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ARTICLE
Received 9 Aug 2016 |Accepted 1 Nov 2016 |Published 16 Dec 2016
Global repositioning of transcription start sites
in a plant-fermenting bacterium
Magali Boutard1,2, Laurence Ettwiller3, Tristan Cerisy1,2,4,5, Adriana Alberti1, Karine Labadie1,
Marcel Salanoubat1,2,4,5, Ira Schildkraut3& Andrew C. Tolonen1,2,4,5
Bacteria respond to their environment by regulating mRNA synthesis, often by altering the
genomic sites at which RNA polymerase initiates transcription. Here, we investigate genome-
wide changes in transcription start site (TSS) usage by Clostridium phytofermentans, a model
bacterium for fermentation of lignocellulosic biomass. We quantify expression of nearly
10,000 TSS at single base resolution by Capp-Switch sequencing, which combines capture of
synthetically capped 50mRNA fragments with template-switching reverse transcription. We
find the locations and expression levels of TSS for hundreds of genes change during meta-
bolism of different plant substrates. We show that TSS reveals riboswitches, non-coding RNA
and novel transcription units. We identify sequence motifs associated with carbon source-
specific TSS and use them for regulon discovery, implicating a LacI/GalR protein in control of
pectin metabolism. We discuss how the high resolution and specificity of Capp-Switch
enables study of condition-specific changes in transcription initiation in bacteria.
DOI: 10.1038/ncomms13783 OPEN
11CEA, DRF, IG, Genoscope, E
´vry 91000, France. 2CNRS-UMR8030, E
´vry 91000, France. 3New England Biolabs, Inc., Ipswich, Massachusetts 01938, USA.
4Universite
´Paris-Saclay, E
´vry 91000, France. 5Universite
´d’E
´vry, E
´vry 91000, France. Correspondence and requests for materials should be addressed to
A.C.T. (email: atolonen@genoscope.cns.fr).
NATURE COMMUNICATIONS | 7:13783 | DOI: 10.1038/ncomms13783 | www.nature.com/naturecommunications 1
Bacteria translate environmental signals into cellular
responses using a network of regulatory RNA and proteins
that control genome-wide transcription patterns. Many of
these regulators affect where RNA polymerase initiates messenger
RNA (mRNA) synthesis at transcription start sites (TSS). As
such, locating and quantifying changes in TSS usage is an
important step to understand bacterial gene regulation. Here, we
investigate TSS architecture in Clostridium phytofermentans ISDg,
a soil bacterium that ferments plant biomass into ethanol, H
2
and
acetate1, and belongs to the Lachnospiraceae family that includes
gut commensals with important roles in host nutrition2,3. This
anaerobic mesophile metabolizes diverse plant components
including cellulose, hemicellulose and pectin by tailoring
expression of many carbohydrate-active enzymes (CAZymes)
and other metabolic enzymes to the available substrate4,5.
C. phytofermentans has a 4.8 Mb genome with 3,926 predicted
protein-encoding genes3, and its ability to alter gene expression in
response to carbon sources and other environmental cues is
mediated by over 300 transcription regulator proteins6and
numerous non-coding RNA including metabolite-sensing
riboswitches7.
We investigate genome-wide patterns of C. phytofermentans
transcription initiation on heterogeneous plant substrates by
demonstrating an approach called Capp-Switch sequencing. The
initiating nucleotide of nascent mRNA is distinguished by a
50triphosphate (50-PPP), which has been exploited for genome-
wide TSS identification with dRNA-seq8by depleting rRNA and
other monophosphorylated transcripts using terminal
exonuclease (TEX). dRNA-seq has been applied to diverse
bacteria9–13, but incomplete and non-specific degradation of
processed RNA requires TSS identification to be based on
statistical comparison of read coverage in þTEX and TEX
samples. Capp-Switch avoids these problems by capturing and
purifying 50mRNA fragments, which are reverse transcribed with
template-switching to tagged cDNA for high-throughput
sequencing (Fig. 1). The 50-PPP of mRNA are modified by
vaccinia capping enzyme (VCE) to bear a biotinylated guanosine
cap that facilitates their capture and purification using
streptavidin magnetic beads. Recently, TSS were identified by
Cappable-Seq14 using VCE to add a desthiobiotin cap for bead-
based capture of 50mRNA, which were then eluted from the
beads and de-capped to ligate adapters for reverse transcription to
tagged cDNA. Capp-Switch streamlines this approach by reverse
transcribing the 50mRNA fragments using template-switching by
Moloney murine leukemia virus reverse (MMLV) transcriptase15.
Template-switching avoids adapter ligation and enables synthesis
of 50-tagged cDNA without releasing RNA from the beads,
permitting use of an irreversible, biotinylated cap to increase
RNA capture affinity. In all, we show Capp-Switch is a robust
method that yields a genome-wide, strand-specific, quantitative
map of TSS at single nucleotide resolution.
