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RESEARCH ARTICLE
The MocR family transcriptional regulator DnfR has
multiple binding sites and regulates Dirammox gene
transcription in Alcaligenes faecalis JQ135
Si-Qiong Xu
1
| Xiao Wang
1
|LuXu
1
| Ke-Xin Wang
1
| Yin-Hu Jiang
1
|
Fu-Yin Zhang
1
| Qing Hong
1
| Jian He
1
| Shuang-Jiang Liu
2,3
|
Ji-Guo Qiu
1
1
Key Laboratory of Agricultural and
Environmental Microbiology, Ministry of
Agriculture and Rural Affairs, College of Life
Sciences, Nanjing Agricultural University,
Nanjing, China
2
State Key Laboratory of Microbial Resources,
and Environmental Microbiology Research
Center at Institute of Microbiology, Chinese
Academy of Sciences, Beijing, China
3
State Key Laboratory of Microbial
Technology, Shandong University, Qingdao,
China
Correspondence
Ji-Guo Qiu, Key Laboratory of Agricultural and
Environmental Microbiology, Ministry of
Agriculture and Rural Affairs, College of Life
Sciences, Nanjing Agricultural University,
Nanjing, China.
Email: qiujiguo@njau.edu.cn
Funding information
National Key R&D Program of China,
Grant/Award Number: 2019YFA0905500;
National Natural Science Foundation of China,
Grant/Award Numbers: 32070092, 32170128
Abstract
Microbial ammonia oxidation is vital to the nitrogen cycle. A biological pro-
cess, called Dirammox (direct ammonia oxidation, NH
3
!NH
2
OH!N
2
), has
been recently identified in Alcaligenes ammonioxydans and Alcaligenes fae-
calis. However, its transcriptional regulatory mechanism has not yet been
fully elucidated. The present study characterized a new MocR-like transcrip-
tion factor DnfR that is involved in the Dirammox process in A. faecalis
strain JQ135. The entire dnf cluster was composed of 10 genes and tran-
scribed as five transcriptional units, that is, dnfIH,dnfR,dnfG,dnfABCDE
and dnfF. DnfR activates the transcription of dnfIH,dnfG and dnfABCDE
genes, and represses its own transcription. The intact 1506-bp dnfR gene
was required for activation of Dirammox. Electrophoretic mobility shift
assays and DNase I footprinting analyses showed that DnfR has one bind-
ing site in the dnfH-dnfR intergenic region and two binding sites in the dnfG-
dnfA intergenic region. Three binding sites of DnfR shared a 6-bp repeated
conserved sequence 50-GGTCTG-N
17
-GGTCTG-30which was essential for
the transcription of downstream target genes. Cysteine and glutamate act
as possible effectors of DnfR to activate the transcription of transcriptional
units of dnfG and dnfABCDE, respectively. This study provided new insights
in the transcriptional regulation mechanism of Dirammox by DnfR in
A. faecalis JQ135.
INTRODUCTION
Nitrogen is an essential component of all living organ-
isms and usually the main limiting nutrient for life on the
earth (Holmes et al., 2019; Kuypers et al., 2018). Micro-
bial ammonia oxidation plays an important role in the
nitrogen cycle (Kartal et al., 2011; Stein, 2011). Ammo-
nia can be oxidized in different microbial processes,
including nitrification, Anammox and Comammox
(Daims et al., 2015; Kessel et al., 2015; Kuenen, 2008;
Stein, 2011). Ammonia oxidation by heterotrophic bac-
teria has been reported a long time ago (Chen, Chen,
et al., 2021; Chen, Xu, et al., 2021; Huang et al., 2018;
Liu et al., 2015; Neerackal et al., 2016; Papen
et al., 1989; Shoda, 2017; Song et al., 2021), however,
its underlying molecular mechanism remains unfruitful
until recently that a new biological process called Dir-
ammox (direct ammonia oxidation, NH
3
!NH
2
OH!N
2
)
was identified in Alcaligenes species (Hou et al., 2022;
Wu et al., 2021; Xu, Qian, et al., 2022). A dinitrogen-
forming (dnf ) gene cluster was cloned from Alcaligenes
ammonioxydans strain HO-1. Escherichia coli cells
contains dnfABC genes converted ammonia to N
2
via
hydroxylamine (Wu et al., 2021). The dnfABC gene
cluster was further identified and characterized by
genetic deletion and complementary mutation in
Received: 14 November 2022 Accepted: 14 December 2022
DOI: 10.1111/1462-2920.16318
© 2022 Applied Microbiology International and John Wiley & Sons Ltd.
Environ Microbiol. 2022;1–14. wileyonlinelibrary.com/journal/emi 1
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Alcaligenes faecalis strain JQ135 (Xu, Qian,
et al., 2022). Recent in vitro enzymatic studies con-
firmed that DnfA could convert hydroxylamine into N
2
in
the presence of NADH and FAD (Wu et al., 2022).
Genomic survey in public databases showed that dnf
cluster was widely distributed in bacteria and responsi-
ble for Dirammox abilities (Hou et al., 2022).
MocR-like transcription factors (MocR-TFs) are a
subdivision of GntR family transcriptional regulators
and are distributed in both Gram-positive and Gram-
negative bacteria (Rigali et al., 2002; Tramonti
et al., 2018). MocR-TFs are chimeric proteins compris-
ing of an N-terminal winged helix-turn-helix DNA bind-
ing domain and a C-terminal aminotransferase-like
(AT-like) domain (Bramucci et al., 2011). To date, only
a few MocR-TFs have been characterized molecu-
larly, most of which are involved in the metabolism of
nitrogen-containing compounds. GabR, for example,
regulates transcription of γ-aminobutyric acid (GABA)
aminotransferase (GabT) gene and promotes the
catabolism of glutamate from GABA in Bacillus subtilis
(Al-Zyoud et al., 2015; Edayathumangalam
et al., 2013;Wuetal.,2017). PdxR, a transcriptional
regulator that activates transcription of the pdxST
genes encoding Pyridoxal 50-phosphate (PLP)
synthase in Bacillus clausii (Qaidi et al., 2013;
Tramonti et al., 2015). EhuR negatively regulated
ectoine uptake and catabolism in Sinorhizobium meli-
loti (Yu et al., 2016). Up to now, most of the reported
MocR-TFs control a single transcriptional unit. The
consensus sequence in the architecture of the DNA
binding sites of MocR-TFs has not been found
(Tramonti et al., 2018).
