Nelson et al. and Breaker
c-di-AMP Riboswitches
Supplementary Information
Riboswitches in eubacteria sense the
second messenger c-di-AMP
James W. Nelson1, Narasimhan Sudarsan2,3, Kazuhiro Furukawa3,*, Zasha
Weinberg2,3, Joy X. Wang3,**, Ronald R. Breaker2,3,4†
1
Department of Chemistry, Yale University, Box 208107, New Haven, CT 06520,
USA 2Howard Hughes Medical Institute, 3Department of Molecular, Cellular and
Developmental Biology, Yale University, Box 208103, New Haven, CT 06520, USA.
4
Department of Molecular Biophysics and Biochemistry, Yale University, Box
208103, New Haven, CT 06520, USA.
Current addresses: *Faculty of Pharmaceutical Sciences, The University of
Tokushima, Tokushima, Japan; **Center for Clinical and Translational
Metagenomics, Brigham and Women's Hospital, Boston, MA 02115
† To whom correspondence should be addressed. E. mail: [email protected]
Dr. Ronald R. Breaker
Tel: (203) 432-9389
Fax: (203) 432-0753
E-mail: [email protected]
Nelson et al. and Breaker
c-di-AMP Riboswitches
Supplementary Results
Supplementary Figure 1│In-line probing of 165 ydaO RNA with yeast extract. a,
Pattern of spontaneous cleavage resulting from equilibrium dialysis/in-line probing of the
B. subtilis 165 ydaO RNA in the absence of any added compounds or in the presence of
specific dilutions of yeast extract. Annotations, including the sites of structural
modulation, are as described in the legend for Fig. 1d. b, Mass spectrometry of an HPLC
fraction that induces 165 ydaO RNA conformational change (active fraction) and an
adjacent fraction that does not induce conformational change (inactive fraction). The
most prominent peak unique to the active fraction (arrow) corresponds to the mass (m/z =
346.0554 for the [M-H]- ion) of adenosine monophosphate. MS/MS analysis (data not
shown) further suggested that this compound was either adenosine 5′ monophosphate, or
one of its close structural isomers (adenosine 2′- or 3′-monophosphate). Given our
previous examination and exclusion of 5´ AMP as a biologically relevant ligand for ydaO
RNAs5, we hypothesized that the natural ligand included an AMP moiety that was not
stable under the ionization conditions used for mass spectrometry, leading us to test a
wide variety of potential ligand candidates (Supplementary Fig. 2), including c-di-AMP.
Nelson et al. and Breaker
c-di-AMP Riboswitches
Supplementary Figure 2│Screening for analogs of 5´ AMP that are bound by the B.
subtilis 165 ydaO RNA. Depicted are the regions of several in-line probing gels
corresponding to the sites of modulation denoted 1 and 2 as described in Fig. 1d. The
concentrations used for each compound in the box are 0.01, 0.1 and 1 mM, and these
results are compared to that for c-di-AMP at 0.01 mM. 5´ IMP denotes inosine-5´monophosphate and A 5´PS denotes adenosyl-5´-phosphosulfate. Other annotations are
as described in the legend to Fig. 1d.
Nelson et al. and Breaker
c-di-AMP Riboswitches
Supplementary Figure 3│In-line probing of the 165 ydaO RNA in the absence and
presence of 100 nM c-di-AMP. This data (see the legend to Fig. 1 for details) was used
in conjunction with the data in Fig. 1d to map the sites of spontaneous cleavage. Note
that some sites of spontaneous cleavage (occurring 3´ of the nucleotide identified) can
only be estimated due to low resolution of the gels (particularly sites closer to the 3´
terminus of the construct).
Nelson et al. and Breaker
c-di-AMP Riboswitches
Supplementary Figure 4 (Part 1)│Ligand binding by the yuaA RNA from B. subtilis.
a, PAGE analysis in-line probing reactions using 150 yuaA RNA from B. subtilis exposed
to various concentrations of c-di-AMP (100 pM to 10 μM) or ATP (17.8 μM to 3.16
mM). Methods and annotations are as described for Fig. 1d.
Nelson et al. and Breaker
c-di-AMP Riboswitches
Supplementary Figure 4 (Part 2)│b, Sequence, secondary structure, and structural
modulation of the 150 yuaA RNA from B. subtilis. Locations were mapped using the data
in a. c, Plot of the fraction of riboswitch RNA bound to ligand versus the logarithm of the
molar concentration of c-di-AMP as inferred from the modulation of spontaneous
cleavage products in a. The KD value of this riboswitch for c-di-AMP is ~450 pM, and
the curve is consistent with that expected for a one-to-one binding interaction between
RNA and ligand.