We apply Capp-Switch sequencing to define a genome-wide
map of 9,457 TSS during C. phytofermentans growth on raw
biomass, heterogeneous polysaccharides (cellulose, hemicellulose
and pectin) and their constituent sugars. We use this TSS map to
investigate features controlling gene regulation, such as RNA
polymerase binding sites, 50untranslated region (UTR) structure,
alternative promoters, operons and non-standard (leaderless and
antisense) transcription. We identify sequence motifs associated
with groups of TSS that are differentially expressed on specific
carbon sources and show these motifs can be used to reconstruct
transcription factor regulons. By integrating Capp-Switch data
with an updated genome annotation, RNA-seq and proteomics,
we discover novel transcriptional units (TU) and protein-
encoding genes. Finally, we discuss how Capp-Switch sequencing
can be applied as a general approach to explore transcription
regulation in prokaryotes.
Results
General transcriptome features. Capp-Switch sequencing
quantified TSS with high reproducibility between duplicate
model substrate (Fig. 2a) and raw biomass (Fig. 2b) cultures. We
identified 9,457 TSS across treatments (Supplementary Data 1),
one-third of which were expressed in both sugar and poly-
saccharide cultures (Fig. 2c). Most reads (74%) contribute to
InterS TSS (Fig. 2d), which we observed upstream of 898 genes.
Among these, 687 genes (77%) are predicted to start operons16
(Supplementary Data 2), supporting these operon predictions and
the existence of many sub-operons. The 50UTR, spanning from
the primary TSS to the start codon, is less than 100 bp for most
genes, but there is no correlation between 50UTR length and TSS
First strand cDNA and
template switching
Fragment
RNA
B
B
Capture 5’
mRNA
Streptavidin beads
DNA
sequencing
DNA
polymerase
Second strand
cDNA
VCE
BB
B
B
B
B
B
B
B
B
B
B
Add 5’ biotin-GTP
cap
MMLV reverse
transcriptase
abc
def
Figure 1 | Overview of the Capp-Switch sequencing approach. Capp-Switch includes (a–c) capture of 50mRNA fragments and (d–f) cDNA synthesis and
sequencing. (a) The mRNA 50triphosphate is capped with biotin-GTP by VCE. (b) RNA is fragmented and (c) the capped 50mRNA fragments are captured
on streptavidin magnetic beads and separated from other RNA. (d) The 50mRNA fragments are reverse transcribed to single-stranded cDNA using MMLV
reverse transcriptase. An oligonucleotide hybridizes to the 30overhang and the complementary sequence is synthesized by the MMLV template-switching
activity. (e) Double-stranded cDNA is synthesized using primers that hybridize to the single-stranded cDNA termini. (f) The cDNA is sequenced on a high-
throughput platform.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13783
2NATURE COMMUNICATIONS | 7:13783 | DOI: 10.1038/ncomms1 3783 | www.nature.com/naturecommunications
strength (Fig. 2e). Studies in other bacteria report many leaderless
mRNA without 50UTR and ribosome binding sites (RBS)11. Four
per cent of InterS TSS are potentially leaderless in C.
phytofermentans, but these genes generally have another
upstream TSS and retain a typical RBS similar to highly
expressed C. phytofermentans genes (Supplementary Fig. 1).
Most genes were expressed from a single, primary TSS on all
substrates (Fig. 2f), but 191 (21%) genes altered their primary TSS
in response to carbon source. Further, genes with substrate-
specific InterS TSS are often differentially expressed on that
carbon source (w2test, Po0.01 for all substrates relative to
glucose) (Fig. 2g), supporting that changing TSS is a widespread
means of transcription regulation. In total, more than a thousand
TSS are specific to each polysaccharide (Supplementary Fig. 2A).
Xylan-specific (Supplementary Fig. 2B) and pectin-specific
(Supplementary Fig. 2C) TSS are primarily associated with
carbohydrate metabolism genes, while the most abundant
functional category of cellulose-specific TSS is prophage genes
(Supplementary Fig. 2D). The C. phytofermentans genome
includes a large prophage island that is not predicted to encode
a viable phage3, but whose transcription is up-regulated on
cellulose and biomass (Supplementary Fig. 3). This burst of
transcriptional initiation at viral genes could indicate prophage
excision was triggered on cellulosic substrates, that is, by low
carbon stress, or that viral proteins contribute to bacterial
fitness17.
