Download emi12978-sup-0001-si

Supplementary Information
Synchronized dynamics of bacterial niche-specific functions during biofilm
development in a cold seep brine pool
Weipeng Zhang1, Yong Wang2, Salim Bougouffa3, Renmao Tian1, Huiluo Cao1, Yongxin Li1 Lin Cai1,
Yue Him Wong1, Gen Zhang1, Xixiang Zhang3, Vladimir B Bajic3, Abdulaziz Al-Suwailem3,
Pei-Yuan Qian1,2#
1
KAUST Global Partnership Program, Division of Life Science, Hong Kong University of Science
and Technology, Clear Water Bay, Hong Kong
2
Sanya Institute of Deep Sea Science and Engineering, Chinese Academy of Sciences, San Ya, Hai
Nan, China
3
King Abdullah University of Science and Technology, Thuwal, The Kingdom of Saudi Arabia
Running title: metagenomics of cold seep biofilms
Keywords: microbial adaptation; extreme condition; inter-species interaction; genome binning
#
Corresponding author:
Pei-Yuan Qian, PhD, Chair professor, Division of Life Science,
The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China
Phone: 0852-2358-7331, Fax: 0852-2358-1559
E-mail: [email protected]
1
Figure S1 Similarity of functional communities from the biofilms illustrated by individual gene
ontology (GOs) and abundance-based PCoA plot. GOs with >0.5% relative abundance in at least one
sample were included in PCoA using the PAST software.
2
Figure S2 The GOs differentiating the brine water and 6d-brine biofilm metagenomes. Comparison
was performed based on Similarity percentage analyses (SIMPER) as described in the Methods
section. Nitrogen cycle, polysaccharide metabolism and proteolysis related functions were
highlighted in blue, red and green respectively.
3
Figure S3 The carbohydrate-Active enZYmes (CAZys) differentiating the 6d-brine and 6d-NBW
biofilm metagenomes. Comparison of 6d-brine versus 6d-NBW biofilms was performed based on
SIMPER analysis. CAZys were presented in the order of their contributions to differences (highest to
lowest,
bottom
to
top).
4
Figure S4 Classification of the transporters in the 6d-brine and 6d-NBW biofilm metagenomes. The
transporters were identified and classified based on BLAST search against the Kyoto Encyclopedia of Genes
and Genomes (KEGG) database with e <10-5.
5
Figure S5 Neighbor-joining phylogenetic organization of the strain deltaproteobacterium sp. nov. and
close-related Deltaproteobacteria strains. The phylogenetic tree was constructed using concatenated 31
essential genes. The reference genomes were obtained form the NCBI database. Bootstrap values based on
1000 replications are shown on the nodes.
6
Figure S6 Neighbor-joining phylogenetic organization of the strain epsilonproteobacterium sp. nov. and
close-related Epsilonproteobacteria strains. The phylogenetic trees was constructed using concatenated 31
essential genes. The reference genomes were obtained form the NCBI database. Bootstrap values based on
1000 replications are shown on the nodes.
7
a
8
b
9
c
Figure S7 Neighbor-joining phylogenetic tree of the ChaB (a) and diguanylate cyclase diguanylate cyclase
(b) in epsilonproteobacterium sp. nov.. Bootstrap values based on 1000 replications are shown on the nodes.
These two genes are phylogenetically close to homologs from Gammaproteobacteria. The adjacent genes
including flagellin, DNA binding protein, histidine kinase and ABC transporter, which are phylogenetically
close to Epsilonproteobacteria, are also shown (c).
10
Figure S8 Network of co-occurring 97% cutoff OTUs based on correlation analysis of the 47 biofilm
samples and 4 water samples. The Spearman’s correlation coefficient of >0.8 and statistically P-value of
<0.01 were used. The size of each node is proportional to the number of connections.
11
Figure S9 Pictures during the Red Sea cruise. (a) Different types of substrates embedded in the slots of the
carousel. The structure of carousel is shown in a previous work (Zhang et al., 2014a). (b) Remotely
Operated Vehicle (ROV) working in the Thuwal seep II during 2013 Red Sea cruise. (c, d) The carousels
were fixed in the frames and then immersed in the water for biofilm development.
12
Figure S10 Rarefaction curves for the ORF counts of the 6d-brine-Ti (blue line) and 6d-NBW-Ti (red line)
biofilm metagenomes and identified GO items. These two metagenomes were selected as representatives
and information of other metagenomes was listed in Table S1.