Nelson et al. and Breaker
c-di-AMP Riboswitches
Supplementary Figure 5│Examining the KD for the 165 ydaO construct by in-line
probing using an RNA concentration near the lower limit of detection (~0.1 nM). a,
PAGE analysis of an in-line probing assay with 165 ydaO RNA from B. subtilis exposed
to various concentrations of c-di-AMP (5 pM to 100 nM). Methods and annotations are
as described for Fig. 1d. b, Plot of the fraction of riboswitch RNA bound to ligand versus
the logarithm of the molar concentration of c-di-AMP as inferred from the modulation of
spontaneous cleavage products in a. Note that half maximal modulation occurs at ~100
pM but that the curve remains steeper than that expected for a 1-to-1 interaction,
suggesting that the RNA concentration remains higher than the KD for ligand binding.
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c-di-AMP Riboswitches
Supplementary Figure 6│In-line probing analysis of the 144 ydaO RNA construct
from B. subtilis. a, Sequence and secondary structure model for the 144 ydaO RNA from
B. subtilis. b, PAGE analysis of an in-line probing assay with 144 ydaO RNA exposed to
various concentrations of c-di-AMP (100 pM to 10 μM) or ATP (17.8 μM to 3.16 mM).
Other annotations are as described in the legend to Fig. 1d. c, Plot of the fraction of
RNAs undergoing structural modulation versus the logarithm of the concentration of cdi-AMP. Values were derived by evaluating the bands undergoing changes in b. Error
bars are the standard deviation of the normalized fraction modulated for bands 1, 2 and 5.
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c-di-AMP Riboswitches
Supplementary Figure 7│Modulation of a c-di-AMP riboswitch from B. subtilis by
c-di-AMPSS, a phosphorothioate-modified analog of c-di-AMP. a, Chemical structure
of c-di-AMPSS. b, PAGE analysis of an in-line probing assay of ydaO 165 exposed to
various concentrations of c-di-AMPSS (100 pM to 100 μM). Annotations are as described
in the legend to Fig. 1d. c, Plot of the fraction of RNAs undergoing structural modulation
versus the logarithm of the concentration of c-di-AMPSS. Values were derived by
evaluating the bands undergoing changes in b. Note that, although the calculated KD is
approximately 7 nM, this is near the expected concentration of RNA in the reaction tubes.
Therefore, the actual KD is most likely lower than this value.
Nelson et al. and Breaker
c-di-AMP Riboswitches
Supplementary Figure 8│Evidence of preferential c-di-AMP binding by a ydaO
RNA representative from Nostoc punctiforme. a, PAGE analysis of an in-line probing
assay with 139 ydaO RNA from Nostoc punctiforme that was exposed to various
concentrations of c-di-AMP or to 1 mM ATP. Vertical lines identify in-line probing
products whose amounts change on addition of c-di-AMP. Methods and other annotations
are as described for Fig. 1d. b, Sequence, secondary structure, and structural modulation
of the 139 ydaO RNA from N. punctiforme. Locations were mapped using the data in a.
c, Plot of the fraction of riboswitch RNA bound to ligand versus the logarithm of the
molar concentration of c-di-AMP as inferred from the modulation of spontaneous
cleavage products in a. The KD value of this riboswitch for c-di-AMP is ~30 nM, and the
curve depicted is that expected for a one-to-one binding interaction between RNA and
ligand.
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c-di-AMP Riboswitches
Supplementary Figure 9│Evidence of preferential c-di-AMP binding by a ydaO
RNA representative from Syntrophus aciditrophicus. a, PAGE analysis of an in-line
probing assay with 137 ydaO RNA from Syntrophus aciditrophicus exposed to various
concentrations of c-di-AMP or 1 mM ATP. Methods and annotations are as described for
Supplementary Fig. 8. b, Sequence, secondary structure, and structural modulation of
the 137 ydaO RNA from S. aciditrophicus. Locations were mapped using the data in a. c,
Plot of the fraction of riboswitch RNA bound to ligand versus the logarithm of the molar
concentration of c-di-AMP as inferred from the modulation of spontaneous cleavage
products in a. The KD value of this riboswitch for c-di-AMP is ~550 pM, and the curve
depicted is that expected for a one-to-one binding interaction between RNA and ligand.