Sequences upstream of primary TSS generally contain the
sigma-A-type consensus 35 and 10 hexamers recognized by
RNA polymerase (RNAP) and associated elements that likely
contribute to promoter function in this organism. An A-rich
region upstream of the -35 hexamer (TTGACA) (Fig. 2h)
resembles the ‘UP element’ that stimulates transcription initiation
by interacting with the RNAP alpha subunit18. Also, the Pribnow
hexamer (TATAAT) has an upstream TG di-nucleotide (Fig. 2i),
which enhances transcription in certain other bacteria19–21 by
interacting with the RNAP sigma-A subunit22. In contrast,
searching upstream of IntraS TSS identified an AT-rich stretch
B10 bp upstream of the TSS lacking RNAP binding sites
(Supplementary Fig. 4A), suggesting IntraS TSS often result
from promiscuous initiation at AT-rich sequences. We observed
IntraS TSS comprised that more than 50% of TSS (Fig. 2d), albeit
with fewer reads per site than InterS TSS. dRNA-seq studies have
rationalized similarly abundant intragenic TSS as resulting from
incomplete TEX degradation12, but our data support these TSS
14
14
12
12
10
10
8
80
400
300
200
100
0
1,200
Reads per million
Genes
900
600
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0
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100 200 300
UTR size (bp) TSS per gene
12345678910
0
1
3
5
7
9
Xlo GalA Cel Xya Hg Sto
400 500
8
Culture 2 log2 (reads per million)
Genes
Bits
2
1
0
Bits
14
2,780
Sugars
5956 TSS
Polysaccharides
6677 TSS
3,175 3,502
100
75
50
25
0
InterS IntraS InterA IntraA
TSS
Percent
Reads
12
10
8
Culture 2 log2 (reads per million)
Log2 (fold change)
Culture 1 log2 (reads per million)
1412108
Culture 1 log2 (reads per million)
ab cd
efg
hi
Figure 2 | General features of TSS identification by Capp-Switch sequencing. Capp-Switch reproducibly quantifies TSS usage in duplicate (a) glucose
(4,399 TSS; R2¼0.96) and (b) stover (1,532 TSS; R2¼0.99) cultures. (c) Venn diagram showing overlap of TSS identified in at least one monosaccharide
and one polysaccharide or biomass treatment. (d) Percentage of reads (purple) and TSS (yellow) classified as InterS, IntraS, InterA or IntraA summed
across treatments. (e) The length of most 50UTR (primary TSS to start codon) is o100 bp (blue bars with left Yaxis), but UTR length does not correlate
with expression strength (black line with right Yaxis). TSS strength is the average reads per million for all TSS in a 20 bp 50UTR size interval. Results show
glucose data. (f) Distribution of the number of InterS TSS per gene for data summed across treatments. (g) Genes with substrate-specific TSS are often
differentially expressed. The Yaxis is the absolute value of log
2
(RPKM substrate/RPKM glucose) from RNA-seq for all genes with InterS TSS specific to
that substrate. Substrates are xylose (Xlo n¼50 genes), galacturonic acid (GalA n¼146 genes), cellulose (Cel n¼94 genes), xylan (Xya n¼91 genes),
pectin (Hg n¼119 genes) and stover (Sto n¼48 genes). Symbols: red triangles are differentially expressed genes, blue circles unchanged genes, box shows
median and interquartile range. Promoter regions upstream of TSS expressed on three sugars and polysaccharides show consensus (h)35 and
(i)10 motifs recognized by RNA polymerase.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13783 ARTICLE
NATURE COMMUNICATIONS | 7:13783 | DOI: 10.1038/ncomms13783 | www.nature.com/naturecommunications 3
bear 50-PPP indicative of transcription initiation. IntraS TSS are
preferentially found in the 50end of genes (Supplementary
Fig. 4B), supporting they are under selective pressure and may
have roles including expression of alternative protein isoforms or
as mimicry molecules to sequester other RNA and ribonucleases
from their mRNA targets9.
Capp-Switch reads (Fig. 3a–d) start at specific positions with
respect to known genes showing TSS at single base resolution,
whereas RNA-seq reads begin throughout genes (Fig. 3e–h). We
observed four common TSS situations: genes with a single
upstream TSS, genes with both upstream and intragenic TSS,
genes with multiple TSS on a single substrate and genes
with substrate-specific TSS. For example, the glyceraldehyde
3-phosphate dehydrogenase (gadph) gene is constitutively trans-
cribed from a single TSS (Fig. 3a). The pyruvate ferredoxin oxido-
reductase (pfor) gene is transcribed from a single, upstream TSS
and another, weaker TSS in the coding sequence (Fig. 3b).
The cel5A cellulase gene23 is simultaneously transcribed from
multiple TSS on cellulose (Fig. 3c), as are other cellulases
(Supplementary Fig. 5). CAZyme expression in C.
phytofermentans is controlled by carbon source24,25 and our
data supports their regulation involves multiple promoters. The
cphy1510 gene encoding the most active xylanase5is transcribed
from three TSS on xylan and a different, upstream TSS on pectin
(Fig. 3d). Similarly, genes for other CAZymes including three
cellulases, one other xylanase, four pectinases and two glycosyl
transferases changed their primary TSS as a function of carbon
source. We confirmed the positions of the primary TSS identified
by Capp-Switch for gadph,pfor (IntraS and primary TSS),
cphy2243 and cphy1510 (xylan and pectin) using 50RACE
(Supplementary Fig. 6).
