Table 1. Oligonucleotide probes used in this studya. wmis: weighted mismatches (ARB probe match tool; http://www.mikro.biologie.tu-muenchen.de)
Probe
Specificity
Probe Sequence (5' 3')
Positionb
FISH
[FA]c
ANME1-305
ANME-1-cluster of uncultured
archaea (distantly related to
Methanomicrobiales/-sarcinales)
AGCCCGGAGATGGGTTCT
305-322
0
ANME1-350
ANME-1-cluster of uncultured
archaea
AGTTTTCGCGCCTGATGC
350-367
40
ANME1-632
ANME-1-cluster of uncultured
archaea
TCAGGGAATACTGCTTGG
632-649
0
ANME1-830
ANME-1-cluster of uncultured
archaea
TCGCAGTAATGCCAACAC
830-847
EelMS240
uncultured archaea BA2H11,
TA1a4;
Methanolobus taylorii;
Methanolobus tindarius,
Methanohalophilus mahii,
Methanohalophilus oregonensis,
Methanococcoides burtonii,
Methanococcoides euhalobius
CCCACTACAACCTGATAG
240-257
cEelMS240
competitor to probe EelMS240
CCCACTACTACCTGATAG
240-257
Reference strainsd
Target organisms
Non-target organisms
not available
Comments
Methanosarcina mazei
Methanosaeta concilii
Methanolobus taylorii
(all 1.5 wmis; probe
signal <30% FA)
Methanosaeta concilii; Methanobacterium
Methanolobus taylorii formicicum;
(0.6 wmis; ≤50% FA) Methanococcus
jannaschii; clones
Ba2H11; TA1a4
(all 1.7 wmis; probe
signal <20% FA)
not available
Microbacterium
lacticum
(3.2 wmis; no signal)
no hybridization with
aggregated archaea at
lowest stringency
0
not available
10
Methanolobus taylorii
(0 wmis; ≤20% FA)
no hybridization with
aggregated archaea at
lowest stringency
probe used with
competitor cEelMS240
weak probe
fluorescence
Hybridization with
aggregated archaea
(probe signal ≤20% FA)
ANME-1 group >5
mismatches
Methanobacterium
formicicum (3.4 wmis;
no signal)
Methanosaeta concilii
(1.1 wmis; probe signal
0% FA);
Methanosarcina mazei
(1.1 wmis; no signal)
no hybridization with
aggregated archaea
(>20% FA)
no hybridization with
aggregated archaea at
lowest stringency
1
Table 1. Oligonucleotide probes used in this studya. wmis: weighted mismatches (ARB probe match tool; http://www.mikro.biologie.tu-muenchen.de)
Probe
Specificity
Probe Sequence (5' 3')
Positionb
FISH
[FA]c
Reference strainsd
Target organisms
Non-target organisms
EelMS538
uncultured archaea BA2H11,
TA1a4
CGGCTACCACTCGGGCCGC
538-556
40
not available
EelMS932
uncultured archaea BA2H11,
TA1a4
(related to Methanosarcinales)
AGCTCCACCCGTTGTAGT
932-949
40
not available
cEelMS932-Mt
competitor to probe EelMS932
targeting Methanolobus taylorii
competitor to probe EelMS932
targeting Methanosaeta concilii
Methanobacterium,
Methanobrevibacter
GGCTCCACCCGTTGTAGT
932-949
AGCTCCACCCGTTGTGGT
932-949
GGACTTAACAGCTTCCCT
859-876
25
Methanobacterium
formicicum
(0 wmis; ≤40% FA)
MSSH859
Methanosarcina, Methanolobus,
Methanosaeta,
Methanohalophilus
TCGCTTCACGGCTTCCCT
859-876
35
Methanosaeta concilii
(0 wmis; ≤70% FA)
MTB499
Methylobacterium
TCCGGTACCGTCATTATC
499-516
40
Methylobacterium
organophilum
(0 wmis; <60% FA)
MTMC701
Methylomonas, Methylobacter,
Methylococcus,
Methylomicrobium
GTGTTCCTTCAGATCTCT
701-718
40
cEelMS932-Mc
MEB859
Comments
Thermococcus stetteri
(0.2 wmis; probe signal
<40% FA)
Hybridization with
aggregated archaea
(≤70% FA), ANME 4.55.1 wmis
Methanolobus taylorii
brightest probe signal of
all EelMS probes;
Methanosaeta concilii
(both 1.1 wmis; probe
hybridization with
signal ≤60%, but clearly aggregated archaea
weaker signal when
(≤70% FA);
using with competitor
probes cEelMS932-Mt/Mc)
Methanococcus
jannaschii
(1.1 wmis; probe signal
<10% FA)
Haloarcula japonica
(1.1 wmis; probe signal
<10% FA)
Acidobacterium
capsulatum
(1.1 wmis; probe signal
<30% FA)
Thiobacillus thiooxidans
(0.2 wmis; probe signal
<40% FA)
below detection limit
below detection limit
below detection limit
below detection limit
2
1. Other probes were used as published previously: ARCH91516, EUB33817, DSR65118, DSS65818, DSV21418, DSV69818, DSB98518, DSS22519, Sval42820, 66021, 22121
2. Position in the 16S rRNA of E.coli
3. Formamide (FA) concentrations in the hybridization buffer in % (v/v)
4. Pure cultures of reference organisms were obtained from the DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany): Acidobacterium
capsulatum(DSM 11244); Corynebacterium urealyticum (DSM 7109); Haloarcula japonica (DSM 6131) Methanobacterium formicicum (DSM 1535); Methanococcus jannaschii
(DSM 2661); Methanolobus taylorii (DSM 9005); Methanosaeta concilii (DSM 3671); Methanosarcina mazei (DSM 2053); Methylobacterium organophilum (DSM 760);
Microbacterium lacticum(DSM 20427); Thiobacillus thiooxidans (DSM 504), Thermococcus stetteri (DSM 5262)
3