13
Figure S11 Steps in genome binning. Each circle represents a metagenomic contig with size proportional to
length and colored by GC content. Only contigs longer than 500 bp were shown. PCoA clustering on
tetranucleotide frequencies was performed for further binning. (a, b) Genome binning of the
deltaproteobacterium sp. nov.. (c-f) Genome binning of the epsilonproteobacterium sp. nov.. Red color
indicated contigs with essential genes.
14
Table S1 Features of the two binned draft genomes.
Genome feature or gene function
deltaproteobacterium sp. nov.
epsilonproteobacterium sp. nov.
Genome recovery
~99%
~96%
Genome size (Mbp)
2.51
2.19
No. of essential genes
106
103
No. of redundant essential genes
1
2
No. of contigs
121
17
N50 of contigs
208,591 bp
31,011 bp
N90 of contigs
81,478 bp
10,933 bp
No. of ORFs
2443
2244
GC content (%)
49.3%
49.8%
15
Table S2 Conserved single copy gene sets for genome completeness estimation. The completeness of each
population bin was investigated using a suite of hidden Markov models (HMM) covering 107 proteins
conserved in 95% of all sequenced bacteria. The number of the 107 single copy genes in
deltaproteobacterium sp. nov. and epsilonproteobacterium sp. nov. were compared the complete genomes of
their close relatives. 1, deltaproteobacterium sp. nov.; 2, Pelobacter propionicus DSM 2379; 3, Pelobacter
carbinolicus DSM 2380; 4, Geoalkalibacter subterraneus Red1; 5, Geobacter lovleyi SZ; 6,
Anaeromyxobacter sp. K; 7, epsilonproteobacterium sp. nov.; 8, Nitratifractor salsuginis DSM 16511; 9,
Sulfurovum sp. NBC37-1; 10, Nitratiruptor sp. SB155-2; 11, Nautilia profundicola AmH; 12, Wolinella
succinogenes DSM 1740.
HMM name
Gene name
1
2
3
4
5
6
7
8
9
10
11
12
PF00162
phosphoglycerate kinase
1
1
1
1
1
1
1
1
1
1
1
1
PF00276
ribosomal protein L23
1
1
1
1
1
1
1
1
1
1
1
1
PF00281
ribosomal protein L5
1
1
1
1
1
1
1
1
1
1
1
1
PF00297
ribosomal protein L3
1
1
1
1
1
1
1
1
1
1
1
1
PF00347
ribosomal protein L6
1
1
1
1
1
1
1
1
1
1
1
1
PF00366
ribosomal protein S17
1
1
1
1
1
1
0
1
1
1
1
1
PF00380
ribosomal protein S9
1
1
1
1
1
1
1
1
1
1
1
1
PF00410
ribosomal protein S8
1
1
1
1
1
1
1
1
1
1
1
1
PF00411
ribosomal protein S11
1
1
1
1
1
1
1
1
1
1
1
1
PF00416
ribosomal protein S13
1
1
1
1
1
1
1
1
1
1
1
1
PF00466
ribosomal protein L10
1
1
1
1
1
1
1
1
1
1
1
1
PF00573
ribosomal protein L4
1
1
1
1
1
1
1
1
1
1
1
1
PF00750
tRNA synthetases class I
1
2
2
1
2
2
2
2
2
3
2
2
PF01025
GrpE nucleotide exchange factor
1
1
1
1
1
1
1
1
1
1
1
1
PF01795
MraW methylase family
1
2
2
1
1
1
1
1
1
1
1
1
TIGR00001
ribosomal protein L35
1
1
1
1
1
1
1
1
1
0
1
1
TIGR00002
ribosomal protein S16
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00009
ribosomal protein L28
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00012
ribosomal protein L29
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00019
peptide chain release factor 1
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00029
ribosomal protein S20
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00043
rRNA maturation RNase YbeY
1
1
1
1
1
0
1
1
1
1
1
1
TIGR00059
ribosomal protein L17
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00060
ribosomal protein L18
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00061
ribosomal protein L21
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00062
ribosomal protein L27
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00064
signal recognition particle-docking protein
FtsY
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00082
ribosome-binding factor A
1
1
1
1
1
1
1
1
1
1
1
1
16
TIGR00086
SsrA-binding protein
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00092
GTP-binding protein YchF
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00115
trigger factor
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00116
translation elongation factor Ts
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00152
dephospho-CoA kinase
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00158
ribosomal protein L9
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00165
ribosomal protein S18
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00166
ribosomal protein S6
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00168
translation initiation factor IF-3
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00234
tyrosine--tRNA ligase
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00337
CTP synthase
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00344
alanine--tRNA ligase
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00362
chromosomal replication initiator protein
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00388
glycine-tRNA ligase, alpha subunit
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00392
isoleucine--tRNA ligase
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00396
leucine--tRNA ligase
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00408
proline-tRNA ligase
