehydrogenase (Retinol) VNG2555C VNG2555C putative ferredoxin VNG2600G trxA2 Thioredoxin VNG2617G adh2 Alcohol dehydrogenase VNG6270G gldA Sn-glycerol-1-phosphate dehydrogenase VNG6294G perA Peroxidase/catalase

C. Cobalamin biosynthesis Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG1550G cbiT Putative precorrin 8-w decarboxylase (AdoMet methyltransferase).\nInvolved in synthesis of Cobalamin precursors VNG1551G cbiL Precorrin-2 C20 methyltransferase\nsynthesis of Cobalamin precursors VNG1553G cbiF Precorrin 4-methyltransferase\nCobalamin biosynthesis; CbiF involved in synthesis of precursors VNG1554G cbiG Cobalamin biosynthesis VNG1558H VNG1558H VNG1559H VNG1559H VNG1561C cbiX, putative Putative cobaltochelatase with ferredoxin domain\npossible function in cobalamin biosynthesis VNG1562H VNG1562H VNG1564H VNG1564H VNG1566G cobN Putative chelatase involved in cobalt insertion into corrin ring VNG1574G cobA Cobalamin adenosyltransferase

D. Fe uptake and storage systems (including Siderophore biosynthesis) Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG2443G dpsA Starvation induced DNA binding protein VNG6210G gabT Gamma-aminobutyrate aminotransferase VNG6211G bdb L-2,4-diaminobutyrate decarboxylase VNG6212G iucA Iron transport protein A VNG6213G iucB Iron transport protein B VNG6214G hxyA Monooxygenase VNG6216G iucC Iron transport protein C

E. Molybdenum cofactor biosynthesis protein Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG0081G moaE Molybdenum cofactor biosynthesis protein VNG0085G moaA3 Molybdenum cofactor biosynthesis protein VNG0086G moeA2 Molybdenum cofactor biosynthesis protein VNG0207H VNG0207H Molybdenum cofactor biosynthesis protein VNG1273G moaC Molybdenum cofactor biosynthesis protein VNG1735C VNG1735C putative molybdenum cofactor sulfurase, The MOSC domain is predicted to be a sulphur-carrier domain that delivers sulphur, from its conserved cysteine, for the formation of diverse sulphur-metal clusters.

F. Transcription i. RNA polymerase Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG0426G rpoM DNA-directed RNA-polymerase subunit M VNG1136G rpb3 DNA-directed RNA polymerase subunit D VNG1140G rpoN DNA-directed RNA polymerase subunit N VNG1141G rpoK DNA-directed RNA polymerase subunit K VNG1169C VNG1169C RNA polymerase Rpb4 VNG2051G rpoE'' DNA-directed RNA polymerase subunit E'' VNG2053G rpoE' DNA-directed RNA polymerase subunit E' ii. General transcription factors Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG0254G tfbG Transcription Factor B homolog G VNG0315G tfbF Transcription Factor B homolog F VNG0734G tfbB Transcription Factor B homolog B VNG2184G tfbA Transcription Factor B homologA VNG2243G tbpE TATA-Binding Protein E iii. Transcription regulators Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG0019H VNG0019H putative repressor VNG0039H VNG0039H putative transcription regulator (ArsR family). Predicted through de novo structure prediction using Rosetta VNG0040C VNG0040C transcription repressor VNG0101G cspD1 Cold shock protein (putative regulator) VNG0142C VNG0142C putative transcription regulator (MarR family) VNG0176H VNG0176H putative histone acetyl transferase VNG0194H VNG0194H putative transcription regulator of the CopG family VNG0258H VNG0258H putative transcription regulator (PadR family) Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG0293H VNG0293H putative transcription regulator, Rosetta predicted structure matched CATH class 1.10.10.10 VNG0320H VNG0320H Transcription reglator (ArsR family) VNG0451G phoU Transcriptional regulator VNG0458G prp1 Putative Phosphate regulatory protein homolog VNG0511H VNG0511H putative transcription regulator, function predicted by Rosetta VNG0536G sirR Transcription repressor (MntR family) VNG0651G imd1 Hypothetical protein VNG0651G VNG0703H VNG0703H putative transcription regulator, Function assigned on the basis of Rosetta-predicted structure match to CATH class 1.10.10.10 VNG0751C VNG0751C putative transcription regulator (PadR family) VNG0826C dmsR putative transcription regulator involved in anaerobic growth on DMSO and/or TMAO. VNG0890G imd2 putative transcriptional regulator with C-terminal CBS domain, COG2524 VNG1029C VNG1029C putative transcription regulator VNG1123G trh7 putative Lrp-like transcriptional regulator VNG1179C VNG1179C TRASH domain containing regulator VNG1215G pai1 acetyl transferase (histone) VNG1377G asnC Putative transcription regulator VNG1404G trh1 Putative transcription regulator VNG1490H VNG1490H Putative transcription regulator (ArsR family) VNG1776G nirH Putative transcription regulator, structural match to lrp-like transcriptional regulator (e = 1x10E-28), COG1552. VNG1836G cspD2 Cold shock protein VNG2020C VNG2020C predicted transcriptional regulator (marR/padR family) VNG2126C VNG2126C putative transcription regulator VNG2277H VNG2277H putative histone acetyltransferase, COG0454:histone acetyltransferase HPA2 (PET=9)\nPFAM0583:GNAT acetyltransferase family VNG2441G rad3b Helicase VNG2476C VNG2476C putative rad3-related helicase, somewhat weak PDB hit to Bacillus uvrB\nCOG1199 (rad3-like helicases), PET=11 VNG2614H VNG2614H putative transcription regulator VNG2661G nusA NusA protein homolog VNG5028G gvpE1 GvpE protein, cluster A VNG5068G boa3 bacterio-opsin activator-like protein VNG5075C VNG5075C PadR family transcription regulator VNG5144H VNG5144H Transcriptional regulator PadR-like family VNG5176C VNG5176C Transcriptional regulator (ArsR family) VNG5182G arsR predicted transcriptional regulators (ArsR family) VNG6193H VNG6193H putative transcription regulator of the (CopG) VNG6441H VNG6441H putative DNA-binding protein, (Robetta server prediction) metal-binding regulators are shown in red font

