authors.library.caltech.edu · 2013-09-30 · 1 SUPPLEMENTARY INFORMATION . Table of contents Page Materials and Methods 2-14 . Supplementary Tables 15-33 . Supplementary Table 1:

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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 28 associated pellet Supplementary Table 6: Microfluidic chip experiment details 29 Supplementary Table 7: Microfluidic analysis of untreated gut samples (supernatant) 30 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 33 Supplementary Figures 34-46 Supplementary Figure 1: 34 Supplementary Figure 2: 35 Supplementary Figure 3: 36 Supplementary Figure 4: 37 Supplementary Figure 5: 41 Supplementary Figure 6: 42 Supplementary Figure 7: 43 Supplementary Figure 8: 44 Supplementary Figure 9: 45 Supplementary Figure 10: 46 Supplementary References 47

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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 seconds) 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 homogenate (1 mg); this mixture was incubated at room temperature for 15 min. DNA

and total RNA were extracted from 150 µl 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, libraries were built using the

Illumina protocol for RNA-Seq sample preparation V2 (https://icom.illumina.com). Briefly, total RNA (at least

5 µg) 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 MgCl2, 10mM DTT), dNTP (0.3 mM), RNaseH (2 U ⋅ µl-1, Invitrogen) and DNA polymerase I

(Invitrogen) were then added to the first-strand reaction to synthesize second strand cDNA (16°C, 2.5 hours).

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Fragmented second strand cDNA samples were sequenced 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 files, 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 software (4) and viewed on

the Cisgenome browser and on the IGB genomic browser (http://www.affymetrix.com).

RNA-Seq results were validated by cDNA phylogenetic libraries and Real-Time quantitative-PCR (qRT-PCR)

(see specifics below).

cDNA inventories

Separate cDNA libraries for fdhFSec and fdhFCys gene variants were generated from gut cDNA. A forward

primer for fdhFSec (Sec427F, Table 4.1) that targets the selenocysteine FDHH active site was designed manually.

Sec427F was used with 1045R (1) to amplify fdhFSec from gut cDNA. The fdhFCys 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 seconds, 60°C for 1 minute, 72°C for 1 minute), 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 Miniprep Kit, QIAGEN) from 48 randomly chosen clones

and sequenced (Laragen Inc., Los Angeles, CA).

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Quantitative PCR

Quantitative RT-PCR for select FDH genotypes (ZnD2sec, ZnB5sec, T. primitia fdhFSec) 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 fdhFSec (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 109 – 106 copies/reaction) were generated from TOPO-TA

plasmid templates containing the relevant inserts. QPCR reactions (20 µl) contained iQ SYBR Green Supermix

(Bio-Rad laboratories, Irvine, CA), 500 nM forward primer, 500 nM reverse primer, 5 ng cDNA or 10 ng DNA.

All reactions were run in duplicate. Thermocycling conditions on a Bio-Rad DNAEngine thermocycler

(Chromo4 real time detector) were: 3 minutes at 95°C, followed by 44 cycles of 95°C for 15 seconds, and 60°C

for 30 seconds.

Microfluidic multiplex digital PCR

For each microfluidic chip experiment, the entire gut tract of one worker termite was extracted and suspended in

250 µl Synthetic Gut Fluid solution (6) containing 0.5 µg ⋅ 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 µl 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 ddH2O. Pellet samples were then repeatedly pipetted and lightly

vortexed to disrupt large particles and protozoa.

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FDHH genes were surveyed using multiplex digital PCR. PCR reactions (20 µl) 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 designed 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 fdhFSec

and fdhFCys gene variants, respectively. Sec427F targets all fdhFSec genes, whereas Cys538F has a more limited

target range for fdhFCys. 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 minutes at 95°C, 45 cycles of (95°C for 15 seconds, 60°C for 1 minute, 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 µl 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

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electrophoresis. PCR reactions (50 µl) contained iQ Multiple Powermix (Bio-Rad Laboratories), 200 – 300 nM

of each primer, and 2.5 µl of template. Benchtop thermocycling conditions were 2 minutes at 95°C, 30 or 35

cycles of (95°C for 15 seconds, 60°C for 1 minute, 72°C for 1 minute), followed by 10 minutes 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 purifications; 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.