0
0
0
0
0
1
0
0
0
0
0
0
TIGR00414
serine--tRNA ligase
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00418
threonine-tRNA ligase
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00420
tRNA-methyltransferase
1
1
1
1
2
1
1
1
1
1
1
1
TIGR00422
valine--tRNA ligase
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00435
cysteine--tRNA ligase
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00436
GTP-binding protein Era
2
2
2
2
2
2
2
2
2
2
2
2
TIGR00442
histidine-tRNA ligase
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00459
aspartate--tRNA ligase
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00460
methionyl-tRNA formyltransferase
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00468
phenylalanine--tRNA ligase, alpha subunit
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00472
phenylalanine--tRNA ligase, beta subunit
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00487
translation initiation factor IF-2
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00496
ribosome recycling factor
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00575
DNA ligase, NAD-dependent
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00631
excinuclease ABC subunit B
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00663
DNA polymerase III, beta subunit
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00810
preprotein translocase, SecG subunit
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00855
ribosomal protein L12
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00922
NusG antitermination factor
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00952
ribosomal protein S15
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00959
signal recognition particle protein
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00963
preprotein translocase, SecA subunit
1
1
1
1
1
1
1
1
1
1
0
1
17
TIGR00964
preprotein translocase, SecE subunit
1
1
1
1
1
1
0
1
0
1
1
1
TIGR00967
preprotein translocase, SecY subunit
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00981
ribosomal protein S12
1
1
1
1
1
1
0
1
1
1
1
1
TIGR01009
ribosomal protein S3
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01011
ribosomal protein S2
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01017
ribosomal protein S4
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01021
ribosomal protein S5
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01024
ribosomal protein L19
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01029
ribosomal protein S7
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01030
ribosomal protein L34
1
0
1
1
1
1
0
1
0
0
1
0
TIGR01031
ribosomal protein L32
1
0
1
1
0
1
1
1
1
1
1
1
TIGR01032
ribosomal protein L20
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01044
ribosomal protein L22
1
1
1
1
1
1
0
1
1
1
1
1
TIGR01049
ribosomal protein S10
1
1
1
1
1
1
0
1
1
1
1
1
TIGR01050
ribosomal protein S19
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01059
DNA gyrase, B subunit
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01063
DNA gyrase, A subunit
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01066
ribosomal protein L13
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01067
ribosomal protein L14
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01071
ribosomal protein L15
1
0
1
1
1
1
1
1
1
1
1
1
TIGR01079
ribosomal protein L24
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01164
ribosomal protein L16
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01169
ribosomal protein L1
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01171
ribosomal protein L2
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01391
DNA primase
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01393
elongation factor 4
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01632
ribosomal protein L11
1
1
1
1
1
1
1
1
1
1
1
1
TIGR01953
NusA transcription termination factor
1
1
1
1
1
1
1
1
1
1
1
1
TIGR02012
protein RecA
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
TIGR02013
TIGR02027
DNA-directed RNA polymerase, beta
subunit
DNA-directed RNA polymerase, alpha
subunit
TIGR02191
ribonuclease III
1
1
1
1
1
1
1
1
1
1
1
1
TIGR02350
chaperone protein DnaK
1
1
1
1
1
2
1
1
1
1
1
1
TIGR02386
DNA-directed RNA polymerase, beta'
subunit
1
1
1
1
1
1
1
1
1
1
1
1
TIGR02397
DNA polymerase III
1
1
1
1
1
1
1
1
1
1
1
1
TIGR02432
tRNA(Ile)-lysidine synthetase
1
1
1
1
1
1
1
1
1
1
1
1
TIGR02729
Obg family GTPase CgtA
1
1
1
1
1
1
1
1
1
1
1
1
18
TIGR03263
guanylate kinase
1
1
1
1
1
1
1
1
1
1
1
1
TIGR03594
ribosome-associated GTPase EngA
1
1
1
1
1
1
1
1
1
1
1
1
TIGR00409
proline--tRNA ligase
1
1
1
1
1
1
1
1
1
1
1
1
19
Table S3 Properties of the representative enzymes involved in polysaccharide degradation and fermentation in the deltaproteobacterium sp. nov.. The gene
sequences were searched against KEGG, CAZy and Conserved domain databases (CDD) using BLAST with e <10-5.
