RESEARCH ARTICLE Open Access
Comparative genomics in “Candidatus
Kuenenia stuttgartiensis”reveal high
genomic plasticity in the overall genome
structure, CRISPR loci and surface proteins
Chang Ding
1*
and Lorenz Adrian
1,2
Abstract
Background: Anaerobic ammonium oxidizing bacteria (anammox bacteria) are contributing significantly to the
nitrogen cycle and are successfully used in wastewater treatment. Due to the lack of complete genomes in the
databases, little is known about the stability and variability of their genomes and how the genomes evolve in
response to changing environments.
Results: Here we report the complete genome of the anammox bacterium “Candidatus Kuenenia stuttgartiensis”
strain CSTR1 which was enriched planktonically in a semi-continuous stirred-tank reactor. A comparison of the
genome of strain CSTR1 with the genome of “Ca. Kuenenia stuttgartiensis”MBR1 and the draft genome of KUST
showed > 99% average nucleotide identity among all. Rearrangements of large genomic regions were observed,
most of which were associated with transposase genes. Phylogenetic analysis suggests that strain MBR1 is more
distantly related to the other two strains. Proteomic analysis of actively growing cells of strain CSTR1 (growth rate ~
0.33 d
−1
) failed to detect the annotated cytochrome cd
1
-type nitrite reductase (NirS) although in total 1189 proteins
were found in the proteome. Yet, this NirS was expressed when strain CSTR1 was under stress or starvation (growth
rate < 0.06 d
−1
). We also observed large sequence shifts in the strongly expressed S-layer protein compared to
other “Ca. Kuenenia”strains, indicating the formation of hybrids of genes encoding the surface proteins.
Conclusions: “Ca. Kuenenia”strains appear to be relatively stable in their basic physiological traits, but show high
variability in overall genome structure and surface proteins.
Keywords: “Ca. Kuenenia stuttgartiensis”, Genome sequencing, Proteomics, CRISPR, Nitrite reductase, S-layer protein
Background
Anaerobic ammonium oxidation (anammox) is an import-
ant biological process that contributes significantly to the
global nitrogen cycle [1], and is increasingly popular in
state-of-the-art wastewater treatment due to its low energy
consumption, low material cost, and small reactor footprint
[2]. As described so far, the anammox process is catalyzed
by anammox bacteria belonging to six different genera [3].
Yet, the dominant genera in waste water treatment plants
are usually “Candidatus Kuenenia”or “Ca. Brocadia”[4–6].
Although “Ca. Brocadia anammoxidans”is the first re-
ported anammox species [7], “Ca. Kuenenia stuttgartiensis”
is by far the most extensively studied in terms of cell struc-
ture, physiology and biochemistry [8]. The first draft gen-
ome of an anammox bacterium was determined from a
mixed community by metagenomics resulting in an almost
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1
Helmholtz Centre for Environmental Research –UFZ, Environmental
Biotechnology, Permoserstraße 15, 04318 Leipzig, Germany
Full list of author information is available at the end of the article
Ding and Adrian BMC Genomics (2020) 21:851
https://doi.org/10.1186/s12864-020-07242-1
complete (estimated 98%) genome of a “Ca. Kuenenia stutt-
gartiensis”strain (hereafter referred to as strain KUST) [9].
TheKUSTgenomeencodedahypothesizedanammox
pathway and many other genes encoding versatile meta-
bolic functions. Later, the roles of some key proteins
encoded in the KUST genome were experimentally vali-
dated, e.g., hydrazine synthase (EC 1.7.2.7), hydrazine de-
hydrogenase (EC 1.7.2.8), hydroxylamine dehydrogenase
(EC 1.7.2.6), and an S-layer protein [10–12]. Most notably,
in 2011, it was confirmed by inhibitor/scavenger tests and
fluorescence staining that in “Ca. Kuenenia”the key inter-
mediate hydrazine was produced from nitric oxide instead
of the previously speculated hydroxylamine [13]. In con-
trast, the functions of multiple large membrane-bound
complexes are not yet functionally understood in detail
[14]. For improved biochemical understanding of the ana-
mmox process, complete genome and protein expression
data are crucial.
Currently, at least 37 anammox genome assemblies are
reported (Table S1) with GC content values of 37 to 45%
(median 41.1%), genome sizes of 2.3 to 5.2 Mbp (median
3.8 Mbp), and scaffold numbers of 1 to > 1000 (median
153). The “Ca. Kuenenia”genome KUST assembly was a
high-quality draft containing five supercontigs which was
sequenced in 2002 [9]. The reactor from which the KUST
cells were obtained was maintained for years and an
IonTorrent-based resequencing in 2012 confirmed the
stability of the genome [12]. Interestingly, after only 2 years
in 2014, another resequencing effort using the single-
molecule real-time (SMRT) sequencing technique revealed
the dominance of a new “Ca. Kuenenia stuttgartiensis”
strain MBR1, while the original KUST strain was barely
detectable. The genome of strain MBR1 was so far the only
complete genome among all anammox bacteria [15]. Due
to the availability of only one complete genome for ana-
mmox bacteria, it is currently not possible to make com-
parison on the genome structure or infer conserved
genomic regions among anammox species.
In our previous work, we obtained granules from an
anammox reactor treating landfill leachate at Beijing Uni-
versity of Technology. Initially, this reactor culture con-
tained four anammox genera (“Ca. Kuenenia”,“Ca.
Anammoxoglobus”,“Ca. Brocadia”,and“Ca. Jettenia”,all
together representing 0.5% of the total population). After
three transfers in serum bottles the culture contained only
a single anammox genus (“Ca. Kuenenia”) at 17% of the
total population and became granule-free [16]. This plank-
tonic culture was then transferred to a semi-continuous
stirred-tank reactor (CSTR), and exhibited stable growth
rate of up to 0.33 d
−1
and high anammox activity [17].
