1
SUPPLEMENTARY INFORMATION
Table of contents
Page
Materials and Methods
2-
14
Supplementary Tables
15-
33
Supplementary Table 1:
Overview of Illumina Sequencing datasets
15
Supplementary Table 2:
FDH and FTHFS Scaffold-
Library Datasets
16-25
Supplementary Table 3:
Mapping of Illumina RNA
-Seq Sequences onto FDH dataset and
26
validation by cDNA libraries and qRT
-PCR
Supplementary Table 4:
Mapping
of Illumina RNA
-Seq Sequences onto FTHFS dataset
27
Supplementary Table 5:
QRT
-PCR analysis of termite gut luminal fluid and particle
2
8
associated pellet
Supplementary Table 6:
Microfluidic chip experiment details
29
Supplementary Table 7:
Microfluidic analysis of untreated gut samples (supernatant)
3
0
Supplementary Table 8:
Microfluidic analysis of treated gut pellets
31
Supplementary Table 9:
Microfluidic analysis of treated gut pellets
32
Supplementary Table 10:
Microfluidic analysis of treated gut pellets
3
3
Supplementary Figures
34-
46
Supplementary Figure 1:
3
4
Supplementary Figure 2:
3
5
Supplementary Figure 3:
3
6
Supplementary Figure 4:
3
7
Supplementary Figure 5:
41
Supplementary Figure 6:
4
2
Supplementary Figure 7:
4
3
Supplementary Figure 8:
4
4
Supplementary Figure 9:
4
5
Supplementary Fi
gure
10:
4
6
Supplementary References 47
2
Materials and Methods
Termite collection
Worker specimens of the dampwood termite
Zootermopsis nevadensis
were collected in the
Chilao campground
in the
San Gabriel Mountains of California. Some
termites
were
processed within 24 hours of harvest, and the
rest were
maintained
for up to 6 months after collection
in plastic boxes at 95% humidity in foil
-covered glass
aquaria in the laboratory. The entire gut tracts of ~5 worker termites were preserved in 50 –
200
μl of RNA
stabilization buffer (
RNA
Protect Bacteria Reagent
, QIAGEN, Valencia, CA) at -
80°C until nucleic acid
extraction for RNA
-Seq and inventory experiments.
Termite gut nucleic acid extraction
100
μl of TE buffer (1 mM Tris
-HCl, 0.1 mM EDTA, pH 8.0) was added to an ice
-thawed tube
containing
worker guts. Guts were then homogenized (3 x 30 sec
onds
) by bead beating with sterile zirconia/silica beads
(0.1 mm) using a MiniBeadbeater
-8 (BioSpec Products, Inc., Bartlesville, OK). Lysozyme (Sigma, St. Louis,
MO) was added to the homogen
ate (1 mg); this mixture was incubated at room temperature for 15 min. DNA
and total RNA were extracted from 150 μ
l aliquots of gut homogenate using a DNeasy Tissue Kit (QIAGEN)
and RNeasy Kit (QIAGEN), respectively
, as previously described (
1
) . Total RNA was used for Illumina RNA
-
Seq and cDNA library experiments.
RNA-
Seq: Processing and sequencing
Samples
from total RNA
were prepared
as previously described (
2
) . Briefly, librarie
s were built
using the
Illumina protocol for RNA
-Seq sample preparation V2 (https://icom.illumina.com). Briefly, total RNA (at least
5
μ
g) was fragmented using an Ambion RNA fragmentation kit and then converted to single
-strand cDNA
using an Invitrogen SuperScript II kit (Invitrogen, Carlsbad, CA). Second Strand Buffer (500 mM Tris
-HCl, pH
7.8, 50 mM MgCl
2
, 10mM DTT), dNTP (0.3 mM), RNaseH (2 U
⋅
μ
l
-1
, Invitrogen) and DNA polymerase I
(Invitrogen) were then added to the first
-strand reaction to synthesize s
econd strand cDNA (16
°C
, 2.5 hours).
3
Fragmented second strand cDNA samples were s
equenced as 37-
mers using the standard Illumina protocol and
pipeline at Caltech’s Sequencing Core Facility (Pasadena, CA).
RNA-
Seq Data Analysis
Illumina raw data
FASTQ fil
es, obtained using GERALD (a software package within the Illumina pipeline),
was aligned to a FASTA file containing FDH gene sequences (Table 4.4, Appendix) with the Maq short read
aligning program (
3
). Samples were analyzed for perfect matches only. Signal intensities were visualized
graphically by converting Maq aligned reads into a .BAR file using the Cisgenome softwa
re
(
4
) and viewed on
the Cisgenome browser and on the IGB genomic browser (http://www.affymetrix.com).
RNA-
Seq results were validated b
y cDNA phylogenetic libraries and Real
-Time quantitative
-PCR (qRT
-PCR)
(see specifics below).
cDNA inventories
Separate cDNA libraries for
fdhF
Sec
and fdhF
Cys
gene
variants were generated from gut cDNA. A forward
primer for
fdhF
Sec
(Sec427F, Table 4.1) that targets the selenocysteine FDH
H
active site was designed manually.
Sec427F was used with 1045R
(
1
) to amplify
fdhF
Sec
from gut cDNA. The
fdhF
Cys
cDNA library was
constructed with primers Cys499F1b and 1045R (
1
) . PCR reactions
contained
200 nM forward primer
(Sec427F or Cys499F1b), 200 nM 1045R, 1X FAILSAFE Premix D
(
EPICENTRE, Madison, WI), 0.07 U
⋅
μl
-1
of EXPAND High Fidelity polymerase (Roche Applied Science,
Indianapolis, IN
), and 0.5 ng
⋅
μl
-1
gut cDNA.
Thermocycling conditions on a Mastercycler Model 5331 thermocycler (Eppendorf, Westbury, NY) were 2 min
at 95
°C, 30 cycles of (95
°C for 30 sec
onds
, 60°C for 1 min
ute
, 72°C for 1 min
ute
), followed by 10 min at 72°C.
Amplicon size was checked on 1.5% agarose gels (Invitrogen) and the products were TOPO
-TA cloned
(Invitrogen). Plasmids were extracted (
QIAprep Spin Minipre
p Kit, QIAGEN) from 48 randomly chosen clones
and sequenced (Laragen Inc., Los Angeles, CA).
4
Quantitative PCR
Quantitative RT
-PCR for select FDH genotypes (ZnD2sec, ZnB5sec, T. primitia
fdhF
Sec
) was performed on
termite gut cDNA and DNA. Quantitative
PCR primers for these genotypes were: ZnD2sec (ZnO
-1636F, 5’
–
ACT ATG ACC GGC AAT TGT CGC CTG TT –3’
; ZnO
-1729R, 5’
– TCA GAC CCA TAT CAC GGC AAA GTT
–3’
), ZnB5sec (ZnB5
-1636F, 5’
– ACG ATG ACG GGC AAC TGC CGG ATG TT –3’
; ZnB5
-1729R, 5’
– TAT
GCC GAG AGC ATT
GGC ATC TT –3’
), and T. primitia
fdhF
Sec
(ZAS
-1636F, 5’
–
ACC ATG ACC GGT AAC
TGC CGG ACC CT –3’
; ZAS
-1729R, 5’
–
TTA TAC CGA GCT TTT CCG CAT CCC –3’
). Primers were
designed with Primer3 software (
5
) and amplify the same region in fdhF genes to avoid primer site biases.
Standard curves (10-
fold dilutions ranging from 10
9
– 10
6
copies/reaction) were generated from TOPO
-TA
plasmid templates containing the relevant inserts. QPCR reactions (20 μ
l) contained iQ SYBR Green Supermix
(Bio
-Rad laboratories, Irvine, CA), 500 nM forward primer, 500 nM reverse primer, 5 ng cDNA or 10 ng DN
A.
