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SUPPLEMENTARY TEXT.
Systems analysis with the Gaggle
mRNA level changes were analyzed simultaneously with gene/protein 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 genes 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).
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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
ycdH , encode subunits of transport systems for PO42-
, 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
cofactor sulfurase (VNG1735C ) implicated in delivering sulphur to diverse metal-sulphur
clusters.
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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. Bacteria 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 protein family (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 roleof 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
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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 binding site 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 UVRadiation 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
archaea Halobacterium sp. NRC-1. Genome Biol 5: R52.
de Lorenzo, V. and J.B. Neilands. 1986. Characterization of iucA and iucC genes of theaerobactin 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-homology method
for the prediction of operons in prokaryotes. Bioinformatics 18 Suppl 1: S329-
336.
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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 datamanagement 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. Protein Data Bank (PDB): database of three-dimensional structuralinformation 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 evolution. 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.
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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. Cell 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
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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.
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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.
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35h
32h 35h
0
0.2
0.4
0.6
0.8
1
1.2
1.4
14h 17h 20h 23h 26h 29h 32h 35h
∆yvgX ∆yvgX (0.6mM Cu)∆ura3∆ura3 (0.6mM Cu)
O . D .
( 6 0 0 n m )
Time (hours)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
14h 17h 20h 23h 26h 29h 32h
∆VNG1179C
∆ura3∆ura3 (0.6mM Cu)
∆VNG1179C (0.6mM Cu)
O . D .
( 6 0 0 n m )
Time (hours)
0
0.2
0.4
0.6
0.8
1
1.2
14h 17h 20h 23h 26h 29h 32h 35h
∆zntA∆
zntA (0.005mM Zn)∆ura3∆ura3 (0.005mM Zn)
O . D .
( 6 0 0 n m )
Time (hours)
0
0.2
0.4
0.6
0.8
1
1.2
∆sirR ∆sirR (0.4mM Mn)∆ura3∆ura3 (0.4mM Mn)
14h 17h 20h 23h 26h 29h
O . D .
( 6 0 0 n m )
Time (hours)
Growth rate assay
i ii
iii iv
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).
Dilution
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
30 minutes
10-4 10-5 10-6 10-4 10-5 10-6
90 minutes
10-4 10-5 10-6
3 hours
10-4 10-5 10-6
6 hours
10-4 10-5 10-6
27 hours
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.
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
1 1 1 21 31 41 51 61 71 81 91 101 111 1 21 131 141 1 51 161 1 71 181 191 2 01 211 221 2 31 241
VNG0700G (yvgX )
VNG1179C
Cu
Zn Zn
Experiments
m R N A
l e v e s ( l o g 1 0 r a t i o s )
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.
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VNG0037H VNG0037H
VNG0293H VNG0293H putative transcription regulator, Rosetta prediction
VNG0452G pstB2 Phosphate transport ATP-binding
VNG0453G pstA2 Phosphate ABC transporter permease
VNG0457G phoX Phosphate ABC transporter periplasmic PO4-binding
