Download In-situ expressions of comammox Nitrospira along the Yangtze River

Water Research 200 (2021) 117241
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In-situ expressions of comammox Nitrospira along the Yangtze River
Shufeng Liu a,b,1, Hetong Cai a,b,1, Jiawen Wang a,b, Haiying Wang a, Tong Zheng a,b,c,
Qian Chen a,b,d, Jinren Ni a,b,d,∗
a
Key Laboratory of Water and Sediment Sciences, Ministry of Education, College of Environmental Sciences and Engineering, Peking University, Beijing
100871, China
b
State Environmental Protection Key Laboratory of All Material Fluxes in River Ecosystems, Peking University, Beijing 100871, China
c
South China Institute of Environmental Sciences, Ministry of Environmental Protection (MEP), Guangzhou 510655, China
d
State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, Xining 810016, China
a r t i c l e
i n f o
Article history:
Received 8 February 2021
Revised 7 May 2021
Accepted 9 May 2021
Available online 13 May 2021
Key words:
genome-centric metatranscriptomics
complete nitrifiers
comammox Nitrospira
in-situ expression patterns
environmental drivers
Yangtze River
a b s t r a c t
The recent discovery of comammox Nitrospira as complete nitrifiers has significantly enriched our understanding on the nitrogen cycle, yet little is known about their metabolic transcripts in natural aquatic
ecosystems. Using the genome-centric metatranscriptomics, we provided the first in-situ expression patterns of comammox Nitrospira along the Yangtze River. Our study confirmed widespread expressions of
comammox Nitrospira, with the highest transcription accounting for 33.3% and 63.8% of amoA and nxrAB
genes expressed in ammonia-oxidizing prokaryotes (AOPs) and Nitrospira sublineages I/II, respectively.
Moreover, comammox two clades differed in nitrification, with clade A acting as the dominator to ammonia oxidation in comammox, and clade B contributing more transcripts to nitrite oxidation than to
ammonia oxidation. Compared to canonical Nitrospira, comammox community had lower expressions of
ammonia/nitrite transporters and nitrogen assimilatory genes, but far higher expressions in urea transport and hydrolysis, facilitating to derivation of ammonia and energy mainly through intracellular ureolytic metabolism. This suggests no need for “reciprocal-feeding” between canonical Nitrospira and AOPs
in a natural river. Aerobic mixotrophy of comammox bacteria was suggested by expressions of genes
coding for respiratory complexes I-V, oxidative/reductive TCA cycle, oxygen stress defenses, and transport/catabolism of simple carbohydrates and low-biosynthetic-cost amino acids. Intriguingly, significant
positive correlations among expressions of ammonia monooxygenases, hydroxylamine dehydrogenase and
copper-dependent nitrite reductase indicated that comammox Nitrospira had the potential of converting
nitrite to nitric oxide accompanied by ammonia oxidation under low-C/N and aerobic conditions, while
gene expressions in this pathway were significantly and positively associated with pH. Overall, this study
illustrated novel transcriptional characteristics of comammox Nitrospira, and highlighted the necessity of
reassessing their contributions to biogeochemical carbon and nitrogen cycling with perspective of in-situ
meta-omics as well as culture experiments.
© 2021 Elsevier Ltd. All rights reserved.
1. Introduction
Nitrification has been assumed as a two-step process mediated
by distinct chemolithoautotrophic microbes: ammonia-oxidizing
archaea (AOA) (Könneke et al., 2005) or bacteria (AOB), followed
by nitrite-oxidizing bacteria (NOB) (Teske et al., 1994). However,
the recently discovered complete ammonia-oxidizing (comammox)
Nitrospira, catalyzing ammonia oxidation to nitrate in single organisms, have fundamentally updated the long-held dogma of two∗
Corresponding author: Jinren Ni, Peking University, No. 5 Yiheyuan Road, Beijing
100871, China, Telephone number: +86-10-62751185
E-mail address: [email protected] (J. Ni).
a
The first two authors contributed equally to this work.
https://doi.org/10.1016/j.watres.2021.117241
0043-1354/© 2021 Elsevier Ltd. All rights reserved.
step nitrification (Daims et al., 2015; van Kessel et al., 2015; Pinto
et al., 2016; Palomo et al., 2016). So far, all known comammox
bacteria belong to sublineage II of Nitrospira, which represents
the most diverse NOB clade (Poghosyan et al., 2019). Comammox Nitrospira harbor a full series of ammonia monooxygenases
(AMO), hydroxylamine dehydrogenases (HAO) and nitrite oxidoreductases (NXR) driving complete nitrification (Daims et al., 2015;
van Kessel et al., 2015). The amo operons of comammox Nitrospira
form two divergent clades, named as comammox clades A and B,
which are phylogenetically distant from homologs of AOA and AOB
(Daims et al., 2015; van Kessel et al., 2015).
Comammox Nitrospira are the third group of ammoniaoxidizing prokaryotes (AOPs), contributing largely to nitrifier abundances and activity on Earth. For example, comammox Nitrospira
S. Liu, H. Cai, J. Wang et al.
Water Research 200 (2021) 117241
have been observed to be the most abundant AOPs or occupy
high proportions in overall Nitrospira of engineered habitats, including drinking water systems (Wang et al., 2017), groundwater
wells (Daims et al., 2015), groundwater-fed rapid gravity sand filters (Palomo et al., 2016; Fowler et al., 2018), recirculating aquaculture systems (Bartelme et al., 2017) and biological nutrient removal systems (Daims et al., 2015; Camejo et al., 2017; Zhao et al.,
2019; Cotto et al., 2020). Global-, continental- or region-scale studies have indicated that comammox Nitrospira outnumber canonical AOPs or Nitrospira in natural ecosystems including freshwater (Palomo et al., 2019), soils (Hu and He, 2017; Palomo et al.,
2019; Osburn and Barrett, 2020) and sediments (Shi et al., 2020; Y.
Xu et al., 2020; Zhang et al., 2020). Higher ammonia-oxidation activity of comammox Nitrospira than those of either AOA or AOB has
been demonstrated in some wastewater treatment plants (WWTPs)
(Zheng et al., 2019) as well as pasture, arable, forest and paddy
soils (Li et al., 2019; Wang et al., 2019).
In recent years, genome-centric metagenomics has been utilized
to gain the taxonomic and functional information of uncultured
species from complex microbial assemblages (Speth et al., 2016).
To date, multiple high-quality draft genomes of comammox Nitrospira have been reconstructed to interpret their taxonomic affiliations, metabolic potentials, evolutionary history besides relative abundances in environments (Daims et al., 2015; van Kessel
et al., 2015; Pinto et al., 2016; Camejo et al., 2017; Wang et al.,
2017; Lawson and Lücker, 2018; Palomo et al., 2018; Koch et al.,
2019). Comparative genomics has demonstrated the niche specialization among comammox clades and canonical nitrifiers in carbon and nitrogen metabolisms, alternative electron donors, energy conservation and transduction, and stress response and defense mechanisms (Camejo et al., 2017; Lawson and Lücker, 2018;
Palomo et al., 2018; Koch et al., 2019). Comammox Nitrospira might
utilize mixotrophic metabolisms as well as tolerate microaerobic
and oligotrophic conditions (Palomo et al., 2018). The identification of novel metabolic pathways and interactions among key nitrifiers has revolutionized our understandings of nitrogen-cycling
microbes, requiring further validation at the transcriptional level
(Lawson and Lücker, 2018; Koch et al., 2019). Genome-centric
metatranscriptomics can provide exact information about the insitu expression patterns of a vast number of functional genes comprehensively. It is urgent to characterize the community-wide expression profiles of comammox Nitrospira by a combination of genomic and transcriptomic data in the environments.