E. Protein Synthesis and Degradation i. Proteases Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG0166G psmA2 Proteasome alpha subunit VNG0303G lon Putative protease La homolog type VNG0321G ids putative membrane-associated protease VNG0329G caaX Zinc metalloproteinase homolog VNG0409C VNG0409C Prolyl Oligopeptidase VNG0446G gcd Glucose dehydrogenase VNG0510G prrIV2 Proteasome-activating nucleotidase 2 VNG0557H VNG0557H putative protease VNG0723G pepQ1 Probable peptidase VNG0875C VNG0875C M50 family peptidase (metalloprotease) VNG0880G psmA1 Proteasome, subunit alpha VNG1233G pepQ2 X-pro aminopeptidase homolog VNG2302G yuxL Acylaminoacyl-peptidase VNG2323H VNG2323H putative zinc-binding CAAX amino terminal protease VNG2324H VNG2324H VNG2416G sec11 Signal sequence peptidase VNG2449G pepB2 Aminopeptidase homolog VNG2465C VNG2465C Prefoldin alpha subunit (GimC alpha subunit) VNG2546G pepB3 Aminopeptidase homolog VNG2616G cxp Probable carboxypeptidase VNG6201G hsp5 Heat shock protease protein VNG6361G npa Neutral proteinase ii. factors Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG0401G epf2 mRNA 3'-end processing factor homolog VNG0549G eif2a translation initiation factor 2 alpha subunit (eIF-2-alpha) VNG1262G eif2b translation initiation factor 2 beta subunit (eIF-2-beta) VNG1756G eif1a2 translation initiation factor 1A-2 (aIF-1A-2) VNG1853G eif2ba Translation initiation factor eIF-2B subunit alpha VNG2056G eif2g translation initiation factor 2 gamma subunit (eIF-2-gamma) VNG2247G hisG ATP phosphoribosyltransferase VNG2584C VNG2584C Translation initiation factor SUI1 VNG2654G eef2 Translation elongation factor eEF-2 iii. Ribosomal proteins Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG0177G rpl15e 50S ribosomal protein L15e VNG0548C VNG0548C Nucleolar RNA-binding protein VNG0550G rps27e 30S ribosomal protein S27e VNG0551G rpl44e 50S ribosomal protein L44E VNG0787G rps3e 30S ribosomal protein S3Ae VNG0790G rps15p 30S ribosomal protein S15P VNG1103G rpl12p 50S ribosomal protein L12P ('A' type) (HL20) VNG1104G rpl10p Acidic ribosomal protein P0 homolog (L10E) VNG1105G rpl1p 50S ribosomal protein L1P (HL8) VNG1108G rpl11p 50S ribosomal protein L11P VNG1132G rps13p 30S ribosomal protein S13P/S18E (HS13) VNG1133G rps4p 30S ribosomal protein S4P VNG1134G rps11p 30S ribosomal protein S11P Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG1137G rpl18e 50S ribosomal protein L18e (HeL18) VNG1138G rpl13p 50S ribosomal protein L13P VNG1139G rps9p 30S ribosomal protein S9P VNG1143G rps2p 30S ribosomal protein S2P VNG1157G rphs6 50S ribosomal protein L7Ae VNG1159G rpl24e 50S ribosomal protein L24E (LSU ribosomal protein L24E) VNG1168C VNG1168C predicted RNA binding protein VNG1170G rpl21e 50S ribosomal protein L21e VNG1433G rps17e 30S ribosomal protein S17e VNG1494G rpl37e 50S ribosomal protein L37e VNG1688C VNG1688C Predicted SAM-dependent methylase VNG1689G rpl3p 50S ribosomal protein L13P VNG1690G rpl4e 50S ribosomal protein L4E VNG1691G rpl23p 50S ribosomal protein L23P VNG1692G rpl2p 50S ribosomal protein L2P VNG1693G rps19p 30S ribosomal protein S19P (HHAS19) VNG1695G rpl22p 50S ribosomal protein L22P (HHAL22) VNG1697G rps3p 30S ribosomal protein S3P (HS4) (HHAS3) VNG1698G rpl29p 50S ribosomal protein L29P (HHAL29) VNG1699C VNG1699C putative RNAse P residing within ribosomal operon VNG1700G rps17p 30S ribosomal protein S17 (HHAS17) VNG1701G rpl14p 50S ribosomal protein L14P (HHAL14) VNG1702G rpl24p 50S ribosomal protein L24P VNG1703G rps4e 30S ribosomal protein S4e VNG1705G rpl5p 50S ribosomal protein L5P (HSal5) VNG1706G rps14p 30S ribosomal protein S14P VNG1707G rps8p 30S ribosomal protein S8P VNG1709G rpl6p 50S ribosomal protein L6P VNG1711G rpl32e 50S ribosomal protein L32E VNG1713G rpl19e Ribosomal protein L19 VNG1714G rpl18p 50S ribosomal protein L18P (HSal18) VNG1715G rps5p 30S ribosomal protein S5P VNG1716G rpl30p 50S ribosomal protein L30P VNG1718G rpl15p 50S ribosomal protein L15P VNG2047G rps27ae 30S ribosomal protein S27E VNG2048G rps24e 30S ribosomal protein S24e VNG2076G rpl40e 50S ribosomal protein L40E VNG2467G rpl31e 50S ribosomal protein L31e VNG2469G rpl39e 50S ribosomal protein L39e VNG2648G rps10p 30S ribosomal protein S10P VNG2657G rps7p 30S ribosomal protein S7P VNG2658G rps12p 30S ribosomal protein S12P (HmaS12)

