1
2
3
Anoxic carbon flux in photosynthetic microbial mats as revealed by
metatranscriptomics
4
5
6
7
8
9
10
11
12
13
14
15
16
Luke C. Burow1,2,‡, Dagmar Woebken1,2,^‡, Ian P.G. Marshall1, Erika Lindquist3, Brad M.
Bebout2, Leslie Prufert-Bebout2, Tori M. Hoehler2, Susannah G. Tringe3, Jennifer PettRidge4, Peter K. Weber4, Alfred M. Spormann1, Steven W. Singer5
1
Department of Chemical Engineering and Department of Civil and Environmental
Engineering, Stanford University, Stanford, CA; 2Exobiology Branch, NASA Ames
Research Center, Moffett Field, CA, USA; 3Joint Genome Institute, Walnut Creek, CA
USA; 4Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory,
Livermore, CA, USA; 5Earth Sciences Division, Lawrence Berkeley National
Laboratory, Berkeley, CA USA
Supplemental Information
1
2
Amplification, sequencing and sequence analysis of 16S rRNA genes and transcripts
3
For construction of 454 pyrotag amplicon libraries from RNA samples, total RNA
4
was converted to double-stranded cDNA using the SuperScript® Double-Stranded cDNA
5
Synthesis Kit (Invitrogen). For 454 pyrotag libraries from the EN (7:00 am) DNA
6
sample, DNA was extracted using the QIAmp DNA Mini Kit in combination with the
7
above-mentioned homogenization and phenol-chloroform extraction. The V8 region of
8
cDNA or DNA was PCR amplified using the universal primers 926F and 1392R (Lane et
9
al., 1985, Weisburg et al., 1991). The reverse primer included the adaptor sequence and a
10
five-base barcode. Amplicons of the 16S rRNA V8 hypervariable region were
11
constructed and sequenced following standard procedure at the Department of Energy
12
Joint Genome Institute using the GS FLX Titanium Series Reagents (454 Life Sciences,
13
Branford, CT, USA) (Engelbrektson et al., 2010).
14
processed (sorting, trimming, removing reads of low quality and classification) using the
15
RDP pipeline with standard settings (http://pyro.cme.msu.edu).
Pyrosequencing amplicons were
16
Nearly full-length 16S rRNA clone libraries were constructed from double-stranded
17
cDNA by amplification with the broadly inclusive bacterial primers 27F and 1391R
18
(Weisburg et al., 1991). PCR products of triplicate PCR reactions were pooled and
19
purified using the Qiagen Min Elute PCR Purification Kit (Qiagen). Ligation,
20
transformation, Sanger sequencing, as well as sequence quality check and contig
21
formation were conducted following standard procedure at the Department of Energy
22
Joint
23
(http://my.jgi.doe.gov/general/index.html). The 16S rRNA transcript sequences were
24
taxonomically
Genome
Institute
classified
(JGI,
using
Walnut
the
Creek,
RDP
CA,
USA)
pipeline
1
(http://rdp.cme.msu.edu/classifier/classifier.jsp). For phylogenetic analysis, sequences
2
were aligned using the SILVA website (http://www.arb-silva.de) and imported in the
3
ARB program (Ludwig et al., 2004) with the SILVA 94 Ref database. The database was
4
supplemented for the analysis of sequences of particular interest (Cyanobacteria and
5
Chloroflexi) by adding the closest relatives to the Elkhorn Slough sequences as well as
6
sequences
7
(http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic trees were calculated using
8
maximum likelihood, maximum parsimony and neighbor joining algorithms, with and
9
without a 50% position variability filter. Bootstrap values were calculated in Geneious
10
11
from
cultured
representatives
from
SILVA
5.0.3 with the PhyML algorithm, using 100 bootstrap trees.