Nelson et al. and Breaker
c-di-AMP Riboswitches
Supplementary Figure 10│Evidence of preferential c-di-AMP binding by a ydaO
RNA representative from Clostridium acetobutylicum. a, PAGE analysis of an in-line
probing assay with 130 ydaO RNA from Clostridium acetobutylicum exposed to various
concentrations of c-di-AMP or 1 mM ATP. Methods and annotations are as described for
Supplementary Fig. 8.b, Sequence, secondary structure, and structural modulation of the
130 ydaO RNA from C. acetobutylicum. Locations were mapped using the data in a. c,
Plot of the fraction of riboswitch RNA bound to ligand versus the logarithm of the molar
concentration of c-di-AMP as inferred from the modulation of spontaneous cleavage
products in a. The KD value of this riboswitch for c-di-AMP is ~1 nM, and the curve
depicted is that expected for a one-to-one binding interaction between RNA and ligand.
Nelson et al. and Breaker
c-di-AMP Riboswitches
Supplementary Figure 11│In-line probing analysis of the 165 ydaO RNA construct
from B. subtilis under sub-optimal conditions. a, PAGE analysis of an in-line probing
assay with ydaO RNA from B. subtilis exposed to various concentrations of c-di-AMP
and ATP with 10 mM MgCl2 and at 37 °C for 16 hours. Methods and other annotations
are as described for Fig. 1d. b, Plot of the fraction of riboswitch RNA bound to ligand
versus the logarithm of the molar concentration of c-di-AMP as inferred from the
modulation of spontaneous cleavage products in a at sites 1, 2, and 3. Not unexpectedly,
the KD of c-di-AMP has increased relative to that obtained under standard in-line probing
conditions to 10 nM. Importantly, however, no consistent modulation with ATP is
observed at concentrations even as high as 5 mM.
Nelson et al. and Breaker
c-di-AMP Riboswitches
Supplementary Figure 12│Riboswitch-mediated gene expression with constructs
driven by different promoters. a, Construct design wherein the promoter (P) is either
the native promoter for the ydaO gene, or the promoter for the B. subtilis lysC gene. b,
Predicted intrinsic transcription terminator stem for the B. subtilis ydaO riboswitch and
its fusion to the lacZ reporter gene. c, Plot of the amount of reporter gene (see b)
expression observed in B. subtilis YP79 cells carrying a knock-out of the disA gene,
normalized to the level observed with unaltered B. subtilis cells, grown in lysogeny broth.
Error bars are the standard deviation of three independent measurements.
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c-di-AMP Riboswitches
Nelson et al. and Breaker
c-di-AMP Riboswitches
Supplementary Figure 13 (previous page)│Genomic locations of c-di-AMP
riboswitches in several organisms. Each riboswitch occurs singularly (denoted by hairpin
annotation), but in some instances appears to control the expression of several genes in an
operon. For representative 3 in B. anthracis, the gene with the asterisk indicates the
presence of an unannotated pseudogene with sequence similarity to a portion of potE (a
putrescine transporter). This possible gene is followed by a large intergenic region and
then the gene for hemolytic enterotoxin HBL (not depicted). The gene name for yuaA
(now ktrA) has been updated in response to recent protein function studies61, while ydaO
has been renamed potE due to its similarity to this gene as determined by ProteinBLAST.
The riboswitch locations in these organisms are typical of the patterns of associations
observed between c-di-AMP riboswitches and the genes they likely control among
diverse bacterial lineages. For example, c-di-AMP riboswitches almost exclusively
control genes involved in cell wall metabolism in Actinobacteria, whereas members of
this same riboswitch class in Cyanobacteria and Bacillales appear to control genes more
directly related to overcoming osmotic stress.
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c-di-AMP Riboswitches
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c-di-AMP Riboswitches
Supplementary Figure 14 (previous page)│In-line probing of the 165 ydaO RNA in
increasing concentrations of c-di-AMP or ATP. Polyacrylamide gel electrophoresis
(PAGE) analysis of an in-line probing assay with 165 ydaO RNA exposed to various
concentrations of c-di-AMP (100 pM to 10 μM in half-log intervals) or ATP (17.8 μM to
3.16 mM in quarter-log intervals). NR, T1 and ‒OH designate no reaction, partial
digestion with either RNase T1 (cleaves after guanosine nucleotides) or hydroxide ions
(cleaves after any nucleotide),. Precursor RNA (Pre) and certain RNase T1 cleavage
product bands are identified. Locations of spontaneous RNA cleavage changes brought
about by c-di-AMP (regions 1 through 6) are identified by asterisks. This is the full
length gel presented in part in Fig. 1d in the main text.
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c-di-AMP Riboswitches
Supplementary Figure 15 │Mutation of conserved nucleotides in the 165 ydaO RNA
abolishes c-di-AMP binding. In-line probing analyses of WT and M1 through M4
RNAs in the absence (‒) or presence of 10 nM c-di-AMP. This is the full length image of
the gel sections presented in Fig. 2c and Fig. 2d.
