genes NIF

Biotechnol Lett (2015) 37:1999–2004
DOI 10.1007/s10529-015-1874-5
ORIGINAL RESEARCH PAPER
Expression of the N2 fixation gene operon of Paenibacillus
sp. WLY78 under the control of the T7 promoter
in Escherichia coli BL21
Lihong Zhang . Xiaomeng Liu .
Xinxin Li . Sanfeng Chen
Received: 14 January 2015 / Accepted: 26 May 2015 / Published online: 9 June 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract
Objective To investigate the transcription and translation and nitrogenase activity of the nine N2-fixinggene (nif) operon (nifBHDKENXhesAnifX) of Paenibacillus sp. WLY78 under the control of the T7
promoter in Escherichia coli BL21 under different
conditions.
Results The Paenibacillus nif operon under the control of the T7 promoter is significantly transcribed and
effectively translated in E. coli BL21 when grown in
medium containing organic N compounds (yeast extract
and Tryptone) or NH4? by using RT-PCR and Western
blot analysis. Transcription and translation of foreign nif
genes in E. coli are not inhibited by environmental
organic or inorganic N compounds or O2. However,
contrary to transcription and translation, nitrogenase
activity is 4 % lower in the recombinant E. coli 78–32
compared to the native Paenibacillus sp. WLY78.
Conclusion The Paenibacillus nif operon under the
control of T7 promoter enables E. coli BL21 to
synthesize active nitrogenase. This study shows how
the nif gene operon can be transferred to non-N2-fixing
bacteria or to eukaryotic organelles.
Keywords Escherichia coli Nif gene operon Nitrogenase activity Paenibacillus T7 promoter
Electronic supplementary material The online version of
this article (doi:10.1007/s10529-015-1874-5) contains supplementary material, which is available to authorized users.
Introduction
L. Zhang X. Liu X. Li S. Chen (&)
State Key Laboratory for Agrobiotechnology and College
of Biological Sciences, China Agricultural University,
Yuanmingyuan West Road No. 2, Haidian District,
Beijing 100193, People’s Republic of China
e-mail: sanfengchen0@163.com; chensf@cau.edu.cn
L. Zhang
e-mail: zhangyongyuan0@163.com
X. Liu
e-mail: liuxiaomeng_1978@163.com
X. Li
e-mail: lixinxin_1978@163.com
L. Zhang
College of Life Science, Shanxi Normal University,
Linfen 041000, Shanxi, China
Biological N2 fixation, the conversion of atmospheric
N2 to NH3, offers a natural means of providing fixed N
for plants (Falkowski 1997). Most biological N2
fixation is catalyzed by a molybdenum-dependent
nitrogenase, which is found in some bacteria and
archaea. The enzyme is composed of two component
proteins: a MoFe protein and a Fe protein. The MoFe
protein component is an a2b2 heterotetramer (a
encoded by nifD and b encoded by nifK) that contains
two metalloclusters (FeMo-co and the P-cluster). The
Fe protein (encoded by nifH) is a homodimer bridged
by an [4Fe–4S] cluster that serves as the obligate
electron donor to the MoFe protein. Apart from nifH,
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nifD and nifK, several other genes (nifE, N, X B, Q, V,
Y, S, U, M, Z, T and W) are required for nitrogenase
biosynthesis (Dixon et al. 1997).
The organization and numbers of nif genes vary
amongst diazotrophic species. In Klebsiella oxytoca,
20 nif genes are co-located within a 24 kb cluster
(Arnold et al. 1988), whereas in Azotobacter vinelandii the nif genes are dispersed and distributed as two
clusters (Jacobson et al. 1989). However, some N2fixing organisms possess a restricted nif gene set. For
example, Paenibacillus sp. WLY78 contains nine nif
genes nifBHDKENXhesAnifV, which are organized as
an operon under a r70-dependent promoter (Wang
et al. 2013a; Xie et al. 2014). The nif gene operon
under the native r70-dependent promoter enabled the
synthesis of catalytically active nitrogenase in E. coli
JM109 (Wang et al. 2013a). In order to escape the
native regulatory factors, the nif gene operon was
placed under the control of T7 promoter and then was
transferred into E. coli BL21, yielding the recombinant E. coli 78–32 (Wang et al. 2013a). However, the
nitrogenase activity of E. coli 78–32 is much lower
than that of Paenibacillus sp. WLY78. The transcription and translation of nif gene operon in E. coli 78–32
have not been studied.