Illumina-based amplicon sequencing of 16S rRNA
genes and fluorescence in-situ hybridization revealed
the dominance of “Ca. Kuenenia”(87% of the total
population) [17].
In our present study, we determined the complete gen-
ome sequence of the dominant “Ca. Kuenenia”species
from our CSTR reactor and refer to it as strain CSTR1.
The genome of strain CSTR1 is the second complete ana-
mmox genome to date, and unveils how variable the ge-
nomes are among the closely related “Ca. Kuenenia”
strains. CRISPR analysis also hints at the strain differenti-
ation history among these strains. Finally, information was
obtained on possible functions of key genes in the ana-
mmox genome via expression analyses using shotgun
proteomics.
Results
Complete genome of “Ca. Kuenenia stuttgartiensis”strain
CSTR1
The longest contig assembled from SMRT sequencing of
the CSTR reactor effluent had a coverage of ~ 93 and a
length of 4.3 Mbp (Table S2). All other contigs had a
coverage of < 35 and a size of < 55 kbp, and were mostly
affiliated with Methyloversatilis, consistent with ampli-
con sequencing results of the same reactor (5 months
before the sampling for genome sequencing) showing
Methyloversatilis as the second most abundant popula-
tion in the community [17].
The longest contig of 4.3 Mbp was circularized and was
foundtocontainasingle16SrRNAgenesequencewhich
shared 100% identity with those of “Ca. Kuenenia stuttgar-
tiensis”strains MBR1 and KUST over 1577 bp. Therefore,
the circularized contig was confirmed to be the genome of
strain CSTR1. The genome had a size of 4,334,932 bp, with
a GC content of 41.03% (Table 1). Three copies of dnaA
genes were found in the genome, and one of the three
dnaA genes (locus tag KsCSTR_00010) was chosen as the
origin of replication (ori) on the genome based on the cu-
mulative GC skew graph (Fig. S1C). This dnaA is the only
dnaA gene that has dnaN (KsCSTR_00020) and gyrB
(KsCSTR_00040) genes at its immediate proximity. The
genome sequence was deposited in the National Center for
Biotechnology Information (NCBI) database (BioProject:
PRJNA603163; BioSample: SAMN13921532; reads archive
number: SRR11213620; genome accession number
CP049055).
Structural variations among “Ca. Kuenenia”strains as
revealed by genome comparison
We compared the overall genome structures of strain
CSTR1 and strain MBR1 as these are the only two avail-
able complete anammox genomes to date. The cumula-
tive GC skew graph of the MBR1 genome (Fig. S1A)
suggested that KSMBR1_2151 is the ori-defining dnaA
gene, which is in contrast to its original description. The
use of KSMBR1_2151 as the ori-defining dnaA gene was
further supported by the high sequence similarity of
KSMBR1_2151 with KsCSTR_00010 defining the ori in
Ding and Adrian BMC Genomics (2020) 21:851 Page 2 of 11
strain CSTR1 and the vicinity of dnaN and gyrB. To fa-
cilitate the comparison between strain MBR1 and strain
CSTR1, we generated a reverse complement of the gen-
ome sequence MBR1, chose the first base pair of
KSMBR1_2151 as position 1, and called the converted
genome MBR1b (GC skew as shown in Fig. S1B).
Mauve alignment of the two circularized genomes
MBR1b and CSTR1 showed strong synteny between the
two genomes, dividing the genome sequences into 20
conserved regions, referred to as ‘locally collinear blocks’
(LCB) (Fig. 1). LCB 1 and 20 (combined) are syntenic
and located around the ori (~ 1.4 Mbp in length). Also
LCB 5 (~ 0.6 Mbp in length, containing the rRNA gene
operon) and LCB 17 (~ 0.7 Mbp in length) are large
LCBs conserved in orientation and genome position.
Considerable rearrangements were found between many
other LCBs of genome MBR1b and strain CSTR1 (Fig.
1). A total of 11 reversal steps were necessary in order to
convert between the genome of strain CSTR1 and the
genome MBR1b as calculated using GRIMM (Table S3).
A close examination of the flanks of the rearranged
genomic blocks almost always identified transposase
genes corroborating genomic motility (Table S4).
Phylogenetic analyses of the three “Ca. Kuenenia
stuttgartiensis”strains showing strain MBR1 is
phylogenetically more distant
The three genomes KUST, MBR1 and CSTR1 are highly
similar, as indicated by high average nucleotide identity
(ANI) values and tetranucleotide signature correlations
(Table 2). ANI values showed that strain KUST and strain
CSTR1 are slightly closer with each other than with strain
MBR1. In terms of rRNA gene sequences, strain KUST
and strain CSTR1 are 100% identical in 5S–16S-23S rRNA
concatenated sequences, while MBR1 has one mismatch at
23S rRNA. We also employed concatenated conserved
protein analysis [9] to obtain a higher resolution in phylo-
genetic relationship among the strains. In the alignment
with a length of 31,842 nt we counted the number of
nucleotide positions where one of the strains had a differ-
ent nucleotide while the other two had the same nucleo-
tide, suggesting a mutation in the strain with the
Table 1 Comparison of the genomes of “Ca. Kuenenia stuttgartiensis”strain KUST, strain MBR1, and strain CSTR1
Strain KUST [9] MBR1 [15] CSTR1 (this study)
Status High quality draft (5 supercontigs) Complete genome Complete genome
Size 4,218,325 4,406,132 4,334,932
Sequencing technology Sanger sequencing SMRT sequencing SMRT sequencing
GC % 41.0 41.1 41.0
Prediction of coding sequence dps/orpheus, AMIGene, glimmer/rbsfinder
and genemarks/genemark.