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Supplementary Figure 2. 16S rRNA (middle panel) and fdhF phylogeny (left panel, fdhFSec; right panel,

fdhFCys) of microfluidic chip sequences are depicted. Grouped clades are composed of chip 16S rRNA

sequences that were re-amplified and 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 added in by parsimony using 600 aligned nucleotides. A PhyML-maximum

likelihood (11) fdhF tree was constructed using 1818 aligned nucleotides from fdhFSec and fdhFCys genes. Chip

fdhF sequences were added in by parsimony using 380 aligned nucleotides using ARB. The tree was then split

into fdhFSec and fdhFCys clades for ease of viewing.

In situ HCR Probe and HCR amplifier synthesis

RNA probes were transcribed and purified by Molecular Instruments (www.molecularinstruments.org) at

Caltech (Pasadena, CA) as described previously (12). Each probe was synthesized with 4 HCR 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 Technologies (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 S9 and S10).

http://www.molecularinstruments.org/

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Probe sequences

ZnD2sec mRNA

Probe targets the formate dehydrogenase (fdhF) gene retrieved from environmental 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 S9). 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 gACUACUgAUAACUggAUUgCCUUAg

AAUUU ACAAAUAAAAgAgCggCgUCUUCUAUUUCgUggACUCCAUUACUCAUUgC

2, 3, S4, S5, S7, S8

2 HCR3-Alexa546

UACgCCCUAAgAAUCCgAACCCUAUg

AAAUA UUUUUCUCCUCAgUUUCgUgCAgAACgUAgUgAggCACUUgAgUgAgAgg

S8

2 HCR4-Alexa647

gACUACUgAUAACUggAUUgCCUUAg

AAAUA UUUUUCUCCUCAgUUUCgUgCAgAACgUAgUgAggCACUUgAgUgAgAgg

S7

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).


Alexa594 CCgAAUACAAAgCAUCAACgACUAgA

AAAAA gUCACCCUgUCCCCgAAgggAAAAACAACUUUCgUUgUCggUCAggggAU

2, 3, S4, S5, S9, 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 S10).


Alexa546 UACgCCCUAAgAAUCCgAACCCUAUg

AAAUA CCgUAUUACCgCggCUgCUggCACggAgUUAgCCggUgCUUCCUUUCCCg

S8

“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 S10).



AAAUA gCUggCUCCCgUAggAgU 2, 3, S4, S5, 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)

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(Supplementary Fig. S7. 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 S9).



AAAUA gCgAACUUgCCgUCCUUgUgCAggUAgACUgUgCCCAAAUCgUUCggUCC

S7

2 HCR3-Alexa647

UACgCCCUAAgAAUCCgAACCCUAUg

AAAUA UUCCUgUAUCgCCgggACggCAACgCUUUggUAgCCgggAUACACgUUgg

S7

HCR amplifier sequences Each HCR amplifier (12) 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 UACgCCCUAAgAAUCCgAACCCUAUg

H1 CAUAgggUUCggAUUCUUAgggCgUAgCAgCAUCAAUACgCCCUAAgAAUCC /C9-dye-3’/

H2 /5’-dye-C12/ UACgCCCUAAgAAUCCgAACCCUAUgggAUUCUUAgggCgUAUUgAUgCUgC

HCR4

I gACUACUgAUAACUggAUUgCCUUAg

H1 CUAAggCAAUCCAgUUAUCAgUAgUCUgACACgACUgACUACUgAUAACUgg /C9-dye-3’/

H2 /5’-dye-C12/ gACUACUgAUAACUggAUUgCCUUAgCCAgUUAUCAgUAgUCAgUCgUgUCA

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In situ HCR protocol

Sample preparation

Intact protozoal samples: Termite gut samples (Fig. 3 and Supplementary Figs S5-S6) were prepared as

described previously (3). To summarize, 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 S4, S8, S9),

termite gut contents were re-suspended and vortexed in double-distilled H2O. 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 S7) 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 ethanol 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

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tube for 16 hr at 55 ºC. After overnight incubation, excess probes were removed by immersing slides in 35 mL

of the following 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 approach 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 prepared 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.