Motifs associated with TSS clusters. We clustered TSS based
on expression across carbon sources and searched sequences
surrounding TSS for overrepresented motifs (Supplementary
Fig. 7; Supplementary Data 3), revealing TSS clusters that share
motifs with potential regulatory functions (Fig. 4). For example,
the TSS cluster up-regulated on galacturonic acid and homo-
galacturonan (HG) (Fig. 4c) has a palindromic motif resembling
the cre operator (TGAAAGCGCTTTCA) bound by B. subtilis
CcpA26,27, a LacI/GalR regulator of numerous carbon metabolism
genes. LacI/GalR genes often have upstream copies of their
operators to auto-repress transcription28, and we found three
copies of the galacturonic acid cluster motif in the 50UTR of
cphy2742, a LacI/GalR gene specifically up-regulated on
galacturonic acid (Fig. 5a). Further, three of the six LacI/GalR
genes with detected primary TSS have upstream variants of the
cre operator that are conserved in their orthologs from related
species (Fig. 5b–d), leading us to propose C.phytofermentans
LacI/GalR regulators recognize related, but distinct, operators
to control separate regulons. Supportingly, the putative
Cphy2742 operator (Fig. 5b) is upstream of 22 genes in the
C. phytofermentans genome (Supplementary Table 1) including
3 CAZymes (PL9 pectin lyases) that degrade HG to galacturonic
acid5and transcription units containing all genes needed to
assimilate galacturonic acid29 (Supplementary Fig. 8).
The putative Cphy2742 operator sites are co-located with
or downstream of TSS for HG degradation and galacturonic
acid metabolism genes (Fig. 5e), supporting Cphy2742 binds
these sites to block transcription. Transcription of the
pl9 genes cphy2919 and cphy3869 switches to upstream primary
TSS on galacturonic acid relative to HG, but all TSS are close
enough to be potentially regulated by Cphy2742 operators. The
pta-ackA (cphy1326-7) acetate synthesis operon also has a
Cphy2742 operator and both pta-ackA expression and acetate
formation are elevated on galacturonic acid (Supplementary
Fig. 9). While B. subtilis CcpA represses most of its targets,
it activates pta and ackA transcription30,31 by binding upstream
of their promoters32. The Cphy2742 operator is also upstream
of the pta gene TSS, suggesting Cphy2742 may similarly activate
transcription of the pta-ackA operon as well as the glycolytic
gene ppdK and the hydrolase gene cphy0367. Collectively, we
propose Cphy2742 represses a comprehensive set of pectin
fermentation genes by binding a conserved palindrome at
or downstream of their TSS to block transcription. In response
to a galacturonic acid-based signal, Cphy2742 de-represses itself
and its targets, and may activate transcription of acetate synthesis
and other aspects of carbon metabolism by binding upstream
of TSS.
cphy2876 (gapdh)cphy3558 (pfor)cphy3202 (cel5A)cphy1510
4
2
0
0
1
2
3
0
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40
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Reads per thousand
Reads per million
Reads per million
Reads per million
Reads per million
Reads per million
Reads per million
Reads per million
0
10
20
3,528,700 3,529,700
Genome position (bp)
4,391,800
78 –546
4,395,400
Genome position (bp)
3,900,000 3,900,650 1,859,0001,858,460
97,78,74
Genome position (bp) Genome position (bp)
3,528,700 3,529,700
Genome position (bp)
4,391,800 4,395,400
Genome position (bp)
3,900,000 3,900,650 1,859,000
1,858,460
Genome position (bp) Genome position (bp)
170
7, 24, 45
130
ab c d
ef gh
Figure 3 | Capp-Switch reads start at specific genome positions corresponding to putative TSS. The number of reads starting at each genome position
are shown for Capp-Switch (a–d) and RNA-seq (e–h). The cphy2876 gapdh gene (a,e) has a single TSS (glucose data shown). The cphy3558 pfor gene
(b,f) has an upstream TSS and an intragenic sense TSS (glucose data shown). The cphy3202 cel5A cellulase gene (c,g) has three TSS during growth on
cellulose. The cphy1510 xylanase gene (d,h) is expressed from three TSS on xylan (red bars) and a single, upstream TSS on pectin (purple). Plots show the
number of reads starting at each genome position with forward strand reads on the positive Yaxis and reverse strand reads on the negative Yaxis. Distance
to the start codon is shown at the base of TSS peaks.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13783
4NATURE COMMUNICATIONS | 7:13783 | DOI: 10.1038/ncomms1 3783 | www.nature.com/naturecommunications
Antisense and novel transcripts. Recent studies found 30–40% of