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c-di-AMP Riboswitches
Supplementary Figure 16│Deletion of the pseudoknot in 165 ydaO RNA abolishes
ATP binding and modestly reduces c-di-AMP binding. In-line probing analysis of M5
in the absence (‒) of ligand, or presence of increasing c-di-AMP or 1 mM ATP. The gel
images depict the region encompassing sites 1 through 6 with annotations as described
for Fig. 1d. This is the full gel presented in Fig. 2d.
Nelson et al. and Breaker
c-di-AMP Riboswitches
Supplementary Figure 17│In vitro transcription termination of the yuaA RNA.
PAGE analysis of an in vitro transcription termination assay using the yuaA riboswitch
from B. subtilis. T is the riboswitch-terminated RNA transcript and FL is the full-length
run-off transcript. M is a marker lane comprising the transcription products from a similar
DNA template encoding the riboswitch plus six additional nucleotides beyond the
predicted terminator site. This is the full length gel presented in Fig. 3a. Note that the
additional lane at the right of the gel is not present in the cropped image, as it is simply
the same reaction presented in the marker lane.
Nelson et al. and Breaker
c-di-AMP Riboswitches
Supplementary Table 1. Category assignment of genes for protein domains
controlled by c-di-AMP riboswitches. Genes predicted to be controlled by the c-diAMP riboswitch were manually assigned to one of several categories listed below based
on the description of domain(s) within that gene from the conserved domain database
(see Materials and Methods). Genes that are not grouped into these categories are not
shown here, but can be found in the Supplementary Online Material file. These categories
are the basis for Fig. 4.
Category
Amino Acid
Metabolism
Amino Acid
Transporters
Domain
accession
cd00245
Brief description (from the Conserved Domain Database)
Coenzyme B12-dependent glutamate mutase epsilon
subunit-like family; contains proteins similar to
Clostridium cochlearium glutamate mutase (Glm) and
Streptomyces tendae Tu901 NikV.
cd00712
Glutamine amidotransferases class-II (GATase)
asparagine synthase_B type.
cd01991
The C-terminal domain of Asparagine Synthase B.
cd04732
HisA.
COG0289 Dihydrodipicolinate reductase [Amino acid transport and
metabolism]
COG1509 Lysine 2,3-aminomutase [Amino acid transport and
metabolism]
TIGR00036 dihydrodipicolinate reductase.
TIGR00653 glutamine synthetase, type I.
cd03262
HisP and GlnQ are the ATP-binding components of the
bacterial periplasmic histidine and glutamine permeases,
respectively.
COG0531 Amino acid transporters [Amino acid transport and
metabolism]
COG0833 Amino acid transporters [Amino acid transport and
metabolism]
COG1174 ABC-type proline/glycine betaine transport systems,
permease component [Amino acid transport and
metabolism]
COG1732 Periplasmic glycine betaine/choline-binding (lipo)protein
of an ABC-type transport system (osmoprotectant binding
protein) [Cell envelope biogenesis, outer membrane]
COG2113 ABC-type proline/glycine betaine transport systems,
periplasmic components [Amino acid transport and
metabolism]
pfam04069 Substrate binding domain of ABC-type glycine betaine
transport system.
Nelson et al. and Breaker
Anion
Transporter
TIGR00909 amino acid transporter.
cd00625
Anion permease ArsB/NhaD.
cd02035
Cell Wall
Metabolism
c-di-AMP Riboswitches
ArsA ATPase functions as an efflux pump located on the
inner membrane of the cell.
cd03293
NrtD and SsuB are the ATP-binding subunits of the
bacterial ABC-type nitrate and sulfonate transport
systems, respectively.
cd07042
Sulfate Transporter and Anti-Sigma factor antagonist
domain of SulP-like sulfate transporters, plays a role in
the function and regulation of the transport activity,
proposed general NTP binding function.
cd07043
Sulfate Transporter and Anti-Sigma factor antagonist)
domain of anti-anti-sigma factors, key regulators of antisigma factors by phosphorylation.