Ref
104
and
NCBI
1
2
Additional capture probes used in MICROBEnrich/MICROBExpress Kits
3
Cyano capture probe 1:
4
5’-AAAAAAAAAAAAAAAAAATCCCCACTGCTGCCTCCCGTAGG-3’
5
Cyano capture probe 2:
6
5’-AAAAAAAAAAAAAAAAAACAACATCTCACGACACGAGCTGA-3’
7
Cyano capture probe 3:
8
5’-AAAAAAAAAAAAAAAAAAGGTTCTTTTCACCTTTCCCTCGC-3’
9
Cyano capture probe 4:
10
5’-AAAAAAAAAAAAAAAAAAACTTACCCGACAAGGAATTTCGC-3’
11
Cyano capture probe 5:
12
5’-AAAAAAAAAAAAAAAAAAGAGCCGACATCGAGGTGCCAAAC-3’
13
Euk probe 1:
14
5’-AAAAAAAAAAAAAAAAAAACCGCGGCTGCTGGCACCAGACT-3’
15
Euk probe 2:
16
5’-AAAAAAAAAAAAAAAAAATTTCCCGTGTTGAGTCAAATTAA-3’
17
Euk probe 3:
18
5’-AAAAAAAAAAAAAAAAAAAACTAAGAACGGCCATGCACCAC-3’
19
Euk probe 4:
20
5’-AAAAAAAAAAAAAAAAAACAACTTTCCCTCACGGTACTTGT-3’
21
Euk probe 5:
22
5’-AAAAAAAAAAAAAAAAAATGTTTTAATTAGACAGTCGGATT-3’
23
Euk probe 6:
24
5’-AAAAAAAAAAAAAAAAAATGGTAACTTTTCTGACACCTCTT-3’
25
Euk probe 7:
26
5’-AAAAAAAAAAAAAAAAAATCGCAGAATTCACTACGACGCCA-3’
27
28
1
Supplemental Tables
2
Supplemental Table 1 Overview of V8 pyrotag sequences derived from the upper most
3
2 mm of Elkhorn Slough microbial mats before and after quality assessment.
Sample
BN, 12 Jan 2009 (9:00 pm), cDNA
EN, 13 Jan 2009 (7:00 am), cDNA
EN, 13 Jan 2009 (7:00 am), DNA
4
5
No. of total
reads
No. of trimmed
reads
Ave. read length
after trimming
10850
10902
24862
9217
9338
20068
384
396
404
1
2
Supplemental Table 2 Statistics of the metatranscriptome reads of Elkhorn Slough
3
microbial mats at 9:00 pm (12th January 2009, BN) and 7:00 am (13th January 2009, EN).
4
5
6
Hits to database (% of de-replicated non rRNA reads)
No. of total
reads
Ave. read
length (bp)
% of non
rRNA reads
No. of passing
reads
No. of dereplicated reads
BN, 12 Jan 2009 (9:00 pm)
590783
396
43
250418
244004
157525 (64.6)
92370 (37.9)
EN, 13 Jan 2009 (7:00 am)
492302
386
32
156554
144017
98894 (68.7)
58178 (40.4)
Sample
RefSeq (microbial)
SEED
1
2
Supplemental Table 3 Estimations of coverage based on reads assigned to microbial
3
RefSeq sequences and unambiguous assignment by MEGAN to SEED subsystems and
4
NCBI species. BN = 21:00, EN = 7:00
Microbial RefSeq
5
SEED Functional Roles
MEGAN Species
BN
EN
BN
EN
BN
EN
Total Reads Assigned
157525
98894
92370
58178
55935
22466
Observed Clusters
53478
34463
1698
1522
422
343
ACE-predicted Clusters
238381
150851
1885
1719
422
343
ACE Coverage %
22.4%
22.8%
90.0%
88.5%
100.0%
100.0%
Chao-predicted Clusters
137799
87429
1936
1724
422
343
Chao Coverage %
38.8%
39.4%
87.7%
88.3%
100.0%
100.0%
1
2
Supplemental Table 4 Gene assignments and SEED functional categories for
3
Microcoleus spp. fermentation pathway and glycogen to polyhydroxyalkonoate
4
conversion by the Chloroflexales.