Rivers are of primary importance in coupling the biogeochemical cycle between continents and oceans (Li et al., 2020). Global
rivers transport millions of tons of bioavailable nitrogen annually
(Meybeck, 1982), in which human activities contribute to increasing nitrogen input (Koch et al., 2015). Nitrifiers, associated with
denitrifiers and anammox bacteria, are crucial to maintain the balance of nitrogen load in large rivers (Huang et al., 2018). Previous studies have demonstrated the widespread existence of comammox Nitrospira, and the significant influences of environmental factors on their relative abundances and metabolic functions in
rivers (Black and Just, 2018; Liu et al., 2020; Zhang et al., 2020).
Moreover, newly recovered comammox Nitrospira genomes have
improved the phylogenomic resolution to facilitate further research
on biogeography (Liu et al., 2020). Nevertheless, the lack of knowledge so far about the in-situ expression profiles of comammox Nitrospira in large rivers restrains our understanding of the ecological
significances of complete nitrification in complex natural freshwater habitats.
To address this issue, we implemented synchronous monitoring
along the Yangtze River, the largest river in Asia, and obtained the
first in-situ expression profiles of comammox Nitrospira using the
genome-centric metatranscriptomics. Our study demonstrated the
widespread expressions of comammox Nitrospira, with the high-
est transcriptional level in the most upstream site of study area.
Particularly, a difference of nitrification characteristics between comammox clade A and B was found. Compared to canonical Nitrospira, comammox community had lower expressions of ammonia/nitrite transporters and nitrogen assimilatory genes but higher
expressions in urea degradation, enabling them to derive ammonia and energy intracellularly. This implied no need for “reciprocalfeeding” between canonical Nitrospira and AOPs. Comammox bacteria might potentially transform nitrite to nitric oxide (NO) during ammonia oxidation under low-C/N and aerobic conditions, and
gene expressions in this pathway were closely related with pH.
2. Materials and Methods
2.1. Sample collection and environmental data measurement
The nitrogen load along the mainbody of the Yangtze River has
drawn great attention (Liu et al., 2018). A thorough understanding
of metabolic characteristics of comammox Nitrospira and canonical nitrifiers is critical for optimizing nitrogen-load management
strategies therein. In March 2018, planktonic microbial biomass for
metatranscriptomic sequencing was synchronously sampled in ten
national hydrologic stations from YiBin (YB) in the upper reach to
XuLiuJing (XLJ) in the estuary (Fig. 1a; Table S1), where we could
be aided with sufficient equipment and licenses from Changjiang
Water Resources Commission for the RNA sampling campaign. The
longitude and latitude of each site were recorded by GPS device
(Magellan, USA), and the curvilinear distances between pairwise
sites were calculated using ArcGIS (v 10.3). No extreme weather
took place during the sampling period. At each site, 20 L surface
water was collected in the midstream position with a depth of
0.5 m, then was divided into several parts at a time and passed
through multiple 0.22 μm polycarbonate membranes (Millipore,
USA) to capture the biomass immediately. All filtered membranes
were kept in 50 mL sterile and RNase-free tubes, and snap-frozen
in liquid nitrogen until RNA extraction. Total time from the beginning of filtration to snap-freezing was ~12 min. Another 5 L surface water was collected for measurements of environmental factors, including temperature, pH, dissolved oxygen (DO), chemical
oxygen demand (COD), total nitrogen (TN), ammonia (NH3 -N), nitrite (NO2 -N), nitrate (NO3 -N), total phosphorus (TP), conductivity,
hardness and suspended solid (SS) concentration according to the
issued protocols by Ministry of Ecology and Environment of China.
The environmental factors of each water sample were displayed in
Fig. S1.
2.2. RNA extraction, metatranscriptomic sequencing and
bioinformatic pipeline
The 0.22-μm filtered membranes were shattered and added to
extraction tubes. Total RNA of each sample was extracted from
biomass in multiple times using the FastRNA®Pro Soil-Direct Kit
(MP Biomedicals, USA) according to the manufacturer’s instruction. Residual genomic DNA was removed from total RNA using
the PureLink®DNase set (Thermo Fisher Scientific, USA). Replicated RNA extracts of individual samples were mixed for RNA
quality and quantity evaluation by the NanoDrop ND-20 0 0 instrument (Thermo Fisher Scientific, USA). RNA integrity was checked
using the Bioanalyzer RNA 60 0 0 Nano kit (Agilent Technologies,
USA). High-quality nucleic acids (> 2 μg and 100 ng μL−1 ) were
obtained for downstream processing. Ribosomal RNA (rRNA) was
removed using the Ribo-Zero Magnetic Kit (Epicenter, USA). The
enriched non-rRNA samples went through library preparation for
complementary DNA (cDNA) synthesis using the TruSeqTM RNA
Sample Prep Kit (Illumina, USA). The cDNA was sequenced on
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Water Research 200 (2021) 117241
Figure 1. In-situ transcriptional abundances of the key ammonia- or nitrite-oxidation genes in comammox Nitrospira and canonical nitrifiers along the Yangtze River. (a) The
sampling sites for metatranscriptomic shotgun sequencing over a 2,750 km continuum along the Yangtze River. Detailed information about the sampling sites is listed in Table
S1. (b) Expressions of amoA genes in AOPs and nxrAB genes in Nitrospira representatives; (c) Clade-level percentages of expressions of amoABC and nxrAB genes in comammox
Nitrospira; (d) Genome-level percentages of expressions of amoABC and nxrAB genes in comammox Nitrospira. The expressions of amoA genes in AOPs are calculated based
on the normalized gene hits into the AOPs-amoA database. The expressions of nxrAB genes in Nitrospira genera, and the clade- and genome-level percentages of amoABC and
nxrAB genes in comammox Nitrospira are calculated based on the normalized hits into the corresponding genes of 36 Nitrospira genomes.
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Water Research 200 (2021) 117241
the Illumina HiSeq X Ten platform (Majorbio Company, Shanghai, China) to generate 150 bp paired-end reads with an insert size of 300 bp. All raw metatranscriptomic datasets can be
found at the NCBI Sequence Read Archive under accession numbers SRR13151498-SRR13151507.
Raw paired-end reads were initially pretreated using Sickle (v
1.200) (-q 20 -l 50) (https://github.com/najoshi/sickle) and NGS
QC Toolkit (v 2.3.3) (-l 70 -s 20) (Patel and Jain, 2012). Then,
rRNA sequences were trimmed off using SortMeRNA (v 2.0) (–
num_alignments 1 -e 10−10 ) based on SILVA database for bacterial, archaeal and eukaryotic sequences (Kopylova et al., 2012).