All reactions were run in duplicate. Thermocycling conditions on a Bio-
Rad DNAEngine thermocycler
(Chromo4 real time detector) were: 3 min
utes
at 95
°C, followed by 44 cycles of 95°C for 15 sec
onds
, and 60°
C
for 30 sec
onds
.
Microfluidic multiplex digit
al
PCR
For each microfluidic chip experiment, the entire gut tract of one worker termite was extracted and suspended in
250
μ
l Synthetic Gut Fluid solution
(
6
) cont
aining
0.5
μ
g
⋅
mL
-1
Dnase-
free RNase (Roche Applied Science).
Cells were released from the gut tract by aspirating the sample 3 –
5 times with a sterile 200
μ
l pipet tip. Large
particles were allowed to sediment for ~5 sec. Cell dilutions (10
-5
– 2.5 x 10
-6
range) were added (1:20 v/v) to
PCR reactions.
In experiments in which particulates (pellet) samples were processed the supernatant was
discarded and the pellet was resuspended ddH
2
O. Pellet samples were then repeatedly pipetted and
lightly
vortex
ed to
disrupt large particles and protozoa.
5
FDH
H
genes were surveyed using multiplex digital PCR. PCR reactions (20
μ
l) contained iQ Multiple
Powermix (Bio
-Rad Laboratories, Discontinued Cat. No. 170-
8848), 0.1% Tween-
20, and 75 –
175 nM ROX
standard. Final reaction concentrations of primer and probes (Table 4.1) were 100 –
400 nM. Specific
concentrations for each chip experiment are described in Supplementary Table 5 (
Below
). 16S SSU rRNA
primers and a general bacterial 16S SSU rRNA probe (1389Prb) were de
signed by Ottesen
et al.
(
7
) . A new 16S
SSU rRNA probe specific for spirochetes (1409RaPrb) was designed based on 1409Ra,
a spirochete-
specific
primer
, and used in some experiments
(
7
) . Functional gene primers (Cys499F1b, 1045R) for formate
dehydrogenase genes have been described in (
1
) . Sec427F and Cys538F primers were designed to target
fdhF
Sec
and
fdhF
Cys
gene variants, respectively. Sec427F targets al
l
fdhF
Sec
genes, whereas Cys538F has a more limited
target range for
fdhF
Cys
.
PCR reactions were loaded on microfluidic chips (Biomark 12.765 Digital Array
series) purchased from Fluidigm Corporation (San Francisco, CA). Microfluidic chip thermocycling conditions
were: 2 min
utes
at 95
°C, 45 cycles of (95°C for 15 sec
onds
, 60°C for 1 min
ute
, 72°C for 1 minute
), followed by
10 minutes
at 72
°C.
Samples were retrieved based on amplification of spirochete
or general
-bacteria DNA.
Fluorescence above
background
for amplification
-positive wells was typically detected <
cycle 35. Total bacterial concentration in
panels sampled for retrieval was inferred from the total number of positive 16S SSU gene amplifications
observed in a separate panel loaded with template at the same dilution and general bacterial 16S SSU rRNA
primers/probes. Only spirochete
panels that corresponded to template dilutions resulting in < 250 all bacteria
hits (~1/3 of all chambers) were sampled for retrieval. Samples were manually retrieved into 10
μ
l TE from
chip chambers using a dissecting microscope and 30 gauge needles (Becton, Dickinson, and Company, Franklin
Lakes, NJ) as described by Ottensen
et al.
(
6)
.
Chip samples were screened for 16S rRNA and
fdhF
gene products via simplex PCR with microfluidic chip
primers on a Mastercycler Model 5331 thermocycler (Eppendorf, Westbury, NY) and agarose gel
6
electrop
horesis.
PCR reactions (50
μ
l) contained i
Q Multiple Powermix (Bio
-Rad Laboratories), 200 –
300 nM
of each primer, and 2.5
μ
l of template. Benchtop thermocycling conditions were 2 minutes
at 95
°C, 30 or 35
cycles of (95
°C for 15 sec
onds
, 60°C for 1 min
ute
, 72°C for 1 minute
), followed by 10 minute
s at 72
°C.
Products from samples that yielded both 16S SSU rRNA and
fdhF
amplicons were PCR purified (QIAquick
PCR purification, QIAGEN). 16S SSU rRNA PCR products were cloned in TOPO
-TA vectors (TOPO
-TA
cloning kit, Invitrogen) for low
-yield PCR purifica
tions; plasmids from 8 randomly chosen clones were purified
(QIAprep Spin Miniprep, QIAGEN). 16S SSU rRNA PCR products and plasmids were sequenced with the
internal primers 533F and 1100R
(
6
) ;
fdhF
products were sequenced with microfluidic chip primers. All
sequencing reactions were performed at Laragen, Inc. (Los Angeles, CA).
Sequence Analysis
Sequences were assembled and edited using DNA
-Star Lasagene software (
Madison, WI). The software
DOTUR was used to group sequences into operational taxonomic units (OTU) based on 8% Phylip DNA
distance between OTUs, a cutoff which corresponds to the definition (3% amino acid distance) used to
distinguish protein phylotypes
(
8
) . Phylogenetic trees were constructed
using algorithms implemented within
the ARB software environment (
9
) . Tree construction details for main figure trees are as follows:
Figure 1.
A
PhyML
-maximum likelihood protein tree (panel A) was constructed using 595 aligned amino acids in
hydrogenase linked FDH sequences from lower and higher termites and wood-
roach. Numbers at nodes are
bootstrap numbers based on parsimony analysis of the data set and 100 resamplings. Numbers within grouped
clades indicate total number sequences in clade.
Filled circles indicate nodes supported by maximum
-
likelihood, parsimony
(PROTPARS; >60 of 100 bootstrap resamplings), and distance (Fitch) tree construction
methods. Unfilled circles indicate nodes supported by only 2 tree construction methods
. The accession numbers
of sequences used for phylogenetic analysis appear in Table Supplementary 2.
7
Supplementary
Figure
2.
16S
rRNA (middle panel) and
fdhF
phylogeny (left panel,
fdhF
Sec
; right panel,
fdhF
Cys
) of microfluidic chip sequences
are depicted
. Grouped clades are composed of chip 16S rRNA
sequences that were re
-amplified a
nd cloned into plasmids prior to sequencing; all other sequences were from
PCR products. 16S rRNA tree was constructed using the neighbor joining algorithm implemented in ARB (
9
)
based on 705 SINA (SILVA Incremental aligner) aligned nucleotides (
10
)
. ZnChp5-
84, ZnChp2-
1, and
ZnChp4-
10 sequences were ad
ded in by parsimony using 600 aligned nucleotides. A PhyML
-maximum
likelihood
(
11
)
fdhF
tree was constructed using 1818 aligned nucleotides from
fdhF
Sec
and
fdhF
Cys
genes. Chip
fdhF
sequences were added in by parsimony using 380 aligned nucleotides using ARB. The tree was then split
into
fdhF
Sec
and
fdhF
Cys
clades for ease of viewing.
In situ HCR
Probe and HCR amplif
ier synthesis
RNA probes were transcribed and purified by Molecular Instruments
(www.molecularinstruments.org
) at
Caltech (Pasadena, CA) as described previously (1
2).
Each probe was synthesized with 4 HC
R initiators
(separated by 5
-nt spacers) 5’ of the probe sequence.
HCR hairpins were fluorophore
-labeled and purified by
Molecular Instruments as described previously (12
), following synthesis of each RNA hairpin as a single strand
by Integrated DNA Techno
logies (IDT).
Probe design
Typically we target each mRNA or rRNA with a single RNA probe. To maximize the odds of specific staining
in environmental samples containing unknown bacterial species, probes are designed to be complementary to a
subsequence of
the target RNA, while maximizing the number of mismatches to other known sequences in the
BLAST nr/nt database (
Supplementary Tables S
9 and S
10).