V NG0962G flaB3 Flagell in B 3 precursor
VNG0974G cheY CHEY and CHEB genes (Chemotaxis protein)
VNG0997G acs2 A cetyl-CoA synthetase
VNG1047H VNG1047H
VNG1121G aspC2 Aspartate aminotransferase
VNG1295H VNG1295H
V NG1332G sod2 S uperoxide dismutase [Mn] 2
VNG1380H VNG1380H
VNG1529G mmdA Methylmalonyl-CoA decarboxylase, subunit alpha
VNG1794C VNG1794C Staphylococcal nuclease homolog
VNG1801G h sp 1 S ma ll h ea t s ho ck p ro te in
VNG1806H VNG1806H
VNG1912G trpD2 P hosphoribosyl transferase
VNG1973H VNG1973H
VNG2109H VNG2109HVNG2246H VNG2246H
V NG2410G gbp3 GTP -binding protein homolog
VNG2431C VNG2431C
VNG2433H VNG2433H
V NG2436G argH A rgininosuccinate lyase
VNG2437G argG A rgininosuccinate synthetase
VNG2473G radA1 DNA repair and recombination protein radA
VNG2482G pstB1 Phosphate ABC transporter ATP-binding
VNG2484G pstC1 Phosphate transporter permease
VNG2486G yqgG Phosphate ABC transporter binding
VNG2508C VNG2508C
V NG2624G ribC Riboflavin synthase alpha subunit
VNG6162H VNG6162H
VNG6241G gvpA2 Gas vesicle structural protein 2 (GVP) (C-VAC)
VNG6262G zurM A BC transporter, permease protein
VNG6264G zurA ABC transporter, ATP-binding protein
VNG6265G y cd H A dh es io n p ro te in
V NG 14 06 G r hl p ut at iv e D NA h el ic as e
V NG5028G gvpE1 GvpE protein, cluster A
V NG5029G gvpD1 GvpD protein, cluster A
VNG5032G gvpC1 GvpC protein, cluster A
VNG5033G gvpN1 GvpN protein, cluster A
VNG5183G arsC
I Genes directly or indirectly repressed by SirR
VNG1653H VNG1653H transposase
VNG2118G pyrE2 Orotate phosphoribosyltransferase
VNG2634H VNG2634H
VNG6181H VNG6181H
VNG6182H VNG6182H IS200-like transposase
VNG6255C VNG6255C
V NG 62 56 G l ip B L ip oa te p ro te in l ig as e
VNG6305C VNG6305C radical SAM superfamily protein
VNG5044H VNG5044H transposase
VNG2059H VNG2059H
VNG0101G cspD1 Cold shock protein (putative regulator)
VNG0146H VNG0146H
VNG0285C VNG0285C transposase
VNG0394C VNG0394C
VNG0426G rpoM DNA-directed RNA-polymerase subunit M
V NG0491G dnaK Chaperone protein dnaK (Hsp70)
VNG0659H VNG0659H
VNG0702H VNG0702H heavy metal transport protein
VNG0765H VNG0765H
VNG0863H VNG0863H
VNG1007H VNG1007H
VNG1132G rps13p 30S ribosomal protein S13P/S18E (HS13)
VNG1289H VNG1289H
V NG1320G cbp Calcium-binding protein homology
VNG1433G rps17e 30S ribosomal protein S17e
VNG1541G sucC Succinyl-CoA synthetase beta chain
VNG1542G sucD Succinyl-CoA synthetase alpha chain
VNG1591H VNG1591H
V NG 16 24 G m dh M al at e d eh yd ro ge na se
VNG1692G rpl2p 50S ribosomal protein L2PVNG1695G rpl22p 50S ribosomal protein L22P (HHAL22)
VNG1698G rpl29p 50S ribosomal protein L29P (HHAL29)
VNG1699C VNG1699C putative RNAse P encoded in ribosomal operon
VNG1727G c mk Cyti dyl ate k in ase
VNG1830G p yr G C TP s yn th as e/ GA Ta se
VNG2273H VNG2273H
VNG2467G rpl31e 50S ribosomal protein L31e
VNG2469G rpl39e 50S ribosomal protein L39e
VNG2643H VNG2643H
V NG2657G rps7p 30S ribosomal protein S 7P
V NG 26 61 G n us A N us A p ro te in h om ol og
VNG6332H VNG6332H
VNG0287H VNG0287H
VNG2664G rpoA DNA-directed RNA polymerase subunit A
VNG5008H VNG5008H
V NG5019G gvpM1 GvpM protein, cluster A
VNG5071C VNG5071C sugar (and other) transporter
II Genes directly or indirectly activated by SirR
A B
I
II
SAM analysis on 352 genes with significant (λ >15.0) change in at least two microarray experiments.
change shared with ∆sirR
change inverse to ∆sirR
Protease/nuclease/chaperone
Dehydrogenase/oxidoreductase/redoxins
Putative transcription regulators
-2.5
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
Fe
wt
Mn
wt
Mn
∆ura3∆sirR
Mn
∆ura3
0 0
m R N A ( l o g 1 0 r a t i o )
zurM ycdH zurA
AND
VNG1405C
Mn(II), PO42-
and Co(II)
transport
VNG2476C
KaiC
SirR
Prp1
0.15
0.12
-0.14
PhoU
0.12
C
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.