Clean non-rRNA reads from large datasets were randomly split
into small pieces, and individually de novo assembled into transcript sequences with a minimum length of 500 bp using Trinity (v 2.8.1) (Grabherr et al., 2011). Statistics of raw RNA reads,
clean non-rRNA reads and assemblies of each dataset were summarized in Table S2. Open reading frames (ORFs) were predicted on the assembled sequences using TransGeneScan (v 1.2.1)
(Ismail et al., 2014). Clean non-rRNA reads were mapped back
to the predicted nucleotide ORFs using BBMap (v 37.48) (https:
//sourceforge.net/projects/bbmap) with minimum identity of 90%.
Read counts were calculated for each ORF and normalized as RPKM
values (Reads per kilobase per million mapped reads). All ORFs
were searched against Kyoto Encyclopedia of Genes and Genomes
(KEGG) database for functional annotation using DIAMOND (v
0.8.38) with the parameters “-e 1e-5 -f 6 -k 1” (Buchfink et al.,
2015). Key ammonia-oxidation transcript sequences were queried
against the NCBI non-redundant (nr) database using BLASTP (v
2.2.31+) for manual inspection.
related genes were identified by profile Hidden Markov Model
searches against Carbohydrate-Active enZYmes (CAZy) database
(Lombard et al., 2014). Metabolic reconstruction was based on
KEGG pathway maps. The subcellular localization of the identified
glycoside hydrolases and peptidases were predicted using CELLO (v
2.5) (Yu et al., 2004). Clean non-rRNA reads of each dataset were
searched against the NCBI nr database using DIAMOND, with the
following analysis in MEGAN (v 6.11.1) (Huson et al., 2007) using
the lowest common ancestor algorithm to extract Nitrospira reads
based on the taxonomy (Black and Just, 2018). BBMap was applied
to align all these Nitrospira reads against the genomic nucleotide
ORFs with a minimum identity of 90%. Such two-stage alignments
might facilitate to extract comammox reads more accurately. According to Liu et al. (2020), Cotto et al. (2020) and controlled experiments in this study (see Supplementary Information), a minimum alignment identity of 90% could reduce cross-assignments
of reads between comammox and canonical Nitrospira genomes
used herein. Especially, no cross-assignments was observed for
key nitrogen-cycling genes between comammox and canonical Nitrospira. For comparisons of the ammonia- or nitrite-oxidation
transcriptional abundances among distinct Nitrospira communities,
read counts for the Nitrospira amoABC or nxrAB genes, respectively,
were normalized by sequence depths and expressed as the hit
numbers per 108 clean non-rRNA reads. For profiling the expressions across different functional genes, read counts were calculated for each genomic ORF and normalized as the RPKM values by
mapping reads and ORF lengths. Functional-gene expression levels
were inferred based on the overall RPKM values of ORFs assigned
to a given orthologous gene of KEGG or COG.
2.3. Phylogenetic analysis of key ammonia-oxidation transcript
sequences
2.5. Statistical analysis
All statistical analyses were performed based on the
community-wide expression profiles of comammox Nitrospira and canonical nitrifiers using R language (v 3.5.0;
https://www.r-project.org/) or OriginPro 2018. p < 0.05 (with
999 permutations) was considered significant for all statistical
tests. Non-metric multidimensional scaling (NMDS) (“metaMDS”
function in vegan) was used to visualize the Bray-Curtis dissimilarities of orthologous-gene expression compositions between
comammox and canonical Nitrospira. Analysis of similarity
(ANOSIM) (“anosim” function in vegan) was then conducted
to test the significance of difference. Distance-based redundancy
analysis (dbRDA) (“capscale” function in vegan) was executed to
quantify the overall effects of environmental factors and geographic distance on orthologous-gene expression compositions of
comammox and canonical Nitrospira (Supplementary Information).
Pairwise Spearman’s (“rcorr” function in Hmisc) or Pearson’s
correlations among the expressions of key nitrification-related
genes and environmental factors were calculated to identify niche
preferences.
All key amo and hao amino-acid transcript ORFs of comammox
Nitrospira were identified based on the above functional annotation
results. Some reference and outgroup sequences were also downloaded from the NCBI protein database. All sequences were aligned
using MAFFT (v 7.310) (Katoh and Standley, 2013). The alignments
were then trimmed using TrimAl (v 1.3) (Capella-Gutiérrez et al.,
2009) to remove poorly aligned regions (i.e., columns composed
of over 90% gaps). Maximum-likelihood phylogenetic trees were
built based on 1,0 0 0 bootstraps using FastTree (v 2.1) (Price et al.,
2010), where a Jones-Taylor-Thornton evolutionary model and CAT
approximation with 20 rate categories were employed.
2.4. Evaluation of in-situ expressions of comammox Nitrospira and
canonical nitrifier communities
To compare the ammonia-oxidation transcriptional abundances
among comammox Nitrospira, AOA and AOB communities, a hybrid annotation pipeline (Yang et al., 2014) was conducted to
screen clean non-rRNA reads against our manually constructed
amoA database. Detailed procedures and parameters refer to our
previous study (Liu et al., 2020). The amoA-like hits were normalized by sequence depths among different samples, which were expressed as the hit numbers per 108 clean non-rRNA reads.
To obtain the expression profiles of comammox versus canonical Nitrospira, genome-centric metatranscriptomics strategies were
executed as follows. A wide range of 36 Nitrospira genomes comprising of 27 separate species were downloaded from the available
databases to form the reference pool (Table S3), which were the
representatives covering the most concerning metabolic features
of Nitrospira. ORFs were predicted for all genomes using Prodigal (v 2.6.3) (Hyatt et al., 2010), and queried against the KEGG
and Clusters of Orthologous Groups (COG) databases using DIAMOND as described above. Additionally, a set of carbohydrate-
3. Results and discussion
3.1. Evidence for transcription of comammox Nitrospira
Comammox Nitrospira transcription or activity has been detected in natural ecosystems such as soils and freshwater sediments, whereas most studies relied on enrichment cultures or microcosm experiments instead of in-situ (Yu et al., 2018; Wang et al.,
2019; Li et al., 2019). To deepen our understandings, metatranscriptomics was utilized here to validate the in-situ expressions of
comammox Nitrospira in the Yangtze River. For all samples, 547
transcript ORFs were assigned to comammox Nitrospira, including 55 ORFs affiliated with comammox amoABC and haoA genes
(Table S4). Both amo and hao operons are reliable biomarkers
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Water Research 200 (2021) 117241
for comammox identification (Yu et al., 2018). Phylogenetic trees
showed that all amoABC and haoA transcript ORFs fell into comammox clade A clusters, and were closely related to the genes
on clade-A-I genomes (e.g., YR-WHU/YR-JJ/YR-XLJ-3) (Fig. S2; Table S3). A total of 50 amoABC and haoA transcript ORFs were assembled from the most upstream site YB of study area with exceptionally higher expressions (average RPKM = 115.8 and 30.4),
whereas only five amoC transcript ORFs were from other sites with
lower expressions (average RPKM = 47.4). This might suggest a relatively high potential of ammonia-oxidation activity of comammox
Nitrospira in the upper reach of the Yangtze. The number and expression of comammox-related amoC transcript ORFs were larger
than those of amoA and amoB, which was consistent with the finding of a previous study (Yu et al., 2018) and could be attributed
to multicopy of amoC in most comammox genomes (Koch et al.,
2019). Comammox-affiliated transcript ORFs were detected at all
sites, suggesting wide expressions of complete nitrifiers along the
Yangtze River.