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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

filter 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 Alexa 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 S4, S8, and S9, 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 excluded 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 S4–S9 contain additional control experiments.

Supplementary Figure S4 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).

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Supplementary Figure S5 shows data for two additional samples for the experiments of Fig. 3.

Supplementary Figure S6 illustrates two sources of background fluorescence when using in situ HCR within

termite-gut protozoa.

Supplementary Figure S7 demonstrates that neither ZnD2sec probe (probe 1 or probe 2) stain cultured bacteria

lacking the target mRNA.

Supplementary Figure S8 demonstrates redundant detection of the ZnD2sec mRNA using two probes. Based on

analysis of known bacterial mRNA sequences, we expect probe 1 to be somewhat more selective than probe 2

(Supplementary Table S9). 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. S8). Notably,

for some bacterial clusters, we observe pixel distributions with multiple slopes (Sample 1 shows the greatest

diversity in slopes, Sample 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 S9 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 S10). Again, the scatter plots reveal distributions with multiple slopes (panel F of Supp.

Fig. S9), suggesting that both probes are binding to more than one target sequence (and indeed, the

proteobacteria probe is designed to do just this).

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In comparison to the studies of Supplementary Figures S8 and S9, it is striking that the pixel distributions of

Fig. 2 and Supplementary Figure S4 (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).

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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

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Supplementary Table 2: FDH (2A) and FTHFS (2B) Scaffold-Library Datasets Table 2A. RNA-Seq scaffold 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 Variant FDH type Nucleotide Accession1

Cryptocercus puntulatus gut clone Cp10sec Sec FDH-H GU563433







Cryptocercus puntulatus gut clone Cp72cys Cys FDH-H GU563437




Cryptocercus puntulatus gut clone Cp9cys Cys FDH-H GU563441

Cryptocercus puntulatus gut clone CpB10sec Sec FDH-H GU563442



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 CpH1cys Cys FDH-H GU563438

Incisitermes minor gut clone Im10sec Sec FDH-H GQ922349

Incisitermes minor gut clone Im11cys Cys FDH-H GQ922364

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Reticulitermes hesperus gut clone Rh15cys Cys FDH-H GQ922398

Reticulitermes hesperus gut clone Rh24sec Sec FDH-H GQ922383












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

Treponema 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 Zn2cysRT Cys FDH-H GU563472

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Zootermopsis nevadensis gut clone Zn51sec Sec FDH-H GQ922423
















Zootermopsis nevadensis gut clone ZnA4cys Cys FDH-H GU563456

Zootermopsis nevadensis gut clone ZnB3cys 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

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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

Zootermopsis 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_ABWL01000021.1::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

Clostridium 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 turicensis 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 dadantii 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 zeae 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

Escherichia 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_ACYJ01000001.1:122540..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 wasabiae 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

Providencia 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

Psychromonas 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 Typhimurium 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:c1650442..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 ATCC 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

Yersinia 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 Zootermopsis 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 AY162295 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 Zootermopsis 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 nevadensis 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 Zootermopsis 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 Zas8 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 JX974516 Zootermopsis nevadensis znf_8 JX974518

26

Supplementary Table 3: Mapping of Illumina RNA-Seq Sequences onto FDH dataset and validation by cDNA libraries and qRT-PCR

Phylotype (FDH category)1RNA-Seq Unique hits (% total)2

FDHFsec cDNA library

(% )3

fdhF Cys cDNA

library (% )3

qRT-PCR (copies/ng gut cDNA)4

qPCR pellet (fluoresc. units)

qPCR Supernatant (fluorescence units)

