1
Supplementary Figure Captions for
“Metabolite stress and tolerance in the production of biofuels and chemicals: Geneexpression-based systems analysis of butanol, butyrate and acetate stresses in the
anaerobe Clostridium acetobutylicum”
by K. V. Alsaker, C. Paredes, and E. T. Papoutsakis
Supplementary Figure S1. Comparison of microarray (black points and lines) against
Q-RT-PCR (blue or green or red points and lines) data. Panel 1: Response to acetate
stress. Panel 2: Response to butyrate stress. We carried out Q-RT-PCR analyses of
similar (but on purpose not identical in order to examine the qualitative reproducibility of
the expression responses to various levels of stress (Tomas et al. 2004) ) biological
experiments with acetate and butyrate stress for genes CAC0393, CAC0456, CAC0877,
CAC3189, and CAC3288, which were selected based on the data presented in the main
paper. For these experiments, we used the medium which includes 30 mM of acetate (i.e.,
CGM supplemented with 80 g/liter glucose and 30 mM acetate), as explained in the main
text. The stressants (acetate and butyrate, separately) were added at the levels shown in
Fig. 1 (45 min acetate; 30 mM butyrate), which are on top of the 30 mM acetate in the
starting medium. The experiments of Fig. 1 and of all the microarray data were carried
out without this initial 30 mM acetate. In order to assess the error in the Q-RT-PCR data,
we carried out separate technical duplicate or triplicate sets of analyses, all done at
different days from the same RNA extractions. Note that each set includes triplicate
analyses for each sample and time point. The data show that there are frequently larger
differences between Q-RT-PCR data from the same biological sample, than differences
between Q-RT-PCR and microarray data from different samples. Second, by necessity,
the Q-RT-PCR data must be normalized by a chosen “housekeeping” gene. We have used
the pollulanase gene for this role based on an extensive set of prior microarray data
(Sillers et al. 2009), but as can be seen from the last panel for each of the acetate and
butyrate stress experiments, the pollulanase expression pattern is not all that invariable,
and this likely leads to some discrepancies in the comparison of the two types of data. In
view of these two qualifiers and despite the presence of the initial 30 mM acetate in the
medium used for the collection of the Q-RT-PCR data, the agreement between the Q-RT-
2
PCR and the microarray patterns is good. Most patterns (acetate stress: CAC0393,
CAP102, CAC0456, CAC0877, CAC3288; butyrate stress: CAC0877, CAC3189,
CAC3288) show good quantitative agreement, while the rest show the correct qualitative
agreement. The sole exception is CAC2543 under butyrate stress; this is likely due to the
fact that the presence of 30 mM acetate induces the upregulation of this gene (see the
corresponding acetate stress graph), with the added butyrate having no further
upregulation effect according to the Q-RT-PCR data. We note that the most consistent
data is for the acetate stress. It appears that the presence of 30 mM acetate in the medium
prior to the butyrate stress attenuates the impact of butyrate as can be seen in the Q-RTPCR data for genes CAC0393, CAP0102, and CAC0456.
Supplementary Figure S2: Expression profiles of a genetic locus that was
predominately upregulated following acetate, butanol, and butyrate stresses. RNA
samples for microarray hybridization were taken 10, 30, 45, 60, and 120 min poststress.
The genes are listed in the predicted order of transcription. Gene CAC1943 is on the
opposite strand compared to the rest of the genes. Other genes in the locus are not shown
because there were not present on the microarray (CAC1919, CAC1937, CAC1941), had
signal too close to background at too many time points (CAC1916, CAC1926,
CAC1929), or exhibited dye bias (CAC1942). Abbreviations: hyp., hypothetical;
transcript. reg., transcriptional regulator.
Supplementary Figure S3: Expression profiles of putative genes related to purine
biosynthesis and the predicted pathways. RNA samples for microarray hybridization
were taken 10, 30, 45, 60, and 120 min poststress. Gene names are based on genome
annotation (Nölling et al. 2001) and homology to B. subtilis orthologs. Abbreviations:
PRPP, 5-phospho-D-ribosyl-, 1-pyrophosphate; PRA, 5-phospho--D-ribosylamine;
GAR, 5-phosphoribosylglycinamide; FGAR, 5’-phophoribosyl-N-formylglcinamide;
FGAM, 5’phosphoribosyl-N-formylglycinamidine; AIR, 5’-phosphoribosyl-5aminomidazole; CAIR, 5’-phosphoribosyl-5-carboxyaminoimidazole; SAICAR, 5’phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazle; AICAR, 5-phosphoribosyl4-carboxamide-5-aminoimidazole; FAICAR, 5’-phosphoribosyl-4-carboxamide-5-
3
formamidolimidazole; IMP, inosine 5’-monophosphate; XMP, xanthosine
monophosphate. Sources: KEGG Genes Database and (Switzer et al. 2002).