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Supplementary Figure 5. Investigation of mechanistic basis of VNG1179C function. A. Differences in normalized (mean=0, variance=1) mRNA
changes in Halobacterium NRC-1 ∆ura3 and Halobacterium NRC-1 ∆ura3 ∆VNG1179C were determined using the SAM algorithm by Tusher et al 2
B. Significant differences among the two sets were hierarchically clustered to reveal two distinct groups. C. Among the two groups are those that ap
to be under negative control of VNG1179C (Group I) and those that are under positive control of VNG1179C (Group II). The inset plot shows mR
profile for YvgX a Cu-efflux P1-type ATPase in ∆ura3 and ∆ura3 ∆VNG1179C backgrounds in presence of Cu.
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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.
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
1 11 21 31 41 51 61 71 81 91 101 111 121 131 141 151 161 171 181 191 201 211 221 231 241 251
VNG0536G (sirR )
VNG6262G (zurM )
VNG6264G (zurA)
VNG6265G (ycdH )
Experiments
m R N A l e v e l s
l o g 1 0 r a t i o
-0.304 -0.243 -0.182 -0.122 -0.061 0.061 0.122 0.182 0.243 0.3
Axes 2 and 3
Fe (2.0)
Fe (4.0)
Fe (6.0)
Fe (7.0)
Cu (0.7)
Cu (1.0) Cu (0.85)
Co (0.2)
Ni (0.75)Ni (0.5)
Ni (1.5)
Co (0.3)
Co (0.5)
Zn (0.005)
Zn (0.01)
Zn (0.02)
Mn (0.8)
Mn (1.0)Mn (1.5)
0.221
0.177
0.133
0.089
0.044
-0.044
-0.089
-0.133
-0.177
-0.221
-0.304 -0.243 -0.182 -0.122 -0.061 0.061 0.122 0.182 0.243 0.304
0.221
0.177
0.133
0.089
0.044
-0.044
-0.089
-0.133
-0.177
-0.221
Co (0.5)
Co (0.3)
Zn (0.005)
Zn (0.01)
Zn (0.02)
Co (0.2)Cu (0.85)
Cu (1.0)Fe (7.0)
Fe (6.0)
Fe (4.0)
Fe (2.0)
Mn (0.8)
Mn (1.0)Mn (1.5)
Ni (1.5)
Ni (0.5) Ni (0.75)
Cu (0.7)
Axes 1 and 3
Ribosomal proteinsGasVesicle biogenesisRadA1Ferredoxin/quinoneoxidoreductaseSod2
A B
C
ZntA,YvgX,CpXArsenic resistance genesFbr (putative fibrillarin)plastocyaninAldehyde reductase (Zn-binding)Zn-binding proteaseslom protease,proteasome
Siderophore biosynthesisIron transport
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 highe
concentration of Co2+ appear to be similar to mRNA level changes at 0.05mM Zn 2+. Eac
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.
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Metal mM up down
Mn 0.8 76 186
1 111 124
1.5 182 167
Fe 2 43 54
4 75 31
6 61 267 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
Supplementary Table 1. Numbers of genes that changed (�
> 15) in at least one concentration of each of the six metals
The values for Fe do not include changes in the time series experiment
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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 hydrolase, Creatinine amidohydrolase (EC:3.5.2.10), or
creatininase, catalyses the hydrolysis of creatinine to creatine.