We compared the transcriptional abundances of comammox Nitrospira and canonical nitrifiers by searching clean non-rRNA reads
against two constructed reference databases. It should be noted
that numbers of comammox reads obtained using the AOP-amoA
amino-acid database and amoABC-gene sequences on all Nitrospira reference genomes were significantly correlated (Spearman’s
r = 0.97, p < 0.001) (Fig. S3), suggesting uniformity of these two
methods in assessing comammox ammonia-oxidation transcripts.
The nxr gene sequences have also been utilized to investigate the
contribution of comammox to overall Nitrospira in nitrite oxidation (Black and Just, 2018; Yu et al., 2018; Fowler et al., 2018),
though more ideal methods are needed. Comammox Nitrospira had
relatively high transcriptional abundances in site YB, constituting
33.3% and 63.8% of AOPs-amoA and Nitrospira-nxrAB gene expressions, respectively (Fig. 1b). At those sites downstream YB, the
transcriptional abundances of canonical AOA/AOB and Nitrospira
sublineages I/II outnumbered comammox Nitrospira by 4.0~69.6
and 14.7~149.9 folds, separately. These results suggested that comammox Nitrospira might contribute more to nitrifying activity in
the upper reach, though they were found expressed widely, which
was consistent with the biogeographic pattern of comammox relative abundance obtained by metagenomics (Liu et al., 2020). Comammox Nitrospira were less abundant and expressed than either AOA or AOB in a few middle- and lower-reach locations
along the Yangtze River. Within comammox Nitrospira, clade AI was the most expressed community in ammonia oxidation, accounting for an average of 79.5% (50~100%) of comammox-amoABC
gene expressions (Fig. 1c). This was generally uniform with the
above results obtained through ORFs identification. Clade A-I was
also the most abundant comammox community over the mainbody of this large river (Liu et al., 2020). Li et al. (2019) and
Zheng et al. (2019) reported that clade A were the most active
comammox in some agricultural soils (N. inopinata cluster in AI) and WWTPs (Ca. N. nitrosa cluster in A-II), but the dominant active species were quite different from those in the Yangtze
River (YR-WHU/YR-JJ/YR-XLJ-3/SG-bin2 in A-I) (Fig. 1d; Table S3).
These findings confirmed broad and flexible niches of comammox clade A in natural and engineered environments, as also suggested by Palomo et al. (2019) based on the global survey. The
most expressed clade-A species were all associated with groundwater, drinking water and riverine ecosystems (Fig. 1d; Table S3),
indicating similar active comammox species among these environments. Notably, clade B contributed far more to comammox-nxrAB
gene expressions (average 28.6%, up to 60.6%) than to amoABC
gene expressions (average 1.3%), and outcompeted clade A in two
sampling sites (Fig. 1c), which suggested a more critical role of
clade B in nitrite oxidation than in ammonia oxidation. No cladeB amoABC ORFs were found on the de novo assembled transcript
Figure 2. Spearman’s correlations (r) between the nitrite-oxidation (nxrAB) transcriptional abundance of comammox clade B, and ammonia- (amoA) or nitriteoxidation (nxrAB) transcriptional abundances of canonical nitrifiers. Color solid lines
indicate the ordinary least square linear regressions, with the shaded area representing 95% confidence intervals. The r and p values of Spearman’s correlations are
stated.
contigs, either (Fig. S2). Moreover, the clade-B nxrAB expressions
and its percentage in comammox nxrAB expressions were all significantly and positively correlated with the amoA and nxrAB expressions of canonical AOPs and Nitrospira (Spearman’s r = 0.82~0.95,
p ≤ 0.003) (Fig. 2). Thus, clade B were speculated to perform the
similar nitrifying feature as canonical Nitrospira in this river reach,
where they might be fed by AOA and AOB. On one hand, distinct
nitrifying niches of comammox two clades might be due to the
fact that they harbored different ammonia uptake systems. Clade
B encode MEP-type ammonia transporters with higher affinity and
lower uptake capacity, while clade A encode Rh-type transporters
with lower affinity and higher uptake capacity (Palomo et al.,
2018). Clade A, rather than clade B, might adapt to the mainstream of the Yangtze River with relatively higher ammonia contents (Liu et al., 2020). On the other hand, high dissimilarity of
amoA gene sequences between clades A and B might be another
reason for their distinct niches in the ammonia-oxidation process
(Palomo et al., 2018). For instance, clade B possessed more abundant amoA genes than clade A in relatively oligotrophic groundwater systems and Upper Yangtze River (Fowler et al., 2018; Liu et al.,
2020). Zheng et al. (2019) detected no ammonia-oxidation activity
of clade B but largely active clade A in various ammonia-enriched
WWTPs. Wang et al. (2019) revealed that clade B could autotrophically grow in forest and paddy soils only in the absence of ammonia amendment. Theoretically, shortening nitrification pathway
with loss of ammonia-oxidation capacity might result in an increased ATP production rate (other than yield) for clade B, a phylogenetic group residing between canonical Nitrospira sublineages
I and II (Palomo et al., 2018). Although more efforts are needed to
illustrate the ammonia- and nitrite-oxidation kinetics of yet uncultured clade B, the comammox two clades may differ in nitrification
characteristics along the Yangtze River, and the nitrite-oxidation
ability of clade B should be highlighted.
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Water Research 200 (2021) 117241
Figure 3. Predicted expression patterns of comammox and canonical Nitrospira communities in the Yangtze River. Pathway arrows or gene names related to carbon
metabolism, aromatic compound cleavage, oxidative phosphorylation, hydrogen metabolism, nitrogen metabolism, stress response and resistance, and membrane transporters are labeled in distinct colors, respectively. Dashed color arrows indicate the incomplete pathways due to expression loss of a few key genes. Solid grey arrows
indicate near-complete pathways with expression loss of only one gene. Pathway or gene expression levels are illustrated by color gradients drawn on various shapes (e.g.
circles, squares, ellipses and polygons). For shapes with two separate parts, the color gradients of the left (or upper) and right (or lower) parts indicate the expression levels
of corresponding pathways or genes in comammox and canonical Nitrospira communities, respectively. For individual shapes with no division, the color gradients indicate
the expression level of corresponding pathways or genes in comammox Nitrospira. Details of expression levels refer to Tables 1, S5 and S6. For clarity of the schematic, the
subcellular locations of genes were ignored unless indicated. Abbreviations of amino acids are labeled in bold fonts with light-grey background. “??”, unconfirmed pathway;
G6P, glucose 6-phosphate; G1P, glucose 1-phosphate; F6P, fructose 6-phosphate; G3P, glyceraldehyde 3-phosphate; PEP, phosphoenolpyruvate; Ru5P, ribulose 5-phosphate;
X5P, xylulose 5-phosphate; R5P, ribose 5-phosphate; PRPP, 5-phosphoribosyl diphosphate; S7P, sedoheptulose 7-phosphate; E4P, erythrose 4-phosphate; Succ, succinate; Fum,
fumarate; Oxa, oxaloacetate; Cit, citrate; Iso, isocitrate; 2-Oxo, 2-oxoglutarate; LPS, lipopolysaccharide; LPT, lipoprotein; TRX, thioredoxin; UDP, uridine diphosphate; GlcNAc,
N-acetylglucosamine; Pi, inorganic phosphate; PPP, pentose phosphate pathway.