TSS are antisense in other bacteria8,9,13. However, antisense
transcription appears rare in C. phytofermentans: o1% of TSS
were antisense either between (InterA) or within genes (IntraA)
(Fig. 2d). To further investigate whether diffuse antisense
transcription was underestimated by our TSS thresholds, we
classified all mapped read starts, including those not meeting TSS
thresholds. Even then, InterA and IntraA classes together
comprise o4% reads. This dearth of antisense transcription
may relate to the early evolutionary divergence of the
Clostridiales33. Alternatively, we would not detect antisense
transcripts that were processed to remove 50-PPP or that are
below the 200 bp size threshold of our cDNA libraries, but studies
in other bacteria using larger size thresholds found antisense TSS
Bits
2
1
0
Bits
2
1
0
Bits
2
1
0
Bits
2
1
0
Bits
2
1
0
Glc Xlo GalA HG Xya Sto Cel Glc Xlo GalA HG Xya Sto Cel
Expression
0.5 0 –0.5
Glc Xlo GalA HG Xya Sto Cel Glc Xlo GalA HG Xya Sto Cel Glc Xlo GalA HG Xya Sto Cel
abcde
Figure 4 | TSS in carbon source-specific clusters share DNA sequence motifs. TSS clusters differentially expressed on (a,b) glucose, (c) galacturonic acid
and HG, (d) xylan and (e) stover and cellulose are shown along with their associated sequence motifs. Rows are expression of a TSS cluster member and
columns are duplicate glucose (Glc), xylose (Xlo), galacturonic acid (GalA), homogalacturonan (HG), xylan (Xya), stover (Sto) and cellulose (Cel) cultures.
Colours show TSS expression as log
2
-transformed read counts scaled to a median of zero for each TSS.
100
50
0
0
1
2
Bits
0
1
2
Bits
0
215 190 166 140
245 218 184
181
182
146
145
125
128
237 199
161158
212 68
82 31
47 44
67
90
217
227
201 139
165 156
98
47
cphy2742 (lacl )
cphy1888 (pI9)
cphy2919 (pI9)
cphy3869 (pI9)
cphy0054 (kdgA)
cphy2534 (kduD)
cphy2741 (uxaB)
cphy0431
cphy0750
cphy3058
cphy0367
cphy0561 (ppdk)
cphy1326 (pta)
cphy2743 (uxaC )
1
2
Bits
Glucose
Xylose
Galacturonic
acid
Cellulose
Xylan
Homo-
galacturonan
Stover
mRNA expression
a
b
c
d
e
Figure 5 | The role of the LacI/GalR regulator Cphy2742 in galacturonic acid and pectin metabolism. (a) Transcription of the LacI/GalR gene cphy2742
is up-regulated on galacturonic acid relative to other carbon sources. Bars shows average RNA-seq RPKM of duplicate cultures; error bars are one s.d.
(b–d) Upstream palindromes resembling cre operator sites found upstream of C. phytofermentans LacI/GalR genes and their orthologs from related
genomes (b)cphy2742 (motif e¼1.1 10 8), (c)cphy2467 (motif e¼2.4 10 8) and (d)cphy1883 (motif e¼8.9 10 2). (e) Twelve genes have both
TSS (blue triangles) and putative Cphy2742 operators (red ovals) including genes for pectin lyases (green), galacturonic acid metabolism (purple), general
carbon metabolism (yellow) and other or unknown (grey). The distance from the translation start is shown for each site.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13783 ARTICLE
NATURE COMMUNICATIONS | 7:13783 | DOI: 10.1038/ncomms13783 | www.nature.com/naturecommunications 5
in B35% of genes10. While comparatively rare, antisense
transcription appears to have important cellular functions. For
example, we observed an antisense TSS in the 50UTR of the
sporulation regulator spoOA (cphy2497) that also opposes
transcription of the spoIVB peptidase (cphy2498) (Fig. 6a). This
TSS was expressed on all sugars, but not polysaccharides,
supporting antisense transcription has a role in repressing
sporulation during log growth in sugar-replete conditions.
TSS reveal novel transcriptional features such as a TU
downstream of the glycoside hydrolase cphy2658 that is
up-regulated to have the strongest initiation site in the genome
on cellulose and corn stover (Fig. 6b). This region contains a
hypothetical open-reading frame (ORF) in the MaGe annotation
(clops3132) that has no similar sequences in Genbank, but the
ORF lacks an ribosome binding site (RBS), and we did not detect
any expressed peptides from this region by mass spectrometry,
suggesting it is a non-coding RNA. The most highly expressed
ABC transporter on glucose is a putative operon (cphy2241-3)
with a single TSS (Supplementary Fig. 5C,F). On all other carbon
sources, we observed repression of cphy2241-3 along with
appearance of an upstream, antisense TU (Fig. 6c) that has no
mapped peptides or predicted ORF. Non-coding RNA are often
associated with ABC transporters in clostridia34, and they may
also regulate ABC transport in this organism.