8
Probe sequences
ZnD2sec mRNA
Probe targets the formate dehydrogenase (fdhF) gene retrieved from environmen
tal termite samples (Genbank
accession number GU563467). The probe is designed to have at least 9 mismatches to other termite gut
sequences in the BLAST nr/nt database (Supplementary Table S
9). For this target, we also designed an alternate
probe (Probe 2)
, which is expected to be less selective, having at least 6 mismatches to other termite gut
sequences in the BLAST nr/nt database.
Probe
Amplifier
Initiator
Spacer
Probe Sequence (50 nt)
Figures
1
HCR4
-
Alexa647
gACUACUgAUAACUggA
UUgCCUUAg
AAUUU
ACAAAUAAAA
gAgCggCgUCUUCUAUUUCgUg
gACUCCAUUACUCAUUgC
2, 3, S
4
, S
5
, S
7
, S
8
2
HCR3
-
Alexa546
UACgCCCUAAgAAUCCg
AACCCUAUg
AAAUA
UUUUUCUCCUCAgUUUCgUgCAgAACgUAgUg
AggCACUUgAgUgAgAgg
S
8
2
HCR4
-
Alexa647
gACUACUgAUAACUggA
UUgCCUUAg
AAAUA
UUUUUCUCCUCAgUUUCgUgCAgAACgUAgUg
AggCACUU
gAgUgAgAgg
S
7
ZnDP
-F1
rRNA
Probe targets the deltaproteobacterial small subunit ribosomal RNA (16S) FG18 (Genbank accession number
DQ420255) with at least 7 mismatches to other sequences in the BLAST nr/nt database (Supplementary Table
S10).
Probe
Am
plifier
Initiator
Spacer
Probe Sequence (50 nt)
Figures
1
HCR2
-
Alexa594
CCgAAUACAAAgCAUCA
ACgACUAgA
AAAAA
gUCACCCUgUCCCCgAAgggAAAAACAACUUU
CgUUgUCggUCAggggAU
2, 3, S
4
, S
5
, S
9
, s10
Proteobacteria rRNA
Probe targets the proteobacteria small subunit ribosomal RNA (16S) with at least 7 mismatches to a selection of
other bacteria (Supplementary Table S
10).
Probe
Amplifier
Initiator
Spacer
Probe Sequence (50 nt)
Figures
1
HCR3
-
Alexa546
UACgCCCUAAgAAUCCg
AACCCUAUg
AAAUA
CCgUAUUACCgCggCUgCUggCACggAgUUAg
CCggUgCUUCC
UUUCCCg
S
8
“All
-bacteria” rRNA
A well
-characterized 18
-nt universal “all bacteria” probe EUB338 (
13) targets a consensus sequence in the
bacterial SSU rRNA (16S) (Supplementary Table S
10).
Probe
Amplifier
Initiator
Spacer
Probe Sequence (18 nt)
Figure
s
1
HCR3
-
Alexa546
UACgCCCUAAgAAUCCg
AACCCUAUg
AAAUA
gCUggCUCCCgUAggAgU
2, 3, S
4
, S
5
, S10
ZAS2sec mRNA
Two probes targeting the ZAS2sec mRNA were used to validate HCR in a pure culture of
Treponema primitia
(Genbank accession NC_015578)
and
Treponema azotonutricium ZAS9
(Genbank accession NC_015577)
9
(Supplementary Fig. S
7. Probes 1 and 2 have 100% homology to the sec
-containing formate
-dehydrogenase
gene of
T. primitia
ZAS2.
T. azotonutrucium
ZAS9 does not contain an fdhF gene (Supplementary Table S
9).
Probe
Amplifier
Initiator
Spacer
Probe Sequence (50 nt)
Figures
1
HCR3
-
Alexa647
UACgCCCUAAgAAUCC
gAACCCUAUg
AAAUA
gCgAACUUgCCgUCCUUgUgCAggUAgACUgU
gCCCAAAUCgUUCggUCC
S
7
2
HCR3
-
Alexa647
UACgCCCUAAgAAUCC
gAACCCUAUg
AAAUA
UUCCUgUAUCgCCgggACggCAACgCUUUggU
AgC
CgggAUACACgUUgg
S
7
HCR amplifier sequences
Each HCR amplifier
(1
2)
comprises two hairpins (H1 and H2) that undergo conditional polymerization in
response to detection of an initiator sequence (I).
/5’-dye-C12/:
5’ Alexa Fluor modification with a C12 spacer
/C9-dye-3’/:
3’ Alexa Fluor modification with a C9 spacer
HCR2
I
CCgAAUACAAAgCAUCAACgACUAgA
H1
UCUAgUCgUUgAUgCUUUgUAUUCggCgACAgAUAACCgAAUACAAAgCAUC /C9
-
dye
-
3’/
H2
/5’
-
dye
-
C12/CCgAAUACAAAgCAUCAACgACUAgAgAUgCUUUgUAUUCggUUAUCUgUCg
HCR3
I
UACgCCCUAA
gAAUCCgAACCCUAUg
H1
CAUAgggUUCggAUUCUUAgggCgUAgCAgCAUCAAUACgCCCUAAgAAUCC /C9
-
dye
-
3’/
H2
/5’
-
dye
-
C12/ UACgCCCUAAgAAUCCgAACCCUAUgggAUUCUUAgggCgUAUUgAUgCUgC
HCR4
I
gACUACUgAUAACUggAUUgCCUUAg
H1
CUAAggCAAUCCAgUUAUCAgUAgUCUgACACgACUgACUACUgAUAACUgg /C9
-
d
ye
-
3’/
H2
/5’
-
dye
-
C12/ gACUACUgAUAACUggAUUgCCUUAgCCAgUUAUCAgUAgUCAgUCgUgUCA
10
In situ HCR protocol
Sample preparation
Intact protozoal samples: Termite gut samples (Fig. 3 and Supplementary Figs S
5- S6
) were prepared as
described previously (3).
To summ
arize, termite gut contents were fixed in 1% formaldehyde for 12 hr at 4 ºC.
The samples were then re-
suspended and stored in 50% EtOH / 50% 1× PBS overnight at 4 ºC. After
permeabilization, samples were applied onto poly
-L-lysine coated microscope slides (Sigma, St. Louis Mo.) and
left to dehydrate at RT. Sample slides were used for experiments within 48 hr after preparation.
Lysed protozoal samples: To prepare lysed protozoal samples (Fig. 2 and Supplementary Figs S
4, S
8, S
9),
termite gut contents were re
-suspended and vortexed in double
-distilled H
2
O. The samples were then fixed,
permeabilized, and dehydrated on slides as described above.
Cultured bacterial samples: Treponema primitia ZAS2 and Treponema azotonutricium ZAS9 were cultured
(Supplementary
Figure S
7) at room temperature in balch tubes containing 4 YACO media. The cultures were
incubated to OD 0.5, at which point 0.5 mL cells were fixed with 1% formaldehyde overnight in a 4
○
C
refrigerator. The fixed samples were washed twice in PBS and subsequently re-
suspended in PBS containing
50% ethanol. Samples were fixed to glass slides coated with poly
-L-lysine by dehydration, and were washed
and dehydrated in increasingly stringent e
thanol solutions: 50%, 75% and 98%.
Detection stage: probe hybridization
Our approach is adapted from (12). Probe solution was prepared by adding 6 pmol of each probe to 300 μL of
50% hybridization buffer (50% HB) at 55 ºC. The probe solution was then added to the sample slide and a
coverslip was placed on top to minimize evaporation. The slides were incubated in a humidified 50 mL Falcon
11
tube for 16 hr at 55 ºC. After overnight incubation, excess probes were removed by immersing slides in 35 mL
of the fol
lowing solutions at 55 ºC (10 min for each wash):
(a)
75% of 50% HB / 25% of 2× SSC
(b)
50% of 50% HB / 50% of 2× SSC
(c)
25% of 50% HB / 75% of 2× SSC
(d)
100% 2× SSC.