Ratio Pellet:Sup

ZnD2sec (fdhF Sec ) 381 (41.4) 67 – 131 2.7 x 10-6 3.1 x 10-886 : 1

ZnHcys (fdhF Cys ) 129 (14.0) – – –Zn2cys (fdhF cys ) 84 (9.1) – 54 –T. primitia (fdhF sec ) 67 (7.3) – – 44 3.8 x 10-7 2.5 x 10-6

1 : 6.5

T. primitia (fdhF cys ) 24 (6.5) – 2 –Zn61sec (fdhF Sec ) 41 (4.5) – – –ZnF7sec (fdhF Sec ) 28 (3.0) – – –ZnB5sec (fdhF Sec ) 27 (2.9) 2 – NCD

ZnB9cys (fdhF cys ) 24 (2.6) – – –ZnLsec (fdhF Sec ) 20 (2.2) – – –ZnB8sec (fdhF Sec ) 13 (1.4) – – –ZnD3cys (fdhF cys ) 11 (1.2) – – –ZnC6sec (fdhF Sec ) 8 (0.9) – – –Zn36sec RT (fdhF Sec ) 7 (0.8) – – –Zn51sec (fdhF Sec ) 6 (0.7) – – –ZnPcys (fdhF cys ) 5 (0.5) – – –Zn75cysRT (fdhF cys ) 4 (0.4) – – –Zn72secRT (fdhF Sec ) 4 (0.4) – – –ZnH8cys (fdhF cys ) 1 (0.1) – – – reads mapping "non-Zootermopsis " sequences

Organism / Host Phylotype RNA-Seq

Unique hits Nasutitermes sp. cst4_3I 1Cryptocercus punctulatus CpN1_T 1Cryptocercus punctulatus CpA1_8I 1Cryptocercus punctulatus CpA1_8I 1Cryptocercus punctulatus CpN1_3F 1Cryptocercus punctulatus CpN1_44L 1Incisitermes sp. Inc_K 1Amitermes sp. JT2 JT2WGJ 1Amitermes sp. JT2 JT2WGK 1Amitermes sp. JT2 JT2WGL 1Amitermes sp. JT2 JT2WGA12 1Amitermes sp. JT2 JT2WGG3 1Reticulitermes flavipes Re_A 1Pseudomonas fluorescens fdhF Sec 3

Escherichia coli fdhF Sec 1 To validate RNA-Seq results, cDNA libraries for fdhFSec and fdhFCys genes were constructed and SYBR-green qPCR assays were performed. Both assays used genotype specific primers designed to limit bias. These experiments focused on the ZnD2sec and ZN2cys phylotypes, which were highly encountered phylotypes in RNA-Seq. Analysis of the fdhFSec cDNA inventory from lab-maintained termite guts indicated the ZnD2sec phylotype accounts for 67% of all clone sequences. This number is similar to the RNA-seq results. Likewise, comparison of ZnD2sec transcription with that of ZnB5sec and T. primitia fdhFSec using SYBR green RT-qPCR are consistent with the cDNA and RNA-Seq transcriptional patterns: Overall, the different methods provided agreement on the abundance of ZnD2sec and Zn2cys (ZnHcys was not observed). The row highlighted in orange associates with a protozoa-associated deltaproteobacterium in microfluidic digital-PCR experiments. The rows highlighted in green all associate with treponemal SSU RNA in microfluidic digital-PCR experiments or in pure culture isolates.