COG0659 Sulfate permease and related transporters (MFS
superfamily) [Inorganic ion transport and metabolism]
COG0715 ABC-type nitrate/sulfonate/bicarbonate transport systems,
periplasmic components [Inorganic ion transport and
metabolism]
COG1392 Phosphate transport regulator (distant homolog of PhoU)
[Inorganic ion transport and metabolism]
TIGR01183 nitrate ABC transporter, permease protein.
cd00118
Lysin domain, found in a variety of enzymes involved in
bacterial cell wall degradation.
cd00254
Lytic Transglycosylase (LT) and Goose Egg White
Lysozyme (GEWL) domain.
cd00761
Glycosyltransferase family A (GT-A) includes diverse
families of glycosyl transferases with a common GT-A
type structural fold.
cd01635
Glycosyltransferases catalyze the transfer of sugar
moieties from activated donor molecules to specific
acceptor molecules, forming glycosidic bonds.
cd02541
Prokaryotic UGPase catalyzes the synthesis of UDPglucose.
cd02549
A sub-family of peptidase family C39.
cd02874
Cortical fragment-lytic enzyme (CFLE) is a peptidoglycan
hydrolase involved in bacterial endospore germination.
cd03255
This family is comprised of MJ0796 ATP-binding
cassette, macrolide-specific ABC-type efflux carrier
(MacAB), and proteins involved in cell division (FtsE),
and release of lipoproteins from the cytoplasmic
membrane (LolCDE).
Nelson et al. and Breaker
COG0308
pfam00062
pfam00877
pfam01435
pfam01464
pfam01471
pfam01551
pfam01943
pfam06737
pfam07486
PRK13914
TIGR00413
TIGR02869
TIGR02884
TIGR02899
TIGR02917
cd01427
Aminopeptidase N [Amino acid transport and
metabolism]
Predicted nucleoside-diphosphate-sugar epimerases [Cell
envelope biogenesis, outer membrane / Carbohydrate
transport and metabolism]
Membrane proteins related to metalloendopeptidases [Cell
envelope biogenesis, outer membrane]
Cell wall-associated hydrolases (invasion-associated
proteins) [Cell envelope biogenesis, outer membrane]
Lipoproteins [Cell envelope biogenesis, outer membrane]
Predicted sugar nucleotidyltransferases [Cell envelope
biogenesis, outer membrane]
Uncharacterized protein containing LysM domain
[Function unknown]
Zn-dependent carboxypeptidase [Amino acid transport
and metabolism]
Putative peptidoglycan-binding domain-containing protein
[Cell envelope biogenesis, outer membrane]
Cell wall hydrolyses involved in spore germination [Cell
envelope biogenesis, outer membrane]
Membrane-bound metallopeptidase [Cell division and
chromosome partitioning]
C-type lysozyme/alpha-lactalbumin family.
NlpC/P60 family.
Peptidase family M48.
Transglycosylase SLT domain.
Putative peptidoglycan binding domain.
Peptidase family M23.
Polysaccharide biosynthesis protein.
Transglycosylase-like domain.
Cell Wall Hydrolase.
invasion associated secreted endopeptidase; Provisional
rare lipoprotein A.
spore cortex-lytic enzyme.
delta-lactam-biosynthetic de-N-acetylase.
spore coat assembly protein SafA.
putative PEP-CTERM system TPR-repeat lipoprotein.
Haloacid dehalogenase-like hydrolases.
cd03788
Trehalose-6-Phosphate Synthase (TPS) is a
COG0702
COG0739
COG0791
COG0797
COG1213
COG1652
COG2317
COG3409
COG3773
COG4942
Hypothetical
Osmoprotectant
c-di-AMP Riboswitches
Nelson et al. and Breaker
Misc.
Transporters
c-di-AMP Riboswitches
glycosyltransferase that catalyzes the synthesis of
alpha,alpha-1,1-trehalose-6-phosphate from glucose-6phosphate using a UDP-glucose donor.
COG1877 Trehalose-6-phosphatase [Carbohydrate transport and
metabolism]
pfam08472 Sucrose-6-phosphate phosphohydrolase C-terminal.
TIGR00685 trehalose-phosphatase.
cd00134
Bacterial periplasmic transport systems use membranebound complexes and substrate-bound, membraneassociated, periplasmic binding proteins (PBPs) to
transport a wide variety of substrates, such as, amino
acids, peptides, sugars, vitamins and inorganic ions.
cd00267
ABC (ATP-binding cassette) transporter nucleotidebinding domain; ABC transporters are a large family of
proteins involved in the transport of a wide variety of
different compounds, like sugars, ions, peptides, and more
complex organic molecules.
cd00995
The substrate-binding domain of an ABC-type
nickel/oligopeptide-like import system contains the type 2
periplasmic binding fold.
cd01115
Permease SLC13 (solute carrier 13).
cd01116
Permease P (pink-eyed dilution).
cd03228
The MRP (Multidrug Resistance Protein)-like transporters
are involved in drug, peptide, and lipid export.