hmm
Prodigal [18] in Prokka pipeline [19] AMIGene in MaGe pipeline [20]
# Coding sequences (CDSs) 4664 4043 4965
Coding density % 85.9 81.2 85.7
Average gene length 776 902 751
# rRNA operons (5S, 16S, 23S) 1 1 1
# tRNAs 44 45 45
MBR1 has much fewer predicted CDSs than the other two genomes and therefore a lower coding density and a higher average gene length due to usage of a
different coding sequence prediction tool (Prodigal)
Fig. 1 Visualized genome alignment of the genome of “Ca. Kuenenia stuttgartiensis”strain CSTR1 and MBR1b. In total, 20 locally collinear blocks
(LCBs) were identified by Mauve and labelled in the figure. Histograms within the LCBs indicate sequence similarity within the regions
Ding and Adrian BMC Genomics (2020) 21:851 Page 3 of 11
nucleotide divergence. This analysis gave 2 nucleotide
changes in KUST, 31 in MBR1, and 4 in CSTR1. When
doing the same analysis with amino acid sequences of the
concatenated protein sequences (10,564 aa length) we
found 8 changes in MBR1, and none in the other two
strains. Therefore, based on the analyses of ANI, rRNA
sequences, and concatenated conserved proteins, strain
MBR1 is phylogenetically more distant from strain KUST
and strain CSTR1.
The “Ca. Kuenenia”strain from which the KUST genome
was obtained in 2002 was re-sequenced in 2012 [12](here-
after KUST2012). We assembled the IonTorrent reads of
KUST2012 (Additional file 1).Therewasatotalofsixmuta-
tions in concatenated conserved gene sequence in
KUST2012 compared to KUST, but all were either insertion
or deletion that caused frame shifts and therefore were
probably sequencing or assembly errors. Therefore, there
were probably no changes in gene sequences of the analyzed
conserved proteins after ten years cultivation of KUST.
Analysis of a shared CRISPR locus suggesting strain KUST
might have differentiated earlier among the three strains
All three “Ca. Kuenenia”genomes contain several
CRISPR elements and CRISPR associated genes (Cas)
(Table S5). Among these CRISPR elements one large
CRISPR locus with more than 5 kbp and 80 or more
spacers near a type I-B Cas gene cluster are shared
by all three genomes (Table S6,Fig.2). This type I-B
CRISPR locus (coordinate: 1.18 Mbp in CSTR1,
3.78Mbp in MBR1b) located within LCB 4 which was
flipped and relocated when comparing the genomes
of CSTR1 and MBR1b (Fig. 1). The 30 bp repeat of
the CRISPR locus is conserved among all three ge-
nomes but has no perfect match in the NCBI nt data-
base to other genomes. Matches to the repeat
sequence with an e-value of 0.12 were present to
CRISPR loci in many other genomes, indicating only
distant relationship. None of the spacer sequences
(210 spacers in total) had a 100% match in the NCBI
nt database other than from the three genomes them-
selves. 45 spacers are shared by all three “Ca. Kuene-
nia”genomes and all are located at the 3′end of the
CRISPR locus (Fig. 2). In total, 19, 35, and 85 spacers
areuniquetothegenomesofstrainKUST,strain
MBR1, and strain CSTR1, respectively, and most of
these unique spacers are located between the leader
sequence (arrow in Fig. 2) and the common spacers.
It is known that newly acquired spacers are inserted
at the leader end of the CRISPR locus [24,25].
Spacers seem to pop out at random locations (gaps in
Fig. 2), the cause of which is unknown. Apart from
the 45 common spacers (orange colored in Fig. 2),
strain MBR1 and strain CSTR1 also shared 12 more
spacers (blue colored in Fig. 2)thatarelocatednext
to the 45 common spacers. This suggested that strain
KUST might have differentiated first from the com-
mon ancestor of the three strains, and the common
ancestor of strain MBR1 and strain CSTR1 continued
to acquire the blue-colored spacers before they even-
tually diversified into two strains. We also examined
changes in the CRISPR locus in KUST2012 compared
to the original KUST genome A nearly identical locus
was found in Node 4 of the KUST2012 assembly
(Additional file 1) with 78 spacers. Compared to the
CRISPR locus in the original KUST genome, two
spacers (the 31
st
and 32
nd
counting from the leader
end) were absent while no new spacers were found.
Table 2 Genome sequence similarity among the genomes of
“Ca. Kuenenia stuttgartiensis”KUST, strain MBR1, and strain
CSTR1
KUST MBR1 CSTR1
KUST Tetra 0.9981 Tetra 0.9985
MBR1 ANI 0.9940 Tetra 0.9994
CSTR1 ANI 0.9960 ANI 0.9922
ANI - average nucleotide identity between genomes as calculated using the
algorithm OrthoANIu [21,22]
Tetra - correlation of tetranucleotide signatures between genomes as
calculated using the program Tetra v1.02 [23]
Fig. 2 Visualized alignment of the largest CRISPR locus in “Ca. Kuenenia stuttgartiensis”genomes KUST, MBR1b, and CSTR1. The CRISPR locus in
KUST2012 is almost identical to that in the KUST, except that the 31
st
and 32
nd
spacers were not present in KUST2012 (counting from the leader
sequence end) and is therefore not shown. Orange: identical spacers among the three genomes; Blue/Green/Purple: identical spacers between
two of the three genomes; Yellow: unique spacers; White: gap in the alignment; Brown: type I-B Cas gene clusters. These Cas gene clusters span
a length of ~ 6 kbp and are not drawn to scale. Arrows indicate the location of a 138-bp leader sequence
Ding and Adrian BMC Genomics (2020) 21:851 Page 4 of 11
Overview of the annotated genome of strain CSTR1
The annotated genome of strain CSTR1 contains 4965
protein-coding genes, one ribosomal RNA operon (5S,
16S, and 23S), one transfer-messenger RNA gene, and
45 tRNA genes (Table 1). The genome of strain CSTR1
contains genes encoding all described ribosomal pro-
teins [26] except the two non-essential proteins L30
and L34. A total of 2849 genes encoding proteins of un-
known functions were found, making up 57% of the
total genes. Of the 4965 protein-coding genes in strain
CSTR1, 4557 genes (92%) have homologs in strain
KUST, 4215 genes (85%) have homologs in strain
MBR1, and 4076 genes (82%) are found in both strain
KUST and strain MBR1, based on a sequence identity
threshold of 40% (homologs include matches with six-
frame translations of genome sequences of strains
KUST and MBR1) (Table S7). Pangenome analysis
based on re-annotated genomes of strain KUST, MBR1,
and CSTR1 showed that 2966 genes were shared by all
three genomes, and numbers of specific genes are 122,
281, and 186, respectively (Fig. S2).