27

Supplementary Table 4: RNA-Seq mapped onto FTHFS scaffold-library

Phylotype (FTHFS)

RNA-Seq Unique hits % total

P37_7A 526 33.6 ZAS_2 383 24.5 P35_1C 133 8.5 znf_8 131 8.4 Cl_P (Zn-F10) 110 7.0 P37_3E 46 2.9 Clone_N 31 2.0 P37_9C 29 1.9 Zas1 (Zn-F16) 28 1.8 Clone_G 25 1.6 P37_2F 20 1.3 Zas5 20 1.3 Zas8 20 1.3 P35_1E 17 1.1 P37_1E 16 1.0 Clone_C 11 0.7 Clone_L 7 0.4 W3_6D 7 0.4 ZAS_9 6 0.4

Sequences in green denote spirochete culture isolates or genes associating with spirochetes in microfluidic digital-PCR

28

Supplementary Table 5: QRT-PCR analysis of termite gut luminal fluid and particle associated pellet

Phylotype Gut fraction Transcript amount (arbitrary units)

Ratio luminal fluid / particle associated pellet

ZAS-2 fdhFsec Luminal Fluid 2.47*10-6 6.46/1 ZAS-2 fdhFsec Particle-Associated Pellet 3.82*10-7

ZnD2sec Luminal Fluid 3.1*10-8 1/86.1 ZnD2sec Particle-Associated Pellet 2.67*10-6

29

Table 6. Microfluidic chip experiment details.

Chip Exp.

Fluidigm Chip No. Primers and Probes (µM) Targets

Spirochete Specificity via

1 1151-005-038

357F (200 nM), 1409Ra (200 nM), 1389Prb (300 nM), Sec427F (200 nM), Cys538F (200 nM), 1045R (200 nM)

Spirochete 16S rRNA, fdhFSec, fdhFCys

Spirochete specific primers, All Bacteria Probe

2 1151-026-033

357F (200 nM), 1409Ra (200 nM), 1389Prb (300 nM), Sec427F (200 nM), Cys538F (200 nM), 1045R (200 nM)


Spirochete specific primers, All Bacteria Probe

3a 1151-067-035 357F (200 nM), 1492RL2D (200 nM), 1409RaPrb (300 nM), Sec427F (200 nM), 1045R (175 nM)

Spirochete 16S rRNA, fdhFSec,

All Bacteria primers, Spirochete specific Probe

3b 1151-067-035

357F (200 nM), 1492RL2D (200 nM), 1409RaPrb (300 nM), Sec427F (200 nM), Cys499F1b (125 nM), 1045R (175 nM)



4 1151-067-038 357F (200 nM), 1492RL2D (200 nM), 1409RaPrb (300 nM), Sec427F (200 nM), 1045R (200 nM)



5 1151-067-041 357F (200 nM), 1492RL2D (200 nM), 1409RaPrb (300 nM), Sec427F (200 nM), 1045R (200 nM)



6 1151-067-047 1409RaPrb (300 nM), Sec427F (200 nM), 1045R (200 nM) 16S rRNA All Bacteria primers

All Bacteria probes


All Bacteria probes


All Bacteria probes

30

Supplementary Table 7: Microfluidic analysis of untreated gut samples (supernatant)

Chip Experiment Targets

Spirochete with fdhF (%)

1 16S rRNA – fdhFSec – fdhFCys 9.1 2 16S rRNA – fdhFSec – fdhFCys 10 3a 16S rRNA – fdhFSec 16.7 3b 16S rRNA – fdhFSec – fdhFCys 40 4 16S rRNA – fdhFSec 5.3

5 16S rRNA – fdhFSec 8.3

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Supplementary Table 8: Microfluidic analysis of treated gut pellets

Chip Experiment Targets

Sequences Retrieved

(proteobacteria seq.s)

% retrieved proteobacteria

6 16S rRNA – fdhFSec.ZnD2sec 4 (2) 50 7 16S rRNA – fdhFSec.ZnD2sec 1 (1) 100 8* 16S rRNA – fdhFSec.ZnD2sec 2 (2) 100

Cumulative 16S rRNA – fdhFSec.ZnD2sec 7 (5) 71 * Performed on partially enriched cultures from whole guts

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Supplementary Table S9. Mismatches between probes and mRNA sequences for selected bacteria in the

BLAST nr/nt database.