3.2. Expression patterns of comammox Nitrospira community
greater than those of complete nitrifiers. Similar to betaproteobacterial AOB, all studied comammox genomes harbor genes for either heme exporter (ccmABCD) or cytochrome c-biogenesis (ccmEFGH) proteins, which are absent in most canonical Nitrospira sublineages I/II (Fig. S6). The expressions of all these genes were observed for comammox instead of canonical Nitrospira. Comammox
amo, hao and ccm operons are located in the proximate genomic
region, and the expressions of almost full series of ccm genes may
confer an advantage in biosynthesis or activation of AMO and HAO
(van Kessel et al., 2015). Compared to canonical Nitrospira, comammox Nitrospira had a higher diversity and transcriptional level of
expressed copper-homeostasis genes (e.g., copACD). Such gene expressions might confer increasing Cu2+ uptake or higher Cu2+ tolerance in large rivers, as comammox bacteria may demand more
copper as cofactor for AMO biosynthesis than canonical Nitrospira
(Palomo et al., 2018).
Besides nitrification-related genes, all analyzed Nitrospira
genomes encode copper-dependent nitrite reductases (nirK) that
form NO from nitrite in denitrifying organisms and some other
nitrifiers (Fig. S6). Interestingly, comammox Nitrospira possessed
a high expression for nirK (average RPKM = 3871.0), which was
the fourth most transcribed gene in comammox transcripts and
exhibited 55.0-fold larger than that of canonical Nitrospira (Fig.
S5; Table 1). A NO-responsive regulator (nnrS) associated with nitrosative stress resistance was also expressed. Similarly, Black and
Just (2018) observed highly abundant nirK genes of comammox
We obtained an integrated transcriptional atlas of comammox
Nitrospira by gathering the data from all samples. NMDS showed
that the orthologous-gene expression compositions of comammox species were different from those of canonical Nitrospira,
which were further confirmed by ANOSIM (r = 0.28, p = 0.004)
(Fig. S4). Comammox and canonical Nitrospira had 1,546 and
1,585 transcribed orthologous genes, respectively, in which clade A
(n = 1,539) had a higher diversity of transcribed orthologous genes
than clade B (n = 158). Clade-A active genes recruited 75.8-fold
higher number of transcriptional sequences than clade B.
3.2.1. Nitrogen metabolism
Distinct expression patterns of the key nitrogen-cycling genes
were observed between comammox and canonical Nitrospira, depicting niche specialization among nitrifiers in this freshwater
ecosystem (Fig. 3; Table 1). Comammox Nitrospira had large expressions of all key genes involved in complete nitrification, occupying 11.0% of comammox transcripts. AmoC was the most transcribed gene for comammox (Fig. S5). Besides low amoABC expressions, clade B also possessed a low expression level of haoA gene
(average RPKM = 4.5), and were then confirmed to play a teeny
role in ammonia and hydroxylamine oxidation. For canonical Nitrospira, nxrAB were the most transcribed genes, the expressions
of which accounted for 13.7% of total transcripts and displayed
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Water Research 200 (2021) 117241
Table 1
Distinct expression patterns of the key nitrogen-cycling genes of comammox and canonical Nitrospira communities in
the Yangtze River.
Orthologous genes in KEGG or COG databases
Comammox Nitrospira
RPKM
ammonia monooxygenase subunit B (amoA)
ammonia monooxygenase subunit B (amoB)
ammonia monooxygenase subunit C (amoC)
hydroxylamine dehydrogenase (haoA)
nitrite oxidoreductase, alpha subunit (nxrA)
nitrite oxidoreductase, beta subunit (nxrB)
heme exporter protein A (ccmA)
heme exporter protein B (ccmB)
heme exporter protein C (ccmC)
heme exporter protein D (ccmD)
cytochrome c-type biogenesis protein ccmE
cytochrome c-type biogenesis protein ccmF
cytochrome c-type biogenesis protein ccmG
cytochrome c-type biogenesis protein ccmH
copper exporting P-type ATPase (copA)
copper resistance protein C (copC)
copper resistance protein D (copD)
putative copper export protein (COG1276) c
copper-dependent nitrite reductase
(NO-forming) (nirK)
nnrS protein involved in response to NO
NO reductase norQ protein
NO reductase activation protein (COG4548) c
regulator of NO reductase transcription
(COG3901) c
Rh-type ammonia transporter
MEP-type ammonia transporter
urea transport system substrate-binding
protein urtA
urea transport system permease protein urtB
urea transport system permease protein urtC
urea transport system ATP-binding protein
urtD
urea transport system ATP-binding protein
urtE
urease gamma subunit ureA
urease beta subunit ureB
urease alpha subunit ureC
urease accessory protein ureF
urease accessory protein ureG
urease accessory protein ureH
carbamate kinase (arcC)
glutamate synthase (NADPH) large chain (gltB)
glutamate synthase (NADPH) small chain
(gltD)
glutamate dehydrogenase (NAD(P)+) (gdhA)
glutamine synthetase (glnA)
nitrogen regulatory protein PII (COG0347) c
nitrate/nitrite transport system
substrate-binding protein nrtA
nitrate/nitrite transport system permease
protein nrtB
nitrate/nitrite transport system ATP-binding
protein nrtC
nitrate/nitrite transporter narK
nitrite transporter nirC
nitrite reductase (NADH) small subunit (nirD)
ferredoxin-nitrite reductase (nirA)
a
Percentage (%)
Canonical Nitrospira
b
RPKM
a
Percentage (%)
2414.6
2102.3
26489.4
1923.1
2771.2
1630.4
20.6
74.9
391.0
619.1
258.7
207.0
211.9
343.9
90.7
26.3
17.4
62.2
3871.0
0.71
0.62
7.79
0.57
0.81
0.48
0.0061
0.022
0.11
0.18
0.076
0.061
0.062
0.10
0.027
0.0077
0.0051
0.018
1.14
NA
NA
NA
NA
54730.0
44394.8
ND
ND
ND
ND
ND
ND
ND
367.5
59.4
NA
NA
ND
70.4
NA
NA
NA
NA
7.54
6.12
ND
ND
ND
ND
ND
ND
ND
0.051
0.0082
NA
NA
ND
0.0097
44.8
104.8
21.9
12.2
0.013
0.031
0.0065
0.0036
ND
314.8
81.5
269.5
ND
0.043
0.011
0.037
12.2
ND
2881.0
0.0036
ND
0.85
NA
58.6
ND
NA
0.0081
ND
232.4
62.6
178.7
0.068
0.018
0.054
ND
ND
ND
ND
ND
ND
47.2
0.014
ND
ND
3.6
126.7
88.5
40.6
16.1
630.6
527.8
72.8
62.2
0.0001
0.037
0.026
0.012
0.0047
0.19
0.16
0.021
0.018
ND
ND
ND
ND
ND
ND
ND
385.2
201.9
ND
ND
ND
ND
ND
ND
ND
0.053
0.028
42.6
141.5
50.0
NA
0.013
0.042
0.015
NA
115.5
1063.6
391.8
42.0
0.016
0.15
0.054
0.0058
NA
NA
7.9
0.0011
NA
NA
4.5
0.0006
ND
NA
28.2
NA
ND
NA
0.0083
NA
6.5
32.3
531.9
19.8
0.0009
0.0045
0.073
0.0027
b
“NA”, the corresponding genes are not available on studied genomes (Fig. S6); “ND”, the expressions of corresponding
genes are not detected.
a
Average values among all samples.
b
Relativizing the RPKM of each orthologous gene by the overall RPKM calculated for total ORFs.
c
Orthologous genes identified from the COG database.