In the dnf cluster, a MocR-family transcriptional
gene dnfR was identified as an transcriptional activator
of dnfABC genes in A. faecalis JQ135 (Xu, Qian,
et al., 2022). However, the organization of dnf cluster
and its regulatory roles of DnfR on the entire dnf cluster
and on itself remain unclear. In the present study, the
function of DnfR was further investigated to better
understand the regulatory mechanism of Dirammox,
which expands the regulatory mechanism in the MocR-
like transcription factors (MocR-TFs).
EXPERIMENTAL PROCEDURES
Chemicals and media
All chemicals used for this experiment are commercially
available. Enzymes were purchased from Vazyme Bio-
tech. The heterotrophic nitrification medium (HNM)
used for bacteria cultivation was described as before
(Xu, Qian, et al., 2022). HNM2 medium was HNM with-
out carbon and nitrogen sources. Luria-Bertani
(LB) broth consisted of the following components:
tryptone (10.0 g/L), yeast extract (5.0 g/L), and NaCl
(10.0 g/L) at pH 7.0.
Bacterial strains and growth conditions
All bacterial strains and plasmids used in this study are
listed in Table 1.A. faecalis JQ135 was previously iden-
tified as an efficient nitrogen heterocyclic compounds
degrading bacterium (Mu et al., 2022; Qiu et al., 2018;
Zhang et al., 2018). E. coli strains were grown at 37C,
and other strains were grown at 30C. Media were sup-
plemented with ampicillin (Amp, 100 μg/ml), chloram-
phenicol (Cm 12.5 μg/ml), gentamicin (Gm, 50 μg/ml),
kanamycin (Km, 50 μg/ml), streptomycin (Str, 50 μg/ml),
or tetracycline (Tet, 40 μg/ml) as required.
Analytical methods
The concentrations of NH
4
+
and NO
2
were determined
by standard methods (APHA, 1998). Hydroxylamine
was determined according to the method of Frear and
Burrell (Frear & Burrell, 1955).
15
N
2
was determined by
GC/MS as previously reported (Xu, Qian, et al., 2022).
Bacterial cells were spectrophotometrically measured at
600 nm (OD
600
) (Wu et al., 2021).
Construction of plasmids and recombinant
strains JQ135ΔdnfR/dnfR
tr
The plasmid pBBR-dnfR
tr
which is 33 bp less than the
plasmid pBBR-dnfR was constructed for gene comple-
mentation. Gene dnfR
tr
was amplified using the primers
dnfRtr-F and dnfRtr-R and pBBR-dnfR used as the tem-
plate and then using the ClonExpress MultiS One Step
Cloning Kit (Vazyme Biotech), thus generating pBBR-
dnfR
tr
. The pBBR-dnfR
tr
plasmid was then transferred
into the JQ135ΔdnfR mutant to generate the comple-
mented strain JQ135ΔdnfR/dnfR
tr
. All primers were
listed in Table S1.
RNA extraction and RT-PCR
The method for culturing A. faecalis JQ135 and its
derivative strains, extraction and reverse transcription
of RNA from A. faecalis JQ135 and its derivative strains
was as described previously (Xu, Qian, et al., 2022).
The primers used for dnfIHRGABCDEF in this experi-
ment were described in Table S1. The transcriptional
level of the 16 S rRNA gene was used as internal stan-
dard, and the data in each column were calculated with
the 2
ΔΔCT
threshold cycle (C
T
) method using three
replicates. All samples were run in triplicates.
2XU ET AL.
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TABLE 1 Strains and plasmids used in this study
Strains and plasmids Description Source
Strains
A. faecalis JQ135 Str
r
; wild type; metabolically versatile bacterium on nitrogen compounds (Xu, Qian,
et al., 2022)
JQ135ΔdnfR Str
r
,Km
r
;dnfR-deletion mutant of JQ135; dnfR::Km
r
(Xu, Qian,
et al., 2022)
JQ135ΔdnfR/dnfR Str
r
,Km
r
,Gm
r
; JQ135ΔdnfR containing pBBR-dnfR (Xu, Qian,
et al., 2022)
JQ135ΔdnfR/dnfR
tr
Str
r
,Km
r
,Gm
r
; JQ135ΔdnfR containing pBBR-dnfR
tr
This study
JQ135/pME6522-P
dnfA
Str
r
,Tc
r
; JQ135 containing pME6522-P
dnfA
This study
JQ135ΔdnfR/
pME6522-P
dnfA
Str
r
,Km
r
,Tc
r
; JQ135ΔdnfR containing pME6522-P
dnfA
This study
JQ135/pME6522-P
dnfH
Str
r
,Tc
r
; JQ135 containing pME6522-P
dnfH
This study
JQ135ΔdnfR/
pME6522-P
dnfH
Str
r
,Km
r
,Tc
r
; JQ135ΔdnfR containing pME6522-P
dnfH
This study
JQ135/pME6522-P
dnfG
Str
r
,Tc
r
; JQ135 containing pME6522-P
dnfG
This study
JQ135ΔdnfR/
pME6522-P
dnfG
Str
r
,Km
r
,Tc
r
; JQ135ΔdnfR containing pME6522-P
dnfG
This study
JQ135/pME6522-P
dnfR
Str
r
,Tc
r
; JQ135 containing pME6522-P
dnfR
This study
JQ135ΔdnfR/
pME6522-P
dnfR
Str
r
,Km
r
,Tc
r
; JQ135ΔdnfR containing pME6522-P
dnfR
This study
JQ135/pME6522-P
dnfR-m1
Str
r
,Tc
r
; JQ135 containing pME6522-P
dnfR-m1
This study
JQ135ΔdnfR/