ZnD2sec fdhF mRNA probe 1 (50 nt) ZnD2sec fdhF mRNA probe 2 (50 nt)FdhF (Bacterial species) Substituted Inserted (nt) Deleted (nt) Substituted (nt) Inserted (nt) Deleted (nt)ZnD2Sec (Ectoparvobacter) 0 0 0 0 0 0Uncultured prokaryote clone Zn76sec 9 0 0 6 0 0Uncultured prokaryote clone Zn5sec 17 0 0 9 0 0Uncultured prokaryote clone Rh9sec 18 0 0 9 0 0Uncultured prokaryote clone ZnB5sec 17 0 0 10 0 0Uncultured prokaryote clone ZnF7 18 0 0 11 0 0Treponema primitia ZAS2 fdhF Sec 19 0 0 13 0 0Treponema azotonutricium NA NA NA NA NA NA

ZAS2 fdhF mRNA probe 1 (50 nt) ZAS2 fdhF mRNA probe 2 (50 nt)FdhF (Bacterial species) Substituted Inserted (nt) Deleted (nt) Substituted (nt) Inserted (nt) Deleted (nt)ZnD2Sec (Ectoparvobacter) 20 0 0 20 0 0Uncultured prokaryote clone Zn76sec 20 0 0 22 0 0Uncultured prokaryote clone Zn5sec 13 0 0 12 0 0Uncultured prokaryote clone Rh9sec 15 0 0 10 0 0Uncultured prokaryote clone ZnB5sec 14 0 0 12 0 0Uncultured prokaryote clone ZnF7 14 0 0 7 0 0Treponema primitia ZAS2 fdhF Sec 0 0 0 0 0 0Treponema azotonutricium NA NA NA NA NA NA

33

Supplementary Table S10. Mismatches between HCR probes and rRNA sequences for selected bacteria in the BLAST nr/nt database. The number of nucleotides substituted, deleted, and inserted in the target are noted for representative deltaproteobacteria, other proteobacteria, and other bacteria. The first 14 deltaproteobacteriaspecies are taken from Figure 1 in the main text, with organisms closer to the top being closer relatives of ZnDP-F1.

"All-bacteria" rRNA probe (18 nt)Bacterial species Substituted (nt) Inserted (nt) Deleted (nt) Substituted (nt) Inserted (nt) Deleted (nt) Mismatches (nt)Ectoparvobacter 0 0 0 0 0 0 0EF454719 (uncultured termite gut deltaproteobacteria) 8 0 0 1 0 0 0EF454687 (uncultured termite gut deltaproteobacteria) 7 0 0 1 0 0 0AB192069 (uncultured termite gut deltaproteobacteria) 15 0 0 0 0 0 0AB089106 (uncultured termite gut deltaproteobacteria) 9 0 0 0 0 0 0AB288881 (uncultured termite gut deltaproteobacteria) 12 0 0 1 0 0 0AY344410 (uncultured lake sediment deltaproteobacteria) 12 1 1 3 0 0 0U85477 (uncultured salt marsh sediment deltaproteobacteria) 10 0 2 4 0 0 0AJ576343 (Cetenoid beetle deltaproteobacteria) 13 0 0 3 0 0 0Desulfoarculus baarsii DSM 2075 13 0 2 2 0 0 0Desulfobacterium vacuolatum DSM 3385 22 0 0 3 0 0 0Desulfotalea psychrophila strain LSv54 22 1 0 3 0 0 0Syntrophus buswellii strain DM-2 25 2 0 2 0 0 0AB089104 (termite protozoa associated sulfate reducing deltaproteo 18 2 0 4 0 0 0Desulfovibrio termitidis 22 5 0 3 0 0 0Desulfovibrio intestinalis strain KMS2 17 0 2 3 0 0 0Desulfovibrio alaskensis NR_029338.1 18 1 0 9 0 0 0Uncultured desulfovibrio AB444082 26 1 0 3 0 0 0 Desulfovibrio sp. ZIRB-2 AY532164 19 2 0 3 0 0 0Neisseria gonorrhoeae strain 49226 23 0 0 6 0 0 0Vibrio harveyi strain C11A3 22 0 2 5 0 0 0Rhizobium leguminosarum strain NMR2 20 0 2 7 0 0 0Escherichia coli 20 0 0 4 0 0 0AB434802 (candidatus endomicrobia) 21 2 0 10 0 0 NAAB434801 (candidatus endomicrobia) 22 2 0 10 0 0 NACandidatus Azobacteroides pseudotrichonymphae 23 7 0 9 0 0 0Treponema primitia strain ZAS-2 31 0 0 13 0 0 0Treponema azotonutricium strain ZAS-9 25 3 0 13 0 0 0Bacillus subtilis 168 18 0 0 10 0 0 0Anabaena variabilis 18 10 0 7 0 0 0Mycobacterium bovis 13 4 0 9 0 0 0Bacteroides fragilis strain DSM 9671 22 6 0 11 0 0 0Acetonema longum DSM 6540 17 0 0 8 0 0 0