Nitrospira within the mussel sediment of upper Mississippi River
(UMR). Kits et al. (2019) suggested that the purely cultured comammox N. inopinata could release NO under oxic conditions, and
nirK was the second most abundant protein in its proteome, which
supported the above finding in oxygen-saturated river water. Consequently, all metagenomic, metatranscriptomic and kinetic studies
implied that nitrate was not the only obligate product during ni-
trification in comammox Nitrospira, and the denitrification could
lead to NO accumulation. As for other denitrifying genes, all Nitrospira lack the key NO reductases norBC, but encode either a NO
reductase activation protein or transcriptional regulator (Fig. S6).
Comammox Nitrospira had 3.7~22.1-fold lower expressions of these
genes than canonical Nitrospira in the Yangtze River. They are possibly involved in NO detoxification. It was reported that the iso7
S. Liu, H. Cai, J. Wang et al.
Water Research 200 (2021) 117241
Besides H+ -translocating NADH:ubiquinone oxidoreductases (nuo),
ten comammox genomes harbor an additional type of complex
I, i.e., Na+ -transporting NADH:ubiquinone oxidoreductases (nqr),
though the expressions were relatively low. The prototype of nqr
operons is from the marine aerobic bacteria Vibrio alginolyticus
(Steuber, 2001), and all identified nqrA sequences on comammox genomes are most closely related to β -AOB by BLASTP to
NCBI nr database (identity: 68.2~71.5%), implying acquisition by
horizontal gene transfer. Additionally, comammox Nitrospira possessed 8.1-fold higher expressions of hydrogenase-related genes
(e.g., hypF/hyfR) than canonical Nitrospira. These genes might confer comammox bacteria the potential of hydrogen oxidation coupled to sulfur reduction (Camejo et al., 2017).
Glycoside hydrolases, glycosyltransferases and carbohydrate
esterases dominated the transcriptional pool of carbohydrateactive enzymes in Nitrospira (Fig. 3; Table S5). For comammox,
GH23, GH109 and GH74 were the most transcribed glycoside
hydrolase families, encoding chitin/peptidoglycan lyase, acetylgalactosaminidase/hexosaminidase or glucanase, etc. Genes coding
for glucosidase (GH3), amylomaltase (GH77) and amylase (GH13)
were also expressed. Although most of them were predicted to be
cytoplasmic, some GH23 and GH74 could play important roles in
periplasmic or extracellular environments (Fig. 3; Fig. S7). Thus,
complete nitrifiers might participate in carbohydrate breakdown
for riverine bacterial community. Notably, whether microbes can
take up and utilize carbohydrates should depend on transporter
activity (Lücker et al., 2010). We indeed detected the expression
of a monosaccharide ATP-dependent transport system in comammox rather than canonical Nitrospira, indicating a potential of metabolizing simple sugars. GT2, GT4 and GT51 were the most expressed glycosyltransferase families for comammox Nitrospira, acting on the formation of glycosidic bonds. Particularly, comammox
bacteria expressed almost full genes for cellulose, glycogen, DAPtype peptidoglycan or KDO2 -lipid A (a precursor of lipopolysaccharide) biosynthesis, while genes encoding lipoprotein-releasing and
lipopolysaccharide-export systems were also transcribed, which
suggested carbon storage as cell walls, membranes, intracellular and extracellular polymeric substances. Moreover, comammox bacteria expressed genes encoding catechol 2,3-dioxygenase,
monomeric catechol outer-membrane receptor and protocatechuate 3,4-dioxygenase. The ability of hydrolyzing aromatic rings has
been also demonstrated for N. inopinata by Han et al. (2019), gaining fundamental insights into micropollutant transformation by nitrifiers.
Comammox and canonical Nitrospira expressed almost full
genes encoding central carbon metabolic pathways, including the
glycolysis/gluconeogenesis, pentose phosphate pathway and TCA
cycle (Fig. 3; Table S5), suggesting they should be able to metabolize hexose sugars (Lücker et al., 2010). They could also use
ethanol, acetate and pyruvate for replenishing TCA cycle intermediates. High expressions of pyruvate ferredoxin oxidoreductases
were observed (average RPKM = 200.4~5384.9). Some uncultured
Nitrospira have been reported to take up pyruvate in wastewater treatment plants (Daims et al., 2001). The oxidative TCA cycle
shares most enzymes with the reductive TCA (rTCA) cycle except
for citrate synthase (CS) and 2-oxoglutarate dehydrogenase complex (OGDC), while the corresponding key enzymes for rTCA cycle were ATP-citrate lyase (ACL) and 2-oxoacid ferredoxin oxidoreductase (OFOR), respectively (Lücker et al., 2010). We found that
comammox bacteria possessed 5.7-fold higher expression of ACL
than CS, and 6.0-fold higher activity of OFOR than OGDC. Similar results were observed for canonical Nitrospira. Although complete nitrifiers might benefit from a mixotrophic lifestyle by utilizing extracellular urea or carbohydrates, their autotrophic process
by CO2 fixation through rTCA cycle was putatively dominant in the
Yangtze River.
lated comammox N. inopinata could not denitrify NO to nitrous
oxide (N2 O), but produced low N2 O by abiotic conversion of hydroxylamine (Kits et al., 2019). More roles of these NO reduction
chaperone genes in Nitrospira remain to be determined in-depth.
For ammonia uptake, the expression of Rh-type transporter encoded by comammox clade A was 4.8-fold lower than that of MEPtype transporter of canonical Nitrospira, and its contribution to comammox transcripts (0.0036%) was also lower than that to canonical Nitrospira transcripts (0.0081%) (Table 1). The expression of
clade-B ammonia transporter was not detected. It was thus hypothesized that comammox bacteria had relatively weak capacity
of utilizing extracellular ammonia sources in the Yangtze River.
However, ammonia can also originate from intracellular urea hydrolysis. We detected relatively high expressions of high-affinity
urea transporters (urtABCDE), ureases (ureABCFGH) and carbamate
kinase (arcC) in comammox Nitrospira, meanwhile, no expressions
of the corresponding genes were observed for canonical Nitrospira.