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 proteinVNG0525C 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
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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 dehydrogenaseVNG2218G 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(i ncluding 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 proteinVNG1735C 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)
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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 homologVNG2465C 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. Translation 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
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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 nucleic acid 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 transposaseVNG0286C 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
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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 6VNG6187G 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
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G e n e
O R F
M n
F e_ s t
F e_
t s
C o
N i
C u
Z n
P f a m
f u n c t i o
n / p f a m
C O G
f u n c t i o n / C O G
M a t c h i n p d b
V N G 0 9 4 3 C
C O G 0 6 4 0
A r s R
- P r e d i c t e d t r a n s c r i p t i o n a l r e g u l a t o r s
V N G 0 9 6 4 C
P F 0 4 2 8 3
U n k n o w
n f u n c t i o n f a m i l y
C O G 2 4 6 9
U n c h
a r a c t e r i z e d A C R
V N G 0 9 9 1
V N G 0 9 9 5
V N G 1 0 2 0 C
P F 0 1 0 6 6
C D P - a l c o h o l p h o s p h a t i d y l t r a n s f e r a s e
C O G 0 5 5 8
P h o s p h a t i d y l g l y c e r o p h o s p h a t e s y n t h a s e
V N G 1 0 2 1 C
P F 0 5 1 6 5
G T P c y
c l o h y d r o l a s e I I I
C O G 2 4 2 9
U n c h
a r a c t e r i z e d A C R
V N G 1 0 2 1 C
P F 0 5 1 6 5
G T P c y
c l o h y d r o l a s e I I I
C O G 2 4 2 9
V N G 1 0 2 3 C
P F 0 0 1 0 7
Z i n c - b i n
d i n g d e h y d r o g e n a s e
C O G 1 0 6 3
T h r e o
n i n e d e h y d r o g e n a s e a n d r e l a t e d Z n - d e p e n d e n t d e h y d r o g e n a s e s
V N G 1 0 2 4 C
C O G 0 7 2 0
6 - p y r u v o y l - t e t r a h y d r o p t e r i n s y n t h a s e - m a y b e i n c o e n z y m e m e t a b o l i s m
V N G 1 0 2 4 C
C O G 0 7 2 0
6 - p y r u v o y l - t e t r a h y d r o p t e r i n s y n t h a s e
V N G 1 0 2 5
V N G 1 0 2 6
V N G 1 0 4 7
V N G 1 0 5 2
V N G 1 0 8 5
V N G 1 0 8 6 C
P F 0 1 8 9 3
C O G 1 7 4 5
U n c h
a r a c t e r i z e d A r C R ; p r o b a b l y m e t a l - b i n d i n g
V N G 1 0 8 8 C
C O G 3 3 8 8
U n c h
a r a c t e r i z e d A r C R
V N G 1 0 9 3 C
P F 0 1 9 0 3
C b i X -
C h e l a t a s e S u p e r f a m i l y - m i g h t b e i n v o l v e d i n
m e t a l c h e l a t i o n
C O G 2 1 3 8
V N G 1 1 1 5
P F 0 1 9 8 0
C O G 1 7 2 0
U n c h
a r a c t e r i z e d A C R
V N G 1 1 6 8 C
P F 0 4 9 1 9
C O G 1 4 9 1
P r e d i c t e d R N A - b i n d i n g p r o t e i n
V N G 1 1 9 3 C
C O G 0 6 4 2
S i g n a
l t r a n s d u c t i o n h i s t i d i n e k i n a s e
V N G 1 2 4 4 C
P F 0 1 8 1 7
C h o r i s m
a t e m u t a s e t y p e I I - i n t h e p a t h w a y o f t y r o s i n e
a n d p h e
n y l a l a n i n e b i o s y n t h e s i s .