In multiplexed experiments where the “all bacteria” probe was used, a second set of lower
-stringency
probe
hybridization and washing steps was added after the above washes due to the shorter probe length. Lower
-
stringency probe hybridization and washes were done in 40% hybridization buffer (40% HB) at 45 ºC.
Amplification stage: HCR polymerization
Our a
pproach is adapted from (12). Each fluorescently labeled HCR hairpin (10 pmol) was snap cooled in 5 μL
of TE buffer with 150 mM of NaCl (heat at 95 ºC for 90 seconds and cool to room temperature on the benchtop
for 30 min). HCR amplifier solution was prepa
red by introducing all snap-
cooled hairpins to 500 μL of 40% HB
pre
-warmed to 45 ºC. The amplifier solution was then added to the sample slide and a coverslip was placed on
top to minimize evaporation. The slides were incubated in a humidified 50 mL Falcon
tube for 16 hr at 45 ºC.
After overnight incubation, excess hairpins were removed by immersing slides in 35 mL of the following
solutions at 45 ºC (10 min for each wash):
(a)
75% of 40% HB / 25% of 2× SSC
(b)
50% of 40% HB / 50% of 2× SSC
(c)
25% of 40% HB / 75% of
2× SSC
(d)
100% 2× SSC.
12
Image acquisition
Sample slides were mounted on Slowfade Gold Antifade reagent with or without DAPI (Invitrogen, Carlsbad,
CA) and visualized on a Nikon Ti
-E fluorescence microscope equipped with a 100-
W mercury arc lamp. Three
fil ter cubes (Semrock) were used to image the staining generated by fluorophore
-labeled HCR amplifiers.
Fluorophore
Excitation
Emission
Alexa 546
535 nm ± 10 nm bandpass
572 nm ± 14 nm bandpass
Alexa 594
586 nm ± 10 nm bandpass
628 nm ± 10 nm bandpass
A
lexa 647
640 nm ± 7 nm bandpass
676 nm ± 14.5 nm bandpass
Pixel intensity scatter plots
For the pixel intensity scatter plots in Fig. 2 and Supplementary Figs S
4, S
8, and S
9, the background threshold
for each channel was manually selected so as to retain
only those pixels with fluorescence intensity clearly
above background. A pixel is included in all three 2-
channel scatter plots if it is above threshold in any of the
three channels. For these samples, most pixels do not contain bacteria, and are hence e
xcluded from the scatter
plots by this thresholding process, improving our ability to draw conclusions about those pixels that do generate
signal in at least one channel.
Additional in situ HCR controls
Supplementary Figs S
4–S
9 contain additional control
experiments.
Supplementary Figure S
4 shows data for four additional samples for the experiments of Fig. 2. Note that the
background for Channel 3 is systematically higher at the top of the image than at the bottom of the image (due
to an imperfection in
the microscope), which accounts for the shift in the vertical baseline signal between the
blue and orange distributions in panels K and L (noticeable for Samples 1, 3, 4 where one rectangle is near the
top of the image and one rectangle is near the bottom
of the image).
13
Supplementary Figure S
5 shows data for two additional samples for the experiments of Fig. 3.
Supplementary Figure S
6 illustrates two sources of background fluorescence when using in situ HCR within
termite
-gut protozoa.
Supplementary
Figure S
7 demonstrates that neither ZnD2sec probe (probe 1 or probe 2) stain cultured bacteria
lacking the target mRNA.
Supplementary Figure S
8 demonstrates redundant detection of the ZnD2sec mRNA using two probes. Based on
analysis of known bacterial mR
NA sequences, we expect probe 1 to be somewhat more selective than probe 2
(Supplementary Table S
9). Qualitatively, strong signal co
-localization is observed (panels A
-C of Supp. Fig.
S8) and composite images reveal bacteria that are free of signal in both
channels, indicating that the observed
signal co
-localization is not simply due to staining of all bacteria. If probes bind to off
-target ZnD2sec
sequences in other bacteria, they will typically do so with altered hybridization yields. In turn, for each different
detection yield, we would expect a different slope in the pixel distribution of panel F (Supp. Fig. S
8). Notably,
for some bacterial clusters, we observe pixel distributions with multiple slopes (Sample 1 shows the greatest
diversity in slopes, Sa
mple 2 shows predominantly 1 slope for two clusters). These results suggest that both
probes are binding measurably to more than one target sequence.
Supplementary Figure S
9 similarly demonstrates redundant detection of the ZnDP
-F1
rRNA using two probes
:
one designed to be specific and one designed to be selective for the rRNA of many proteobacteria
(Supplementary Table S
10). Again, the scatter plots reveal distributions with multiple slopes (panel F of Supp.
Fig. S
9), suggesting that both probes are
bin
ding to more than one target sequence (and indeed, the
proteobacteria probe is designed to do just this).
14
In comparison to the studies of Supplementary Figures S
8 and S
9, it is striking that the pixel distributions of
Fig. 2 and Supplementary Figure S
4 (showing four additional samples) show signs of predominantly one linear
distribution (with one slope) for bacterial clusters stained for both ZnD2sec and
ZnDP
-F1
rRNA (orange
clusters in panels J
-L). If the probe for either target were binding multiple sequences, we would expect to see
multiple distributions with different slopes. These data increase our confidence that both probes are selective for
their intended targets. The vertical distribution using the ZnD2sec probe and the “all bacteria” rRNA probe
(blue scatter in panel K) provides additional evidence for the selectivity of the ZnD2sec probe. The slight
deviation from vertical for the distribution using the ZnDP
-F1
rRNA probe and the “all bacteria” probe (blue
scatter in panel L) suggests that there is weak binding of an off
-target sequence by the
ZnDP
-F1
probe.
Supplementary Figure S10 demonstrates that ZnDP
-F1 rRNA probe yields minimal staining of cultured
Desulfovibrio alaskensis
that contains 18 substitutions and 1 insertion (See Table S10).
15
Supplementary Table 1:
Overview of Illumina Sequencing datasets
Sample
Total
sequences
Sequences
mapping to
FDH library i
Sequences
mapping to
FDH library
ii
Sequences
mapping to
FDHf library
iii
Sequences
mapping
to FTHFS
library
Field
collected
14,043,698
54
54
55
45
Lab maintained
13,913,270
15
15
15
43
combined
libraries
27,956,968
69
69
70
88
16
Supplementary Table 2:
FDH
(2A) and FTHFS
(2B) Scaffold-
Library D
atasets
Table 2A
.
RNA-
Seq scaff
old data set. FDH nucleotide sequences are categorized into Sec and Cys enzyme
variants and major FDH types (FDH
-H, hydrogen
-linked; FDH
-NADH, NADH-
linked; FDH
-N and FDH-
O,
respiratory chain linked).