Comammox organisms were reported to have an order of magnitude greater potentials for urea degradation than canonical Nitrospira in mussel habitats of UMR (Black and Just, 2018), which was
similar with our results. Urea transport and hydrolysis are more
common metabolisms in comammox than in canonical Nitrospira
(Fig. S6). The observed niche separation in genomes and transcriptomes indicated that complete nitrifiers had advantages in deriving
ammonia and energy through ureolytic activity in a natural large
river. Koch et al. (2015) had proposed a “reciprocal-feeding” interaction between NOB and AOPs, in which Nitrospira could supply
ammonia to AOPs by hydrolyzing urea, while AOPs would subsequently oxidize ammonia to nitrite, offering energy sources for Nitrospira. In the Yangtze River, such reciprocal-feeding seemed not
established due to much higher expressions of ureolytic genes in
comammox bacteria compared to canonical Nitrospira. A significant and positive correlation was observed between expressions
of canonical AOPs-amoA and Nitrospira-nxrAB genes (Pearson’s adjusted R2 = 0.80, p < 0.001, data source from Fig. 1b). Therefore,
canonical AOPs might oxidize ammonia to nitrite, providing canonical Nitrospira with energy sources, but no ammonia was produced
by canonical Nitrospira through ureolytic activity possibly due to
the existence of comammox bacteria in the Yangtze River.
For ammonia assimilation, comammox Nitrospira possessed
3.2~7.8-fold lower expressions of glutamate synthase (gltBD), glutamine synthase (glnA) and nitrogen regulatory protein P-II than
canonical Nitrospira. The contributions of such gene expressions
to comammox transcripts (0.096%) were also lower than those to
canonical Nitrospira transcripts (0.28%). Kits et al. (2017) suggested
that the growth rate of comammox N. inopinata was slower than
those of canonical nitrifiers in oligotrophic and dynamic habitats.
More energy production through ammonia oxidation might help
comammox organisms (especially clade A) to survive in various environments with many competitors, e.g., canonical nitrifiers.
For nitrite uptake/assimilation, no expression was observed for
comammox nitrite transporter (narK), but a range of nitrite transporters (i.e., nrtABC, nirC and narK) of canonical Nitrospira were all
expressed. More importantly, all studied comammox Nitrospira lack
the assimilatory nitrite reductase (nirA) and cytochrome c nitrite
reductases (nrfAH) (Fig. S6), and canonical Nitrospira harbored an
18.9-fold higher expression of dissimilatory nitrite reductase (nirD)
than comammox organisms. These transcriptomic findings demonstrated that comammox Nitrospira could not utilize nitrite as sole
nitrogen source for growth, as proposed by Palomo et al. (2018).
3.2.2. Energy and carbon metabolisms
Genes coding for respiratory complexes I-V are highly conserved in Nitrospira (Poghosyan et al., 2019). In the Yangtze River,
they were more expressed in canonical Nitrospira (2.0% of transcripts) than in comammox Nitrospira (1.4%) (Fig. 3; Table S5).
8
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Water Research 200 (2021) 117241
Peptides and amino acids are important carbon or energy
sources for organisms. Although comammox and canonical Nitrospira expressed many cytoplasmic peptidases, some murein endopeptidases, Zn-dependent carboxypeptidases and aminopeptidases were predicted to be located in outer-membrane, periplasmic or extracellular environments (Fig. 3; Fig. S7). Furthermore,
comammox bacteria expressed genes for a range of amino acid
transporters and catabolic pathways linking alanine, aspartate, asparagine, glutamate, glutamine, glycine, serine, threonine, cysteine
and proline to central metabolisms (Fig. 3; Table S5). This could allow them to utilize proteins and amino acids as alternative carbon
or energy sources. Four most highly-biosynthetic-cost amino acids,
i.e., phenylalanine, tyrosine, tryptophan and histidine (Akashi and
Gojobori, 2002), could be synthesized by comammox bacteria, but
the degradation pathways were incomplete and not that expressing
for all genomes. This suggested that complete nitrifiers have been
selected to potentially degrade low-biosynthetic-cost amino acids.
Interestingly, the expressions of alanine dehydrogenase, glutamate
dehydrogenase, L-aspartate oxidase, glycine dehydrogenase as well
as predicted amidohydrolases (average RPKM = 20.7~306.8) (Table
S5) involved in deamination suggested that amino acids were the
critical nitrogen and energy source besides urea and free ammonia for comammox Nitrospira. Seven newly recovered comammox
genomes from the Yangtze River harbored 1~3 peptidases within
their ammonia-oxidation gene clusters for possible co-expressions
of peptidases, AMO and HAO (Liu et al., 2020), which would provide genomic evidence for the above transcriptomic findings.
Yangtze River. The dbRDA showed that pH and NO2 -N were significantly associated with the compositional variation of orthologousgene expressions of both comammox and canonical Nitrospira
(p = 0.002~0.038), while temperature and TN were only related
to that of comammox Nitrospira (p < 0.020) (Table S7). The above
environmental factors explained a total of 53.2% and 34.5% of expression variation for comammox and canonical Nitrospira, respectively, which were much higher than the overall explanatory proportions by geographic distance (Fig. S8). Moreover, the pure effect (49.3%) of environmental factors on comammox Nitrospira was
about 2-fold higher than that (25.1%) on canonical Nitrospira. These
observations suggested a critical role of environmental selection in
shaping the expression pattern of comammox, rather than canonical Nitrospira. Combined with our metagenomic study (Liu et al.,
2020), we confirmed that environmental selection determined the
community, abundance and expression variation of comammox
Nitrospira along the Yangtze River. Furthermore, specific associations between the expressions of comammox nitrification-related
genes and environmental variables were investigated (Fig. 4; Fig.
S9). We found significant and positive correlations between pH
and the expressions of comammox-amoA, amoC, amoABC, haoA and
nirK genes (Spearman’s r = 0.72~0.89, p < 0.020), while no significant correlations were identified between these gene expressions and any other factors including nutrients (p > 0.11). No
significant relationships were found between comammox nitriteoxidation expression level and all analyzed environmental factors
(p > 0.13). Comammox-amoA abundance was also significantly and
positively correlated with pH in the Yangtze River (Liu et al., 2020).