C O G 1 6 0 5
C h o r i s m a t e m u t a s e
V N G 1 2 6 3 C
P F 0 1 8 9 3
C O G 1 7 4 5
s o m e
i n t e r a c t i o n w i t h v n g 1 0 8 6 C - U n c h a r a c t e r i z e d A r C R ; p r o b a b l y m e t a
l -
b i n d i n g
V N G 1 2 9 5
V N G 1 3 1 4
V N G 1 3 1 5
V N G 1 3 1 8
V N G 1 3 2 3 C
P F 0 0 8 0 1
P K D d o
m a i n
V N G 1 3 2 4 C
P F 0 4 1 3 8
G t r A - l i k
e p r o t e i n
C O G 2 2 4 6
U n c h
a r a c t e r i z e d m e m b r a n e p r o t e i n
V N G 1 3 3 9 C
P F 0 0 5 0 1
A M P - b i n d i n g e n z y m e
C O G 0 3 1 8
A c y l - C o A s y n t h e t a s e s ( A M P - f o r m i n g ) / A M P - a c i d l i g a s e s I I - m a y b e i n
M E N A Q U I N O N E B I O S Y N T H E S I S
V N G 1 3 4 3 C
P F 0 1 9 7 3
C O G 1 6 3 4
U n c h
a r a c t e r i z e d R o s s m a n n f o l d e n z y m e
V N G 1 3 6 5 C
P F 0 4 2 5 8
S i g n a l p
e p t i d e p e p t i d a s e
C O G 3 3 8 9
V N G 1 3 6 6
C O G 3 2 7 7
R N A - b i n d i n g p r o t e i n i n v o l v e d i n r R N A p r o c e s s i n g
V N G 1 3 7 2 C
P F 0 5 2 9 9
M 6 1 g l y
c y l a m i n o p e p t i d a s e
V N G 1 3 7 6
V N G 1 3 8 0
V N G 1 3 8 1
V N G 1 4 1 3
V N G 1 4 3 8
C O G 0 6 4 0
A r s R
- P r e d i c t e d t r a n s c r i p t i o n a l r e g u l a t o r s
V N G 1 4 7 1 C
C O G 1 7 1 1
U n c h
a r a c t e r i z e d A r C R
V N G 1 4 7 3
V N G 1 5 3 4
V N G 1 5 5 8
C O G 1 1 4 1
F e r r e
d o x i n 1
V N G 1 5 5 9
V N G 1 5 5 9
V N G 1 5 6 2
V N G 1 5 6 4 H
m a t c h t o 1 e r j ( p d b ) - t r a n s c r i p t i o n
i n h i b i t o r
V N G 1 5 6 4 H
m a t c h t o 1 e r j ( p d b ) - t r a n s c r i p t i o n
i n h i b i t o r
V N G 1 6 1 3
V N G 1 6 4 2
V N G 1 6 6 3 C
P F 0 0 5 7 1
C B S d o
m a i n
C O G 0 5 1 7
C B S
d o m a i n s - p l a y a r e g u l a t o r y r o l e m a k i n g p r o t e i n s s e n s i t i v e t o a d e n o
s y l
c a r r y i n g l i g a n d s
m a t c h t o 1 z f j ( p d b ) I n o s i n e
M o n o p h o s p h a t e D e h y d r o g e n a s e
V N G 1 6 6 4
V N G 1 6 7 9
V N G 1 6 8 8 C
C O G 2 1 0 6
U n c h
a r a c t e r i z e d A C R
V N G 1 7 2 0
V N G 1 7 3 7
V N G 1 7 3 7
V N G 1 7 4 0 C
V N G 1 7 4 0 C
V N G 1 7 4 4
P F 0 2 5 3 5
Z I P Z i n c t r a n s p o r t e r
C O G 0 4 2 8
P r e d i c t e d d i v a l e n t h e a v y - m e t a l c a t i o n s t r a n s p o r t e r
V N G 1 7 4 6 C
C O G 3 3 6 1
U n c h
a r a c t e r i z e d A C R
V N G 1 8 0 0
V N G 1 8 0 6
V N G 1 8 2 0
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G e n e
O R F
M n
F e_ s t
F e_
t s
C o
N i
C u
Z n
P f a m
f u n c t i o n
/ p f a m
C O G
f u n c t i o n / C O G
M a t c h i n p d b
V N G 1 8 2 0 H
V N G 1 8 6 1 C
P F 0 4 0 5 5 /
P F 0 0 9 1 9 /
P F 0 1 9 3 8
R a d i c a l S
A M s u p e r f a m i l y / n o k n o w n f u n c t i o n / T R A M
d o m a i n
C O G 0 6 2 1
2 - m e t h
y l t h i o a d e n i n e s y n t h e t a s e