cd03230
This family of ATP-binding proteins belongs to a
multisubunit transporter involved in drug resistance (BcrA
and DrrA), nodulation, lipid transport, and lantibiotic
immunity.
cd03257
The ABC transporter subfamily specific for the transport
of dipeptides, oligopeptides (OppD), and nickel (NikDE).
cd03295
OpuCA is the ATP binding component of a bacterial
solute transporter that serves a protective role to cells
growing in a hyperosmolar environment.
cd06174
The Major Facilitator Superfamily (MFS) is a large and
diverse group of secondary transporters that includes
uniporters, symporters, and antiporters.
cd06261
Transmembrane subunit (TM) found in Periplasmic
Binding Protein (PBP)-dependent ATP-Binding Cassette
(ABC) transporters which generally bind type 2 PBPs.
COG1277 ABC-type transport system involved in multi-copper
enzyme maturation, permease component [General
function prediction only]
COG3932 Uncharacterized ABC-type transport system, permease
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+
+
Na or K
Transporters
cd00293
cd01987
cd01988
COG0475
COG0569
COG0591
COG1055
COG2060
COG2156
COG2216
COG3158
COG3263
pfam00375
pfam00582
pfam00924
pfam00999
pfam02702
pfam03814
TIGR00931
TIGR00932
Signal
Transduction
TIGR00933
TIGR02121
cd00075
c-di-AMP Riboswitches
components [General function prediction only]
USP: Universal stress protein family.
USP domain is located between the N-terminal sensor
domain and C-terminal catalytic domain of this
Osmosensitive K+ channel histidine kinase family.
The C-terminal domain of a subfamily of Na+ /H+
antiporter existed in bacteria and archaea.
Kef-type K+ transport systems, membrane components
[Inorganic ion transport and metabolism]
K+ transport systems, NAD-binding component
[Inorganic ion transport and metabolism]
Na+/proline symporter [Amino acid transport and
metabolism / General function prediction only]
Na+/H+ antiporter NhaD and related arsenite permeases
[Inorganic ion transport and metabolism]
K+-transporting ATPase, A chain [Inorganic ion transport
and metabolism]
K+-transporting ATPase, C chain [Inorganic ion transport
and metabolism]
High-affinity K+ transport system, ATPase chain B
[Inorganic ion transport and metabolism]
K+ transporter [Inorganic ion transport and metabolism]
NhaP-type Na+/H+ and K+/H+ antiporters with a unique
C-terminal domain [Inorganic ion transport and
metabolism]
Sodium:dicarboxylate symporter family.
Universal stress protein family.
Mechanosensitive ion channel.
Sodium/hydrogen exchanger family.
Osmosensitive K+ channel His kinase sensor domain.
Potassium-transporting ATPase A subunit.
Na+/H+ antiporter NhaC.
transporter, monovalent cation:proton antiporter-2 (CPA2)
family.
potassium uptake protein, TrkH family.
sodium/proline symporter.
Histidine kinase-like ATPases; This family includes
several ATP-binding proteins for example: histidine
kinase, DNA gyrase B, topoisomerases, heat shock protein
HSP90, phytochrome-like ATPases and DNA mismatch
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B12-Related
SAM-Related
tRNA
Synthetase
c-di-AMP Riboswitches
repair proteins
cd00082
Histidine Kinase A (dimerization/phosphoacceptor)
domain; Histidine Kinase A dimers are formed through
parallel association of 2 domains creating 4-helix bundles;
usually these domains contain a conserved His residue and
are activated via trans-autophosphorylation by the
catalytic domain of the histidine kinase.
cd00130
PAS domain; PAS motifs appear in archaea, eubacteria
and eukarya.
cd00156
Signal receiver domain; originally thought to be unique to
bacteria (CheY, OmpR, NtrC, and PhoB), now recently
identified in eukaryotes ETR1 Arabidopsis thaliana; this
domain receives the signal from the sensor partner in a
two-component systems; contains a phosphoacceptor site
that is phosphorylated by histidine kinase homologs;
usually found N-terminal to a DNA binding effector
domain; forms homodimers
cd06225
Histidine kinase, Adenylyl cyclase, Methyl-accepting
protein, and Phosphatase (HAMP) domain.