F. Replication, Repair and recombination Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG0133G rpa Replication A related protein VNG1255C VNG1255C replication protein A ortholog VNG1406G rhl putative DNA helicase VNG1408G ush 5'-nucleotidase/2',3'-cyclic phosphodiesterase and related esterases;\nUDP-sugar hydrolase VNG2160C VNG2160C replication protein A ortholog VNG2173G rad24a DNA repair protein VNG2213G brr2 Pre-mRNA splicing helicase VNG2333C VNG2333C recJ-like phosphoesterase (endonuclease) VNG2441G rad3b Helicase VNG2473G radA1 DNA repair and recombination protein radA VNG2476C VNG2476C putative rad3-related helicase, somewhat weak PDB hit to Bacillus uvrB\nCOG1199 (rad3-like helicases), PET=11 VNG2620G uvrD Repair helicase

G. Transposase Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG0042G ntp putative transposase VNG0213H VNG0213H putative transposase VNG0285C VNG0285C putative transposase VNG0286C VNG0286C probable transposase VNG1653H VNG1653H putative transposase VNG5042H VNG5042H putative transposase VNG5044H VNG5044H putative transposase VNG6148H VNG6148H predicted transposase VNG6182H VNG6182H IS200-like transposase

H. Energy metabolism Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG0150H VNG0150H cytochrome C biogenesis protein VNG0151C VNG0151C profilin-like contractile protein VNG0582C VNG0582C putative cytochrome bc1\n VNG0583G cyb Cytochrome b6 VNG0665G coxB1 Cytochrome c oxidase subunit II VNG1257H VNG1257H putative cytochrome oxidase VNG2138G atpB V-type ATP synthase beta chain VNG2139G atpA V-type ATP synthase alpha chain VNG2140G atpF V-type ATP synthase subunit F VNG2141G atpC V-type ATP synthase subunit C VNG2142G atpE V-type ATP synthase subunit E VNG2143G atpK H+-transporting ATP synthase subunit K VNG2144G atpI V-type ATP synthase subunit I VNG2150G etfB Electron transfer flavoprotein subunit beta VNG2151G etfA Electron transfer flavoprotein subunit alpha VNG2193G coxA1 Cytochrome c oxidase subunit I VNG2195G coxB2 Cytochrome c oxidase subunit II VNG5055G cydA cytochrome d oxidase chain I VNG5057G cydB cytochrome d oxidase chain II

I. Cell division/Flagellin/Chemotaxis Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG0192G ftsZ2 Cell division protein ftsZ homolog VNG0265G ftsZ4 Cell division protein VNG0949G gspE3 Type II secretion system protein VNG0950G fapH Flagella-related protein H VNG0953C VNG0953C archaeal flagellin-associated protein Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG0954C VNG0954C Flagellar Accessory protein D or E VNG0960G flaB1 Flagellin B1 precursor VNG0961G flaB2 Flagellin B2 precursor VNG0962G flaB3 Flagellin B3 precursor VNG0966G cheR Chemotaxis protein VNG0967G cheD Chemotaxis protein VNG0971G cheA Chemotaxis protein VNG0973G cheB Protein-glutamate methylesterase VNG0974G cheY CHEY and CHEB genes (Chemotaxis protein) VNG0976G cheW1 Chemotaxis protein VNG1008G flaA1a Flagellin A1 precursor VNG1009G flaA2 Flagellin A2 precursor VNG1933G ftsZ3 Cell division protein VNG2181G mcm MCM / cell division control protein 21 VNG2271G orc6 Orc / cell division control protein 6 VNG6150G orc1 Orc / cell division control protein 6 VNG6187G orc3 Orc / cell division control protein 6 VNG6260G ftsZ5 Cell division protein