The genome of strain CSTR1 contains all reported es-
sential genes related to anammox metabolism, including
two identical gene sets for the heterotrimeric hydrazine
synthase (KsCSTR_28210 / KsCSTR_28190 / KsCSTR_
28200 and KsCSTR_12690 / KsCSTR_12670 / KsCSTR_
12680), hydrazine dehydrogenase genes KsCSTR_46980
/ KsCSTR_11820, hydroxylamine dehydrogenase gene
KsCSTR_43280, and the alpha, beta and gamma sub-
units of nitrite:nitrate oxidoreductase (KsCSTR_08000 /
KsCSTR_07970 / KsCSTR_07960).
Just as strain KUST and strain MBR1, strain CSTR1
also contains four different types of ATPase genes,
three complex III genes, a complete set of genes
encoding the reductive acetyl-CoA pathway for CO
2
fixation, and two sets of complex I genes (both con-
taining 14 subunits: A to N including the peripheral
NADH input module nuoEFG) (KsCSTR_25990-
KsCSTR_26120 and KsCSTR_45490-KsCSTR_45710).
The complex I gene cluster KsCSTR_45490-KsCSTR_
45710 was speculated to couple NADH oxidation to
CO
2
and menaquinone reduction [27]. The type 3b
(sulf) hydrogenase operon in strain MBR1 (KSMBR1_
3671–3674), which is absent in the genome KUST, is
present in strain CSTR (KsCSTR_28360–28,390). This
operon was speculated to be involved in hydrogen
metabolism [15].
The abundance of transposase genes in the genome of
strain CSTR1 as well as in strain KUST and strain
MBR1 (> 5% of all genes) is significantly higher than
average abundance (1.1%) in prokaryotic genomes [28],
with > 200 full length or remnant transposase genes
identified (Table S8,Table S9). Although there are as
many as 38 groups of transposase genes in the three
anammox genomes, the top 10 groups account for >
60% of all transposase genes. Interestingly, the group 13
transposase, which is a IS1634 family transposase, exists
in 20 full copies in strain MBR1, but is absent in both
strain KUST and strain CSTR1.
Overall expression analysis of genes in strain CSTR1 by
shotgun proteomics
The expressed proteome of strain CSTR1 was evaluated
using triplicate samples from a semi-CSTR reactor run-
ning at 3 days hydraulic retention time (HRT). In total,
20,800 PSMs were found, leading to 5429 identified pep-
tides and 1189 proteins, of which 1168 were quantified.
The most abundant proteins in the proteomes were
similar to those found in previous studies (Table S7)[14,
29], including most of the key enzymes involved in the
anammox pathway such as hydrazine synthase, hydra-
zine dehydrogenase, nitrite:nitrate oxidoreductase, and
hydroxylamine oxidase (Table S7). The genome of strain
CSTR1 encodes two highly similar hydrazine dehydro-
genase, KsCSTR_46980 (kustc0694, KSMBR1_2369)
(gene tags in brackets are homologs in strains KUST and
MBR1, same below) and KsCSTR_11820 (kustd1340,
KSMBR1_1220). Abundances of unique peptides belong-
ing to KsCSTR_46980 indicated that KsCSTR_46980
was highly expressed (Table S10), agreeing with the pre-
vious finding [11]. A nitrite transporter KsCSTR_14610
(kuste3055, KSMBR1_1070) (0.019%, 441) (abundance,
abundance rank; same for further data given below) and
an ammonium transporter KsCSTR_43840 (kustc1009,
KSMBR1_2627) (0.0039%, 790) were expressed, while
the previously characterized ammonium sensor
KsCSTR_21800 (kuste3690, KSMBR1_3866) [30] was
not found in the proteome.
Expression of putative respiratory complexes was simi-
lar to what was reported before, including the expression
of complex I genes, ATPase genes, and Rieske/cytb com-
plex genes [14]. Although the reactor was running at an
HRT of 3 days and therefore cells were actively dividing,
the previously reported division ring protein KsCSTR_
10780 (kustd1438, KSMBR1_1135) [31] was not found
in the proteome. One of the CRISPR-associated genes
(KsCSTR_13270) near the large CRISPR locus described
above was expressed at a level of 0.10% of the whole
proteome (rank 134). The group 15 transposase
KsCSTR_47830 (rank 1021) is the only transposase that
is detected in the proteome.
One of the top 50 abundant proteins KsCSTR_28510
(KSMBR1_3708) (0.54% of total proteins, rank 27), an-
notated as a hypothetical exported protein, was not
found in the genome of KUST including its six-frame
translation, while all the other top 50 abundant proteins
had homologs in the genome of strain MBR1 and KUST.
Ding and Adrian BMC Genomics (2020) 21:851 Page 5 of 11
Expression of putative nitrite reductase genes
The identity of the nitrite reductase gene in “Ca. Kuene-
nia”genomes is unclear. It was initially hypothesized
that the annotated cytochrome cd
1
-type nitrite reductase
(NirS) kuste4136 (KSMBR_0452 / KsCSTR_33370) con-
verts nitrite to nitric oxide –a key step in the anammox
metabolism [13]. However, a further study indicated that
this NirS was barely detectable in the proteome of “Ca.