Sequence Source
Vari
ant
FDH type
Nucleotide Accession
1
Cryptocercus puntulatus
gut clone Cp10sec
Sec
FDH
-H
GU563433
Cryptocercus puntulatus
gut clone Cp14sec
Sec
FDH
-H
GU563436
Cryptocercus puntulatus
gut clone Cp16sec
Sec
FDH
-H
GU563432
Cryptocercus puntulatus
gut clone Cp24sec
Sec
FDH
-H
GU563451
Cryptoc
ercus puntulatus
gut clone Cp28sec
Sec
FDH
-H
GU563450
Cryptocercus puntulatus
gut clone Cp34sec
Sec
FDH
-H
GU563452
Cryptocercus puntulatus
gut clone Cp3sec
Sec
FDH
-H
GU563434
Cryptocercus puntulatus
gut clone Cp72cys
Cys
FDH
-H
GU563437
Cryptocercus puntulatus
gut clone Cp78sec
Sec
FDH
-H
GU563453
Cryptocercus puntulatus
gut clone Cp82sec
Sec
FDH
-H
GU563454
Cryptocercus puntulatus
gut clone Cp94sec
Sec
FDH
-H
GU563455
Cryptocercus puntulatus
gut clone Cp9cys
Cys
FDH
-H
GU563441
Cryptocercus puntulatus
gut clone CpB10sec
Sec
FDH
-H
GU563442
Cryptocercus puntulatus
gut clone CpB2sec
Sec
FDH
-H
GU563446
Cryptocercus puntulatus
gut clone CpB3sec
Sec
FDH
-H
GU563440
Cryptocercus puntulatus
gut clone CpC1cys
Cys
FDH
-H
GU563444
Cryptocercus puntulatus
gut clone CpC3sec
Sec
FDH
-H
GU563443
Cryptocercus puntulatus
gut clone CpD1cys
Cys
FDH
-H
GU563445
Cryptocercus puntulatus
gut clone CpD8sec
Sec
FDH
-H
GU563439
Cryptocercus puntulatus
gut clone CpE8cys
Cys
FDH
-H
GU563447
Cryptocercus puntulatus
gut clone CpF1cys
Cys
FDH
-H
GU563435
Cryptocercus puntulatus
gut clone CpF8cys
Cys
FDH
-H
GU563449
Cryptocercus puntulatus
gut clone CpF9cys
Cys
FDH
-H
GU563448
Cryptocercus puntulatus
gut clone CpH1cys
Cys
FDH
-H
GU563438
Incisitermes minor
gut clone Im10sec
Sec
FDH
-H
GQ922349
Incisitermes minor
gut clone Im11cys
Cys
FDH
-H
GQ922364
17
Incisitermes minor
gut clone Im15sec
Sec
FDH
-H
GQ922351
Incisitermes minor
gut clone Im22sec
Sec
FDH
-H
GQ922353
Incisitermes minor
gut clone Im24cys
Cys
FDH
-H
GQ922369
Incisitermes minor
gut clone Im26sec
Sec
FDH
-H
GQ922354
Incisitermes minor
gut clone Im27sec
Sec
FDH
-H
GQ922355
Incisitermes minor
gut clone Im3sec
Sec
FDH
-H
GQ922356
Incisitermes minor
gut clone Im42cys
Cys
FDH
-H
GQ922371
Incisitermes minor
gut clone Im5cys
Cys
FDH
-H
GQ922373
Incisitermes minor
gut clone Im63sec
Sec
FDH
-H
GQ922361
Reticulitermes hesperus
gut clone Rh15cys
Cys
FDH
-H
GQ922398
Reticulitermes hesperus
gut clone Rh24sec
Sec
FDH
-H
GQ922383
Reticulitermes hesperus
gut clone Rh2sec
Sec
FDH
-H
GQ922381
Reticu
litermes hesperus
gut clone Rh35sec
Sec
FDH
-H
GQ922385
Reticulitermes hesperus
gut clone Rh36cys
Cys
FDH
-H
GQ922410
Reticulitermes hesperus
gut clone Rh41sec
Sec
FDH
-H
GQ922386
Reticulitermes hesperus
gut clone Rh47cys
Cys
FDH
-H
GQ922402
Reticulitermes
hesperus
gut clone Rh53sec
Sec
FDH
-H
GQ922389
Reticulitermes hesperus
gut clone Rh54cys
Cys
FDH
-H
GQ922404
Reticulitermes hesperus
gut clone Rh65cys
Cys
FDH
-H
GQ922406
Reticulitermes hesperus
gut clone Rh71sec
Sec
FDH
-H
GQ922391
Reticulitermes hesperu
s
gut clone Rh93cys
Cys
FDH
-H
GQ922409
Reticulitermes hesperus
gut clone Rh9sec
Sec
FDH
-H
GQ922397
Treponema primitia
str. ZAS
-1
Cys
FDH
-H
GQ922450
Treponema primitia
str. ZAS
-1
Sec
FDH
-H
GQ922449
Treponema primitia
str. ZAS
-2
Cys
FDH
-H
FJ479767
Trep
onema primitia
str. ZAS
-2
Sec
FDH
-H
FJ479767
Zootermopsis nevadensis
gut clone Zn13cys
Cys
FDH
-H
GQ922430
Zootermopsis nevadensis
gut clone Zn16secRT
Sec
FDH
-H
GU563476
Zootermopsis nevadensis
gut clone Zn25secRT
Sec
FDH
-H
GU563475
Zootermopsis nevadensis
gut clone Zn2cys
Cys
FDH
-H
GQ922431
Zootermopsis nevadensis
gut clone Zn2cysRT
Cys
FDH
-H
GU563472
18
Zootermopsis nevadensis
gut clone Zn36secRT
Sec
FDH
-H
GU563477
Zootermopsis nevadensis
gut clone Zn51sec
Sec
FDH
-H
GQ922423
Zootermopsis nevadensis
gu
t clone Zn51secRT
Sec
FDH
-H
GU563478
Zootermopsis nevadensis
gut clone Zn55secRT
Sec
FDH
-H
GU563479
Zootermopsis nevadensis
gut clone Zn56secRT
Sec
FDH
-H
GU563473
Zootermopsis nevadensis
gut clone Zn5secRT
Sec
FDH
-H
GU563471
Zootermopsis nevadensis
gut
clone Zn61sec
Sec
FDH
-H
GQ922426
Zootermopsis nevadensis
gut clone Zn61secRT
Sec
FDH
-H
GU563480
Zootermopsis nevadensis
gut clone Zn62sec
Sec
FDH
-H
GQ922427
Zootermopsis nevadensis
gut clone Zn67cysRT
Cys
FDH
-H
GU563482
Zootermopsis nevadensis
gut clo
ne Zn70sec
Sec
FDH
-H
GQ922428
Zootermopsis nevadensis
gut clone Zn71cysRT
Cys
FDH
-H
GU563483
Zootermopsis nevadensis
gut clone Zn72secRT
Sec
FDH
-H
GU563484
Zootermopsis nevadensis
gut clone Zn75cysRT
Cys
FDH
-H
GU563481
Zootermopsis nevadensis
gut clone
Zn76secRT
Sec
FDH
-H
GU563485
Zootermopsis nevadensis
gut clone Zn9cys
Cys
FDH
-H
GQ922435
Zootermopsis nevadensis
gut clone Zn9cysRT
Cys
FDH
-H
GU563474
Zootermopsis nevadensis
gut clone ZnA4cys
Cys
FDH
-H
GU563456
Zootermopsis nevadensis
gut clone ZnB3c
ys
Cys
FDH
-H
GU563459
Zootermopsis nevadensis
gut clone ZnB5sec
Sec
FDH
-H
GU563460
Zootermopsis nevadensis
gut clone ZnB8sec
Sec
FDH
-H
GU563461
Zootermopsis nevadensis
gut clone ZnB9cys
Cys
FDH
-H
GU563462
Zootermopsis nevadensis
gut clone ZnC11cys
Cys
FDH
-H
GU563466
Zootermopsis nevadensis
gut clone ZnC1cys
Cys
FDH
-H
GU563463
Zootermopsis nevadensis
gut clone ZnC6sec
Cys
FDH
-H
GU563464
Zootermopsis nevadensis
gut clone ZnC8sec
Sec
FDH
-H
GU563465
Zootermopsis nevadensis
gut clone ZnD2sec
Sec
FDH
-H
GU563467
Zootermopsis nevadensis
gut clone ZnD3cys
Cys
FDH
-H
GU563468
Zootermopsis nevadensis
gut clone ZnE2cys
Cys
FDH
-H
GU563469
Zootermopsis nevadensis
gut clone ZnF7sec
Sec
FDH
-H
GU563458
Zootermopsis nevadensis
gut clone ZnH6cys
Cys
FDH
-H
GU563457
19
Zootermopsis nevadensis
gut clone ZnH8cys
Cys
FDH
-H
GU563470
Zootermopsis nevadensis
gut clone ZnHcys
Cys
FDH
-H
GQ922420
Zootermopsis nevadensis
gut clone ZnJcys
Cys
FDH
-H
GQ922417
Zootermopsis nevadensis
gut clone ZnKcys
Cys
FDH
-H
GQ922418
Zootermopsi
s nevadensis
gut clone ZnLsec
Sec
FDH
-H
GQ922412
Zootermopsis nevadensis
gut clone ZnMsec
Sec
FDH
-H
GQ922413
Zootermopsis nevadensis
gut clone ZnOsec
Sec
FDH
-H
GQ922415
Zootermopsis nevadensis
gut clone ZnPcys
Cys
FDH
-H
GQ922419
Aeromonas salmonicida
subsp.