The C. phytofermentans genome may encode significantly
more genes than in the NCBI Genbank annotation. Classifying
TSS using the MaGe annotation showed 735 (7%) TSS map to
MaGe-specific clops genes of unknown function (Supplementary
Data 4), including 64 clops genes with InterS TSS. We examined
which of these novel TU encode proteins by mapping
C. phytofermentans MS/MS peptide spectra to the genome
translated in all frames, identifying peptides outside the predicted
proteome in 21 InterS, 13 IntraS, 5 InterA and 25 IntraA regions
(Supplementary Data 5). The combination of TSS and expressed
peptides supports ORFs with N-terminal extensions such as
cphy0891 (Supplementary Fig. 10A) and the existence of novel
ORFs. For example, clops3461, which overlaps with cphy2929 on
the opposite strand (Fig. 6d), and an antisense overlapping ORF
in cphy1953 encoding the ComEA competence protein
(Supplementary Fig. 10B).
TSS also show mechanisms of RNA-mediated gene regulation.
Comparative genomics with other clostridia detected a putative
T-box upstream of the C. phytofermentans trp operon34. In low
tryptophan conditions, the T-box promotes antitermination of
the trp operon by base pairing with uncharged tRNAtrp (ref. 35).
We observed transcription halted abruptly in the 50UTR of the
trp operon in glucose cultures (Fig. 6e), consistent with T-box-
mediated repression. In cellulose cultures, antitermination in the
T-box enabled trp operon mRNA expression, potentially enabling
translation of the trytophan-rich carbohydrate binding modules
in cellulases and other CAZymes. TSS also support riboswitches
associated with genes for metabolism of flavin mononucleotide
(FMN), cobalamin, thiamine pyrophosphate (TPP) and lysine
(Supplementary Data 6). For example, C. phytofermentans is
auxotrophic for thiamine, which it uptakes by a thiamine
transporter, Cphy0729 (ref. 36). The cphy0729 gene has a
single, constitutive TSS with an extended 50UTR containing a
putative TPP-sensing riboswitch (Fig. 6f) that could regulate
transporter expression in response to intracellular TPP levels37.
Discussion
The strategy presented here to quantify condition-specific
changes in transcription initiation by Capp-Switch sequencing
could be generally applied to dissect the regulation of complex
bacterial phenotypes. In this study, we explored the transcrip-
tional programme enabling C. phytofermentans to ferment the
cellulosic, hemicellulosic and pectic components of plant biomass.
130 4×105
10
360 357
60
50
6
3
0
3
5
0
5
cphy2497 (spoOA)
cphy2929
clops3461
cphy2498 (spoIVB)cphy2658 cphy2659 cphy2243 cphy2244
60
389
10
Reads per million
Reads per million
Reads per million
Reads per million
Reads per million
Reads per million
0
40
80
1,300
75 0
0.5
1
70
567
274
tRNATrp- TPP
0
0.2
0.4
600
4
2
0
3,597,700
50
4,711,500
4,712,000
4,712,500
4,713,000
4,713,500
943,400
943,600
943,800
944,000
943,200
3,597,800
3,597,900
3,598,000
3,598,100
3,067,000 3,068,000
Genome position (bp)
Genome position (bp) Genome position (bp) Genome position (bp)
3,069,000 3,246,000 3,247,000 3,248,000 3,249,000 2,763,500 2,764,000 2,764,500 2,765,000 2,765,500
Genome position (bp) Genome position (bp)
cphy3848 (trpE)cphy0729
ab c
de f
Figure 6 | TSS show genome features. (a) The cphy2497 spoOA gene has both a primary TSS and an antisense TSS in the 50UTR (grey arrow) that were
observed on all sugars (glucose data shown). (b) A novel transcription unit (grey arrow) is up-regulated to be the most highly expressed TSS on biomass.
(c) Induction of a transcription unit (grey arrow) upstream of the ABC transporter cphy2243 is associated with repression of the transporter. This TSS was
observed on all substrates except glucose (cellulose data shown). (d) A primary TSS, RNA-seq reads, and three in-frame peptides expressed on cellulose
support the MaGe-predicted clops3461 gene rather than the annotated cphy2929 gene. Positions of peptides detected by mass spectrometry (purple) are
shown. (e) The trpE (cphy3848) gene has an upstream T-box that terminates transcription in the 50UTR during log-phase growth on glucose. (f) The
thiamine transporter (cphy0729) has an extended 50UTR containing a TPP-binding riboswitch. All plots show the number of reads starting at each genome
position for RNA-seq (blue) and Capp-Switch (red). Numbers at base of TSS peaks are distances to start codons of (a)cphy2497,(c)cphy2243,(d)
clops3461, (e)cphy3848,(f)cphy0729 and (b) the cphy2659 stop codon.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13783
6NATURE COMMUNICATIONS | 7:13783 | DOI: 10.1038/ncomms1 3783 | www.nature.com/naturecommunications
We found that growth on these different carbon sources entailed
widespread TSS changes, including use of substrate-specific TSS
for genes encoding biomass-degrading enzymes such as cellulases,
xylanases and pectinases. Substrate-specific TSS could enable
tuning of expression by changing promoters or the regulatory
properties (that is, binding sites or secondary structure) of the
50UTR. We observed that genes encoding cellulases and other
enzymes are simultaneously expressed from more than one TSS.