Kuenenia”[14], while others detected NirS with as much
as 28% sequence coverage [29]. This led to the question
if the product of kuste4136 is the actual nitrite reduc-
tase, or if there might be another enzyme with nitrite
reductase activity. Candidates for such an enzyme
include two putative hydroxylamine oxidoreductase
genes (KsCSTR_49490 / KSMBR1_2163 / kustc0458 and
KsCSTR_29630 / KSMBR1_3792 / kuste4574) [27].
In our proteome datasets that came from samples
taken from the actively running reactor at 3 d HRT, the
three subunits of hydrazine synthase and hydrazine de-
hydrogenase represented the four most abundantly
expressed proteins. Also the three subunits of nitrite:ni-
trate oxidoreductase and the two putative hydroxylamine
oxidoreductases KsCSTR_49490 (1.19%, rank 14) and
KsCSTR_29630 (0.35%, rank 46) were among the most
abundant proteins in the proteome. In contrast, no NirS
(KsCSTR_33370) was detected. Only when we examined
multiple sets of whole proteome data over a period of al-
most 3 years (all were planktonic cells), we found that
NirS appeared in some datasets (Table S11), all at rela-
tively long HRTs (≥15d). The inconsistent detection of
NirS suggested that it was at least dispensable for growth
and energy conservation in “Ca. Kuenenia”strains. The
other two nitrite reductase candidates KsCSTR_49490
and KsCSTR_29630 were consistently expressed in all
examined datasets (Table S11).
Sequence evolution of the gene encoding the S-layer
protein
The gene kustd1514 in the genome KUST was character-
ized as a heavily glycosylated protein forming the S-layer of
the bacterium [12,32]. The homologs of this gene in strain
MBR1 and strain CSTR1 are KSMBR1_1301 and KsCSTR_
09970, respectively, both are located within LCB 1 (coord-
inate: 0.91 Mbp in CSTR1 and 0.94 Mbp in MBR1b). Just
as kustd1514, KsCSTR_09970 was also highly expressed in
thegenomeofstrainCSTR1(Table S7,rank8).Interest-
ingly, while KSMBR1_1301 shares 99% amino acid identity
with kustd1514, KsCSTR_09970 shares only 53% identity
with kustd1514. In fact, in the top 50 abundant proteins in
strain CSTR1, all the other proteins exhibited > 95% amino
acid sequence identity with their homologs in the genome
of strain MBR1 and KUST (Table S7).
The re-sequenced KUST genome in 2012 [12] revealed
a kustd1514 with significantly changed amino acid
sequence (hereafter referred to as kustd1514b). The loci
kustd1514 and KSMBR1_1301 share almost 100% iden-
tity across the whole length of the sequence (Fig. 3B). In
contrast, kustd1514/KSMBR1_1301, kustd1514b, and
KsCSTR_09970 shared some regions with almost 100%
identities and some regions with only 20–60% identities.
It seems that kustd1514b is a hybrid of kustd1514 and
KsCSTR_09970. A search in the NCBI nr database only
found six more proteins (all in Planctomycetes) that were
distantly related to the three proteins mentioned here
and which had a homologous region of at least 50% of
the query sequence (Fig. S3).
In the resequencing data of KUST in 2012 where
kustd1514b was found, a unique partial sequence of the
original kustd1514 (between amino acid position 155
and position 726) was found in two short and low-
coverage contigs 318 and 458 (coverage 1.3 ~ 1.5, overall
coverage was ~ 74) (Additional file 1). Therefore, it
seems that the population that contained the original
kustd1514 still existed when the microbial community
was re-sequenced in 2012.
Although we did not analyze glycosylation of KsCSTR_
09970, proteomic data suggested that the protein KsCSTR_
09970 was similarly glycosylated as kustd1514b. In a total
of 10,045 peptide spectrum matches (PSMs) obtained from
a large collection of proteomic data of strain CSTR1 which
were associated with 46 tryptic peptides in KsCSTR_09970
(Table S12) only one single PSM (for the peptide at pos-
ition 590–624) had an 11-aa overlap with one of the eight
peptides that were reported to be glycosylated [32]andthat
were shared between kustd1514b and KsCSTR_09970 (Fig.
S4). This is in agreement with the observation by van
Teeseling et al. [32] who found almost no non-glycosylated
variants of the detected peptides in kustd1514b.
Discussion
The analysis of the complete genome of “Ca. Kuenenia
stuttgartiensis”strain CSTR1 provides evidence for
strong genome structure conservation in bacteria of the
species “Ca. Kuenenia stuttgartiensis”. By comparing the
CSTR1 genome with the genome of strain MBR1, we
found that three regions (0.6–1.4 Mbp in length) are
conserved in both their orientations and locations (Fig.
1). Apart from the conserved regions, we observed con-
siderable rearrangements, and almost always found
transposase genes at the flanks of the rearranged genom-
ics blocks, which was consistent with previous findings
showing evidence of high transposase activities in “Ca.
Kuenenia”strains [15,33]. Abundant transposases were
also detected in the proteome of “Ca. Brocadia”[34],
suggesting similar transposase activities in “Ca. Broca-
dia”. Since transposase activities facilitate genetic ex-
changes and mutations which could be beneficial to the
microorganisms [28], the anammox bacteria in the
Ding and Adrian BMC Genomics (2020) 21:851 Page 6 of 11
natural environment may require such activities to adapt
themselves to changing environment.
Comparison of the longest CRISPR locus in the ge-
nomes of strain KUST, strain MBR1, and strain CSTR1
suggested that strain KUST might have differentiated
from the common ancestor of the three strains earlier
than when strain MBR1 and strain CSTR1 differentiated
from each other. This is in contrast to phylogenetic ana-
lyses with rRNA gene sequences, ANI, and concatenated
conserved proteins that suggest that CSTR1 and KUST
are more similar to each other than both are to strain
MBR1. Such discrepancy can be explained by a horizontal
gene transfer event of the whole CRISPR locus, meaning
that the CRISPR locus does not necessarily represent the
immune history in the strain. A second explanation could
be that strain MBR1 evolved faster than the other two
strains due to faster growth or higher mutation rates.