Enzyme name
Length (AA)
KEGG
CAZy family
Domain
Beta-1,4-endoglucanase
355
K01179
-
FrvX
xylanase/chitin deacetylase
347
K01506
CE4
CE4_SF superfamily
Starch phosphorylase
845
K00688
GT35
Alpha amylase
714
-
GH57
GH38_57_N_lamB_ydjl_SF
Alpha amylase
1559
-
GH13
AmyAc_1
Acetate kinase
422
K00925
-
Acetate kinase superfamily
Alcohol dehydrogenase
345
K13953
-
PTZ00354
Alcohol dehydrogenase
334
K00001
-
PTZ00354
Domain organization
Glycosyltransferase_GTB_type
superfamily
20
Table S4 Number of genes involved in carbohydrate metabolism, nitrogen metabolism and sulfur metabolism in 1, deltaproteobacterium sp. nov.; 2, Pelobacter
propionicus DSM 2379; 3, Pelobacter carbinolicus DSM 2380; 4, Geoalkalibacter subterraneus Red1; 5, Geobacter bemidjiensis Bem; 6, Geobacter daltonii
FRC-32; 7, Geobacter sp. M21; 8, Geobacter uraniireducens Rf4; 9, Geobacter lovleyi SZ; 10, Anaeromyxobacter sp. K; 11, Anaeromyxobacter sp. Fw109-5;
12, Myxococcus xanthus DK 1622; 13, Desulfarculus baarsii DSM 2075; 14, Desulfurivibrio alkaliphilus AHT2; 15, Syntrophobacter fumaroxidans MPOB.
The gene annotation was based on the KEGG database.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Glycolysis / Gluconeogenesis
28
27
26
12
23
24
23
24
26
11
10
18
19
11
26
Citrate cycle (TCA cycle)
25
20
20
13
21
22
20
20
19
8
8
24
15
8
15
Pentose phosphate pathway
15
22
16
14
16
7
8
9
15
5
7
23
15
15
15
Pentose and glucuronate interconversions
4
3
2
0
4
5
3
2
5
0
1
6
3
2
2
Fructose and mannose metabolism
14
13
14
6
13
13
12
12
15
6
5
16
13
14
15
Galactose metabolism
3
3
3
0
7
3
7
3
4
5
5
6
3
2
4
Ascorbate and aldarate metabolism
3
3
1
0
2
2
3
1
3
0
0
7
2
0
3
Starch and sucrose metabolism
7
6
6
3
10
7
5
6
5
3
3
15
2
12
10
Amino sugar and nucleotide sugar metabolism
20
18
12
5
19
12
19
11
19
12
13
21
23
15
24
Pyruvate metabolism
22
21
11
4
12
15
15
12
19
6
6
17
23
12
24
Glyoxylate and dicarboxylate metabolism
18
14
12
5
20
12
17
12
15
6
8
16
15
6
14
Propanoate metabolism
19
17
6
4
7
6
7
5
9
7
8
14
14
1
15
Butanoate metabolism
17
16
9
2
12
7
12
8
12
6
6
14
15
3
20
C5-Branched dibasic acid metabolism
7
6
3
1
4
5
2
3
5
0
0
6
6
5
6
Inositol phosphate metabolism
0
5
3
0
5
4
3
3
5
0
0
7
4
4
6
Nitrogen metabolism
3
17
8
7
9
9
11
8
16
6
7
10
11
6
11
Sulfur metabolism
3
5
6
6
7
9
6
6
9
4
5
10
11
9
12
21
Table S5 Number of genes involved in energy metabolism in 1, epsilonproteobacterium sp. nov.; 2, Nitratifractor salsuginis DSM 16511; 3, Sulfurovum sp.