J. Miscellaenous Metalloproteins Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Putative Function VNG0249G fbr Copper binding proteins/plastocyanin/azurin VNG0684G fbp Fructose-bisphosphatase VNG0795G hcpC Halocyanin precursor-like VNG1197G bcp Bacterioferritin comigrating protein VNG2196G hcpB Halocyanin precursor-like Supplementary Table 2. II. Genes with unknown function Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Pfam function/pfam COG function/COG Match in pdb VNG0013C PF00301 Rubredoxin COG0675 Predicted transposases VNG0037H COG1598 Uncharacterized ACR VNG0041C VNG0043H PF01797 Transposase IS200 like COG1943 Predicted transposase - may be in DNA replication, recombination and repair

VNG0049H COG0500 SAM-dependent methyltransferases match to 1dl5 (pdb) - Protein-L- Isoaspartate O-Methyltransferase - A Catalyst for Protein Repair

VNG0053H VNG0132C VNG0219H VNG0222C PF01882 Unknown function family COG1721 Uncharacterized ACR VNG0233H VNG0248C VNG0261H VNG0282H VNG0289H PF03692 Uncharacterised protein family (UPF0153) - contain 8 COG0727 Predicted Fe-S-cluster oxidoreductase conserved cysteines that may form a metal binding site - function might be an Fe-S cluster as part of an oxidoreductase complex VNG0291H PF00753 Metallo-beta-lactamase superfamily COG0491 Zn-dependent , including glyoxylases match to 1qh3 (pdb) - Human Glyoxalase II - requires Zn, Mn and Cl ions VNG0352H VNG0378C PF04414 Unknown function family VNG0394C COG1340 Uncharacterized archaeal coiled-coil domain match to 1jch (pdb) - Ribosome inhibitor, hydrolase VNG0402H VNG0439C PF00070 Pyridine nucleotide-disulphide oxidoreductase COG0644 ������������������������������ VNG0468C PF00070/ Pyridine nucleotide-disulphide oxidoreductase/FAD COG0644 ������������������������������ PF01494 binding domain VNG0518H VNG0520H VNG0525C PF01458 induced by oxidative stress and iron deprivation. Essential for COG0719 Predicted membrane components of an uncharacterized iron-regulated ABC- iron acquisition via chrysobactin, a siderophore type transporter SufB

VNG0527C PF01458 Unknown function family COG0719 Predicted membrane components of an uncharacterized iron-regulated ABC- type transporter SufB VNG0535C PF01169 Unknown function family COG2119 Predicted membrane protein VNG0546C PF01908 Unknown function family COG2047 ����������������������������������������������������� VNG0584H PF00355 Rieske [2Fe-2S] domain COG0723 ������������������� VNG0585H VNG0586C PF00127 Copper binding proteins, plastocyanin/azurin family COG3794 ������������ VNG0617H VNG0642C COG0839 NADH:ubiquinone oxidoreductase subunit 6 (chain J) - energy metabolism