COG2205 Osmosensitive K+ channel histidine kinase [Signal
transduction mechanisms]
COG3103 SH3 domain protein [Signal transduction mechanisms]
pfam08239 Bacterial SH3 domain.
smart00287 Bacterial SH3 domain homologues.
cd00245
Coenzyme B12-dependent glutamate mutase epsilon
subunit-like family; contains proteins similar to
Clostridium cochlearium glutamate mutase (Glm) and
Streptomyces tendae Tu901 NikV.
cd00512
Coenzyme B12-dependent-methylmalonyl coenzyme A
(CoA) mutase (MCM)-like family; contains proteins
similar to MCM, and the large subunit of Streptomyces
coenzyme B12-dependent isobutyryl-CoA mutase (ICM).
cd02065
B12 binding domain (B12-BD).
pfam02310 B12 binding domain.
cd01335
Radical SAM superfamily.
cd02440
S-adenosylmethionine-dependent methyltransferases
(SAM or AdoMet-MTase), class I; AdoMet-MTases are
enzymes that use S-adenosyl-L-methionine (SAM or
AdoMet) as a substrate for methyl-transfer, creating the
product S-adenosyl-L-homocysteine (AdoHcy).
cd00165
S4/Hsp/ tRNA synthetase RNA-binding domain; The
domain surface is populated by conserved, charged
residues that define a likely RNA-binding site; Found in
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cd00673
COG0013
c-di-AMP Riboswitches
stress proteins, ribosomal proteins and tRNA synthetases;
This may imply a hitherto unrecognized functional
similarity between these three protein classes.
Alanyl-tRNA synthetase (AlaRS) class II core catalytic
domain.
Alanyl-tRNA synthetase [Translation, ribosomal structure
and biogenesis]
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c-di-AMP Riboswitches
Supplementary Table 2. DNA primers used in this study.
Primer
Name
Originally
from
Sequence (5´ to 3´)
Purpose
ydaO1
Ref. 5
TAATACGACTCACTATAGGGAA
AACAAATCGCTTAATC
ydaO2
Ref. 5
CATCGAACACAGGTTTCATTC
ydaO3
This study
ATCTCCTCTCACGAACGC
ydaO4
This study
ydaO5
This study
ydaO6
This study
TAATACGACTCACTATAGGGTC
TCTTTTACCGCTTAATCAAACA
CG
CCTCCTCTCTGTATACGCCTAC
G
ATCTCCTCTCACGAACGC
Forward primer for amplifying
WT 165, WT 144, M3, M4, and
M5 ydaO from B. subtilis
Reverse primer for amplifying
WT, M3, and M4 ydaO 165 from
B. subtilis
Reverse primer for amplifying
WT 144 from B. subtilis
Forward primer for amplifying
WT yuaA 148 from B. subtilis
ydaO7
This study
ydaO8
This study
ydaO9
This study
ydaO10
This study
ydaO11
This study
TAATACGACTCACTATAGGGTA
ATGCATGGCGCTTAATCCTCGA
TTCAAGAGGAGCGGGGGACCC
GTTCTCCTGGGGCGTATCGCC
ydaO12
This study
GAGTCCCCTCATCGATGCCTGC
GAGGTTAGCTGACGGGCTCGG
GCCGAAGAGGTGCCCTACGCC
AAATGGCGATACGCCCCAGG
Reverse primer for primer
extension of ydaO from S.
aciditrophicus
ydaO13
This study
Forward primer for amplification
of ydaO from C. acetobutylicum
ydaO14
This study
TAATACGACTCACTATAGGGTA
ATATCATTAGCCTAAATTTTAA
A
AATCTTCCTCCTTTTATTCTACG
TAATACGACTCACTATAGGGAA
AACAAATCGCTTAATCTGAAAT
CAGAGCGGGGGACCCAATAGA
ACGGCTTTTTGCCGTTGGGGTG
AATCCTTTTTAGG
CATCGAACACAGGTTTCATTCA
TCTCCTCTCACGAACGCTTACG
AGGTTAGCTGACCGATTCGGGC
ATATGAGAGTTAGCCCTACCTA
AAAAGGATTCACCC
CATCGAACACAGGTTTCATTCA
TCTCCTCTCACGAACGCTTACG
AGGTTCGCTGACGGATTCGGGC
ATATGAGAGTTAGCCCTACCTA
AAAAGGATTCACCC
ACGAACGCTTACGAGG
Reverse primer for amplifying
WT yuaA 148 from B. subtilis
Reverse primer for amplifying
WT ydaO 144 from B. subtilis
Forward primer for primer
extension of M1 and M2 ydaO
165
Reverse primer for primer
extension of M1 ydaO 165
Reverse primer for primer
extension of M2 WT ydaO 165
Reverse primer for amplification
of M5 ydaO RNA
Forward primer for primer
extension of ydaO from S.