pME6522-P
dnfR-m1
Str
r
,Km
r
,Tc
r
; JQ135ΔdnfR containing pME6522-P
dnfR-m1
This study
JQ135/pME6522-P
dnfR-m2
Str
r
,Tc
r
; JQ135 containing pME6522-P
dnfR-m2
This study
JQ135ΔdnfR/
pME6522-P
dnfR-m2
Str
r
,Km
r
,Tc
r
; JQ135ΔdnfR containing pME6522-P
dnfR-m2
This study
JQ135/pME6522-P
dnfR-m3
Str
r
,Tc
r
; JQ135 containing pME6522-P
dnfR-m3
This study
JQ135ΔdnfR/
pME6522-P
dnfR-m3
Str
r
,Km
r
,Tc
r
; JQ135ΔdnfR containing pME6522-P
dnfR-m3
This study
JQ135/pME6522-P
dnfH-m1
Str
r
,Tc
r
; JQ135 containing pME6522-P
dnfH-m1
This study
JQ135ΔdnfR/
pME6522-P
dnfH-m1
Str
r
,Km
r
,Tc
r
; JQ135ΔdnfR containing pME6522-P
dnfH-m1
This study
JQ135/pME6522-P
dnfH-m2
Str
r
,Tc
r
; JQ135 containing pME6522-P
dnfH-m2
This study
JQ135ΔdnfR/
pME6522-P
dnfH-m2
Str
r
,Km
r
,Tc
r
; JQ135ΔdnfR containing pME6522-P
dnfH-m2
This study
JQ135/pME6522-P
dnfH-m3
Str
r
,Tc
r
; JQ135 containing pME6522-P
dnfH-m3
This study
JQ135ΔdnfR/
pME6522-P
dnfH-m3
Str
r
,Km
r
,Tc
r
; JQ135ΔdnfR containing pME6522-P
dnfH-m3
This study
JQ135/pME6522-P
dnfG2
Str
r
,Tc
r
; JQ135 containing pME6522-P
dnfG2
This study
JQ135/pME6522-P
dnfG3
Str
r
,Tc
r
; JQ135 containing pME6522-P
dnfG3
This study
JQ135/pME6522-P
dnfG4
Str
r
,Tc
r
; JQ135 containing pME6522-P
dnfG4
This study
DH5αCloning host Lab stock
HB101(pRK2013) Help strain for parental mating Lab stock
Plasmids
pBBR1MCS-5 Gm
r
; broad-host-range cloning plasmid Lab stock
pMD19-T Cloning plasmid Takara
pCE2 TA/Blunt-Zero Cloning plasmid for transcriptional start site Vazyme
pBBR-dnfR Gm
r
; pBBR1MCS-5 harbouring dnfR gene (Xu, Qian,
et al., 2022)
pBBR-dnfR
tr
Gm
r
; pBBR1MCS-5 harbouring dnfR
tr
gene This study
pET-dnfR Km
r
; NdeI-XhoI fragment containing dnfR inserted into pET29a(+) This study
(Continues)
MocR FAMILY TRANSCRIPTIONAL REGULATOR DnfR HAS MULTIPLE BINDING 3
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EMSA
All DNA fragments used in EMSA were PCR amplified
from A. faecalis JQ135 genomic DNA using the primers
shown in Table S1. Approximately 20 ng of a promoter
probe was mixed with 140 nM purified DnfR in a binding
buffer (50 mM Tris–HCl [pH 7.0], 30 mM KCl, 5% [vol/vol]
glycerol, 1 mM EDTA, and 5 mM DTT). The effects of
ammonia, hydroxylamine, nitrite, nitrate and 20 essential
amino acids on the binding of DnfR to the promoter
probes were evaluated by adding 0.1 mM of these sub-
strate to the reaction system respectively. The mixture
was incubated at 25C for 30 min and was then sepa-
rated by 6% (vol/vol) native polyacrylamide gel electro-
phoresis in 0.5Tris-glycine-EDTA. Gels were stained
with 4 S Green Plus Nucleic Acid Stain (Sangon Biotech),
and DNA fragments were visualized under UV light.
DNase I footprinting
The promoter region P
dnfG
(F
cl
-2) and P
dnfH-dnfR
(Fb) were PCR amplified and inserted into the plasmid
pMD19-T (TaKaRa) to create the plasmids pMD19T-Fb
and pMD19T-F
cl
-2, respectively. Then the specific
method for DNase I footprinting was described as
before (Xu, Qian, et al., 2022).
β-galactosidase assays
The primers of lacZ-dnfH/R/G-PstI/EcoRI listed in the
TableS1wereusedtoamplifiedthefragmentsof
promoters of the dnfH/R/Gto create the plasmids of
pME6522-P
dnfH/R/G
-lacZ. The primers of lacZ-P
dnfH/R-
m1/2/3
-PstI/EcoRI were used to amplified the fragments
of mutated promoters of the dnfH/Rto create the of a
series of mutated plasmids, in which, the nucleotide
‘G’was exchanged with ‘A’; the nucleotide ‘T’was
exchanged with ‘C’. The primers of lacZ-P
dnfG2/3/4
-
PstI/EcoRI were used to amplified the different length
of fragments containing the promoters of the dnfG to
create the plasmids of pME6522-P
dnfG2/3/4
-lacZ.Take
P
dnfG
for example. The PCR products were digested
with EcoRI and PstI and ligated into the EcoRI/PstI-
digested pME6522 to generate plasmid
pME6522-P
dnfG
. The resulting plasmids containing
P
dnfG
-lacZ fusions were transformed into A. faecalis
JQ135 and JQ135ΔdnfR, yielding JQ135/
pME6522-P
dnfG
and JQ135ΔdnfR/pME6522-P
dnfG
,
respectively. The plasmids of pME6522-P
dnfH/R-m1/2/3
and pME6522-P
dnfG2/3/4
were only transformed into
strain A. faecalis JQ135. β-galactosidase assays were
performed as before (Xu, Qian, et al., 2022). One
Miller unit of enzyme activity was defined as the
amount of enzyme required to catalyse o-nitrophenyl-
β-D-galactopyranoside to produce 1 μmol o-
nitrophenol per min (Sambrook & Russell, 2001). All
samples were tested in triplicates.