salmonicida
A449
Sec
FDH
-H
NC_009348.1:1906100-
1908244
Aggregatibacter aphrophilus
NJ8700
Sec
FDH
-H
NC_012913.1: c1159571-
1157412
Acetonema longum APO
-1
Sec
FDH
-H
GQ922445
Buttiauxiella
SN1
Sec
FDH
-H
GQ922446
Carboxydothermus hydrogenoformans
Z
-2901
Sec
FDH
-NAD
NC_007503.1:646163-
648844
Carboxydothermus hydrogenoformans
Z
-2901
Sec
FDH
-O
NC_007503.1:702113-
705121
Citrobacter koseri
ATCC BAA
-895
Cys
FDH
-H
NC_009792.1:1727418-
1729565
Citrobacter koseri
ATCC BAA
-895
Sec
FDH
-H
NC_009792.1:3531364-
3533511
Citrobacter rodentium
ICC168 fdhFsec
Sec
FDH
-H
NC_013716.1:c3662542-
3660395
Citrobacter rodentium
ICC168 fdhFsec
Sec
FDH
-H
NC_013716.1:c3568359-
3566212
Citrobacter
str. TSA
-1
Sec
FDH
-H
GQ922447
Citrobacter
sp. 30_2
Cys
FDH
-H
NZ_GG657366.1:c1094197-
1096347
Citrobacter
sp. 30_2
Sec
FDH
-H
NZ_GG657366.1:c93031-
90884
Citrobacter
sp. 30_2
Sec
FDH
-N
NZ_GG657366.1:c1468196-
1465035
Citrobacter
sp. 30_2
Sec
FDH
-O
NZ_GG657366.1:c37521-
34471
Citrobacter youngae
ATCC 29220
Sec
FDH
-H
NZ_ABWL01
000021.1::c93031-
90884
Citrobacter youngae
ATCC 29220
Cys
FDH
-H
NZ_ABWL01000021.1:c24883-
27030
Citrobacter youngae
ATCC 29220
Sec
FDH
-O
NZ_ABWL01000021.1:c43554-
40504
Clostridium bartlettii
DSM 16795
Cys
FDH
-NAD
NZ_ABEZ02000007.1:22423
-25119
Clostri
dium bartlettii
DSM 16795
Sec
FDH
-H
NZ_ABEZ02000007.1:c36324-
34174
Clostridium beijerinckii
NCIMB 8052
Cys
FDH
-H
NC_009617.1:c4364248-
4366389
Clostridium bolteae
ATCC BAA
-613
Cys
FDH
-H
NZ_ABCC02000017.1:93731
-95716
Clostridium carboxidivorans
P7
Sec
FDH
-H
NZ_ACVI01000105.1:231-
2378
20
Clostridium carboxidivorans
P7
Cys
FDH
-H
NZ_ACVI01000010.1:36001-
38157
Clostridium difficile
630
Sec
FDH
-H
NC_009089.1:c3884230-
3882086
Cronobacter turicensis
Cys
FDH
-H
NC_013282.1:1996635-
1998845
Cronobacter turicensi
s
Sec
FDH
-H
NC_013282.1:2002311-
2004458
Cronobacter turicensis
Cys
FDH
-NAD
NC_013282.1:c1009687-
1006715
Desulfitobacterium hafniense
DCB
-2
Sec
FDH
-NAD
NC_011830.1:1504497-
1507178
Dickeya dadantii
Ech586
Cys
FDH
-N
NC_013592.1:c3063358-
3066408
Dickeya da
dantii
Ech586
Cys
FDH
-H
NC_013592.1:2958853-
2961003
Dickeya dadantii
Ech703
Cys
FDH
-H
NC_012880.1:c1450903-
1453053
Dickeya dadantii
Ech703
Cys
FDH
-N
NC_012880.1:c2955857-
2958907
Dickeya dadantii
Ech703
Cys
FDH
-O
NC_012880.1:c1523376-
1526423
Dickeya zea
e
Ech1591
Cys
FDH
-H
NC_012912.1:3084906-
3087056
Desulfatibacillum alkenivorans
AK-
01
Sec
FDH
-NAD
NC_011768.1:5447766-
5450528
Desulfobacterium autotrophicum
HRM2
Cys
FDH
-NAD
NC_012108.1:1930486-
1933251
Desulfotomaculum acetoxidans
5575
Sec
FDH
-NAD
NC_013216.1:c3713225-
3715906
Escherichia coli
O157:H7 str. FRIK2000
Sec
FDH
-H
NZ_ACXO01000060.1:c38313-
36585
Escherichia coli
O157:H7 str. FRIK966
Sec
FDH
-H
NZ_ACXN01000050.1:79269
-81416
Escherichia coli
83972
Sec
FDH
-H
NZ_ACGN01000114.1:89871
-92018
Escher
ichia coli
APEC O1
Sec
FDH
-H
NC_008563.1:c4646031-
4643884
Escherichia coli
O157:H7 str. EC4024
Sec
FDH
-H
NZ_ABJT01000004.1:c104404-
106551
Escherichia coli
O157:H7 str. TW14588
Sec
FDH
-H
NZ_ABKY02000001.1:164635648497
Escherichia
sp. 4_1_40B
Sec
FDH
-H
NZ_ACDM01000067.1:c85542-
83814
Escherichia coli
BL21(DE3)
Sec
FDH
-H
NC_012947.1:4135920-
4138067
Escherichia coli
SE11
Sec
FDH
-H
NC_011415.1:c4568500-
4570647
Escherichia coli
UMN026
Sec
FDH
-H
NC_011751.1:c4792216-
4790069
Edwardsiella ictaluri
93-
146
Sec
FDH
-H
NC_012779.1:3156478-
3158622
Edwardsiella tarda
EIB202
Sec
FDH
-H
NC_013508.1:3053142-
3055286
Eggerthella lenta
VPI 0255
Cys
FDH
-H
NC_013204.1:c3320160..3322586
Cronobacter) sakazakii
ATCC BAA
-894
Cys
FDH
-NAD
NC_009778.1:2900970-
2903942
Cronobacter sakazakii
ATCC BAA
-894
Cys
FDH
-H
NC_009778.1:c1996280-
1998430
Enterobacter
sp. 638
Sec
FDH
-H
NC_009436.1:c 329787-
331934
21
Enterobacter
sp. 638
Cys
FDH
-H
NC_009436.1:c1907448-
1909598
Enterobacter cancerogenus
ATCC 35316
Sec
FDH
-H
NZ_ABWM02000022.1:
21042
-23189
Enterococcus faecalis
V583
Cys
FDH
-NAD
NC_004668.1:1367291-
1370011
Escherichia fergusonii
ATCC 35469
Sec
FDH
-H
NC_011740.1:4397249-
4399396
Escherichia fergusonii
ATCC 35469
Sec
FDH
-N
NC_011740.1:1525306..1528353
Escherichia fergusonii
ATCC
35469
Sec
FDH
-O
NC_011740.1:3984322..3987372
Escherichia coli
str. K
-12 substr. MG1655
Sec
FDH
-N