S. Xu et al. (2020) demonstrated that comammox Nitrospira preferred to grow in slightly alkaline environments. The optimal pH
values for AMO and HAO of canonical AOPs are around 7.0~8.0,
however, it remains unknown about that of comammox Nitrospira
(Blum et al., 2018). For the freshwater ammonia oxidizers, the influence of pH (7.0~8.5) on the reaction activity should not be ignored (Jones and Hood, 1980). As relatively rare species, comammox bacteria could be sensitive to environmental variation. Although further experiments are needed to verify our observations,
pH could be an important indicator for comammox amo and hao
expressions in large rivers. No directly significant correlations were
observed between TN or NH3 -N and the ammonia-oxidation transcriptional abundances of comammox Nitrospira, because comammox bacteria were likely to have multiple nitrogen sources like
ammonia, urea and amino acids from extracellular or intracellular environments. However, we found that the overall expressions
of ccmABCDEFGH genes were significantly and negatively correlated
with nutrient gradients such as TN, NH3 -N and TP (Spearman’s
r = -0.66~-0.82, p < 0.040), which indicated oligotrophic dynamics of heme export and cytochrome c-type biogenesis processes in
comammox Nitrospira. These genes are involved in energy transduction (e.g., respiration), CO2 binding and hydroxylamine oxidation, as well as other cellular processes (Sanders et al., 2010). Intriguingly, we obtained significant and positive correlations among
expressions of comammox-amoA, amoB, amoC, amoABC, haoA and
nirK genes (Spearman’s r = 0.73~0.93, p < 0.020) (Fig. 4). Meanwhile, the expressions of these genes were less or not significantly correlated with comammox-nxrA, nxrB and nxrAB expressions (Spearman’s r = -0.33~0.66, p = 0.038~0.987). Significant
positive correlations among amoABC, haoA and nirK expressions
implied that comammox Nitrospira had the potential to transform
nitrite to NO accompanied by ammonia oxidation under low-C/N
(<1.2) and aerobic conditions along the Yangtze River. Despite previous studies did not propose this hypothesis for riverine comammox Nitrospira, the newly recovered bins YR-WHU and YR-XLJ3 from the Yangtze River indeed encode a “fkpA-cytochrome cnirK” gene set in proximate to amo or hao operons within their
ammonia-oxidation gene clusters possibly for stable co-expressions
3.2.3. Stress response, defense and resistance
For response to environmental stress, ~50 genes encoding oxygen stress defense, multidrug efflux system, metal resistance, salt
resistance, and cold and heat shock resistance were mostly expressed in comammox and canonical Nitrospira (Fig. 3; Table S6).
Planktonic Nitrospira were exposed to a plethora of DO in water
of the Yangtze River (monitoring data: 10.0±0.5 mg/L). Accordingly, catalases, peroxidases, peroxiredoxins, Cu-Zn family superoxide dismutase, chlorite dismutase, or bacterioferritin were highly
expressed (1.3% of transcripts), protecting comammox Nitrospira
from radicals and reactive oxygen species (Palomo et al., 2018).
Compared to comammox, no catalase or superoxide dismutase was
transcribed in canonical Nitrospira. Both comammox and canonical
Nitrospira also possessed a very high expression of cytochrome bdtype quinol oxidase (average RPKM = 1292.9 and 3153.1, respectively) (Table S5). All studied genomes encode 4~10 copies of this
high-affinity enzyme, some of which not only allows organisms to
respire oxygen under aerobic states, but also has evolved for oxidative stress protection (Richardson, 20 0 0). Moreover, for response
to temperature fluctuation (10.4~15.0°C), the heat and cold shock
resistance genes were highly expressed in Nitrospira (0.56~1.1% of
transcripts). Comammox Nitrospira had 3.4-fold lower transcripts of
cold shock resistance genes but higher expressions of heat shock
resistance genes than canonical Nitrospira, suggesting distinct optimum metabolizing temperatures between them. Indeed, our previous study demonstrated that temperature was one of the key
factors determining niche separation between planktonic comammox and canonical Nitrospira (Liu et al., 2020). Relatively low expressions of the multidrug efflux system, metal and salt resistance
genes were observed for Nitrospira, because the potentially toxic
substances (e.g., antibiotics and metals) were not so enriched in
fluvial water as sewage (Lücker et al., 2010).
3.3. Environmental effects on expressions of comammox Nitrospira
community
We further explored how environmental factors influenced the
in-situ expressions of comammox Nitrospira community along the
9
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Water Research 200 (2021) 117241
Figure 4. Environmental effects on expressions of comammox Nitrospira community. Heatmap exhibits the pairwise Spearman’s correlations among environmental factors
and expressions of the key nitrification-related genes in comammox Nitrospira community. Scale bars indicate correlation coefficients. Only the significant correlations (p <
0.05) are labeled in circles. The “amo”, “nxr” and “ccm” represent the full series of amoABC, nxrAB and ccmABCDEFGH genes, respectively. Line charts on the top right corner
indicate the spearman’s correlations (r) between transcriptional abundances of comammox amoABC, haoA and nirK genes. Color solid lines indicate the ordinary least square
linear regressions, with the shaded area representing 95% confidence intervals. The r and p values of Spearman’s correlations are stated.
(Liu et al., 2020). Fig. 1d also showed that both YR-WHU and YRXLJ-3 were the major expressed species. These observations suggested that the potential of NO production during ammonia oxidation should not be ignored for comammox Nitrospira in the Yangtze
River. Kits et al. (2019) demonstrated that comammox N. inopinata
does accumulate NO under aerobic conditions, and highlighted the
dependency of NO production on oxygen, however, they did not
indicate co-varied expressions of amoABC, haoA and nirK genes.
AOB mainly perform nitrifier denitrification under oxygen-limited
conditions (Liu et al., 2021), which seems very different from comammox Nitrospira. Although further experiments are needed to
demonstrate the process of nitrite reduction to NO for comammox
Nitrospira in large rivers, we expect a possible cooperation among
comammox Nitrospira, complete and truncated denitrifiers in an
appropriate way, as denitrifiers could transform comammox terminal products NO3 -N, NO or N2 O into N2 . This would facilitate a
re-evaluation of nitrogen transforming processes in natural ecosystems, and even potentially upgrade and retrofit current schemes
for better management of TN.
4. Conclusions
Genome-centric metatranscriptomics was utilized to reveal the
wide in-situ expressions and novel transcriptional characters of
comammox Nitrospira in the Yangtze River. Our study demonstrated a difference of nitrification characteristics between comammox two clades, namely clade A were the most expressed comammox community in ammonia oxidation, while clade B contributed more transcripts to nitrite oxidation than to ammonia
oxidation. Comammox Nitrospira possessed lower expressions of
ammonia/nitrite transporters and nitrogen assimilatory genes but
quite higher expressions in urea transport and degradation than
canonical Nitrospira, which implied the restriction of “reciprocalfeeding” between canonical NOB and AOPs in large rivers. Aerobic
mixotrophic lifestyle of comammox bacteria was suggested based
on the expressions of genes for respiratory complexes I-V, oxidative/reductive TCA cycle, transport and catabolism of simple carbohydrates and amino acids, and oxygen stress defense. Comammox
Nitrospira had the potential of nitrite reduction to NO accompa-
10
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Water Research 200 (2021) 117241
nied by ammonia oxidation under low-C/N and aerobic conditions
of the Yangtze River, while gene expressions in this pathway were
significantly and positively related with pH.
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Poghosyan, L., Koch, H., Lavy, A., Frank, J., van Kessel, M.A.H.J., Jetten, M.S.M., Ban-
Author contributions
JN and SL designed the research. SL and HC performed the research. SL, HC and JN wrote the paper. JW and QC performed the
sampling and sequencing. JW, HW, TZ and QC contributed new
ideas and information. All the authors contributed to interpretation of the findings.
Declaration of Competing Interest
The authors declare no competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
Financial supports are from National Natural Science Foundation of China (51721006 and 91647211), National Key Basic Research Program of China (2016YFC0402102), and China Postdoctoral Science Foundation (2019M660333). Bioinformatic supports
from Majorbio Company and High-performance Computing Platform of Peking University are also acknowledged.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.watres.2021.117241.
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