Transcriptional start site of the dnf cluster
The transcriptional start sites (TSSs) of the dnfH,dnfR
and dnfG genes were determined by 50-rapid amplifica-
tion of cDNA ends (50RACE) using the HiScript-TS 50/30
TABLE 1 (Continued)
Strains and plasmids Description Source
pME6522 Tc
r
; shuttle vector for transcriptional lacZ fusion and promoter probing Lab stock
pME6522-P
dnfA
Tc
r
;P
dnfA
fragment fused into pME6522 This study
pME6522-P
dnfH
Tc
r
;P
dnfH
fragment fused into pME6522 This study
pME6522-P
dnfR
Tc
r
;P
dnfR
fragment fused into pME6522 This study
pME6522-P
dnfG
Tc
r
;P
dnfG
fragment fused into pME6522 This study
pME6522-P
dnfR-m1
Tc
r
;P
dnfR-m1
fragment fused into pME6522 This study
pME6522-P
dnfR-m2
Tc
r
;P
dnfR-m2
fragment fused into pME6522 This study
pME6522-P
dnfR-m3
Tc
r
;P
dnfR-m3
fragment fused into pME6522 This study
pME6522-P
dnfH-m1
Tc
r
;P
dnfH-m1
fragment fused into pME6522 This study
pME6522-P
dnfH-m2
Tc
r
;P
dnfH-m2
fragment fused into pME6522 This study
pME6522-P
dnfH-m3
Tc
r
;P
dnfH-m3
fragment fused into pME6522 This study
pME6522-P
dnfG2
Tc
r
;P
dnfG2
fragment fused into pME6522 This study
pME6522-P
dnfG3
Tc
r
;P
dnfG3
fragment fused into pME6522 This study
pME6522-P
dnfG4
Tc
r
;P
dnfG4
fragment fused into pME6522 This study
pMD19T-Fb Amp
r
; 248-bp fragment, promoter region of dnfR and dnfH, directionally cloned into
pMD19-T
This study
pMD19T-F
cl
-2 Amp
r
; 184-bp fragment, promoter region of dnfG, directionally cloned into pMD19-T This study
4XU ET AL.
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RACE kit (Vazyme Biotech) according to the manufac-
turer’s instructions. Total RNA was used and isolated
as described above. The primers TSS-dnfA/R/H/G-1/2
used in this experiment were listed in the Table S1. The
final PCR product was purified with a gel-extraction kit
(Vazyme Biotech) and then ligated into pCE2 TA/Blunt-
Zero vector using the Blunt-Zero Cloning kit (Vazyme
Biotech) for sequencing. The promoters region of the
four transcriptional units are predicted through the fol-
lowing websites: phiSITE (http://www.phisite.org/main/
index.php?nav=tools&nav_sel=hunter) (Klucar
et al., 2009), BacPP (http://www.bacpp.bioinfoucs.com/
home/) (Silva et al., 2011) and BDGP (https://www.
fruitfly.org/seq_tools/promoter.html) (Reese, 2002).
RESULTS
Organization and transcriptional analysis
of the dnf cluster
Our previous studies have shown that dnfABC and
dnfR genes were essential for Dirammox process (Xu,
Qian, et al., 2022). However, the composition of the dnf
cluster was not experimentally investigated. According
to the previous investigation about the dnfABCR genes,
RT-PCR was carried out to investigate the entire dnf
cluster (Figure 1). Total RNA, cDNA and DNA extrac-
tion from the JQ135 cells were used as the templates
for PCR amplification. The amplified products were
detected by electrophoresis to determine the transcripts
of dnf clusters. As shown in Figure 1B, among the
amplified 11 fragments (2 fragments within genes and
9 fragments across genes), 10 fragments were suc-
cessfully amplified using cDNA as the template except
fragment 5. Therefore, the dnfABC genes were co-
transcribed with two downstream genes (named as
dnfDE). On the other side, two genes (named dnfIH)
opposite to dnfR form a transcription unit. Interestingly,
fragment F3 can also be amplified, which means that
the four genes (named dnfIHRG) seem to be co-tran-
scribed, though dnfR and dnfIHG are divergently orien-
tated. The possible reason might be that the
transcription unit dnfIHRG was also started by the pro-
moter of dnfG (see below). Therefore, the dnf cluster
was organized into five transcriptional units, that is,
FIGURE 1 Tanscriptional analysis of dnf cluster. (A) Organization of dnf cluster. The gene IDs of dnfI to dnfF are AFA_14070 to
AFA_14115, respectively. (B) Transcriptional unit analysis of the dnf gene cluster. PCR amplification of the dnf gene cluster using total RNA (R),
cDNA (cD) and DNA (D) as the templates. The amplified products were detected by electrophoresis. (C) Transcriptional analysis of dnfI,dnfH,
dnfR,dnfG,dnfA,dnfB,dnfC,dnfD,dnfE and dnfF in strains JQ135, JQ135ΔdnfR, JQ135ΔdnfR/dnfR in HNM. Different letters indicate
differences as determined by one-way AVONA (p< 0.05). (D) In vivo dnfH,dnfR,dnfG and dnfA promoter activity assay. β-galactosidase assays
were performed with JQ135 and JQ135ΔdnfR, carrying the plasmids pME6522–P
dnfH
, pME6522–P
dnfR
, pME6522–P
dnfG
and pME6522–P
dnfA
,
respectively, grown in the HNM. P
dnfH
or P
dnfR
is a 155 bp promoter region of dnfH or dnfR, respectively. P
dnfG
or P
dnfA
is a 301 or 381 bp
promoter region of dnfG or dnfA, respectively. β-galactosidase activity was measured as described in materials and methods. Each value is the
mean ± standard deviation (SD) of at least three cultures. Different letters indicate differences as determined by one-way AVONA (p< 0.05)
MocR FAMILY TRANSCRIPTIONAL REGULATOR DnfR HAS MULTIPLE BINDING 5
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dnfHI,dnfR,dnfG,dnfABCDE and dnfF (Figure 1A).
DnfI and DnfG were predicted as serine hydroxymethyl-
transferase while DnfH is described as phosphoserine
aminotransferase. DnfD, DnfE and DnfF are annotated
as pyridoxal kinase, high-affinity choline uptake protein
BetT and redox-sensitive transcriptional activator
SoxR, respectively.
The transcriptions of dnf cluster genes in strain
JQ135 were assessed using quantitative RT-PCR (RT-
qPCR). As shown in Figure 1C, when cultured in HNM,
the transcription levels of 10 dnf genes in strain JQ135
were 35.2 ± 1.5, 15.0 ± 0.8, 5.4 ± 0.6, 8.3 ± 0.8, 248.0
± 5.2, 150.1 ± 2.5, 27.0 ± 0.6, 8.1 ± 0.5, 9.2 ± 1.0 and
4.8 ± 0.4 folds higher, respectively, than those in
JQ135ΔdnfR (Figure 1C). In the complemented strain
JQ135ΔdnfR/dnfR, the transcription levels of 10 dnf
genes were similar to those of the wild-type strain
JQ135. These results indicated that these 10 genes
constitute a cluster regulated by DnfR.