NBC37-1; 4, Nitratiruptor sp. SB155-2; 5, Caminibacter mediatlanticus TB-2; 6, Nautilia profundicola AmH; 7, Lebetimonas sp. JS170; 8, Wolinella
succinogenes DSM 1740; 9, Helicobactercetorum MIT99-5656; 10, Arcobacter sp. L; 11, Campylobacter coli CVM N29710; 12, Campylobacter coli
15-537360; 13, Sulfurospirillum barnesii SES-3; 14, Sulfuricurvum sp. IFRC-1; 15, Sulfurimonas autotrophica DSM 16294.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Oxidative phosphorylation
42
35
40
40
42
37
40
22
18
41
42
42
42
40
40
Photosynthesis (F-type ATPase)
8
8
8
8
16
8
16
8
8
16
8
8
16
16
16
Carbon fixation pathways in prokaryotes
20
14
20
18
14
16
14
12
6
11
19
17
12
11
11
Methane metabolism
17
18
18
15
14
18
18
12
12
15
17
16
16
17
15
Nitrogen metabolism
39
18
30
30
24
23
18
17
2
17
18
16
13
17
20
Sulfur metabolism
12
8
13
13
4
11
10
3
8
11
10
10
11
12
13
22
Table S6 A summary of all the metagenomes of the biofilm and water communities. The average length of the raw reads is 100bp.
Location
Brine pool
NBW
Duration and substrate types
3d-Al
3d-Ti
3d-PVC
6d-Al
6d-Ti
6d-PVC
Water
3d-Al
3d-Ti
3d-PVC
6d-Al
6d-Ti
6d-PVC
Water
No. of raw reads
42,410,000
43,180, 000
32,590, 235
45,034,038
43,045, 105
39,505, 320
43,256,204
21,680,000
23,390, 000
26,046, 780
22,480, 000
22,840, 000
19,221, 704
41,955,586
Trimmed reads
7, 132
7, 601
120, 221
887, 069
234, 721
276, 172
6,750
3, 813
3, 977
220, 629
3, 621
3, 777
62, 991
6,945
Reads assigned to CAZy
2, 995, 329 3, 113, 483 3, 129, 437 3, 145, 173 3, 199, 498 3, 114, 722 2,87,934
1,472, 380
1,555, 587 1,499, 375 1,495, 843 1,477, 342 1,465, 674 1,232,731
Assembled contigs
1, 012, 332
945, 510
N50 of contigs
630
467
Average length of contigs (bp)
588
519
Predicted ORFs
1, 378, 917
1, 026, 605
ORFs could be annotated
868,717 (63%)
657, 027 (64%)
ORFs assigned to GOs
482,620 (35.1%)
287, 449 (28%)
ORFs assigned to KEGGs
303,361(22%)
318, 247 (31%)
23
Table S7 Primer used in quantitative PCR.
Genes
Sequences
deltaproteobacterium sp. nov.
endoglucanase-F
TCGAAGGTATCGCTGATA
endoglucanase-R
ATCATCGACAAACTGCAC
acetate kinase-F
GCACTAAATTGTGGAAGCTC
acetate kinase-R
CAACGCCATGGGTATTGTCGG
alcohol dehydrogenase-F
AGCGCTGCTGGTGGAGCAGCC
alcohol dehydrogenase-R
TCACTCCTTTATTTCCAACTG
Zn-dependent protease-F
AGCGGGGTGTTATCAAATCG
Zn-dependent protease-R
CAACAACAGTCGATCTTCA
epsilonproteobacterium sp. nov.
narG-F
ATGAGTGTAAACGATAATAG
narG-R
AACCGAACTCGTTTCTATAC
narH-F
AGAAACGTATCCGGGCAAG
narH-R
GGGTTTTCCCGTCGGCC
napA-F
CTGTTGGGATGTCCGTTCC
napA-R
TGCATTAAAGTAACCTTTG
napB-F
GACTATCGCTTCTGCCGCG
napB-R
GTACCCTTTAAGCGCTGC
nifD-F
GTTTGCAAAAGAGCGCC
nifD-R
CTCATTACGCCAGGAACG
nifH-F
ATGCAACAACAGTTGGTG
nifH-R
GGGTCACACCCGACGATA
Acetate:CoA ligase-F
AAAGCCGCGATCATCTTCG
Acetate:CoA ligase-R
AGTGGATCGCGCCGATTCTCG
motA-F
GGTCTACTGTTAGGTGCGAT
motA-R
TTAATAAGTTCGTTTACATC
24