Multiple regulators may control transcription of these genes,
reflecting the numerous transcription factors encoded by this
organism (Supplementary Data 7). Genes for biomass-degrading
enzymes in other Clostridiales are regulated by various
transcription factors including a two-component system for
hemicellulases38, a LacI/GalR protein for b-1-3 glucanases39 and
alternative sigma factors for cellulases40. We defined TSS clusters
that were differentially expressed on specific carbon sources and
used them to guide the discovery of sequence motifs with
potential regulatory function, leading us to identify the LacI/GalR
Cphy2742 as a putative regulator of pectin metabolism.
Combining TSS mapping with motif searching could be broadly
applied to LacI/GalR regulators and other types of transcription
factors. For example, each of the 4 TetR regulators for which we
detected TSS also have conserved, TSS-associated palindromes
that resemble operator sites (Supplementary Fig. 11).
We also gained insight into regulatory mechanisms such as
antisense transcription, leaderless transcription and non-coding
RNA. We observed that antisense and leaderless transcription are
much rarer than reported in other bacteria and it will be
interesting to see if they are similarly uncommon in closely-
related bacteria. We also show that integration of Capp-Switch
TSS mapping with RNA-seq and proteomics enables discovery of
novel transcription units and protein-encoding genes. Transcrip-
tion initiation is a complex and important component of gene
regulation for which most of the underlying mechanisms in
C. phytofermentans are yet unknown. Further, these results
illustrate how little we know about gene regulation in plant-
fermenting clostridia, a group of bacteria with important roles in
soil and gut microbiomes that have significant potential to serve
as biocatalysts for industrial transformation of plant biomass.
Methods
Bacterial cultivation.C. phytofermentans ISDg (ATCC 700394) was cultured
anaerobically at 30 °C in GS2 medium41 containing 5 g l 1of either D-( þ)-glucose
(Sigma G5767), D-( þ)-xylose (Sigma X3877), D-galacturonic acid sodium salt
(Sigma 73960), regenerated amorphous cellulose (RAC) from Avicel PH-101
(Sigma 11365), birchwood xylan (Sigma X0502), apple pectin (HG) (Sigma P8471)
or raw corn stover (Qteros Inc) cut in 0.5 3.0 cm strips. RAC was prepared by
phosphoric acid treatment42. Duplicate cultures were sampled in mid-log phase or
after 2 days (RAC) or 3 days (stover). Fermentation products were quantified by
HPLC43.
Capp-Switch library preparation.Total RNA was extracted from duplicate
cultures for each treatment using TRI reagent (Sigma 93289) and treated with
Turbo DNase (Ambion AM2238) at 0.2 U mg1RNA for 30 min at 37 °C. RNA
was purified by Zymo Concentrator-5 (Zymo Research R1015) (4200 bp capture)
into 15 ml water. RNA was 50capped using VCE (NEB M2080) at 3 U mg1RNA
with 0.1 mM SAM and 0.5 mM 30biotin-GTP (NEB N0760) for 30 min at 37 °C
and purified by Zymo Concentrator-5 (4200 bp capture) with two additional
washes into 45 ml water. RNA was fragmented for 30 s at 94 °C using NEBNext
Magnesium-based RNA fragmentation buffer (NEB E6101) and purified by Zymo
Concentrator-5 (total RNA capture) into 100 ml water. Streptavidin magnetic
beads (NEB S1421S) were pre-washed twice with low-salt buffer (10 mM Tris,
50 mM NaCl, 1mM EDTA), twice with binding buffer (10 mM Tris, 500 mM NaCl,
1 mM EDTA) and resuspended at 4 mg ml 1beads in binding buffer. Capped
RNA fragments were bound to strepta vidin beads for 20 min at room temperature
and magnetically separated from other RNA by washing twice with binding buffer
and twice with low-salt buffer to elute non-bound RNA. Beads were washed once
with 1 mM Tris–HCl pH 7.5 and resuspended in 1 mM Tris–HCl pH 7.5.