Identification of the transcription start sites
of transcriptional units of dnfIH,dnfR,
dnfG, and dnfABCDE
In our previous study, we determined the TSS of dnfA
gene (Xu, Qian, et al., 2022). In this study, we contin-
ued to use the total RNA extracted from JQ135 cells
grown in HNM to determine the TSSs of other three
transcriptional units in the dnf cluster by 50RACE. As
shown in Figure 2, the A bases of the ATG start codons
of dnfH,dnfR and dnfG are determined at positions
25, 13 and 89, respectively, relative to their TSSs. A
more detailed analysis of the promoter is shown in
Figure 2. The TSSs of these two set of divergent tran-
scriptional units do not overlap with each other. The
TSSs of dnfH and dnfR genes are close (88 bp), while
the TSSs of dnfG and dnfA genes are separated by a
distance of 300 bp. A more detailed analysis of the four
promoters is shown in Figure 2.
Determination of the binding sites of DnfR
Previous study has showed that DnfR bound to the
upstream region of promoter region of dnfABC genes
and activated its transcriptional expression (Xu, Qian,
et al., 2022). Here, further studies were carried out to
investigate the regulatory mechanism and other pos-
sible binding regions of DnfR. Five intergenic frag-
ments that may contain promoter region with a long
interval in the 10 genes were selected, namely dnfI-
dnfH (fragment Fa, 241 bp), dnfH-dnfR (fragment Fb,
248 bp), dnfG-dnfA (fragment Fc, 688 bp), dnfD-dnfE
(fragment Fd, 227 bp) and the promoter region of dnfF
(fragment Fe, 349 bp). The purified DnfR was tested
for its ability to bind to the above five DNA fragments
using electrophoretic mobility shift assay (EMSA).
Figure 2D showed that fragments Fb and Fc were
shifted while fragments Fa, Fd and Fe were not. In the
fragment Fc, DnfR has been shown to bind to the pro-
moter region of dnfA. Since fragment Fc was a long
space with two divergent genes (dnfG and dnfA), we
further tested whether DnfR could bind to the pro-
moter region of dnfG (206 to +95 relative to the
dnfG TSS, F
cl
) alone. Fragment F
cl
were then subdi-
vided into two subfragments F
cl
-1 to F
cl
-2. EMSA
results in Figure 2E showed that F
cl
and subfragment
F
cl
-2 were shifted in the presence of DnfR, indicating
the presence of DnfR binding site in the promoter
region of dnfG. DNase I footprinting was performed to
identify the DnfR-binding sites of above intergenic
regions fragment Fb and F
cl
-2 (Figure 2). In the dnfH-
dnfR intergenic sequence, DnfR binds to a 48-bp
region which located on the 14 to 34 and 54 to
102 according to the TSSs of dnfR and dnfH,
respectively. In the promoter region of dnfG,DnfR
binds to the 108 to 144 region according to the
TSS of dnfG. A more detailed analysis of the four pro-
moters is shown in Figure 2.
The intact dnfR gene is required for
regulation of Dirammox
As shown in Figure 2A, the binding region towards frag-
ment F
2
covered the A base of ATG of dnfR gene
(1506 bp). Then, a second start codon ATG was found
with 33 bp space downstream the dnfR gene. In order
to check which ATG is the true starting codon, the trun-
cated dnfR
tr
(1473 bp) was supplemented to mutant
strain JQ135ΔdnfR. A new complementary strain
named as JQ135ΔdnfR/dnfR
tr
was constructed. The
results showed that JQ135ΔdnfR/dnfR
tr
had no differ-
ences to JQ135ΔdnfR (Figure S1). These experiments
indicated that the 33 bp sequence of dnfR is
necessary.
The role of DnfR and the conserved
sequences of three binding sites
To verify the roles of these binding regions on transcrip-
tion of dnf genes, a series of promoter fragments con-
taining DnfR protection regions were fused with lacZ
gene and inserted to plasmid pME6522, produced plas-
mids pME6522-P
dnfA
, pME6522-P
dnfG
, pME6522-P
dnfH
,
and pME6522-P
dnfR
, and then transformed them into
strains JQ135 and JQ135ΔdnfR, respectively. The
β-galactosidase assays showed that, similar to dnfA,
promoters of dnfIH and dnfG showed no activities in
mutant strain JQ135ΔdnfR, while expressed high activi-
ties (3.5 ± 0.4, 4.9 ± 0.8 folds higher at 12 h than in
JQ135ΔdnfR) in wild type strain JQ135 (Figure 1D),
6XU ET AL.
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suggesting that DnfR actively regulates the transcrip-
tions of transcriptional units of dnfIH,dnfG and
dnfABCDE. In contrast, β-galactosidase activities of
promoter P
dnfR
in JQ135ΔdnfR were approximately
11.6 ± 0.9 fold at 12 h relative to those in wide type
JQ135, indicating that DnfR negatively regulated itself.
FIGURE 2 Identification of DnfR binding sites. (A) The DNA elements in the promoter regions of dnfH and dnfR. The TSSs of dnfH and dnfR
are indicated in grey and red, respectively, and are indicated with arrowheads. The DNA binding sites of DnfR are shown in purple.
(B) Organization of dnf cluster. The figures below the cluster are fragments used in the EMSA experiments. (C) DNA elements in the promoter
region of dnfG and dnfA. The TSSs of dnfG and dnfA are indicated in grey and green, respectively, and are indicated with arrowheads. The
subfragments used in the EMSA experiments are indicated. The DNA binding sites of DnfR are shown in purple. (D) EMSAs of fragments Fa, Fb,
Fc, Fd and Fe with purified DnfR (140 nM). (E) EMSAs of subfragments F
cl
,F
cl
-1 and F
cl
-2 with purified DnfR (140 nM). (F) and (G) DNase I
footprinting determination of the protected regions in P
dnfH
-
dnfR
and P
dnfG
promoter regions by DnfR, respectively. The DnfR protected regions
were shown in dashed box and the sequence were shown at the bottom
MocR FAMILY TRANSCRIPTIONAL REGULATOR DnfR HAS MULTIPLE BINDING 7
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Comparing three DnfR binding sites of promoter
regions of dnfA,dnfG, and dnfH genes, they share a
consensus sequence of 6-bp direct repeats ‘GGTCTG-
N
17
-GGTCTG’with 17 bp spacer and located upstream
of the TSSs (Figure 3A). In addition, there are also two
sets of perfect palindrome sequences 50-GGTCTG-
N
6
(N
29
)-CAACAC-30in the DnfR binding site in frag-
ment F
2
(intergenic region of dnfH-dnfR), with the inter-
vals of 6 and 29 bp, respectively. In order to determine
the roles of repeats sequences and palindromes
sequences in the promoters, a series of site-directed
mutations (site1, site2, or site3 in Figure 3) of intergenic
region of dnfH-dnfR were ligated into the EcoRI/PstI-
digested pME6522 to generate plasmids
pME6522-P
dnfR-m1
to P
dnfR-m3
(to test the activity of
dnfR promoter) and pME6522-P
dnfR-m4
to P
dnfR-m6
(to test the activity of dnfH promoter) (Figure 3), and
then transformed into wide type JQ135 and mutant
JQ135ΔdnfR, respectively. β-galactosidase activities
driven by P
dnfR-m1
and P
dnfR-m2
promoters in wide type
were 16.9 ± 1.3, 19.2 ± 1.7 fold higher at 12 h than
those in JQ135ΔdnfR, respectively, which are similar to
those by P
dnfR
(in the wide type 11.6 ± 0.9 fold higher
at 12 h than in JQ135ΔdnfR). Very low β-galactosidase
activity (<60 Miller units) of P
dnfR-m3
promoter were
detected at 12 both in strain JQ135 and JQ135ΔdnfR.