VNG0659H VNG0677H VNG0698H VNG0709C PF00753 Metallo-beta-lactamase superfamily COG0491 Zn-dependent hydrolases, including glyoxylases match to 1qh3 (pdb) - Human Glyoxalase II - requires Zn, Mn and Cl ions VNG0746C PF04225 Protein of unknown function (DUF444) COG2718 Uncharacterized BCR VNG0765H VNG0788H VNG0789C VNG0810H VNG0837H VNG0846C VNG0863H VNG0892H PF04229 Unknown function family VNG0903C PF02608 Basic membrane protein COG1744 Surface lipoprotein VNG0920H PF02366 Dolichyl-phosphate-mannose-protein mannosyltransferase VNG0925C PF03214 Reversibly glycosylated polypeptide VNG0932C PF01796 Unknown function family VNG0934H VNG0941C PF01656 CobQ/CobB/MinD/ParA nucleotide binding domain COG0857 BioD-like N-terminal domain of phosphotransacetylase Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Pfam function/pfam COG function/COG Match in pdb VNG0943C COG0640 ArsR - Predicted transcriptional regulators VNG0964C PF04283 Unknown function family COG2469 Uncharacterized ACR VNG0991H VNG0995H VNG1020C PF01066 CDP-alcohol phosphatidyltransferase COG0558 Phosphatidylglycerophosphate synthase VNG1021C PF05165 GTP cyclohydrolase III COG2429 Uncharacterized ACR VNG1021C PF05165 GTP cyclohydrolase III COG2429 VNG1023C PF00107 Zinc-binding dehydrogenase COG1063 Threonine dehydrogenase and related Zn-dependent dehydrogenases VNG1024C COG0720 6-pyruvoyl-tetrahydropterin synthase - may be in coenzyme metabolism VNG1024C COG0720 6-pyruvoyl-tetrahydropterin synthase VNG1025H VNG1026H VNG1047H VNG1052H VNG1085H VNG1086C PF01893 COG1745 Uncharacterized ArCR; probably metal-binding VNG1088C COG3388 Uncharacterized ArCR VNG1093C PF01903 CbiX - Chelatase Superfamily - might be involved in COG2138 metal chelation VNG1115H PF01980 COG1720 Uncharacterized ACR VNG1168C PF04919 COG1491 Predicted RNA-binding protein VNG1193C COG0642 Signal transduction histidine kinase VNG1244C PF01817 Chorismate mutase type II - in the pathway of tyrosine COG1605 Chorismate mutase and phenylalanine biosynthesis. VNG1263C PF01893 COG1745 some interaction with vng1086C - Uncharacterized ArCR; probably metal- binding VNG1295H VNG1314H VNG1315H VNG1318H VNG1323C PF00801 PKD domain VNG1324C PF04138 GtrA-like protein COG2246 Uncharacterized membrane protein VNG1339C PF00501 AMP-binding COG0318 Acyl-CoA synthetases (AMP-forming)/AMP-acid II - may be in MENAQUINONE BIOSYNTHESIS VNG1343C PF01973 COG1634 Uncharacterized Rossmann fold enzyme VNG1365C PF04258 Signal peptide peptidase COG3389 VNG1366H COG3277 RNA-binding protein involved in rRNA processing VNG1372C PF05299 M61 glycyl aminopeptidase VNG1376H VNG1380H VNG1381H VNG1413H VNG1438H COG0640 ArsR - Predicted transcriptional regulators VNG1471C COG1711 Uncharacterized ArCR VNG1473H VNG1534H VNG1558H COG1141 Ferredoxin 1 VNG1559H VNG1559H VNG1562H VNG1564H match to 1erj (pdb) - transcription inhibitor VNG1564H match to 1erj (pdb) - transcription inhibitor VNG1613H VNG1642H VNG1663C PF00571 CBS domain COG0517 CBS domains - play a regulatory role making proteins sensitive to adenosyl match to 1zfj (pdb) Inosine carrying ligands Monophosphate Dehydrogenase VNG1664H VNG1679H VNG1688C COG2106 Uncharacterized ACR VNG1720H VNG1737H VNG1737H VNG1740C VNG1740C VNG1744H PF02535 ZIP Zinc transporter COG0428 Predicted divalent heavy-metal cations transporter VNG1746C COG3361 Uncharacterized ACR VNG1800H VNG1806H VNG1820H Gene ORF Mn Fe_st Fe_ts Co Ni Cu Zn Pfam function/pfam COG function/COG Match in pdb VNG1820H VNG1861C PF04055/ Radical SAM superfamily/no known function/TRAM COG0621 2-methylthioadenine synthetase PF00919/ domain PF01938 VNG1865H VNG1880C PF01979 metal dependent hydrolase superfamily COG1574 Predicted metal-dependent hydrolase with the TIM-barrel fold match to 1Ie7 (pdb) - Amidohydrolase; requires Ca ions for function VNG1898C PF00582 Universal stress protein family COG0589 Universal stress protein UspA and related nucleotide-binding proteins match to 1mjh (pdb) - ATP-binding domain - binds Mn ions VNG1925H VNG1940H VNG1973H VNG2006C PF01171 PP-loop superfamily COG2117 Predicted subunit of tRNA(5-methylaminomethyl-2-thiouridylate) methyltransferase, contains the PP-loop ATPase domain - may be in Translation, ribosomal structure and biogenesis VNG2027H VNG2039H VNG2054H PF01850 PIN domain COG1412 Uncharacterized proteins of PilT N-term./Vapc superfamily VNG2059H VNG2081H VNG2097C COG2311 Uncharacterized membrane protein VNG2191H VNG2259C PF04477 COG1892 Uncharacterized ArCR VNG2260H VNG2273H VNG2298A PF01205 VNG2299H VNG2299H VNG2342H VNG2415H VNG2431C VNG2477H VNG2532H VNG2539H VNG2543C PF01871 AMMECR1 - may have a basic cellular function in either COG2078 Uncharacterized ACR the transcription, replication, repair or translation machinery VNG2556H VNG2582H PF00581 Rhodanese-like domain - involved in cyanide COG0607 Rhodanese-related sulfurtransferases detoxification VNG2619H VNG2642H VNG2644C COG2450 Uncharacterized ACR VNG2652H COG0675 Predicted transposases VNG5008H VNG5038H VNG5069C PF00753/ Metallo-beta-lactamase superfamily - requires zinc ions match to 1qh3 (pdb) - Human PF00581 as cofactor/ Rhodanese-like domain - involved in Glyoxalase II - binds Zn, Mn, Cl cyanide detoxification ions in structure VNG5083H VNG5137H VNG5173H arsA2 VNG5180G PF02374 - Anion-transporting ATPase match to 1f48A (pdb) - Arsenite- arsa2(sbe Translocating ATPase ams) arsD VNG5181G arsC VNG5183G PF01451 - Low molecular weight phosphotyrosine protein match to 1jl3 (pdb) - Arsenate arsC phosphatase Reductase -binds SO42- ions VNG5200H match to 1fnn (pdb) - Cell division control protein 6 VNG6052H VNG6133H VNG6159H VNG6181H COG0675 Predicted transposases VNG6206H VNG6221H COG0675 Predicted transposases VNG6296C PF00561 alpha/beta hydrolase fold COG0596 Predicted hydrolases or acyltransferases (alpha/beta hydrolase superfamily) match to 1cqw (pdb) - Haloalkane Dehalogenase - binds iodine ions in structure Supplementary Table 3. Phenotypes under selected metal stress for in frame single gene deletion strains ORF Knockout_Mn Fe_st Fe_ts Co Ni Cu Zn Function VNG0149G zntA NC NC - D D D D Zinc-transporting ATPase VNG0457G phoX na NC - na NC na NC Phosphate ABC transporter periplasmic phosphate-binding VNG0700G yvgX NC NC - NC NC D NC Molybdenum-binding protein VNG2358G appA NC NC - NC NC NC NC Oligopeptide binding protein VNG2558G fepC NC na - na na na na Ferric enterobactin transport protein VNG2562H VNG2562H NC na - na na na na periplasmic binding protein, probably involved in iron transport VNG6212G iucA NC na - na na na NC Iron transport protein A VNG6265G ycdH NC NC - NC na na na Adhesion protein