Abbreviation
Gene Product
(gene abbrieviation)
SEED Functional Categories
LDH
Lactate dehydrogenase
(ldh, ykgF)
PFL
GP
Pyruvate-formate lyase (pfl)
Glycogen phosphorylase
(glg)
Alcohol/Acetaldehyde
dehydrogenase (adhE)
Acetate kinase (ack)
Pyruvate:Ferredoxin
oxidoreductase (pfo)
Bidirectional [NiFe]
hydrogenase (hoxH)
Bidirectional [NiFe]
hydrogenase (hoxY)
Bidirectional [NiFe]
hydrogenase (hoxU)
Bidirectional [NiFe]
hydrogenase (hoxF)
Bidirectional [NiFe]
hydrogenase (hoxE)
Pyruvate dehydrogenase
complex (pdh)
L-lactate dehydrogenase (EC 1.1.2.3), D-Lactate
dehydrogenase (EC 1.1.1.28), Predicted L-lactate
dehydrogenase, Iron-sulfur cluster-binding subunit YkgF
Pyruvate formate-lyase (EC 2.3.1.54)
Glycogen phosphorylase (EC 2.4.1.1)
ADH
ACK
PFR
HoxH
HoxY
HoxU
HoxF
HoxE
PDC
PhaA
PhaB
PhaC
ActP
LAP
PFK
GPI
FBP
FBA
Acetoacetyl CoA transferase
(phaA)
Acetoacetyl-CoA reductase
(phaB)
Polyhydroxyalkanoate
synthase (phaC)
Acetate permease (actP)
Lactate permease (lldp)
6-phosphofructokinase (pfk)
Glucose-6-phosphate
isomerase (gpi)
Fructose-1,6-bisphosphatase
(fbp)
Fructose-bisphosphate
Alcohol dehydrogenase (EC 1.1.1.1): Acetaldehyde
dehydrogenase (EC 1.2.1.10)
Acetate kinase (EC 2.7.2.1)
Pyruvate-flavodoxin oxidoreductase (EC 1.2.7.-)
NAD-reducing hydrogenase subunit HoxH (EC 1.12.1.2)
NAD-reducing hydrogenase subunit HoxY (EC 1.12.1.2)
NAD-reducing hydrogenase subunit HoxU (EC 1.12.1.2)
NAD-reducing hydrogenase subunit HoxF (EC 1.12.1.2)
NAD-reducing hydrogenase subunit HoxE (EC 1.12.1.2)
Pyruvate dehydrogenase E1 component alpha subunit
(EC 1.2.4.1), Pyruvate dehydrogenase E1 component beta
subunit (EC 1.2.4.1), Dihydrolipoamide acetyltransferase
component of pyruvate dehydrogenase complex (EC
2.3.1.12), Dihydrolipoamide dehydrogenase component
of pyruvate dehydrogenase complex (EC 1.8.1.4)
Acetyl-CoA acetyltransferase (EC 2.3.1.9)
Acetoacetyl-CoA reductase (EC 1.1.1.36)
Polyhydroxyalkanoic acid synthase; poly(R)hydroxyalkanoic acid synthase, class III, PhaC subunit
Acetate permease ActP (cation/acetate symporter)
L-lactate permease
6-phosphofructokinase (EC 2.7.1.11), 6phosphofructokinase, eukaryotic type (EC 2.7.1.11)
Glucose-6-phosphate isomerase (EC 5.3.1.9)
Fructose-1,6-bisphosphatase, type I (EC 3.1.3.11),
Fructose-1,6-bisphosphatase, GlpX type (EC 3.1.3.11)
Fructose-bisphosphate aldolase class II (EC 4.1.2.13),
TPI
G3P
PGK
PGM
ENS
PPS
PYK
1
aldolase (fba)
Triose phosphate isomerase
(tim)
Glyceraldehyde-3-phosphate
dehydrogenase (gap)
Phosphoglycerate kinase
(pgk)
Phosphoglycerate mutase
(pgm)
Enolase (ens)
Phosphoenolpyruvate
synthase (pps)
Pyruvate kinase (pyk)
Fructose-bisphosphate aldolase class I (EC 4.1.2.13)
Triosephosphate isomerase (EC 5.3.1.1)
NAD-dependent glyceraldehyde-3-phosphate
dehydrogenase (EC 1.2.1.12); Glyceraldehyde-3phosphate dehydrogenase (EC 1.2.1.12) (GAPDH)
Phosphoglycerate kinase (EC 2.7.2.3)
Phosphoglycerate mutase (EC 5.4.2.1)
Enolase (EC 4.2.1.11)
Phosphoenolpyruvate synthase (EC 2.7.9.2)
Pyruvate kinase (EC 2.7.1.40)
1
2
Supplemental Table 5 Accession numbers in Microcoleus chthonoplastes PCC 7420 and
3
Chloroflexales genomes (Oscillochloris trichodes DG6, Chloroflexus aggregans DSM
4
9485, Roseiflexus castenholzii DSM 13941, Chloroflexus sp. Y-400-fl) for fermentation
5
and PHA production pathways.