Next, the β-galactosidase activities driven by P
dnfH-m3
promoter in wild type JQ135 were 8.6 ± 1.1 fold higher
FIGURE 3 Features and characterizations of three binding sites of DnfR. (A) Analysis of the three binding sites. The conserved areas of
three binding sites are indicated in blue or orange and marked by vertical lines. (B) Mutational analysis of the binding site of P
dnfH-dnfR
. Mutational
sites are indicated in dotted line. (C) β-galactosidase assays were performed with JQ135 and JQ135ΔdnfR, carrying the plasmids pME6522–
P
dnfH
, pME6522–P
dnfR
and a series of mutant plasmids grown in the HNM. Each value is the mean ± standard deviation (SD) of at least three
cultures. Different letters indicate differences as determined by one-way AVONA (p< 0.05)
8XU ET AL.
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at 12 h than those in JQ135ΔdnfR, which are similar to
P
dnfH
(9.1 ± 0.7 fold higher at 12 h in the wide type than
those in JQ135ΔdnfR); while very low β-galactosidase
activity (<80 Miller units) of P
dnfH-m1
and P
dnfH-m2
pro-
moters were detected at 12 both in strain JQ135 and
JQ135ΔdnfR. These results indicated that the two
direct repeated sequences (site1 and site2, blue parts
in Figure 3) were essential for the activation of the
downstream target genes but has no effect on its
upstream dnfR gene. In contrast, the reverse palin-
drome sequence (site3, orange parts in Figure 3), it
had no effect on the target genes, but was crucial for
the expression of dnfR itself.
Genes dnfGRHI forms as a
transcriptional unit
Figure 2shows that dnfR and dnfIH areasetofdiver-
gently orientated transcriptional units, and their tran-
scription start sites do not overlap, however, RT-PCR
analysis showed that dnfR is co-transcribed with dnfH
(Figure 1). Therefore, it is hypothesized that the pro-
moter of dnfG could transcribe dnfGRHI together. To
verify this hypothesis, a series of fragments with/
without the dnfG promoter but not reaching to the 10
region of gene dnfH were ligated into the
β-galactosidase reporter plasmids pME6522.
β-galactosidase activities driven by P
dnfG2
and P
dnfG3
promoters in wide type JQ135 were 5.5 ± 0.4, 4.7
± 0.4 fold higher at 12 h than those in JQ135ΔdnfR,
respectively, which are similar to P
dnfG
; while very low
β-galactosidase activity (<100 Miller units) of P
dnfG4
plasmids were detected at 12 both in strain JQ135
and JQ135ΔdnfR (Figure 4). The results indicated that
the P
dnfG
was a promoter of transcriptional unit cover-
ing the intergenic region of dnfH-dnfR genes, that is,
dnfGRHI formed as a transcriptional unit.
The possible effectors of DnfR
To explore the possible effectors of DnfR towards dnf
cluster, the EMSA and/or β-galactosidase assays were
performed. EMSA experiments showed that 0.1 mM
ammonia, hydroxylamine, nitrite, and nitrate had no sig-
nificant effects on DnfR binding to the four promoter
regions (P
dnfH-m3
,P
dnfR-m2
,P
dnfG
and P
dnfA
), inferring
that none of them is the efficient effector for DnfR
(Figure S2). Then 20 amino acids were used to investi-
gate whether they were the possible effectors. As shown
in the Figure 5, none of the 20 amino acids (0.1 mM)
could increase the binding of P
dnfH-m3
or P
dnfR-m2
frag-
ments with DnfR, indicating that 20 amino acids may not
be the possible effectors/ligands of DnfR to activate dnfH
or dnfR gene. However, 0.1 mM cysteine and glutamate
could increase the binding of DnfR towards P
dnfG
and
P
dnfA
fragment, respectively (Figure 5). In addition, the
amount of DnfR–P
dnfG
and DnfR–P
dnfA
complex
increased along with the gradient concentration of cyste-
ine and glutamate, respectively (Figure 5I,J). Further-
more, the effects of cysteine and glutamate to DnfR
were investigated using four β-galactosidase activity
reporter plasmids pME6522-P
dnfA
, pME6522-P
dnfG
,
pME6522-P
dnfH
, and pME6522-P
dnfR
into strain JQ135.
The strains were then cultured in HNM2 (HNM without
carbon and nitrogen source) with or without 1 mM cyste-
ine or glutamate. As shown in Figure 6,verylow
β-galactosidase activity (<200 Miller units) of all four
reporter plasmids were detected at 4 or 8 h in strain
JQ135 in HNM2, indicating no transcriptions when no
carbon and nitrogen sources were present in media.