ZnDP-F1 rRNA probe (50 nt) Proteobacteria rRNA probe (50 nt)

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Supplementary Figure 1.

Supplementary Figure 1. Schematic of gene inventory, RNA-Seq, microfluidic PCR work-flow. FDHH gene inventories and NCBI database sequences serve as scaffolds for RNA-Seq read mapping and data analysis. RNA-Seq based identifications of candidate genotypes belonging to transcriptionally important organisms can be corroborated using independent transcriptomic methods (cDNA gene inventory, qRT-PCR). Microfluidic, multiplex digital PCR on single cells can then be employed to obtain more genetic information on these important organisms. Illumina transcript reads of gut community RNA were first mapped to gene inventory and pure culture sequence data. This first step identified highly transcribed acetogenic gene markers (fdhF genotypes seen as arrows leading to 1+2a in Figure SUPP 1). These results were corroborated with two independent methods (cDNA libraries and qPCR). Next, microfluidic PCR was used to uncover the identity of organisms encoding highly transcribed fdhF genotypes (arrows leading to 3 in SUPP Figure 1). Finally, HCR-FISH probes were used to target a highly expressed gene phylotype from an organism identified to be a part of large gut particles by microfluidic and qPCR analyses.

35


Supplementary Figure 2. Microfluidic-dPCR links FDH phylotypes to bacterial hosts. Microfluidic-chip samples are labeled “ZnChp(Chip number)-sample”, bolded, and in colored font (blue, green, orange). Pure culture sequences are bolded. fdhF sequences outlined by black boxes were highly encountered in RNA-Seq and cDNA datasets. Lines connecting sequences highlight 16S rRNA - fdhF colocalizations (duplex gene pairs for all but ZnChp1-1, ZnChp1-2 samples, which contained 16S rRNA, fdhFSec and fdhFCys gene products amplified together in triplex-PCR). Line thickness corresponds to the number of repeated co-localizations and indicates our confidence in the observed associations. Only repeatable associations are reported. Tree construction parameters and sequence accession numbers are reported in supplementary materials and methods.

36


Supplementary Figure 3: FTHFS linked to bacterial SSU Phylotypes Microfluidically associated FTHFS sequences are on the left, connected via line to the corresponding 16S sequences.

37

Supplementary Figure S4

Supplementary Figure S4. Additional examples of co-localization of ZnD2sec mRNA, ZnDP-F1 rRNA, and the rRNA of all bacteria in lysed protozoal preparations (cf. Fig. 2). One sample per page. (A) Channel 1: Signal for ZnD2sec mRNA. (B) Channel 2: Signal for ZnDP-F1 rRNA. (C) Channel 3: Signal for rRNA of all bacteria. (D-F) Composite of each pair of channels with phase. (G-I) White pixels are above a background threshold in a given channel. White pixels within the orange and blue rectangles are used for the scatter plots of panels J-L. (J-L) Pixel intensities for each pair of channels for bacterial clusters with contrasting morphologies and co-staining properties. Scale bar = 5 µm.

38

Supplementary Figure S4 (continued).

39


40


41

Supplementary Figure S5.