The results from proteome analysis in strain CSTR1 were
mostly in agreement with previous findings in terms of the
key enzymes in the anammox metabolism (except the an-
notated cytochrome cd
1
-type nitrite reductase, nirS)and
other abundant proteins. A number of differences to previ-
ous studies were noticed including the division ring protein
and an ammonium sensor protein. The absence of the de-
scribed division ring protein KsCSTR_10780 (kustd1438,
KSMBR1_1135) [31] was puzzling as the culture was at the
highest growth rate for anammox bacteria (0.33 d
−1
)and
the protein is a huge protein (3690 aa) with as many as 65
tryptic peptides that are suitable for mass spectrometry.
Possible reasons for the absence include too low expression
levels, or unknown reasons that prevent tryptic digestion of
the protein, or unknown protein modifications that prevent
peptide identification. Similar to the division ring protein, a
previously characterizedammoniumsensorprotein
KsCSTR_21800 (kuste3690, KSMBR1_3866) [30]wasalso
absent in the proteome.
The S-layer protein sequence in strain CSTR1 (KsCSTR_
09970) differed significantly from that in strain KUST
(kustd1514) and in KUST2012 (kustd1514b). KsCSTR_
09970 is similarly glycosylated as its homolog kustd1514b.
Since KsCSTR_09970 in our analyses and also kustd1514
and kustd1514b [29,32] were all highly expressed, they ap-
pear to fulfill the same function as an S-layer protein in
“Ca. Kuenenia”strains despite their dissimilar sequences.
These suggested that the anammox bacteria may employ
diverse proteins to perform the same functions.
Due to the inconsistent expression level of the anno-
tated cytochrome cd
1
-type nitrite reductase (NirS,
kuste4136), it was hypothesized that two putative hydrox-
ylamine oxidoreductase genes (kustc0458 and kuste4574)
may take the role of producing nitric oxide from nitrite in
“Ca. Kuenenia”[27]. Consistent with the hypothesis, in
our study we did not detect KsCSTR_33370 (analog of
kuste4136) in the proteome of strain CSTR1 at high
growth rates, but found KsCSTR_49490 (analog of
kustc0458) and KsCSTR_29630 (kuste4574) at high ex-
pression levels. Further experiments are needed to verify
whether these two candidates are representing nitrite re-
ductase activity in growing “Ca. Kuenenia”strains. Inter-
estingly, when the anammox cells are under stress of high
temperature, high nitrite, or starvation, we did observe
NirS in the proteome (Table S11). This suggested that the
NirS in strain CSTR1 might be involved in other functions
apart from nitrite reduction or in stress response.
Fig. 3 Amino acid sequence identity comparison among the four S-layer protein homologs in KUST (kustd1514), KUST2012 (kustd1514b), strain
MBR1 (KSMBR1_1301), and strain CSTR1 (KsCSTR_09970). Shown are always comparisons of the two sequences indicated on the right side.
Positions (aa) refer to the amino acid (aa) positions in the alignment of the four proteins. Window size: 20 amino acids. Step: 1 amino acid
Ding and Adrian BMC Genomics (2020) 21:851 Page 7 of 11
Conclusions
In this study, the complete genome of “Ca. Kuenenia stutt-
gartiensis”strain was sequenced and circularized. High
average nucleotide identity was observed between the gen-
ome of strain CSTR1 and the genomes of “Ca. Kuenenia
stuttgartiensis”MBR1 and KUST, while considerable dif-
ferences were observed among the genomes, including
rearrangements of large genomic regions, changes in
CRISPR elements, and sequence shifts in the strongly
expressed S-layer protein. Such differences suggested
that, while “Ca. Kuenenia”strains showed stability of
basic physiological traits, they evolved in other key as-
pects such as phage defense components and surface
properties. Such changes may help the strain to cope
with changing environments but the exact causes and
consequences of the changes are not clear.
The identity of the actual nitrite reductase in “Ca.
Kuenenia”is still unclear. We showed that the annotated
cytochrome cd
1
-type nitrite reductase (NirS) is at least
dispensable in “Ca. Kuenenia”, due to its absence in the
whole proteome of strain CSTR1. The expression of this
NirS under stress or starvation conditions suggested that
it could be variably induced.
Methods
Source of the inoculum
Anammox granules were obtained from an anammox re-
actor treating landfill leachate in Beijing University of Tech-
nology (reactor R11 in [16]). The biomass was cultivated in
anammox medium in serum bottles and became planktonic
after a few transfers of granule-free liquid. The planktonic
culturewaslaterusedtoinoculatea2-lsemi-CSTRana-
mmox reactor fed with 20 mM nitrite and 20 mM ammo-
nium at an hydraulic retention time of 3 days [17].
Cultivation and DNA extraction
Reactor effluent from the 2-l semi-CSTR anammox re-
actor was collected in April 2017. The abundance of the
anammox population in the reactor at the time of collec-
tion was estimated to be around 87% by fluorescence in-
situ hybridization. Cells were pelleted from 2 l of the re-
actor effluent by centrifugation at 5000 g, 4 °C for 15
min. The obtained wet cells (452 mg) were sent with ice
packs to Max Planck-Genome-Centre Cologne for DNA
extraction, library construction, and sequencing.
Sequencing and genome assembly
A genomic DNA library of 20–30 kbp insert size was
prepared with DNA extracted from the wet cells as de-
scribed above, and sequenced on a PacBio RS II sequen-
cer using one SMRT cell with the P6-C4 reagent. In
total, 79,588 reads were generated, amounting to a total
data size of 875 Mbp. Read length N50 was 15,927 bp,
and subread length N50 was 9475 bp. Genome
assembling was done using SMRT Analysis v2.3.0. The
assembly protocol HGAP.2 was used with default
settings.