Next, the level of β-galactosidase of pME6522-P
dnfA
were approximately 2.8 ± 0.5 times (317 ± 9.2 Miller
units) higher at 4 or 8 h induced by glutamate than cyste-
ine or HNM2. Furthermore, β-galactosidase activities of
pME6522-P
dnfG
were 12.1 ± 1.3 times (1742 ± 69
Miller units) and 10.1 ± 1.8 times (1452 ± 41 Miller units)
higher at 4 or 8 h induced by glutamate or cysteine than
that in HNM2, respectively. In contrast, neither glutamate
nor cysteine could induce the expression of the
pME6522-P
dnfR
and -P
dnfH
(β-galactosidase activities
FIGURE 4 In vivo dnfG,dnfG-dnfR,dnfG-dnfH and dnfR-P
dnfH
promoter activity assays. (A) Schematic diagram of the promoter
regions of dnfG,dnfG-dnfR,dnfG-dnfH and dnfR-P
dnfH
. (B) β-
galactosidase assays were performed with JQ135 and JQ135ΔdnfR,
carrying the plasmids pME6522–P
dnfG
, pME6522–P
dnfG2
, pME6522–
P
dnfG3
and pME6522-P
dnfG4
, respectively, grown in the HNM. P
dnfG
,
P
dnfG2
,P
dnfG3
and P
dnfG4
are 301, 1766, 3381 and 389-bp promoter
regions of dnfG, respectively. β-galactosidase activity was measured
as described in materials and methods. Each value is the mean
± standard deviation (SD) of at least three cultures. Different letters
indicate differences as determined by one-way AVONA (p< 0.05)
MocR FAMILY TRANSCRIPTIONAL REGULATOR DnfR HAS MULTIPLE BINDING 9
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FIGURE 5 Determination of effectors of DnfR. (A, B), (C, D), (E, F), and (G, H) are EMSA with P
dnfH-m3
,P
dnfR-m2
,P
dnfG
, and P
dnfA
promoter
sequences (20 nM), respectively, and DnfR (140 nM) in the presence of 0.1 mM 20 amino acids. (): DNA fragment (10 nM) alone: (+): DNA
fragment (10 nM) plus DnfR; (+amino acid) DNA fragment (10 nM) plus DnfR and amino acid. (I) EMSA with P
dnfG
promoter sequence (20 nM)
and DnfR (140 nM) in the presence of different concentrations of cysteine. (J) EMSA with P
dnfA
promoter sequence (20 nM) and DnfR (140 nM)
in the presence of different concentrations of glutamate
10 XU ET AL.
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<200 Miller units) (Figure 6). Based on the results of
EMSA and β-galactosidase assays, cysteine and gluta-
mate could be the effectors of transcriptional units of
dnfG and dnfABCDE,respectively.
DISCUSSION
The present study investigated the organization of dnf
gene cluster in A. faecalis JQ135, and expanded it as
five transcriptional units: dnfHI,dnfR,dnfG,dnfABCDE,
and dnfF. EMSA and DNase I footprinting analyses
showed that DnfR could activate the transcriptions of
dnfHI,dnfG, and dnfABCDE genes but repress the
transcription itself. The three binding sites of DnfR
shared a 6-bp repeated conserved sequence 50-
GGTCTG-N
17
-GGTCTG-30. The cysteine and gluta-
mate could be the effectors of transcriptional units of
dnfG and dnfABCDE, respectively.
MocR-TFs belongs into the GntR-family transcrip-
tional regulators (Taw et al., 2015; Xu, Qian,
et al., 2022). MocR-TFs are special in its chimeric struc-
ture, which consist of an N-terminal DNA-binding
domain and a C-terminal aspartate aminotransferase-
like domain (Bramucci et al., 2011). However, no
reports on its aspartate aminotransferase activities has
been found ever. They often involves in controlling the
metabolism of organic nitrogen compounds
(Belitsky, 2014; Tramonti et al., 2016; Wu et al., 2017).
Previous studies showed that most MocR-TFs encod-
ing genes and their target genes are located together
with divergent orientations. The promoters of the two
genes are located in the intergenic region, and often
overlap with each other. MocR-TFs binds this region to
represses its own expression, but activates the expres-
sion of the structure genes. For example, GabR, regu-
lating GABA metabolism in Bacillus subtilis,isan
autorepressor and transcriptional activator of gabR and
gabT, respectively, which allows the bacterium to use
GABA as nitrogen and carbon sources (Okuda
et al., 2014; Wu et al., 2017). PdxR, involved in the reg-
ulation of pyridoxal phosphate synthesis in Bacillus
clausii, activates the transcription of pdxST genes
encoding PLP synthase while represses its own
expression (Tramonti et al., 2015). However, MocR-
TFs binding to multiple transcriptional units or distant
genes are rarely reported. In this study, DnfR controls
multiple sites of dnf gene cluster in A. faecalis JQ135,
that is, one site in the spacer region of dnfH and dnfR
and two sites in the spacer region of dnfG and dnfA.
These findings of multiple regulatory sites provided
new research paradigm of MocR-TFs.
In previous studies, the binding sites of MocR-TFs
are diverse in DNA length, sequence, and arrange-
ments (Tramonti et al., 2018). In this study, a conserved
sequence of 6-bp direct repeats 50-GGTCTG-N
17
-
GGTCTG-30were shared with three DnfR binding sites
which were essential for the activation of the down-
stream target genes (Figure 4). No binding sequences
of MocR-TFs were identical to this. The most similar
MocR-TFs target DNA was 50-ATACCA-N
34
-ATACCA-
30for GabR binding. The common feature was ACC but
the sequences were reverse complementary, that is,
GGT in DnfR. Nevertheless, some broad common fea-
tures can be drawn out. Like TauR, PdxR and EhuR, a
relevantly common feature is the occurrence of a TG
group of DnfR (Tramonti et al., 2015; Wiethaus
et al., 2008; Yu et al., 2016); Another is the 23 bp dis-
tance of the two direct repeats of DnfR, which is similar
to most MocR-TFs (Tramonti et al., 2018). In addition,
the structure of two direct repeats in binding sites of
MocR-TFs was similar with findings in AraC/XylS family
transcription regulators (Gallegos et al., 1997;
Schleif, 2010). For example, AraC binds a tandem
repeat 50-AGC-N
7
-TCCATA-30motif to activate the
expression of araBAD genes which involved in the
catabolism of L-arabinose; XylS binding sites were two
essential repeat motifs 50-T(C/A)CA-N
4
-TGCA-30which
involved in toluate degradation (Gallegos et al., 1997).
These structural features of direct repeats contrast with
binding sites by members of IclR (Molina-Henares
et al., 2005), LysR (Maddocks & Oyston, 2008), SoxR
(Wang et al., 2021) and TetR (Bertram et al., 2021;
Ramos et al., 2005) family regulators, which recognize
palindromic or inverted repeat sequences.