Supplementary Figure S4: Expression profiles of putative genes related to thiamine
(A), histidine (B) and riboflavin (C) biosynthesis and the predicted pathways. RNA
samples for microarray hybridization were taken 10, 30, 45, 60, and 120 min poststress.
Gene names are based on genome annotation (Nölling et al. 2001) and homology to E.
coli and B. subtilis orthologs. Abbreviations: HMP, hydroxymethylpyrimidine; ThiS,
thiamine biosynthesis sulfur carrier protein, HET, hydroxyethylthiazole; G3P,
glyceraldehyde-3-phosphate; FMN, flavin mononucleotide; FAD, flavin adenine
dinucleotide. Sources: KEGG Genes Database and (Begley et al. 1999; Lawhorn et al.
2004; Perkins and Pero 2002; Rodionov et al. 2002; Vitreschak et al. 2002).
Supplementary Figure S5: Expression profiles of putative genes related to aromatic
amino acid biosynthesis and the predicted pathways. RNA samples for microarray
hybridization were taken 10, 30, 45, 60, and 120 min poststress. Genes are listed in order
of the predicted pathway. Gene names are based on genome annotation (Nölling et al.
2001) and homology to E. coli and B. subtilis orthologs. aroDH is a fusion protein
containing domains for both shikimate dehydrogenase (aroD) and chorismate mutase
(aroH). Glycolytic gene product of eno can apparently also catalyze the same reaction
as aroC (KEGG Genes Database). Abbreviations: Phe. phenylalanine; Tyr., tyrosine;
Trp., tryptophan. Sources: KEGG Genes Database and (Gollnick et al. 2002).
Supplementary Figure S6: Expression profiles of putative genes related to branchedchain amino acid biosynthesis and the predicted pathways. RNA samples for microarray
hybridization were taken 10, 30, 45, 60, and 120 min poststress. Gene names are based
on genome annotation (Nölling et al. 2001) and homology to B. subtilis orthologs.
Source: KEGG Genes Database.
4
Supplementary Figure S1. Panel 1: Acetate stress data.
5
Supplementary Figure S1. Panel 2: Butyrate stress data.
CAC0393 (after pullulanase)
CAP0102 (after pullulanase)
100
100
Ratio
Ratio
10
10
1
0.1
1
0
50
100
time (min)
0
150
50
150
time (min)
CAC0456 (after pullulanase)
CAC0877 (after pullulanase)
100
10
Ratio
Ratio
100
10
1
1
0
50
100
time (min)
150
0
50
100
time (min)
150
CAC3189 (after pullulanase)
CAC2543 (after pullulanase)
100
100
time (min)
Ratio
Ratio
10
10
1
0.1
1
0
50
100
time (min)
0
150
CAC3288 (after pullulanase)
50
100
150
Pullulanase
10
Ratio
Ratio
10
1
0.1
1
0.1
0
50
100
time (min)
150
0
50
100
time (min)
150
6
Supplementary Figure S2
Acetate
stress
Butanol
stress
Butyrate
stress
CAC1946-phage-related transcript. reg.
CAC1945-phage-related antirepressor protein
CAC1944-hyp. protein
CAC1943-hyp. protein
CAC1940-hyp. protein
CAC1939-hyp. protein
CAC1938-predicted hydrolase
CAC1936-hyp. protein
CAC1935-hyp. protein
CAC1934-hyp. protein
CAC1933-DNA replication protein DnaC
CAC1932-hyp. protein
CAC1931-hyp. protein
CAC1930-MazG family protein (uncharacterized)
CAC1928-hyp. protein
CAC1927-CF-31 family hyp. protein
CAC1925-hyp. protein
CAC1924-hyp. protein
CAC1923-hyp. protein
CAC1922-hyp. protein
CAC1921-hyp. protein
CAC1920-Zn-finger containing protein
CAC1918-hyp. protein
CAC1917-hyp. protein
CAC1915-hyp. protein
CAC1914-hyp. protein
fold-lower
expression
after stress
Key
4
1
4
fold-higher
expression
after stress
7
Supplementary Figure S3
Acetate