A total of 183 contigs were obtained after the above-
mentioned automatic assembly pipeline, of which the lon-
gest contig had a length of 4,341,910 bp and an average
coverage of 93 while the other contigs had a maximal
length of 54,306 bp and a maximal coverage of 33 (Table
S2). Initial BLAST search of the longest contig showed
homology to “Ca. Kuenenia”. We then took the longest
contig and circularized the contig using Circlator [35]
(github.com/sanger-pathogens/circlator). The circularized
contig (4,334,644 bp) was then used as a reference genome
to which the original reads were mapped using the rese-
quencing protocol of SMRT Analysis v2.3.0 in order to cor-
rect errors. Average coverage was 104 and was consistent
along the entire contig (Fig. S5). The finalized circular con-
tig had a size of 4,334,932 bp, and contained a single copy
of 16S rRNA gene sequence. This contig was considered to
be the complete genome of the anammox population in the
reactor.
Assembly of the resequencing data of strain KUST in
2012 [12] was done based on the IonTorrent reads
downloaded from ebi.ac.uk (reads archive number:
ERR342261) using SPAdes v3.13.0 under default settings
(Additional file 1).
Sequence analyses
Annotation of the genome of strain CSTR1 was done
through the automatic annotation pipeline provided by
MicroScope [20] with manual curation. Potentially
missed protein coding sequences were added after com-
parison with the genome of strain MBR1 and KUST.
Coding sequences were then refined by applying the
maximum allowed sequence overlap of 60 bp [36]: if two
coding sequences shared at least 60 bp overlap and the
overlap was not caused by misprediction of start codons,
the coding sequence that was shorter was removed un-
less it encoded a protein of which the function was pre-
dicted or it was found in the proteome of strain CSTR1.
For pangenome and core-genome analysis, the three
anammox genomes were re-annotated in Prokka pipe-
line [19] using Prodigal [18] as the coding sequence pre-
diction tool. Pangenome analysis was performed in
KBase [37] using default settings.
CRISPR elements were identified using CRISPRcasFinder
ontheonlineserver(crisprcas.i2bc.paris-saclay.fr)[38].
Correlation of tetranucleotide signatures between ge-
nomes was calculated using the program Tetra v1.02 [23].
Average nucleotide identity (ANI) between genomes was
calculated on the online server (ezbiocloud.net/tools/ani)
with default settings which uses the OrthoANIu algorithm
based on USEARCH [21,22]. Concatenated conserved
protein analysis was done as described previously using a
Ding and Adrian BMC Genomics (2020) 21:851 Page 8 of 11
set of 50 conserved genes including ribosomal proteins,
DNA-directed RNA polymerase subunits, and other pro-
teins [9]. All 50 chosen genes are present in full length in
genomes of “Ca. Kuenenia stuttgartiensis”strains.
Alignment of genomic blocks and visualization was done
using Mauve v2.3.1 with Progressive Mauve algorithm and
a seed weight of 15 [39]. Genome rearrangement steps were
calculated using the online server grimm.ucsd.edu/GRIMM
based on GRIMM v2.01 [40]. Alignment of gene and pro-
tein sequences was done using MEGA7 [41]withsettings
noted in the figure legends.
Proteomics analyses
Samples for proteomics analysis were taken from the la-
boratory semi-CSTR reactor of a liquid volume of 1 l.
The reactor was running at an HRT of 3 days with
inflowing nitrite and ammonium concentrations of 60
mM. Three replicates of samples were taken on the
same day from the reactor. Supernatant was removed
after centrifugation at 5000 g, 16 °C for 10 min, and cell
pellets were resuspended in ammonium bicarbonate buf-
fer (50 mM). Three cycles of freeze/thaw (−80 °C / +
40 °C) were used to disrupt cells. Protein extracts were
treated sequentially with 62.5 mM dithiothreitol and 128
mM 2-iodacetamide before digestion with reductively
methylated trypsin (Promega) overnight.
Digested peptides were desalted using C
18
ziptips (Milli-
pore, Merck), vacuum dried, and reconstituted in 0.1%
formic acid before nano-liquid chromatography–mass
spectrometry (nano-LC-MS) analysis. Protein from
around 3 × 10
6
cells was injected in each nano-LC-MS/
MS run. Nano-LC-MS analysis was done on a nano-LC
system (Dionex Ultimate 3000RSLC, Thermo Scientific)
equipped with an Orbitrap Fusion Tribrid mass spectrom-
eter (Thermo Scientific). Peptides were separated on an
Acclaim PepMap 100 C
18
column (100 Å pore size, 3 μm
particle size, 75 μm × 250 mm, Thermo Scientific) at a
flow rate of 300 nL min
−1
and a column oven temperature
of 35 °C. Mobile phase was mixed from solution A (0.1%
formic acid in water) and solution B (0.08% formic acid in
20% water + 80% acetonitrile). Pump gradient was as fol-
lows: solution B (%) was 4% for 1 min, ramped up to 10%
from 1 min to 5 min, slowly ramped up to 35% from 5 min
to 100 min, ramped up to 55% from 100 min to 120 min,
quickly ramped up to 90% from 120 min to 130 min, held
at 90% from 130 min to 135 min, went down to 4% from
135 min to 137 min, and held at 4% from 137 min to 145
min. Peptides were ionized in TriVersa NanoMate,
Advion electrospray ion source. Mass spectrometry ana-
lysis was performed in positive mode. Both MS1 and MS2
scans were performed on the Orbitrap mass analyzer with
a MS1 resolution of 120,000 and a MS2 resolution of 60,
000. Only ions with a charge state between 2 and 4 were
selected for fragmentation.
Acquired raw data were analyzed using Proteome Discov-
erer (v2.4, Thermo Fisher Scientific). MS2 spectra were
searched using SequestHT against a fasta file containing all
protein sequences in the genome of “Ca. Kuenenia stuttgar-
tiensis”strain CSTR1 (NCBI accession number CP049055).