aciditrophicus
Reverse primer for amplification
of ydaO from C. acetobutylicum
Nelson et al. and Breaker
ydaO15
This study
ydaO16
This study
ydaO17
This study
ydaO18
c-di-AMP Riboswitches
TAATACGACTCACTATAGGGTT
TAGTGGGTAGCCTAATTCAAAT
TCATGAAAGCTGAATTTGGAAC
GGAGGAACCAATGTTTGGGGC
TAATC
GGGGGTTCCTTCCATTGCCTAC
GGAGTTAGCTGACGGGCTAAG
ACTGGAAGGTGTCTCTCATAAG
ATTAGCCCCAAACATTGG
AAGGAATTCAAAAATAATGTT
GTCCTTTTAAATAAGATCTGAT
AAAATGTGAACTAA
AGAAAACAAATCGCTTAATCTG
AAATCAG
Forward primer for primer
extension of ydaO from N.
punctiforme PCC 73102
This study
TTACCGCTAATGTCATGGCAAA
ATTGCC
ydaO19
This study
GAGATTTATCTAATTTCTTTTTT
CTTTTCCTCGTCCTCCAAGATTT
CAGTC
ydaO20
This study
AAGAAAAAAGAAATTAGATAA
ATCTC
Forward primer for amplifying
the region upstream of cdaA orf
for generating the 5flank of the
cdaA KO fragment.
Reverse primer for amplifying the
region upstream of cdaA orf for
generating the 5 flank of the
cdaA KO fragment. Also contains
a region complementary to the
erythromycin forward primer
ydaO20
Forward primer for amplifying
the erythromycin cassette
ydaO21
This study
CACAGCCCAGCGGTTGTTTAAG
AATCTGCGCAAAAGACATAAT
CGATTCAC
ydaO22
This study
GTGAATCGATTATGTCTT
TTGCGCAGATTCTTAAACAA
ydaO23
This study
Reverse primer for primer
extension of ydaO from N.
punctiforme PCC 73102
Primer meant for cloning
the ydaO leader with
containing a lysine promoter
with an engineered EcoRI
site.
Reverse primer for amplifying the
erythromycin cassette
Forward primer for building
the 3 flank of the cdaA KO
CCGCTGGGCTGTG
fragment with the
erythromycin 3
complement.
TCCTTTTCAATCGTTTCATCTGC Reverse primer to generate the 3
GTTTTCCAAATTCAC
flank of the cdaA KO fragment.
Forward primer for cloning the
ydaO promoter region with EcoRI
site (-466 nt from the ydaO
translation start site)
Reverse primer for cloning the
ydaO promoter region with a
BamHI site (-129 nt from the
ydaO translation start site)
Reverse primer for cloning the
ydaO promoter region that creates
a terminator mutation (M6) with a
BamHI site
ydaO24
This study
AACAGAATTCGATTTTAGCCTC
TGTTTTTTTATTTTTGGTAAGTA
AA
ydaO25
This study
AAAGGATCCTGAACAACAAAA
AACACAGAGGCAAACGGATGC
CCCTGTGCC
ydaO26
This study
AAAGGATCCTGAACAACAAAA
AACTTAGAGGCAAACGGATGC
CCCTGTGCC
Nelson et al. and Breaker
c-di-AMP Riboswitches
ydaO27
This study
CCCGAATCCGTCAGCGAACCTC
GTAAGCGTT
Forward primer for construction
of M1 reporter construct
ydaO28
This study
AACGCTTACGAGGTTCGCTGAC
GGATTCGGG
Reverse primer for construction
of M1 reporter construct
ydaO29
This study
TACGACAAATTGCAAAAATAA
TGTTGTCCTTTTAAATAAGATC
TGATAAAATGTGAACTAAATTG
TATATGTTAAATTTCATACAGT
CT
Forward primer containing lysC
promoter from B. subtilis for in
vitro transcription termination
assay of the yuaA riboswitch.
ydaO30
This study
CACGGATCCGGCAAATTGCTT
Reverse primer for amplification
of the yuaA riboswitch (-238 to
+30 with respect to the
translational start site), used in in
vitro transcription
Reverse primer for amplification
of the yuaA riboswitch (-238 to 28 with respect to the
translational start site), the marker
used in in vitro transcription
ATTTTTAATTCTTCCCAA
ydaO31
This study
TTTTTGAAACAAAAAAGATTATT
GCAAAGAATGGCCGATAAT
Nelson et al. and Breaker
c-di-AMP Riboswitches
Supplementary References
63. Holtmann, G., Bakker, E.P., Uozumi, N., Bremer, E. KtrAB and KtrCD: two
K+ uptake systems in Bacillus subtilis and their role in adaptation to
hypertonicity. J. Bacteriol. 185, 1289-1298 (2003).