Supplemental Table 2. Primers and PCR conditions used in this study.
Application
Target
Primers
Sequence (5’-3’)
Anneal temp (oC)/#cycles
/extension (s) at 72oC
MISA1
Round 1
spacer_pmoC606f
GGGGYCAYRCBTTCTGGT
(enrichment)
spacer_pmoA191r
CCARAARTCCCARTCNC
Round 2 (bulk
spacer_pmoC626f
RCBTTCTGGTKBATGGAAGA
amplification)
spacer_pmoA189r
CCARAARTCCCARTCNCC
Group C
GroupC_spacer1f
AGGTAATGAAGCGCATGTCC
52/20/60”
length divergent
GroupC_spacer2f
GCTGTTCAAGCAGCTTAGCC
52/5/60”  48/30/60”
CuMMO-encoding
GroupC_moBr
GCTTTAKCAAASCGTTCAC
GroupO_spacer1f
ATTTACCCCGGGAGGTATTG
52/20/60”
GroupO_spacer2f
TTACCCCGGGAGGTATTGAT
52/5/60”  48/30/60”
GroupO_moBr
GGCCAAAGCGCTAATACACT
GroupW_spacer1f
GCTTTGTAATCCTCGGATGG
52/20/60”
GroupW_spacer2f
CAGACAACCAACGCGAGATA
52/5/60”  48/30/60”
GroupW_moBr
TCGATCGTACCGATCAGTGT
GroupZ_spacer1f
CACCATTACATTGGGGCTTT
52/20/60”
GroupZ_spacer2f
TCCATATTCACGCCATGTTAAC
52/5/60”  48/30/60”
GroupZ_moAr
AACGCAATACAACCCACCAT
Degenerate
moB156r
KHYYKYTCRCCATGYGC
CuMMOB
moB172r
CGCATRCGYATRAANGGYTC
reverse primers
moB175r
TRCGCATRCGYAGRAANGG
moB180r
TACCABTGAAYSGTRCGCATMC
moB185r
TCRTACCABTGAAYSGTRCGCAT
Recovery of full-
gene
52/20/30”
52/5/30”  48/ 20/30”
sequences22
Group O
Group W
Group Z
Recovery of 16S gene
Round 1
T1methph29F1
TTGAACGCTGGCGGYATGCT
candidates, Groups
(enrichment)
T1methph1292R1
AGGACCGGCTTTBTGGGATT
Round 2 (bulk
T1methph44F2
ATGCTTAACACATGCAAGT
amplification
T1methph1279R2A
TGGGATTTGCTGACTTTCGC
from 5% of
T1methph1279R2B
TGGGACTTGCTCCACCTCGC
enrichment)
T1methph1279R2C
TGGGATTAGCTTACTCTCGC
T1methph1279R2D
TGGGATTTGCTGACTCTCGC
T1methph1279R2E
TGGGATTGGCTCACTTTCGC
OPU1 and
52/20/30”
OPU33
52/8/60”  48/25/60”
Recovery of marine
pxm gene
sequences4
Amplification
pxmA_cons_f
TMTTTGCCTAYCACTACTGGAAYT
from sediment
pxmA_cons_r
GTGCCGCGCTCRATGATGCG
Amplification
pxmA550f
CCSASYAAYTGGCCSMTYTT
from water
pxmC310r
AGGWRYYRTCCTGYTCGG
Group OPU1
pmoA_OPU1qPCR_242f
TTACCCCGATCATGCTGGTT
pmoA5
pmoA_OPU1qPCR_312R
GATTCTGAAGTGTTCCCAAACGA
Taqman probe
TTCCCAGCCGCTGTTCAGGCA
Group OPU3
pmoA_OPU3qPCR_492f
TTGCACCTTTACATYTACCTGTTGA
pmoA
pmoA_OPU3qPCR_612r
ACCTTTTTCTACCATTCYGATRTACTC
Taqman probe
ACAACGGCATGATGTTTACTGTTGCTGATTTA
Group OPU1
16S_OPU1qPCR_f
CAATGCCGCGTGTGTGAA
16S rRNA gene
16S_OPU1qPCR_r
CCTCTCTTCCTCCCGACTGAA
candidate
Taqman probe
AGGCCTGCGGGTTGTAAAGCA
Group OPU3
16S_OPU3qPCR_f
AGCACTTTCAATTGGGAGGAAA
16S rRNA gene
16S_OPU3qPCR_r
GCCGGTGCTTCTTCTAAAGGT
candidate
Taqman probe
MAGCTGGGTTAATAGCCCYGCTCTTGACA
Pelagic pxmA
wc_pxmA_qPCR_f
CCGATTGAAGCAGGCAAC
sequence
wc_pxmA_qPCR_r
TGCCAGCACGGATATATTGG
Taqman probe
AGCTAACCTTGGCTGACATGTTCGGCT
pmoA_GroupC_qPCR_f
CCGCGTTTCAGGCGATATT
pmoA_GroupC_qPCR_r
CAGGCGGTCGCACCAA
Taqman probe
TGGTACCTGTTTCGGCTGCC
52/35/30”
52/5/60”  48/ 30/60”
column
qPCR5,6
Group C pmoA
1
60/40
60/40
60/40
60/40
57/40
57/40
MISA, which targets the non-coding region between pmoC and pmoA among ammonia-oxidizing and
methane-oxidizing proteobacteria, was performed as described previously (Tavormina et al., 2010)
with a single primer modification to the reverse primer in the enrichment round, to reduce nonspecific
products in the final bulk amplicon fraction. The modified primer was validated in parallel with the
published assay against ten cultured ammonia-oxidizing and methane-oxidizing cultured strains as
previously described, and provided results indistinguishable from the original assay. The primers used
in this study have been optimized to the detection of proteobacterial Cu-MMO-encoding organisms,
and thus the detection of verrucomicrobial, Gram-positive, or Archaeal Cu-MMO-encoding gene
sequences was beyond the scope of this study. For amplification of fluorescent PCR product,
pmoC_spacer626f includes a fluorescent tag (Well-Red D1, Sigma Proligo). To construct clone libraries,
non-fluorescent MISA fragments were cloned into the pSmart-GC-Kan system (Lucigen, Middleton, WI)
following the manufacturer’s guidelines. This system suppresses expression of insert DNA, and
increases cloning efficiency of the MISA fragment. For each library, more than 48 colonies were
selected for screening. Clones containing inserts of the correct size (between 70 and 95% of each
library; the remainder of the libraries represented cloning of primer-dimers) were digested with RsaI
and separately with HaeIII to assess restriction patterns. Clones demonstrating unique RFLP patterns
were sequenced.
2 To
recover full-length pmoA sequence from Cu-MMO variants, a well-conserved region in pmoB was
targeted for primer design of 6 degenerate reverse primers. For each individual Cu-MMO sequence
targeted, genotype-specific forward primers were designed from MISA fragment sequence. A 20-cycle
enrichment round was performed using appropriate environmental template DNA, one of the
genotype-specific forward primers and each of the degenerate reverse primers in separate reactions.
This enrichment round was followed by qPCR amplification with internal genotype-specific primers on
each of the enrichment products, to determine the most efficient reverse primers for each target.
From this information, nested PCR was performed on the enrichment PCR product, to recover and
sequence a full-length Cu-MMO-A gene fragment. To validate the recovered full-length sequence,
specific reverse primers were designed from the recovered sequence, and paired with specific forward
primers, to amplify the Cu-MMO target from environmental DNA in a single 35-cycle round of PCR, and
the resulting amplicon was sequenced directly.
3 To
identify candidate 16S rRNA gene sequences from groups OPU1 and OPU3, representative
methane-oxidizing 16S rRNA gene sequences (accession numbers: DQ852349, AF150804, NR_043450,
NR_025039, NR_043562, X72771, L20840, NR_025132, U77533) were aligned, and two conserved
regions at positions 29-62 and 1259-1292 (M. capsulatus strain Bath numbering) were targeted for
primer design. Reverse primers for the bulk amplification of 16S rRNA gene targets were combined into
an equimolar cocktail. This strategy targets 16S rRNA gene sequences from gammaproteobacterial
methanotrophs, but may also amplify non-methanotrophic 16 rRNA gene sequences from organisms
including Kangiella and Beggiatoa. Amplicons were generated from environmental DNA from above
Mound 12 (AT15-44 CTD1, 700 meters below sea level, [mbsl]) as well as from a previouslycharacterized bathypelagic DNA sample from the Santa Monica Basin that harbors abundant OPU1 and
OPU3 microorganisms (SMB mound above clambed, (Tavormina et al., 2010). Amplicons were cloned
and analyzed as described previously (Tavormina et al., 2008).
4 To
recover pxm genes from the marine environment, water column (AT15-59 CTD11, Jaco Scarp 1714
mbsl) and sediment (Md. Quepos, 0-3 cm) samples were targeted with consensus primers
pxmA_cons_f and pxmA_cons_r (Table S2). The sediment sample yielded an amplicon whose sequence
affiliated (78% DNA identity, 92% protein similarity) with terrestrial soil and freshwater pxmA
sequences. Alignment of this fragment with existing pxmA sequences allowed the design of a new
degenerate consensus primer, pxmA550f, which when paired with degenerate consensus primer
pxmC310r, resulted in the amplification of a ~2-kb pxmABC-encoding fragment from marine water
column DNA (AT15-59, cast CTD11, 1714 mbsl).
5 Quantitative
PCR assays (Taqman qPCR) for the OPU1 and OPU3 pmoA clades, OPU1 and OPU3
candidate 16S rRNA gene sequences, marine water column pxmA sequence, and for Group C pmoA,
were developed using Primer Express v2.0 (Applied Biosystems, Foster City, CA, USA), based on all
available related sequences (>96% identity), including sequences recovered in this study. The assays in
this study may not reliably enumerate these clades in environments where significant divergent gene
evolution has occurred. Quantitative PCR was performed on a 7300 real-time PCR system (Applied
Biosystems) using a two-step PCR (94oC, 15 s and 57oC or 60oC, 60 s) for 40 cycles. Standard curve
analysis on linearized control templates over 5 orders of magnitude indicated an amplification
efficiency >95% for all assays. Signal from nontarget CuMMO-encoding sequences was below the level
of detection, indicating that each assay is specific for its designated target. For the 16 water samples
collected in AT15-59 CTD11, qPCR was conducted in triplicate. Deviation in estimated abundance
between these triplicate measurements was typically between 10 and 30% for individual samples. For
all other casts and dives (~100 environmental samples), single qPCR assays were conducted for all
targets. qPCR amplicons for pmoA gene targets of groups OPU1, OPU3, and Group C, and 16S rRNA
gene targets, were sequenced bi-directionally to validate the identity of amplified sequences.
6
Sequencing throughout this study was performed using a commercial service (Big Dye chemistry,
Laragen, Culver City, California) and all sequences have been deposited in Genbank (Accession
numbers JX569083-JX569145).