stress
fold-lower
expression
after stress
Butanol
stress
Butyrate
stress
Key
4
1
4
Purine Synthesis
CAC1392-purF
CAC1396-purD
CAC1394-purN
CAC1655-purQL
CAC1393-purM
CAC1390-purE
CAC1391-purC
CAC1821-purB
CAC1395-purH
GTP Synthesis
CAC2701-guaB
CAC2700-guaA
CAC0298-gmk
CAC1718-gmk
CAC1217-gmk
CAC0997-ndk-like
GTP Synthesis
CAC3593-purA
CAC1821-purB
CAC3112-adk
fold-higher
expression
after stress
PRPP
CAC1392
PRA
CAC1396
GAR
CAC1384
FGAR
CAC1655
FGAM
CAC1393
thiamine
biosynthesis
AIR
CAC1390
CAIR
CAC1391
SAICAR
CAC1821
histidine
biosynthesis
AICAR
CAC1395
FAICAR
CAC1395
CAC2701
XMP
CAC2700
GMP
CAC0298,
CAC1718, CAC1217
GDP
IMP
CAC3593
adenylosuccinate
CAC1821
AMP
CAC3112
ADP
CAC0997
GTP
ATP
8
Supplementary Figure S4
(A)
Acetate
stress
Butanol
stress
Butyrate
stress
CAC0495-thiE
CAC0908-thiF
CAC1356-thiH
CAC2077-dxs
CAC2920-thiE
CAC2921-thiH
CAC2922-thiG
CAC2923-thiF
CAC2924-thiS
CAC2971-thiI
CAC3014-thiC
CAC3095-thiMK
CAC3096-thiMK
AIR
ThiS
CAC2923, CAC0908
G3P + pyruvate
CAC2971
CAC3014
CAC2077 (dxs)
ThiS-SH
HMP
1-deoxy-DCAC2922, CAC2921,
xylulose
CAC3095
CAC1356, CAC2924 phosphate amino acid(s)
HMP-P
CAC3095
CAC3096
HMP-PP
HET-P
HET
CAC0495,
CAC2920
thiamine monophosphate
CAC3096
CAC3095
thiamine
(from pentose
phosphate pathway)
(B)
Acetate
stress
(from purine
biosynthesis
pathway)
Butanol
stress
Butyrate
stress
PRPP
CAC0936
CAC0936-hisG
CAC0943-hisE
CAC0942-hisI
CAC0940-hisA
CAC0939-hisH
CAC0941-hisF
CAC0938-hisB
CAC1369-hisC
CAC3031-hisC
CAC2727-hisJ
CAC0937-hisD
phosphoribosyl-ATP
CAC0943
phosphoribosyl-AMP
CAC0942
phosphoribosyl-formimino-AICAR-P
CAC0940
phosphoribulosyl-formimino-AICAR-P
CAC0939, CAC0941
imidazole-glycerol-3P
AICAR
CAC0938
imidazole-acetol-3P
(to purine
CAC1369, CAC3031 metabolism)
histidinol-P
CAC2727
histidinol
CAC0937
(C)
Acetate
stress
Butanol
stress
Butyrate
stress
CAC0592-ribA
CAC0590-ribD
CAC0593-ribH
CAC0591-ribB
CAC1806-ribF
fold-lower
expression
after stress
Key
4
1
4
fold-higher
expression
after stress
histidine
GTP
CAC0592
2,5-diamino-6-hydroxy-4-(5’phophoribosylamino) pyrimidine
CAC0590
5-amino-6-(5’phosphoribosylamino)
uracil
CAC0590
5-amino-6-(5’phosphoribitylylamino)
uracil
CAC0593
6,7-dimethyl-8-ribityllumazine
CAC0591
riboflavin
FMN
FAD
CAC1806
CAC1806
9
Supplementary Figure S5
Acetate
stress
Butanol
stress
Butyrate
stress
Chorismate Formation Genes
CAC0892-aroA
CAC0894-aroB
CAC0899-aroC
CAC0713-eno
CAC0897-aroDH
CAC0898-aroK
CAC0895-aroE
CAC0896-aroF
Phe.-, Tyr.-Formation Genes
erythrose 4-phosphate
+
CAC0897-aroDH
phosphoenolpyruvate
CAC0217-pheA
aroABCDHKEF
CAC0893-tyrA
trpEGDFCAB
quinone/menaquinone
CAC1369-hisC
chorismate
synthesis?
?
CAC3031-hisC
aroDH
Trp.-Formation Genes
prephenate
folate synthesis?
tryptophan
CAC3162-trpE
CAC3163-trpG
CAC3161-trpD
CAC3159-trpF
CAC3157-trpA
CAC3158-trpB
Putative Trp. Transporter
CAC3617
pheA, hisC
tyrA, hisC
phenylalanine
tyrosine
fold-lower
expression
after stress
Key
4
1
4
fold-higher
expression
after stress
10
Supplementary Figure S6
Acetate
stress
Butanol
stress
Butyrate
stress
CAC3172-leuD
CAC3173-leuC
CAC3171-leuB
CAC3176-ilvN
CAC3169-ilvB
CAC0091-ilvC
CAC3170-ilvD
CAC1479-ilvE
fold-lower
expression
after stress
Key
4
1
4
fold-higher
expression
after stress
2-isopropylmalate
leuDCB-ilvE
leucine
pyruvate
ilvNBCDE
valine
isoleucine
11
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