VNG0101G cspD1 NC NC - na na na na Cold shock protein (putative regulator) VNG0536G sirR D NC - NC NC NC NC Transcription repressor VNG0703H VNG0703H na NC - na na NC na putative transcription regulator VNG1179C VNG1179C NC NC - NC NC D NC putative Lrp-like transcription regulator (AsnC family) VNG5176C VNG5176C na na - na na na NC transcriptional regulator, arsR family

VNG1012H VNG1012H na NC - na na na NC glutaredoxin

VNG2652H VNG2652H NC NC - na na na NC putative transposase VNG6182H VNG6182H NC NC - na na na na IS200-like transposase

VNG0402H VNG0402H na NC - na NC na na

Key Phenotype mRNA abundance Fe_st: Fe steady state NC No Change up Fe_ts: Fe time series D Defective growth down na not assayed no significant change

Supplementary Table 4. Transcription factor binding sites in upstream regions of putative metal response regulators tfbA tfbB tfbC tfbD tfbE tfbF tfbG tbpB VNG0019H YY YY VNG0039H Y VNG0040C Y cspD1 Y YY VNG0142C Y VNG0176H YY VNG0194H Y YY VNG0258H Y Y VNG0293H VNG0320H Y Y VNG0511H VNG0703H Y VNG1029C Y trh7 Y VNG1490H Y cspD2 VNG2163H rad3b YY VNG2641H VNG5009H VNG5144H arsR Y VNG6193H Y Y VNG6441H Y Y Y indicates mRNA level of transcription factor changed under the same conditions in which the corresponding TFB mRNA also changed indicates binding site for TFB in the promoter of that gene metal-binding regulators are indicated in red font