The dnfABC genes are responsible for the conver-
sion of ammonia into N
2
via NH
2
OH and considered as
widely distribution in bacteria. Lasted research showed
that DnfABC can enzymatically convert NH
2
OH into N
2
in vitro (Wu et al., 2022). However, the biological pro-
cess of transforming ammonia into NH
2
OH still remains
FIGURE 6 Effects of cysteine and glutamate on four promotors.
β-galactosidase assays were performed with JQ135 carrying the
P
dnfH
-lacZ,P
dnfR
-lacZ,P
dnfG
-lacZ and P
dnfA
-lacZ transcriptional
fusions grown in the presence of 0.5 mM cysteine or glutamate at 0, 4
and 8 h. Each value is the mean ± standard deviation (SD) of at least
three cultures. Different letters indicate differences as determined by
one-way AVONA (p< 0.05)
MocR FAMILY TRANSCRIPTIONAL REGULATOR DnfR HAS MULTIPLE BINDING 11
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mystery. The results of this study showed that
10 genes, including dnfABC gene and dnfDEFGHIR,
formed a gene cluster and were regulated by DnfR.
The dnfDEGI genes are predicted to be involved in
nitrogen-related metabolism. For example, DnfI and
DnfG were predicted as serine hydroxymethyltransfer-
ase while DnfH is described as phosphoserine amino-
transferase. Although the functions of the other seven
genes were not fully identified or investigated
(e.g., gene deletion and complementation) in this study,
we might infer that dnfDEFGHI genes must participate
in the Dirammox process in a certain way. At the same
time, glutamate or cysteine may interact with DnfR to
activate the expression of dnfABCDE and dnfHI.On
this basis, it is possible that the true substrate(s) of Dnf
is nitrogen-containing organic compound(s) but not
ammonia itself. Another possibility is that the Dirammox
is deeply intertwined with primary metabolism
(e.g., amino acid synthesis or degradation), which
increasing the difficulty and complexity to investigate.
During the regulatory processes by transcriptional
proteins, specific effectors or inducers are generally
needed. For example, PLP and GABA trigger GabR-
mediated transcription activation in Bacillus subtilis via
inducing the expression of gabT genes (Wu
et al., 2017); the transcription of transcriptional units of
genKH and genDFM in Corynebacterium glutamicum
can be activated by GenR in the present of effectors
3-hydroxybenzoate or gentisate (Chao & Zhou, 2013);
1-Naphthol as effector to activate mcbBCDEF cluster
transcription encoding the upstream pathway of carba-
ryl degradation in Pseudomonas sp. strain XWY-1 (Ke
et al., 2021). The β-galactosidase activity assays in
Figure 6of this study showed that no promotor activi-
ties were detected in wild-type strain JQ135 when no
carbon and nitrogen sources were present in media,
indicating that the transcription of dnfR,dnfA,dnfG and
dnfH required effectors or inducers. EMSA analyses
showed that 0.1 mM ammonia, hydroxylamine, nitrite,
and nitrate had no significant effect on DnfR binding to
all four promoter regions (Figure S2), suggesting that
none of them is the direct effector for DnfR. The cyste-
ine and glutamate could activate the expression of
dnfG and dnfA genes, respectively, but neither of them
have effects on the expression of dnfH gene. Moreover,
DnfR could repress its own expression and its tran-
scription may not depend on glutamate and cysteine.
Therefore, we may infer that the effectors of DnfR
towards four dnf transcriptional units are different with
each other. Some regulator protein can control multiple
sites using one effector. For example, gentisate can
induce GenR to activate transcriptions of two transcrip-
tional units (genD and genK)inC. glutamicum, while
6-hydroxypicolinic acid can derepress transcriptions of
three transcriptional units (picT,picB and picC) from
PicR in A. faecalis (Chao & Zhou, 2013; Xu, Wang,
et al., 2022). Why are there multiple binding sites of
DnfR in the dnf cluster with different effectors? The
probable reasons might be that these transcriptional
units in the dnf cluster are expressed at different time
or in different order, and response for different effec-
tors, in order to regulate itself more finely to adapt
changing surroundings, nutrients or stresses. These
hypotheses need further deep studies.
AUTHOR CONTRIBUTIONS
Siqiong Xu: Data curation (equal); investigation (equal);
writing –original draft (equal); writing –review and edit-
ing (equal). Xiao Wang: Investigation (equal). Lu Xu:
Investigation (equal). Kexin Wang: Investigation
(equal). Yinhu Jiang: Investigation (equal). Fuyin
Zhang: Investigation (equal). Qing Hong: Funding
acquisition (equal); investigation (equal). Jian He: Fund-
ing acquisition (equal); investigation (equal); supervision
(equal); writing –original draft (equal); writing –review
and editing (equal). Shuang-Jiang Liu: Conceptualiza-
tion (equal); funding acquisition (equal); investigation
(equal); project administration (equal); writing –original
draft (equal); writing –review and editing (equal). Jiguo
Qiu: Conceptualization (equal); funding acquisition
(equal); investigation (equal); project administration
(equal); supervision (equal); writing –original draft
(equal); writing –review and editing (equal).
ACKNOWLEDGEMENTS
This work was supported by grants from the National
Key R&D Program of China (2019YFA0905500) and
the National Natural Science Foundation of China
(No. 32070092 and 32170128).
CONFLICT OF INTEREST
The authors declare that they have no conflict of
interest.
DATA AVAILABILITY STATEMENT
The complete genome sequence of Alcaligenes faeca-
lis JQ135 is available in GenBank at accession number
CP021641. The gene IDs of dnfI to dnfF are
AFA_14070 to AFA_14115, respectively.
ORCID
Qing Hong https://orcid.org/0000-0001-5385-6281
Shuang-Jiang Liu https://orcid.org/0000-0002-7585-
310X
Ji-Guo Qiu https://orcid.org/0000-0002-7633-6227
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SUPPORTING INFORMATION
Additional supporting information can be found online
in the Supporting Information section at the end of this
article.
How to cite this article: Xu, S.-Q., Wang, X.,
Xu, L., Wang, K.-X., Jiang, Y.-H., Zhang, F.-Y.
et al. (2022) The MocR family transcriptional
regulator DnfR has multiple binding sites and
regulates Dirammox gene transcription in
Alcaligenes faecalis JQ135. Environmental
Microbiology,1–14. Available from: https://doi.
org/10.1111/1462-2920.16318
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