Mass tolerance for precursor ion mass and fragment ion
mass was 3 ppm and 0.5 Da, respectively. Maximal two
missed cleavage sites were allowed. Oxidation on methio-
nine residues was set as a dynamic modification and carba-
midomethylation on cysteine residues was set as a fixed
modification. Protein and peptides abundance values were
calculated by intensity-based label free quantification using
theMinoranodeimplementedinProteomeDiscoverer.
Rankings in abundance were calculated by comparing all
protein abundances within one sample and ranking them
according to their MS1 intensity value. The proteomics
data have been deposited to the ProteomeXchange Consor-
tium via the PRIDE partner repository with the dataset
identifier PXD018553.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12864-020-07242-1.
Additional file 1 Fig. S1. Cumulative GC skew along the genomes of
“Ca. Kuenenia stuttgartiensis”strains. Fig. S2. Venn diagram showing the
core genome and the genes specific in “Ca. Kuenenia stuttgartiensis”
strains. Fig. S3. Unrooted phylogenetic trees of ten S-layer homologous
gene constructed based on the Neighbor-Joining method. Fig. S4. Amino
acid sequence of the S-layer protein of strain CSTR1 (KsCSTR_09970). Fig.
S5. Coverage of SMRT sequencing reads along the genome of “Ca. Kue-
nenia stuttgartiensis”strain CSTR1. Table S1. Reported genome assemblies
of anammox bacteria up to this study (as of 02.02.2020). Table S2. List of
the longest 25 contigs obtained by the automatic assembling pipeline in
the PacBio SMRT Analysis software package. Table S3. Hypothetical se-
quential rearrangement events from genome CSTR1 to genome MBR1b
as calculated by GRIMM. Table S4. List of the 20 locally collinear blocks
(LCBs) of the genome of “Ca. Kuenenia stuttgartiensis”strain CSTR1 after
Mauve alignment with MBR1b. Table S5. CRISPR elements in the ana-
mmox genomes KUST, MBR1 and CSTR1. Table S6. Comparison of the
large CRISPR locus near the type I-B CRISPR-Cas cluster in the three stud-
ied anammox genomes. Table S7. Protein-coding genes of “Ca. Kuenenia
stuttgartiensis”strain CSTR1 and their abundance in the proteome. Table
S8. Transposase genes and their classification in the anammox genomes
KUST, MBR1 and CSTR1. Table S9: List of transposase genes and their clas-
sification in the anammox genomes KUST, MBR1 and CSTR1. Table S10.
Abundances of peptides from two highly similar hydrazine dehydroge-
nases KsCSTR_46980 and KsCSTR_11820 in the proteome of “Ca. Kuene-
nia stuttgartiensis”strain CSTR1. Table S11. Detection of three nitrite
reductase gene candidates in the proteome of “Ca. Kuenenia stuttgartien-
sis”strain CSTR1 over time. Table S12. List of peptides detected in the S-
layer protein KsCSTR_09970 in a series of “Ca. Kuenenia stuttgartiensis”
strain CSTR1 samples. Additional file 1. Genome assembly from the
IonTorrent-based resequencing of “Ca. Kuenenia stuttgartiensis”strain
KUST in 2012.
Abbreviations
Anammox: Anaerobic ammonium oxidation; SMRT sequencing: Single-
molecule real-time sequencing; CSTR: Continuous stirred-tank reactor;
CRISPR: Clustered regularly interspaced short palindromic repeats;
bp: Base pair; ori: Origin of replication; ANI: Average nucleotide identity;
LCB: Locally collinear block; HRT: Hydraulic retention time; PSM: Peptide
Ding and Adrian BMC Genomics (2020) 21:851 Page 9 of 11
spectrum match; nano-LC-MS: Nano-liquid chromatography–mass
spectrometry; CDS: Coding sequence; aa: Amino acids.
Acknowledgements
Protein mass spectrometry was done at the Centre for Chemical Microscopy
(ProVIS) at the Helmholtz Centre for Environmental Research, which is
supported by European regional development funds (EFRE—Europe Funds
Saxony) and the Helmholtz Association. Rohit Budhraja, Shubhangi Karande,
Emea Okorafor Ude, and Francis Enyi are thanked for providing strain CSTR1
proteomics data for nitrite reductase search.
Authors’contributions
CD and LA conceived the study. CD did the bioinformatics analyses and lab
experiments. CD and LA wrote the manuscript. Both authors have reviewed
and approved the final manuscript.
Funding
This study was financially supported by Humboldt Research Fellowship for
Postdoctoral Researchers from the Alexander von Humboldt Foundation
(Germany) and the German Research Foundation (DFG) Priority Programme
(SPP1927). The funders had no role in study design, data collection or
analysis, interpretation of data, or in writing the manuscript. Open Access
funding enabled and organized by Projekt DEAL.
Availability of data and materials
The genome sequencing data of “Ca. Kuenenia stuttgartiensis”strain CSTR1
have been submitted to the National Center for Biotechnology Information
(NCBI) in the BioProject PRJNA603163 (BioSample: SAMN13921532; reads
archive number: SRR11213620; genome accession number CP049055). The
mass spectrometry proteomics data have been deposited to the
ProteomeXchange Consortium via the PRIDE partner repository with the
dataset identifier PXD018553. The IonTorrent reads of the resequencing
project for “Ca. Kuenenia stuttgartiensis”strain KUST in 2012 was
downloaded from the European Molecular Biology Laboratory’s European
Bioinformatics Institute (EMBL-EBI) under the reads archive number
ERR342261.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Helmholtz Centre for Environmental Research –UFZ, Environmental
Biotechnology, Permoserstraße 15, 04318 Leipzig, Germany.
2
Chair of
Geobiotechnology, Technische Universität Berlin, Ackerstraße 76, 13355
Berlin, Germany.
Received: 6 July 2020 Accepted: 18 November 2020
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