NC_000913.2:1545425..1548472
Escherichia coli
str. K
-12 substr. MG1655
Sec
FDH
-O
NC_000913.2:c4080795..4083845
Escherichia coli
str. K
-12 substr. MG1655
Sec
FDH
-H
NC_000913.2:c4295242..4297389
Eubacterium acidaminophilum
Sec
FDH
-NAD
AJ312124.1:11347..14028
Eubacterium acidaminophilum
Sec
FDH
-NAD
AJ312125.1:2250..4943
Heliobacterium modesticaldum
Ice1: NC_010337
Cys
FDH
-NAD
NC_010337.2:1747735..1750623
Klebsiella pneumoniae
pneumoniae MGH 78578
Cys
FDH
-H
NC_009648.1:2290424..2292574
Klebsiella pneumoniae
pneumoniae MGH 78578
Sec
FDH
-H
NC_009648.1:c4907710-
4905563
Klebsiella pneumoniae
342
Cys
FDH
-H
NC_011283.1:c2310716-
2308566
Klebsiella pneumoniae
342
Sec
FDH
-H
NC_011283.1:5239144-
5241291
Klebsiella pneumoniae
NTUH-
K2044
Sec
FDH
-O
NC_012731.1:c46019..49069
Klebsiella pneumoniae
NTUH-
K2044
Sec
FDH
-H
NC_012731.1:c358869-
356722
Klebsiella pneumoniae
NTUH-
K2044
Sec
FDH
-N
NC_012731.1:c2794353..2797400
Klebsiella pneumoniae
NTUH-
K2044
Cys
FDH
-H
NC_012731.1:3017444..3019594
Klebsiella pneumoniae
342
Sec
FDH
-N
NC_011283.1:2546701..2549748
Klebsiella pneumoniae
342
Sec
FDH
-O
NC_011283.1:5557641..5560691
Mannheimia succiniciproducens
MBEL55E
Cys
FDH
-NAD
NC_006300.1:684085..686892
Moorella thermoacetica
ATCC 39073
Sec
FDH
-NAD
NC_007644.1:c2432486..2435188
Moorella thermoacetica
ATCC 39073
Sec
FDH
-H
NC_007644.1:c2292497..2294737
Methanococcus maripaludis
S2
Sec
FDH
-F420
BX950229.1:145038..147068
Methanococcus vannielii
SB
Sec
FDH
-F420
CP000742.1:c663600..665624
Natranaerobius thermophilus
JW/NM
-WN
-LF
Sec
FDH
-NAD
NC_010718.1:115206..117887
Oxalobacter formigenes
HOxBLS
Cys
FDH
-H
NZ_GG658151.1:2458842..2460998
Pantoea
sp. At
-9b
Cys
FDH
-H
NZ_ACYJ01000
001.1:122540..124690
Pantoea
sp. At
-9b
Sec
FDH
-O
NZ_ACYJ01000014.1:c128676..13172
22
Pectobacterium carotovorum
carotovorum WPP14
Cys
FDH
-H
NZ_ABVY01000027.1:c9266..11416
Pectobacterium carotovorum
brasiliensis PBR1692
Cys
FDH
-H
NZ_ABVX01000086.1:c2739..4889
Pectobacterium atrosepticum
SCRI1043
Cys
FDH
-H
NC_004547.2:1420602..1422752
Pectobacterium atrosepticum
SCRI1043
Cys
FDH
-H
NC_004547.2:c1752061..1754157
Pectobacterium atrosepticum
SCRI1043
Cys
FDH
-H
BX950851.1:1752061..175415
Pectobacterium wa
sabiae
WPP163
Cys
FDH
-H
NC_013421.1:c1930748..1932898
Photobacterium profundum
3TCK
Sec
FDH
-H
NZ_AAPH01000003.1:97396
-99486
Pelobacter propionicus
DSM 2379
Cys
FDH
-H
NZ_AAJH01000001.1:11892..14606
Proteus mirabilis
ATCC 29906
Cys
FDH
-H
NZ_ACLE01000010.1:50054..52222
Proteus mirabilis
ATCC 29906
Sec
FDH
-H
NZ_ACLE01000010.1:30536
-32701
Providencia alcalifaciens
DSM 30120
Sec
FDH
-H
NZ_ABXW01000042.1:35044-
37197
Providencia alcalifaciens
DSM 30120
Sec
FDH
-NAD
NZ_ABXW01000042.1:c37197-
35044
Provide
ncia alcalifaciens
DSM 30120
Sec
FDH
-O
NZ_ABXW01000042.1:c12952312647
Providencia alcalifaciens
DSM 30120
Sec
FDH
-N
NZ_ABXW01000042.1:235693-
23874
Proteus mirabilis
HI4320
Cys
FDH
-H
NC_010554.1:c3265604..3267772
Proteus mirabilis
HI4320
Sec
FDH
-H
NC
_010554.1:3909884-
3912028
Providencia rettgeri
DSM 1131
Sec
FDH
-N
NZ_ACCI02000030:c33183-
30136
Providencia rustigianii
DSM 4541
Sec
FDH
-H
NZ_ABXV02000023.1:88004
-90157
Providencia rustigianii
DSM 4541
Sec
FDH
-N
NZ_ABXV02000023.1:70811
-73858
Psychrom
onas
sp. CNPT3 fdhFsec
Sec
FDH
-H
NZ_AAPG01000013.1:c5742
-3595
Ruminococcus
sp. 5_1_39B_FAA
Cys
FDH
-NAD
NZ_GG696049.1:c238140..240848
Salmonella enterica serovar
Typhi str. CT18
Sec
FDH
-H
NC_003198.1:4370484..4372631
Salmonella enterica serovar
Typhimur
ium str. LT2
Sec
FDH
-H
AE006468.1:c4525350..4527497
Salmonella enterica serovar
Typhi str. CT18
Sec
FDH
-O
NC_003198.1:3697528..3700578
Salmonella typhimurium
LT2
Sec
FDH
-H
NC_003197.1:c4525350..4527497
Salmonella typhimurium
LT2
Sec
FDH
-N
NC_003197.1:c1
650442..1653489
Salmonella typhimurium
LT2
Sec
FDH
-O
NC_003197.1:c4244758..4247808
Serratia proteamaculans
568
Cys
FDH
-H
NC_009832.1:c2657681..2659837
Serratia proteamaculans
568
Sec
FDH
-N
NC_009832.1:87013..90060
Serratia grimesii
ZFX
-1
Cys
FDH
-H
GQ922448
Shigella flexneri
2a str. 301
Sec
FDH
-O
NC_004337.1:c4098182..4101232
23
Shigella
sp. D9
Sec
FDH
-H
NZ_ACDL01000041:c39372-
37225
Shigella sonnei
Ss046
Sec
FDH
-O
NC_007384.1:c4296262..4299312
Shigella sonnei
Ss046
Sec
FDH
-N
NC_007384.1:c1741118.