Supplementary Figure S5. Additional examples of co-localization of ZnD2sec mRNA and ZnDP-F1 rRNA in association with termite gut protozoa (cf. Fig. 3). One sample per column. (A) Channel 1: Signal for ZnD2sec mRNA. (B) Channel 2: Signal for ZnDP-F1 rRNA. (C) Composite of ZnD2sec mRNA signal and ZnDP-F1 rRNA signal with phase contrast. (D) Channel 3: Signal for rRNA of all bacteria. (E) Composite of ZnDP-F1 rRNA signal and all-bacteria rRNA signal with phase contrast. Scale bar = 10 µm.

42


Supplementary Figure S6. Autofluorescence (AF) and non-specific-amplification (NSA) for in situ HCR in termite gut protozoa (cf. Fig. 3 and Supplementary Fig. S2). These control experiments illustrate two sources of background: autofluorescence (AF) and non-specific amplification (NSA). In termite gut samples, wood chips are a major source of autofluorescence. Non-specific amplification arises if HCR hairpins are retained in the sample that are not contained within polymers tethered to specifically bound probes. AF is characterized by using the in situ protocol while leaving out the probes and the HCR amplifiers. AF + NSA is characterized by using the in situ protocol while leaving out the probes. Scale bar = 10 µm.

43


Supplementary Figure S7. Testing ZnD2sec probe specificity in cultured termite-gut acetogenic Treponema primitia (expressing ZAS2sec but not ZnD2sec) and non-acetogenic Treponema azotonutricium spirochetes (expressing neither ZAS2sec nor ZnD2sec). Treponema primitia cells grown under homo-acetogenic conditions stained (red) using (A) ZAS2 mRNA probes (probe 1 and probe 2) or (B) ZnD2sec mRNA probes (probe 1 and probe 2). Non-acetogenic Treponema azotonutricium stained (red) by (C) ZAS2sec mRNA probes or (D) ZnDsec mRNA probes. Note the presence of signal (red) in panel A and the absence of signal in panels B, C, D. Cells in all panels stained by DAPI (blue). Scale bar = 10 µm.

44


Supplementary Figure S8. Two-channel redundant detection of the ZnD2sec mRNA in lysed protozoal preparations using two probes with differing degrees of selectivity. (A) Channel 1: signal from probe 1. (B) Channel 2: signal from probe 2. (C) Composite of both channels with phase contrast. (D-E) White pixels are above a background threshold in a given channel. White pixels within the orange and blue rectangles are used for the scatter plot of panel F. (F) Pixel intensities for both channels for different bacterial clusters. Scale bar = 10 µm.

45


Supplementary Figure S9. Two-channel redundant detection of the ZnDP-F1 rRNA in lysed protozoal preparations using one probe targeting ZnDP-F1 rRNA and one probe targeting the rRNA of proteobacteria. (A) Channel 1: Signal for ZnDP-F1 rRNA. (B) Channel 2: Signal for Proteobacteria rRNA. (C) Composite of both channels with phase contrast. (D-E) White pixels are above a background threshold in a given channel. White pixels within the orange and blue rectangles are used for the scatter plot of panel F. (F) Pixel intensities for both channels for different bacterial clusters. Scale bar = 10 µm.

46

Supplementary Figure S10

Supplementary Figure S10. Testing ZnDP-F1 rRNA probe specificity in a pure culture of the deltaprotiobacterium Desulfovibrio alaskensis whose rRNA contains 18 substitutions and one insertion within the probe target window (see Table S10). (A) Phase. (B) Channel 1: Signal for ZnDP-F1 rRNA probe. (C) Channel 2: Signal for all-bacterial rRNA probe. (D) Composite of both channels with phase contrast. The ZnDP-F1 probe yields minimal staining.

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authors.library.caltech.edu · 2013-09-30 · 1 SUPPLEMENTARY INFORMATION . Table of contents Page Materials and Methods 2-14 . Supplementary Tables 15-33 . Supplementary Table 1: