1
Supplementary Material for
Uncultivated DPANN archaea are ubiquitous inhabitants of global oxygen
-
deficient zones
with diverse metabolic potential
Irene H. Zhang
1
#
, Benedict Borer
1
,
Rui Zhao
1
, Steven Wilbert
2
, Dianne K. Newman
2,3
, and
Andrew R.
Babbin
1#
1
Department of Earth, Atmospheric and Planetary Sciences, Massachusetts Institute of
Technology, Cambridge,
Massachusetts
, USA
2
Division of
Biology and Biological Engineering
,
California Institute of Technology
, Pasadena,
California, USA
3
Division of
Geological and Planetary Sciences, California Institute of Technology
, Pasadena,
California, USA
#
Address correspondence to Irene H. Zhang,
izhang@mit.edu
and Andrew R. Babbin,
babbin@mit.edu
Table of Contents
Supplementary Methods
Supplementary References
Supplementary Tables S1
–
S2
Supplementary Figures S1
–
S3
2
Supplementary Methods
Sequence selection and g
enetic engineering
DPANN
nos
Z
-
like genes from ODZ
metagenome
-
assembled genomes were extracted and aligned
with MAFFT
-
linsi v7.450
using the
--
leavegappyregion parameters
(1)
. Three representative
sequences were obtained by clustering the DPANN
nos
-
like genes
at 90% nucleotide
identity with
vsearch
(2)
.
P
utative
nos
Z
-
like gene sequences were optimized for the
Pseudomonas
aeruginosa
genome using the IDT codon optimization tool. These fragments were synthesized as
gBlocks from Twist Bioscience. Using gBlocks as template, the fragments were amplified with
homologous overhangs using PCR
primers (Table S1).
Products were cloned into the pJM220
plasmid linearized by KpnI and HindIII digestion using Gibson assembly (NEB). Putative
nos
Z
-
like gene containing plasmids were introduced at the attTn7 site in the PA14(∆
nosZ
) strain genome
(3, 4)
.
Strains with integrated plasmids will drive gene exp
ression using a rhamnose
-
inducible
promotor when grown in the presence of l
-
rhamnose
.
Growth conditions and N
2
O reduction tests
Three
Pseudomonas aeruginosa
PA14
∆
nosZ
strains containing the putative
nos
Z
-
like genes were
grown on LB plates at 3
7
°
C
overnight along with the parent wild
-
type
PA14
(positive control)
and P
A14
∆
nosZ
(negative control). Colonies were inoculated into 10 mL Luria
-
Bertani (LB) broth
within serum vials. Serum vials containing each strain, along with a cell
-
free control, were capped
and sealed post
-
inoculation and grown within a 37
°
C incubator with shaking at 100 rpm. A total
of 4 replicates were performed for each strain. After cultures used up the available oxygen to reach
stationary growth phase, sterile 50
μ
mol L
-
1
nitrate and 0.02% l
-
rhamnose w/v were injected into
each serum bottle to drive denitrification and the expression of the
nos
Z
-
like genes. After 24 hours
of growth at 37
°
C
, N
2
O concentrations were measured using a Unisense N
2
O microelectrode
connected to the Unisense Field Multimeter according to manufacturer protocols. N
2
O
concentrations were normalized to the average N
2
O readout of the P
A14
∆
nosZ
control.
Supplementary References
1.
Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7:
improvements in performance and usability.
Mol Biol Evol
30:772
–
780.
2.
Rognes T, Flouri T, Nichols B, Quince C, Mahé F. 2016. VSEARCH: a versatile open
source tool for metagenomics.
PeerJ
4:e2584.
3.
Choi K
-
H, Schweizer HP. 2006. mini
-
Tn7 insertion in bacteria with single attTn7 sites:
example
Pseudomonas aeruginosa
.
Nat Protoc
1:153
–
161.
4.
Jeske M, Altenbuchner J. 2010. The
Escherichia coli
rhamnose promoter rhaP(BAD) is in
Pseudomonas putida
KT2440 independent of Crp
-
cAMP activation.
Appl Microbiol
Biotechnol
85:1923
–
1933.
3
Supplementary
Tables
putnos1_DDOPOKPK_
00765
_
Opt
putnos2_APMGHGFK_0
0118
_
Opt
putnos3_DAILHJJC_
00524_Opt
Forward
primer
CAGGAATTCCTCGAG
AAGCTTATGAAGAAT
AAAGTCCTCATCATC
CAGGAATTCCTCGAG
AAGCTTATGAAGAAG
TACCTGCTCATC
CAGGAATTCCTCGAG
AAGCTTATGCTCGTC
GGGATCGTGTC
Reverse
primer
GGGAACTGGTGGTCT
CGTAAGGTACCTCGC
GAATCAGAACG
CGTTCTGATTCGCGA
GGTACCTTATTCGACC
ACGAGGGTCC
CGTTCTGATTCGCGA
GGTACCTTACTTCACG
ATCAGCTTCCC
Table S
1
:
Primers used to amplify DPANN
nosZ
-
like gene fragments with overhangs for each of
the 3 representative gene sequences.
Parameter
Values
Producer radius R [μm]
[0.5,
0.75
, 1.0,
1.25
, 1.5]
Consumer radius
r
[μm]
[0.05, 0.1,
0.25, 0.5]
Cell distance d [μm]
[0,
0.
1,
0.5, 1, 2
]
Table S
2
:
List of parameters
used in model
including specific values used for all simulations.
4
Supplementary
Figures
Figure S1
(continued on next page)
5
Figure S1:
(
A
)
Percentage of the total community assigned to the Archaea domain, with DPANN
archaeal phyla grouped together (teal). Other archaea are colored by phylum.
(
B
)
Proportion of
total archaeal reads belonging to the DPANN superphylum vs. other archaeal taxa, with DPANN
archaeal phyla grouped together (orange). Other archaea are colored by phylum. All bars are scaled
to 100%.
6
Figure S2:
Prediction of protein location for
(
A
)
canonical
nosZ
-
encoded protein
(
B
)
DPANN
nosZ
-
like protein
(
C
)
cytochrome c oxidase subunit II. Protein topologies and locations were
predicted
using DeepTHHMM.
Probability scores on the y
-
axis indicate predicted probability of
the protein region as belonging to
a signal peptide sequence (
blue
), cytoplasmic (inside membrane,
p
urple
), membrane
-
associated (
orange
), or outside the membrane such as in the periplasmic space
(
green
).
Amino acid positions are indicated on the x
-
axis.
7
Figure S3:
N
2
O concentrations after 24 hours of anaerobic growth in LB supplemented with 50
μ
mol L
-
1
nitrate and 0.02% rhamnose to drive expression from the DPANN
nosZ
-
like gene within
the
P. aeruginosa
PA14 ∆
nosZ
background. N
2
O concentrations are normalized to the average
N
2
O concentration for the parent
PA14 ∆
nosZ
control (i.e., 25 μmol L
-
1
N
2
O). The putnos1,
putnos2, and putnos3 strains correspond to the 3 representative
nosZ
-
like genes after clustering
DPANN
nosZ
-
like sequences at 90% nucleotide identity. Wild
-
type
PA14 and a cell free control
are also shown.