Supplementary Table 5: Strains used in this study Plasmid used for Strain_name Gene gene replacement Reference Halobacterium NRC-1 (wild type strain) Ng. et al (2000) Halobacterium NRC-1 � ura3 ura3 (parent strain gene deletions) Peck et al (2000) Halobacterium NRC-1 � ura3 � cspD1 cspD1 pNBcspD1d this study Halobacterium NRC-1 � ura3 � zntA zntA pNBzntAd this study Halobacterium NRC-1 � ura3 � VNG0402H VNG0402H pNB0402Hd this study Halobacterium NRC-1 � ura3 � phoX phoX pNBphoXd this study Halobacterium NRC-1 � ura3 � sirR sirR pNBsirRd this study Halobacterium NRC-1 � ura3 � yvgX yvgX pNByvgXd this study Halobacterium NRC-1 � ura3 � VNG0703H VNG0703H pNB0703Hd this study Halobacterium NRC-1 � ura3 � VNG1012H VNG1012H pNB1012Hd this study Halobacterium NRC-1 � ura3 � VNG1179C VNG1179C pNB1179Cd this study Halobacterium NRC-1 � ura3 � appA appA pNBappAd this study Halobacterium NRC-1 � ura3 � fepC fepC pNBfepCd this study Halobacterium NRC-1 � ura3 � VNG2562H VNG2562H pNB2562Hd this study Halobacterium NRC-1 � ura3 � VNG2652H VNG2652H pNB2652Hd this study Halobacterium NRC-1 � ura3 � VNG5176C VNG5176C pNB5176Cd this study Halobacterium NRC-1 � ura3 � VNG6182H VNG6182H pNB6182Hd this study Halobacterium NRC-1 � ura3 � iucA iucA pNBiucAd this study Halobacterium NRC-1 � ura3 � ycdH ycdH pNBycdHd this study Supplementary Table 6. Oligonucleotides used for constructing and evaluating gene knockout strains ORF Gene oligo_name oligo_sequence (5'-3') VNG0101G cspD1 VNG0101G_a CCAGCGCGACGAAGTCCGGGA VNG0101G_b ACTGTGACGCCTCGCACTCAT VNG0101G_c AGTGCGAGGCGTCACAGTTAATCGGGATTCCGTATAGAC VNG0101G_d TCCCGAATGATCTGGTGGGGG VNG0101G_e CAGGCGGTTCAACGTGGACGA VNG0101G_f GCGCTCGACGTTGATCTTGC VNG0101G_g AATGGCGACAGGCGAAGTTG VNG0101G_h GCCTGCTCGATGTCGAACTC VNG0149G zntA VNG0149G_a CTGCGACCGGTGTATCCGTGA VNG0149G_b CGGGTGATCGCGTGAAGACAT VNG0149G_c TCTTCACGCGATCACCCGTAGCCGCCGGCCCAGCGCTAC VNG0149G_d GGAGCCGTCGCCGGCGTGGTG VNG0149G_e GAACGTCGAGATGGACTCCAG VNG0149G_f ACATGAGTGCGCTCCCGTTG VNG0149G_g ACGCCCAGTCGAATCAGACC VNG0149G_h ATGGCCGACACGACTGACAC VNG0402H VNG0402H VNG0402H_a ACTGGGAGAGATACGGCGCGA VNG0402H_b ACCACGTTTCGTGTCGCCCAT VNG0402H_c GGCGACACGAAACGTGGTTGACCGTCCCCGAAACGGTCG VNG0402H_d CTTCCGGAGCTTGGACGCCAG VNG0402H_e AACTTGTCGTGGTCGGGCTTG VNG0402H_f TCGCGCTCCTGTTTGAGGAAG VNG0402H_g AAAGAAGCGCGAACAACTCCG VNG0402H_h GAGTTCGCCGTCCGGGAACTC VNG0457G phoX VNG0457G_a GATCGATGGACTCGGCTTCCA VNG0457G_b TTCGGCGTCGTCTGCTGGCAT VNG0457G_c CCAGCAGACGACGCCGAATAGCGCGTCGCCCCACCCAGC VNG0457G_d GCGGGCGTGATGTAGATGACG VNG0457G_e CAGAGGCGGTCGACGTCGTCG VNG0457G_f GGATGAACGACGAGAAGATG VNG0457G_g CTGACCGTCATCGTCAACAC VNG0457G_h ATGATGGTCCGGTCCTGTTC VNG0536G sirR VNG0536G_a GCTGCCGCCGGGCGACCAGTA VNG0536G_b AACACCGTCGTTTAGATGCAT VNG0536G_c CATCTAAACGACGGTGTTTGAGCGCGTTCACGGAAGTCC VNG0536G_d CAGCATTTCCCCCAGCCAGAT VNG0536G_e GGTGGCGGCGTACGCTGCCGC VNG0536G_f GAACGACACCGGATACTAAC VNG0536G_g ACGCCCTCGAACACCACATC VNG0536G_h CTCGGTGACCGTGAGTTCAG VNG0700G yvgX VNG0700G_a GAACGCGCTGTCCCCGCCGCG VNG0700G_b CCGCGCTGTTCTGGTCGTCAT VNG0700G_c ACGACCAGAACAGCGCGGTAGCCGGGGTGTGGCGGCGCG VNG0700G_d GCTTTTGGGTCAGTCCGCCGT VNG0700G_e GGCTCTGGATCTGGCTGGTGA VNG0700G_f ACCGAACTCGGTCAGTCACTC VNG0700G_g GCGACCGCCTGAAAGTCAAAC VNG0700G_h GACGAAGTACGCGCTGATGC ORF Gene oligo_name oligo_sequence (5'-3') VNG0703H VNG0703H VNG0703H_a CGCGATGGCGGTGTCGAGTGT VNG0703H_b GGTTTCGTCAGTTGAACTCAT VNG0703H_c AGTTCAACTGACGAAACCTAGCGCCTACACGACCGACGC VNG0703H_d GCGCGTCCGGGAGGGGGTGGC VNG0703H_e CGACGACCCCGCGGACGTGAT VNG0703H_f CGTCGTCGGCGAACTACATC VNG0703H_g CGAGTGCGAAGCTTGTACTG VNG0703H_h AGGGCTTCAACGGGCTTGTC VNG1012H VNG1012H VNG1012H_a CTCGTTGGAGCCACCGGGGCA VNG1012H_b CGGTACGGTCGACACGCGCAT VNG1012H_c