RNA was converted to single-strand cDNA by SMARTscribe MMLV reverse
transcriptase (Clontech 634836) at 10 U ml1with 2.5 mM DTT, 1 mM dNTP,
1.2 mM SMARTer stranded oligo and 0.6 mM SMART stranded N6 primer
(Clontech 634836) by incubating 90 min at 42 °C and 10 min at 70 °C. Beads were
collected and the supernatant was combined with the liquid fraction after the beads
were washed with 30 ml 1 mM Tris pH 7.5. The cDNA was twice purified using
1 volume of solid phase reversible immobilization (SPRI) beads (Beckman Coulter
A63880). cDNA was left on beads after the second purification and double-
stranded cDNA was synthesized by 18 cycles PCR using SeqAmp DNA polymerase
(Clontech 638504) with 0.25 mM primers (Universal Forward PCR primer and
indexed Reverse PCR primer) and then SPRI purified with 1 volume of beads.
DNA was sequenced on Illumina MiSeq with 150 bp paired-end reads chemistry.
TSS identification and classification.Sequencing reads were quality filtered44
and the 3 bp MMLV reverse transcriptase 30non-template extension was
removed from the 50end of forward (R1) reads. Reads were mapped to the
C. phytofermentans ISDg genome (NCBI NC_010001.1) using Bowtie 2
(version 2.2.4)45. Alignments showed 87–98% of reads mapped to unique positions
in the C. phytofermentans genome, yielding between 0.4 million (corn stover)
and 3.4 million (glucose) reads per culture (Supplementary Table 2). TSS were
identified using R1 reads by calculating the number of reads starting at each
genomic position, clustering read counts within a 5 bp sliding window, and
retaining the position with the greatest number of reads. TSS were defined as
genome positions with greater than 10 read starts per million reads in both
duplicate cultures. Capp-switch TSS were confirmed by 50RACE (Sigma
03353621001) using primers in Supplementary Table 3 to amplify PCR products,
which were resolved by electrophoresis, excised and sequenced.
Genes in the NCBI and MicroScope (MaGe) annotations46 were used to divide
TSS into four categories: InterS (intergenic TSS with downstream gene in same
orientation), InterA (intergenic TSS with downstream gene opposite orientation),
IntraS (intragenic TSS in gene with same orientation) or IntraA (intragenic TSS
in gene with opposite orientation). The InterS TSS with the most reads for each
gene was defined as the primary TSS. Capp-Switch results were compared with
strand-specific (dUTP) RNA-seq of C. phytofermentans grown in the same
culture conditions5. RNA-seq gene expression was calculated as RPKM using
the Bioconductor47 package ‘easyRNASeq’ and differential expression was defined
as a DESeq48 (version 1.22.1) P-value o0.05 adjusted for multiple testing of the
3,902 genes in C. phytofermentans genome by Bonferroni correction. Peptides
corresponding to novel ORFs were identified by mapping peptide MS/MS spectra
from glucose, xylan and cellulose cultures4to the genome translated in all six
frames. Peptides were identified from spectra using SEQUEST and filtered to a
5% false discovery rate using a target-decoy approach49,50 including a target
database and a decoy of the reversed sequences.
Motif analysis.Sequence motifs were identified using MEME51 with a background
model of di-nucleotide frequencies in the C. phytofermentans genome. Searches for
RNA polymerase binding site motifs included positions 25–50 bp (35 motif) and
5–20 bp ( 10 motif) upstream of all primary TSS expressed on the three sugars
and polysaccharides. The top palindromic motifs associated with LacI/GalR and
TetR regulators were found by searching sequences from 250 (upstream) to
þ50 bp (downstream) relative to the start codon of C. phytofermentans genes
and their putative orthologs from related genomes identified by top reciprocal
BLAST searches (Supplementary Table 4). These motifs were used for genome-
wide scans from 250 to þ50 bp within all C. phytofermentans genes using
MAST52. To cluster TSS by expression, the 1,188 TSS with at least a 30-fold change
in read counts between two conditions were log
2
-transformed and each TSS
was normalized to have a median value of 0 across conditions and scaled so the
sum of the squared expression levels is 1. TSS were separated into 24 clusters
by K-means using the city-block similarity metric. Significant motifs (eo0.001)
associated with individual K-means clusters were identified by searching
100 to þ10 bp with respect to each TSS.
Data availability.The authors confirm that all data underlying the findings are
fully available without restriction. RNA sequencing files in FASTQ format are
available in the European Nucleotide Archive under study accession PRJEB13063.
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Acknowledgements
This work was funded by a CNRS Chaire d’Excellence to A.C.T. and the Genoscope-
CEA. We thank NEB for providing reagents (biotin-GTP, vaccinia capping enzyme and
streptavidin beads), the Genoscope-CEA sequencing platform for RNA sequencing and
the LABGeM group for supporting the MicroScope (MaGe) annotation resource.
Author contributions
L.E., A.A., M.S., I.S. and A.C.T. conceived the project. M.B., T.C. and K.L. collected data.
M.B., L.E., I.S. and A.C.T. analysed the results. A.C.T. wrote the paper.
Additional information
Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
Competing financial interests: The authors declare no competing financial
interests.
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