V N G 1 8 6 5 H
V N G 1 8 8 0 C
P F 0 1 9 7 9
m e t a l d e p e n d e n t h y d r o l a s e s u p e r f a m i l y
C O G 1 5 7 4
P r e d i c t e d m e t a l - d e p e n d e n t h y d r o l a s e w i t h t h e T I M - b a r r e l f o l d
m a t c h t o 1 I e 7 ( p d b ) - U r e a
A m i d o h y d r o l a s e ; r e q u i r e s C a i o n s
f o r f u n c t i o n
V N G 1 8 9 8 C
P F 0 0 5 8 2
U n i v e r s a
l s t r e s s p r o t e i n f a m i l y
C O G 0 5 8 9
U n i v e r
s a l s t r e s s p r o t e i n U s p A a n d r e l a t e d n u c l e o t i d e - b i n d i n g p r o t e i n s
m a t c h t o 1 m j h ( p d b ) - A T P - b i n d i n g
d o m a i n - b i n d s M n i o n s
V N G 1 9 2 5 H
V N G 1 9 4 0 H
V N G 1 9 7 3 H
V N G 2 0 0 6 C
P F 0 1 1 7 1
P P - l o o p s u p e r f a m i l y
C O G 2 1 1 7
P r e d i c t e d s u b u n i t o f t R N A ( 5 - m e t h y l a m i n o m e t h y l - 2 - t h i o u r i d y l a t e )
m e t h y l t r a n s f e r a s e , c o n t a i n s t h e P P - l o o p A T P a s e d o m a i n - m a y b e i n
T r a n s l a t i o n , r i b o s o m a l s t r u c t u r e a n d b i o g e n e s i s
V N G 2 0 2 7 H
V N G 2 0 3 9 H
V N G 2 0 5 4 H
P F 0 1 8 5 0
P I N d o m
a i n
C O G 1 4 1 2
U n c h a
r a c t e r i z e d p r o t e i n s o f P i l T N - t e r m . / V a p c s u p e r f a m i l y
V N G 2 0 5 9 H
V N G 2 0 8 1 H
V N G 2 0 9 7 C
C O G 2 3 1 1
U n c h a
r a c t e r i z e d m e m b r a n e p r o t e i n
V N G 2 1 9 1 H
V N G 2 2 5 9 C
P F 0 4 4 7 7
C O G 1 8 9 2
U n c h a
r a c t e r i z e d A r C R
V N G 2 2 6 0 H
V N G 2 2 7 3 H
V N G 2 2 9 8 A
P F 0 1 2 0 5
V N G 2 2 9 9 H
V N G 2 2 9 9 H
V N G 2 3 4 2 H
V N G 2 4 1 5 H
V N G 2 4 3 1 C
V N G 2 4 7 7 H
V N G 2 5 3 2 H
V N G 2 5 3 9 H
V N G 2 5 4 3 C
P F 0 1 8 7 1
A M M E C R 1 - m a y h a v e a b a s i c c e l l u l a r f u n c t i o n i n e i t h e r
t h e t r a n s
c r i p t i o n , r e p l i c a t i o n , r e p a i r o r t r a n s l a t i o n
m a c h i n e r y
C O G 2 0 7 8
U n c h a
r a c t e r i z e d A C R
V N G 2 5 5 6 H
V N G 2 5 8 2 H
P F 0 0 5 8 1
R h o d a n e
s e - l i k e d o m a i n - i n v o l v e d i n c y a n i d e
d e t o x i f i c a t i o n
C O G 0 6 0 7
R h o d a
n e s e - r e l a t e d s u l f u r t r a n s f e r a s e s
V N G 2 6 1 9 H
V N G 2 6 4 2 H
V N G 2 6 4 4 C
C O G 2 4 5 0
U n c h a
r a c t e r i z e d A C R
V N G 2 6 5 2 H
C O G 0 6 7 5
P r e d i c t e d t r a n s p o s a s e s
V N G 5 0 0 8 H
V N G 5 0 3 8 H
V N G 5 0 6 9 C
P F 0 0 7 5 3 /
P F 0 0 5 8 1
M e t a l l o - b
e t a - l a c t a m a s e s u p e r f a m i l y - r e q u i r e s
z i n c i o n s
a s c o f a c t o r / R h o d a n e s e - l i k e d o m a i n - i n v o l v e d i n
c y a n i d e d e t o x i f i c a t i o n
m a t c h t o 1 q h 3 ( p d b ) - H u m a n
G l y o x a l a s e I I - b i n d s Z n , M n , C l
i o n s i n s t r u c t u r e
V N G 5 0 8 3 H