CGCGTGTCGACCGTACCGTAGCGCGACTGGTCGGATTCC VNG1012H_d ATCGTGGAAGTCATCAACGAC VNG1012H_e GTGGACCGTGACACCGTCGCC VNG1012H_f CAACACGGACAACCTCAAAG VNG1012H_g ACTGCCCGTACTCCCAGAAG VNG1012H_h GGGTTTCGAGGTGGGTGATG VNG1179C VNG1179C VNG1179C_a CCTTCGAGACCTTCGGCGCGA VNG1179C_b ACAGGCCAACAGCACAGCCAT VNG1179C_c GCTGTGCTGTTGGCCTGTTGATGCAGCGCCACCGCTTCG VNG1179C_d GCGTGGACGCCATCGTGCAGG VNG1179C_e CGCAGCCACCAGGATCATCGC VNG1179c_f GGACGCAGTGCTGTATTTGG VNG1179c_g TCTCCGACCGGATCACGAAG VNG1179c_h CTCGAAATCGACGGTGTCTG VNG2358G appA VNG2358G_a ATACCACGGAGATGATCGAGA VNG2358G_b ATGGTTGTCTCTATTTGCCAT VNG2358G_c GCAAATAGAGACAACCATTAAGCCACCAGTTCAGAGGGA VNG2358G_d GATGATGAGCACGAGCGCGAA VNG2358G_e GTGTCGGATCGCGTGGCGGTC VNG2358G_f TCCGGAAGACGTGGTGTATC VNG2358G_g TCGGGAAGGTCGGCATCAAG VNG2358G_h TATCCGGCGCATAGCCGTAG VNG2558G fepC VNG2558G_a ACAACCTCGGGGTCGTGATGT VNG2558G_b GTTCGCGGGTTGTCGTGTCAT VNG2558G_c ACACGACAACCCGCGAACTGAGGGCGGCGGATGCGGACG VNG2558G_d CTCGGCGATGCCCACAACCAC VNG2558G_e GAGCTCGCCAGCCCCTACATC VNG2558G_f CCGACTCACAACGGTTATAC VNG2558G_g TGGACCCACACCACCAACTC VNG2558G_h TGCCGTCCTCGTCGTGTAAC VNG2562H VNG2562H VNG2562H_a GCGGTGGGGGCGGCGGACTCG VNG2562H_b GATCTGCCGTCTTCGCGCCAT VNG2562H_c GCGCGAAGACGGCAGATCTAGGCAGCGATGACAGCCAAC VNG2562H_d TTCTTCCACGCGATCGCGTAC VNG2562H_e TCTCACCGAGCGCAGCCTGAT VNG2562H_f CCGAACCGCACTTTCTCAAC VNG2562H_g CGATTCGTACACGGTGACAG VNG2562H_h CGGCGTAGAAGTTCTCCTTG VNG2652H VNG2652H VNG2652H_a CCGAGAGCATCACGCACTTCT VNG2652H_b ATAATCGTTCCATACTGTCAT VNG2652H_c ACAGTATGGAACGATTATTGAGGGAACTACAAAAGCCTC ORF Gene oligo_name oligo_sequence (5'-3') VNG2652H_d GCGTCCTCCAACGCCCCCACC VNG2652H_e GCTGCGTCCGGCGAGGTGCAG VNG2652H_f TCAACGTGAACTCCACTACC VNG2652H_g CGTGTTCGTGCTGTGGTGAG VNG2652H_h GATGAGGTTGGGCGGCTTAC VNG5176C VNG5176C VNG5176C_a TCGGAAATCGAGTCGGTCACG VNG5176C_b AGCGGTTGCGTCCTGAACCAT VNG5176C_c GTTCAGGACGCAACCGCTTAATGACAACGAGACGATGGT VNG5176C_d TCGAGATGACGACGTTGACGG VNG5176C_e CAGGCGTTGGTCGACGCACGC VNG5176C_f ACTCGGTGCTCTCGTCCTTC VNG5176C_g TCAGCCATGGGCAACGACAC VNG5176c_h TCGTCTCGGTCGGTTCGTAG VNG6182H VNG6182H VNG6182H_a GGAAGAAACGTTCGATCAGGT VNG6182H_b CGCGTGCCGTGTGGTCTTCAT VNG6182H_c AAGACCACACGGCACGCGTGACCGAACTCACGAAGACGC VNG6182H_d GCGTTCCTCTACGTCGCGGGT VNG6182H_e GATTCGTGAACGCGACGCTGT VNG6182H_F CCCGAAGGACTCCACGATAC VNG6182H_G CGAAATAGCCGCCGACAAAG VNG6182H_H TCGACTGTCTCGCTCGAAAC VNG6212G iucA VNG6212G_a AATCCGGCGGGCGACGCCGTC VNG6212G_b CACCGCACCGACACCGGTCAT VNG6212G_c ACCGGTGTCGGTGCGGTGTGACAGCGCCACTGCCCCGAA VNG6212G_d GAACCCGGCGCGTTCGAAGGC VNG6212G_e GAGTATCGATCCCCTGTCGGC VNG6212G_f CGTGAACCGACTCCGTCATC VNG6212G_g GGTTCTACCGGGACAACCAG VNG6212G_h TCCGCCAACGACTCCAGTTC VNG6265G ycdH VNG6265G_a GGGTCGATGTCACTCTAGACA VNG6265G_b CGTGTGTGTCTGCTCGTCCAT VNG6265G_c GACGAGCAGACACACACGTGACACCACACCACGACAACA VNG6265G_d CAGTTCGGCCTCACCGGCGAG VNG6265G_e AAATCCTTCTCGATCACGTTT VNG6265G_f GATCACGTCCGTCATCAGTC VNG6265G_g CCGTCCGCAATCTCATTCCC VNG6265G_h CAACGAGTTCGACGCCATCC Primers a+b and c+d were used to amplify flanking 500bp segments of the target gene. Using the overlap in b and c primers the two 500bp segments were fused in a third PCR reaction using a+d primers. Chromosomal primers e (upstream) and f (downstream) are external to the 500bp flanking segments of the targetr gene. Primers g and h are internal to the intact gene and not present in the deletion copy. Second crossover recombinant colonies were screened for gene deletion using primers a+d or e+d. Colonies positive for a+d/e+d screening were further analyzed by a+d: this confirms knockout; e+d and a+f: these confirm correct chromosomal location of the knockout; and g+h: this confirms absence of intact copy. We also included a ura3 gene specific primer set to rule out presence of plasmid in the cell.