Abbreviation
M chthonoplastes
Accession
Chloroflexales Accession Numbers
Number(s)
LDH
ZP_05029329.1,
ZP_07687075.1, YP_002464022.1, YP_001430946.1,
ZP_05027132.1,
YP_002568646.1, YP_001278741.1
ZP_05024030.1
PFL
ZP_05031046.1
GP
ZP_05024411.1,
YP_002464170.1, YP_002568551.1, YP_001634362.1,
ZP_05024271.1
YP_001431030.1, ZP_07687229.1, YP_001278475.1
ADH
ZP_05027137.1
ACK
ZP_05027154.1
PFR
ZP_05026241.1
YP_001275570.1, YP_001434103.1, ZP_07686769.1
HoxH
ZP_05027024.1
YP_002569037.1, YP_001634797.1, YP_002463799.1,
YP_001431870.1, YP_001276440.1, ZP_07687015.1
HoxY
ZP_05027037.1
HoxU
ZP_05027023.1
HoxF
ZP_05027028.1
HoxE
ZP_05027052.1
PDC
ZP_05028143.1,
YP_002462952.1, YP_002569853.1, YP_001635576.1,
ZP_05027952.1,
ZP_07685943.1, YP_002569855.1, YP_001635578.1,
ZP_05025210.1,
YP_002462954.1, ZP_07684644.1, YP_001275667.1,
ZP_05028044.1,
YP_001431750.1
ZP_05025437.1,
ZP_05025524.1
PhaA
YP_001278635.1, YP_001430842.1, ZP_07684826.1,
YP_002569328.1, YP_001635078.1, YP_002463281.1
PhaB
YP_002569329.1, YP_001635079.1, YP_002463282.1,
YP_001430841.1, YP_001278634.1, ZP_07684827.1
PhaC
YP_002571218.1, YP_002462002.1, YP_001636840.1,
YP_001278834.1, YP_001430259.1, ZP_07684148.1
ActP
ZP_05025509.1
ZP_07686543.1, YP_002461629.1, YP_002571351.1,
YP_001636967.1, ZP_07684384.1
LAP
PFK
YP_001431699.1, YP_001276400.1
ZP_05027350.1,
YP_001432611.1, YP_001276170.1, ZP_07684184.1,
ZP_05028256.1
YP_002568694.1, YP_001634491.1, YP_002464006.1,
ZP_07684183.1, YP_001431984.1, YP_001277056.1,
ZP_07684183.1
GPI
ZP_05030931.1
FBP
ZP_05023982.1,
YP_002571277.1, YP_001636895.1, YP_002462025.1,
ZP_05027047.1
YP_001431328.1, YP_001278423.1, ZP_07685583.1
FBA
ZP_05028766.1
TPI
ZP_05029410.1
YP_001278117.1, YP_001431118.1, ZP_07684429.1,
YP_002571815.1, YP_001637392.1, YP_002462281.1
G3P
ZP_05024661.1
ZP_07687131.1, ZP_07687132.1, YP_002464998.1,
YP_002571711.1, YP_001637296.1
PGK
ZP_05030467.1
YP_002570760.1, YP_002462015.1, YP_001636411.1,
YP_001275819.1, YP_001432174.1, ZP_07685368.1
PGM
ZP_05023660.1
YP_002463031.1, ZP_07684561.1, YP_002569529.1,
YP_001635261.1, YP_001434297.1, YP_002463496.1,
ZP_07684870.1, YP_001430323.1, YP_001278867.1,
YP_002569059.1, YP_001634818.1, ZP_07685097.1,
YP_002464852.1, YP_002568144.1, YP_001633993.1
ENS
ZP_05031224.1
YP_001637375.1, ZP_07686911.1, YP_001430229.1,
YP_001278813.1, YP_002465011.1
PPS
ZP_05028882.1,
ZP_05025507.1,
ZP_05029015.1,
ZP_05029154.1,
ZP_05029107.1
PYK
ZP_05024576.1,
ZP_07684115.1, YP_002571078.1, YP_001636715.1,
ZP_05026799.1,
YP_001431802.1, YP_001275776.1, YP_002461694.1
ZP_05023969.1,
ZP_05023802.1
1
1
2
Supplemental Table 6 Number of metatranscriptomic reads recruited to M.
3
chthonoplastes and Chloroflexales genes for fermentation and PHA production pathways.