V N G 5 1 3 7 H
V N G 5 1 7 3 H
a r s A 2
V N G 5 1 8 0 G
P F 0 2 3 7 4 -
a r s a 2 ( s b e
a m s )
A n i o n - t r a
n s p o r t i n g A T P a s e
m a t c h t o 1 f 4 8 A ( p d b ) - A r s e n i t e -
T r a n s l o c a t i n g A T P a s e
a r s D
V N G 5 1 8 1 G
a r s C
V N G 5 1 8 3 G
P F 0 1 4 5 1 -
a r s C
L o w m o l e c u l a r w e i g h t p h o s p h o t y r o s i n e p r o t e i n
p h o s p h a t a s e
m a t c h t o 1 j l 3 ( p d b ) - A r s e n a t e
R e d u c t a s e - b i n d s S O 4 2 - i o n s
V N G 5 2 0 0 H
m a t c h t o 1 f n n ( p d b ) - C e l l d i v i s i o n
c o n t r o l p r o t e i n 6
V N G 6 0 5 2 H
V N G 6 1 3 3 H
V N G 6 1 5 9 H
V N G 6 1 8 1 H
C O G 0 6 7 5
P r e d i c t e d t r a n s p o s a s e s
V N G 6 2 0 6 H
V N G 6 2 2 1 H
C O G 0 6 7 5
P r e d i c t e d t r a n s p o s a s e s
V N G 6 2 9 6 C
P F 0 0 5 6 1
a l p h a / b e t a h y d r o l a s e f o l d
C O G 0 5 9 6
P r e d i c t e d h y d r o l a s e s o r a c y l t r a n s f e r a s e s ( a l p h a / b e t a h y d r o l a s e s u p e r f a m i l y )
m a t c h t o 1 c q w ( p d b ) - H a l o a l k a n e
D e h a l o g e n a s e - b i n d s i o d i n e i o n s
i n s t r u c t u r e
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Supplementary Table 5: Strains used in this study
Strain_name Gene
Plasmid used for
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 studyHalobacterium 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
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 3. Phenotypes under selected metal stress for in frame single gene deletion strains
tfbA tfbB tfbC tfbD tfbE tfbF tfbG tbpB
VNG0019H Y Y Y YVNG0039H Y
VNG0040C Y
cspD1 Y Y Y
VNG0142C Y
VNG0176H Y Y
VNG0194H Y Y Y
VNG0258H Y Y
VNG0293H
VNG0320H Y Y
VNG0511H
VNG0703H Y
VNG1029C Y
trh7 Y
VNG1490H Y
cspD2
VNG2163H
rad3b Y Y
VNG2641H
VNG5009H VNG5144H
arsR Y
VNG6193H Y Y
VNG6441H Y Y
metal-binding regulators are indicated in red font
Y indicates mRNA level of transcription factor changed under the same conditions in which the
corresponding TFB mRNA also changed
Supplementary Table 4. Transcription factor binding sites in upstream regions of putative metal
response regulators
indicates binding site for TFB in the promoter of that gene
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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
Supplementary Table 6. Oligonucleotides used for constructing and evaluating
gene knockout strains
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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 GCGTGGACGCCATCGTGCAGGVNG1179C_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
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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 CGAAATAGCCGCCGACAAAGVNG6182H_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 presenceof plasmid in the cell.