.1744165
Vibrio angustum
S14
Sec
FDH
-H
NZ_AAOJ01001.1:c1074316.1076460
Yersinia aldovae
ATCC 35236
Cys
FDH
-H
NZ_ACCB01000002.1:136225.138372
Yersinia aldovae
ATCC 35236
Sec
FDH
-O
NZ_ACCB01000003.1:36348..39395
Yersinia bercovieri
ATCC 43970
Cys
FDH
-H
NZ_AALC02000017.1:13658..15805
Yersinia bercovieri
ATCC 43970
Sec
FDH
-O
NZ_AALC02000005.1:103163.106210
Yersinia enterocolitica subsp. enterocolitica
8081
Cys
FDH
-H
NC_008800.1:3050211..3052358
Yersinia enterocolitica subsp. enterocolitica
8081
Sec
FDH
-O
NC_008800.1:c 4525888..4528935
Yersinia frederiksenii
ATCC 33641
Cys
FDH
-H
NZ_AALE02000011.1:c133500.13564
Yersinia frederiksenii
ATCC 33641
Cys
FDH
-H
NZ_AALE02000004.1:63404..6554
Yersinia frederiksenii
ATCC 33641
Sec
FDH
-O
NZ_AALE02000005.1:c136955-
13390
Yersinia intermedia
ATCC 29909
Cys
FDH
-H
NZ_AALF02000015.1:c38542..40698
Yersinia intermedia
ATCC 29909
Sec
FDH
-N
NZ_AALF02000012.1:109282-
112284
Yersinia kristensenii
ATCC 33638
Cys
FDH
-H
NZ_ACCA01000001.1:c40178..42325
Yersinia kristensenii
ATC
C 33638
Cys
FDH
-H
NZ_ACCA01000002.1:c40178..42325
Yersinia kristensenii
ATCC 33638
Sec
FDH
-O
NZ_ACCA01000015.1:8904-
11951
Yersinia mollaretii
ATCC 43969
Cys
FDH
-H
NZ_AALD02000036.1:52..2196
Yersinia mollaretii
ATCC 43969
Cys
FDH
-H
NZ_AALD02000005.1:c25400..27571
Yersinia mollaretii
ATCC 43969
Sec
FDH
-O
NZ_AALD02000033.1:c13893..16940
Yersinia pestis
KIM
Cys
FDH
-H
NC_004088.1:678737..680884
Yersinia pseudotuberculosis
IP 32953
Cys
FDH
-H
NC_006155.1:474164..476311
Yersinia pseudotuberculosis
IP 32953
Cys
FDH
-H
NC_009708.1:c4151279..4153426
Yersinia rohdei
ATCC 43380
Cys
FDH
-H
NZ_ACCD01000002.1:c116227.11837
Yersinia rohdei
ATCC 43380
Sec
FDH
-N
NZ_ACCD01000004.1:c74607-
71605
Yersinia ruckeri
ATCC 29473
Cys
FDH
-H
NZ_ACCC01000020.1:c42838..4496
Yer
sinia ruckeri
ATCC 29473
Sec
FDH
-N
NZ_ACCC01000005.1:93044
-96546
1
‘c’ infront of genome coordinates indicates complement sequence
24
Table 2B.
RNA-
Seq scaffold data set: FTHFS nucleotide sequences
Sequence Source
Sequence name
Nucleotide Accession
Zootermopsis nevadensis
Clone_T
AY162309
Zootermopsis nevadensis
Clone_H
AY162302
Zootermopsis nevadensis
Clone_E2
AY162297
Zootermopsis nevadensis
Clone_U
AY162310
Zootermopsis nevadensis
Clone_T
AY162309
Zootermopsis nevadensis
Clone_H
AY162302
Zo
otermopsis nevadensis
Clone_E2
AY162297
Zootermopsis nevadensis
Clone_U
AY162310
Zootermopsis nevadensis
Clone_F2
AY162299
Zootermopsis nevadensis
Clone_M
AY162305
Zootermopsis nevadensis
Clone_L
AY162304
Zootermopsis nevadensis
Clone_G2
AY162301
Zootermopsis nevadensis
Clone_I
AY162303
Zootermopsis nevadensis
Clone_R
AY162308
Zootermopsis nevadensis
Clone_G
AY162300
Zootermopsis nevadensis
Clone_Z
AY162312
Zootermopsis nevadensis
Clone_P
AY162307
Zootermopsis nevadensis
Clone_C
AY162
295
Zootermopsis nevadensis
ZAS_2
AF093252
Zootermopsis nevadensis
Clone_N
AY162306
Zootermopsis nevadensis
ZAS_9
AF320287
Zootermopsis nevadensis
Clone_A
AY162294
Zootermopsis nevadensis
Zas_1_b
AF093251
Zootermopsis nevadensis
ZAS_1_a
AY162313
Zootermopsis nevadensis
Clone_F
AY162298
Zootermopsis nevadensis
Clone_Y
AY162311
Zootermopsis nevadensis
Clone_E
AY 162311
Zootermopsis nevadensis
ZEG 11.4
DQ420349
Zootermopsis nevadensis
W3_5A
JX974463
Zootermopsis nevadensis
W3_8A
JX974464
Zoot
ermopsis nevadensis
W3_6D
JX974465
Zootermopsis nevadensis
W3_2C
JX974466
Zootermopsis nevadensis
5_13_11
JX974467
Zootermopsis nevadensis
5_15_2
JX974468
Zootermopsis nevadensis
5_15_5
JX974469
Zootermopsis nevadensis
5_13_3
JX974470
Zootermopsis ne
vadensis
5_13_4
JX974471
Zootermopsis nevadensis
5_10_8
JX974472
Zootermopsis nevadensis
5_10_4
JX974473
Zootermopsis nevadensis
8_26_FP
JX974474
Zootermopsis nevadensis
8_26_FI
JX974475
25
Zootermopsis nevadensis
5_10_1
JX974477
Zootermopsis nevadensis
5_13_5
JX974478
Zootermopsis nevadensis
8_26_FK
JX974479
Zootermopsis nevadensis
8_26_FM
JX974480
Zootermopsis nevadensis
5_10_7
JX974481
Zootermopsis nevadensis
5_13_2
JX974482
Zootermopsis nevadensis
5_15_4
JX974483
Zootermopsis nevadensis
5_16_14
JX974484
Zootermopsis nevadensis
5_16_5
JX974485
Zootermopsis nevadensis
5_16_18
JX974486
Zootermopsis nevadensis
5_13_8
JX974487
Zootermopsis nevadensis
5_15_6
JX974488
Zootermopsis nevadensis
5_15_3
JX974489
Zootermopsis nevadensis
5_16_2
JX974490
Zootermopsis nevadensis
5_16_13
JX974491
Zootermopsis nevadensis
5_15_8
JX974492
Zootermopsis nevadensis
5_16_6
JX974493
Zootermopsis nevadensis
5_13_9
JX974494
Zootermopsis nevadensis
5_16_8
JX974495
Zootermopsis nevadensis
5_13_6
JX974496
Zooterm
opsis nevadensis
5_13_16
JX974497
Zootermopsis nevadensis
5_16_11
JX974498
Zootermopsis nevadensis
5_13_7
JX974499
Zootermopsis nevadensis
5_13_10
JX974500
Zootermopsis nevadensis
P35_1A
JX974501
Zootermopsis nevadensis
P35_1C
JX974502
Zootermopsis nevadensis
P35_1D
JX974503
Zootermopsis nevadensis
P35_1E
JX974504
Zootermopsis nevadensis
P35_1F
JX974505
Zootermopsis nevadensis
P35_1G
JX974506
Zootermopsis nevadensis
P35_1H
JX974507
Zootermopsis nevadensis
Zas6
JX974509
Zootermopsis nevadensis
Za
s8
JX974510
Zootermopsis nevadensis
P37_2F
JX974511
Zootermopsis nevadensis
P37_3E
JX974512
Zootermopsis nevadensis
P37_7A
JX974513
Zootermopsis nevadensis
P37_9C
JX974514
Zootermopsis nevadensis
Termi178
JX974515
Zootermopsis nevadensis
Termi179
JX9
74516
Zootermopsis nevadensis
znf_8
JX974518