Abbreviation
LDH
PFL
GP
ADH
ACK
PFR
HoxH
HoxY
HoxU
HoxF
HoxE
PDC
PhaA
PhaB
PhaC
ActP
LAP
PFK
GPI
FBP
FBA
TPI
G3P
PGK
PGM
ENS
PPS
PYK
4
Microcoleus
BN
EN
Reads Reads
4
0
116
27
43
2
125
61
3
0
458
264
20
11
29
4
4
10
31
21
15
17
32
5
0
0
0
0
0
0
1
0
0
0
10
5
1
0
24
2
7
1
3
0
12
3
12
0
2
1
10
1
641
201
16
3
Cyanobacteria
BN
EN
Reads Reads
9
1
177
53
56
11
276
125
7
1
512
296
41
18
32
6
4
11
40
41
16
18
60
16
0
0
0
0
6
29
2
3
1
0
28
11
4
1
59
7
271
57
5
1
17
4
49
2
5
2
26
2
1056
370
38
11
Chloroflexales
BN
EN
Reads Reads
1
4
0
0
31
40
0
0
0
0
17
21
0
1
0
0
0
0
0
0
0
0
3
2
8
5
26
19
41
28
22
8
1
0
11
9
0
0
8
6
0
0
21
30
8
4
6
16
4
14
3
1
0
0
5
9
Total
BN
EN
Reads Reads
23
12
334
198
118
77
300
138
20
11
731
494
45
34
36
18
24
22
83
94
23
26
139
62
36
45
49
48
74
110
132
69
19
12
66
48
21
20
81
24
342
79
44
41
121
45
104
49
18
36
68
38
1117
410
79
32
1
2
3
Supplemental Figure Legend
4
Supplemental Figure 1. Diel production of H2 or organic acids in Elkhorn Slough mats
5
from January 12-13, 2009. Mat core incubations in vials commenced at dawn with H2
6
and organic acids allowed to accumulate over a diel cycle with N2 flushing of the vial at
7
the end of the day and night periods. Vials in which organic acids were measured were
8
reset at dawn by replacement with fresh field site water. Vertical error bars indicate the
9
standard deviation of three replicates. (a) H2 production in different layers of microbial
10
mat. Mat cores were sectioned at different depths, 0-2 mm, 2-4 mm and 4-15 mm.
11
Yellow diamonds denote natural solar irradiance. E, microeinsteins. (b) Organic acid
12
production in intact mat cores (0-15 mm).
13
Supplemental Figure 2. Collector’s curves generated by MOTHUR for reads assigned to
14
self-BLAST clusters (reads overlap by 200 or more base pairs with 90% or greater
15
sequence identity), RefSeq sequences (a), and unambiguous MEGAN assignments to
16
SEED subsystems and species (b).
17
Supplemental Figure 3. Classification of reads for Chloroflexi populations. Within the
18
metatranscriptome reads affiliated with the phylum Chloroflexi, 96% were assigned to the
19
class Chloroflexi at both time points, and ca. 85% of those reads further to the order
20
Chloroflexales (BN; n = 18,769 and EN; n = 26,981, respectively). About 3% of the reads
21
within the class Chloroflexi were assigned to the order Herpetosiphonales, which are
22
non-phototrophic members of the Chloroflexi, while the remaining ~12% of sequences in
23
the class Chloroflexi could not be assigned further. b) In both samples, within the
1
Chloroflexales, ~31% of the reads were assigned to the Chloroflexaceae and further to
2
the genera Chloroflexus and Roseiflexus, while ~12% were assigned to the
3
Oscillochloridaceae and further to the genus Oscillochloris. All the reads within the
4
Oscillochloris could be assigned to Oscillochloris trichoides, while the majority of the
5
reads within the Chloroflexus and Roseiflexus could not be assigned any further than to
6
the genus level. Furthermore, 57% of the Chloroflexales assigned reads could not be
7
classified further than to the order.
8
Supplemental Figure 4. Transcripts classified within different SEED subsystems
9
(functional categories) from the Begin Night (BN; 21:00, January 12, 2009) and End
10
Night (EN; 07:00, January 13, 2009) libraries. Percentage abundances of transcripts
11
within different SEED subsystems were calculated for different taxonomic group: all
12
organisms, Chloroflexales or Microcoleus spp..
13
transcripts for a given taxonomic group are not shown. Unassigned reads represent
14
transcripts that had matches to the RefSeq microbial database but could not be classified
15
within a specific SEED category. See materials and methods for more information
16
regarding taxonomic and functional classifications of transcripts.
17
SEED subsystems with <1% of
1
Supplemental Figure 1
a
1000
250
4 - 15 mm
800
200
150
600
100
400
200
50
0
12:00
3
2 - 4 mm
18:00
0:00
Time
6:00
0
12:00
Irradiance (mE m-2 s-1)
Hydrogen (nmol cm-3)
0 - 2 mm
b
Organic acids (nmol cm-3)
2
300
Day
250
Acetate
200
Propionate
Day
Night
Formate
150
100
50
0
16:30
21:30
0:30
Time
6:30
12:30
1
2
3
4
5
6
7
8
9
Supplemental Figure 2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Supplemental Figure 3
1
Supplemental Figure 4
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27