Here, the transcription and translation of nif genes
under T7 promoter in E. coli BL21 under different
conditions have been studied.
Materials and methods
Bacterial strains, media, and growth conditions
Paenibacillus sp. WLY78 and the recombinant E. coli
78–32 were used. Paenibacillus sp. WLY78 is a N2fixing strain isolated from the rhizosphere of bamboo.
E. coli 78–32 is a derivative of E. coli BL21 carrying
plasmid pET-28b which contains Paenibacillus nif
gene operon (nifBHDKENXhesAnifV) under control of
T7 promoter (Fig. 1a. The both bacteria were routinely grown in LD medium (low salt lysogeny broth
medium containing per liter: 2.5 g NaCl, 5 g yeast
extract and 10 g Tryptone) with shaking at 180 rpm
and 30 °C. When appropriate, 50 lM kanamycin was
added for maintenance of plasmid in the E. coli 78–32.
For transcriptional and translational analysis,
Paenibacillus sp. WLY78 and E. coli 78–32 were
grown in N-deficient medium under N2-fixing
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condition (without O2 and NH4?) or non-N2-fixing
condition (21 % O2 and 100 mM NH4?). N-Deficient
medium contained (per liter) 10.4 g Na2HPO4, 3.4 g
KH2PO4, 26 mg CaCl22H2O, 30 mg MgSO4, 0.3 mg
MnSO4, 36 mg ferric citrate, 7.6 mg Na2MoO42H2O,
10 lg p-aminobenzoic acid, 5 lg biotin, 4 g glucose
as carbon source and 2 mM glutamate as N source.
Nitrogenase activity assays
The nitrogenase activity assays were performed as
described previously (Wang et al. 2013a). The
recombinant cultures of E. coli 78–32 were collected
after 8 h culture in LD medium and washed three
times with deionized water. The pellet was then
resuspended in N-deficient medium to an OD600 value
of 0.2–0.4 in a serum bottle with 2 mM IPTG
containing kanamycin (50 lM). The serum bottle
was evacuated and charged with pure Ar. After 5–6 h,
ethylene at 10% (v/v) of the headspace was injected
into the serum bottle. After 30 min to 1 h, ethylene
was analyzed by GC. After completion of the
acetylene reduction assay, protein concentration of
the resulting suspensions in whole cell lysates was
determined by the Bradford method.
RNA isolation
Culture conditions were centrifuged at 80009g for
5 min. Total RNA was extracted from the cells using
an SV total RNA isolation system (Promega) according to the manufacturer’s instructions and treated with
DNase I (Promega). After ethanol precipitation, the
RNA pellet was resuspended in 25 ll RNase-free
water. The integrity and size distribution of the RNA
were verified by agarose gel electrophoresis. The
concentration and purity of the total RNA were
determined at 260/280 nm, and RNA was stored at
-70°C.
Reverse transcription polymerase chain reaction
(RT-PCR)
Primers are shown in Supplementary Table 1. The
recombinant E. coli 78–32 was grown to OD600 =
0.3–0.4 at different concentrations of NH4? and O2
with 2 mM IPTG containing kanamycin (50 lM).
Cells were harvested at 4°C. RT-PCR was carried out
on DNA-free RNA using an RT-PCR kit (Takara). The
Biotechnol Lett (2015) 37:1999–2004
2001
Fig. 1 The recombinant E. coli strains and their N2 fixation
abilities. a Scheme showing the genetic organization of the nif
gene operon and its promoter in the native Paenibacillus sp.
WLY78 and the recombinant E. coli strains; b nitrogenase
activities of the recombinant strains and the native Paenibacillus
sp. WLY78, with vector 1 and vector 2 as controls. Vector 1
indicates that E. coli JM109 carrying the empty vector plasmid
pHY300PLK, and vector 2 indicates that E. coli BL21 carrying
the empty vector plasmid pET-28b (Position Line 96–97)
genomic DNA from Paenibacillus sp. WLY78 was
used as template in RT-PCR as the positive control.
The negative control PCR reactions using RNA in the
absence of reverse transcriptase showed that the
isolated RNA preparations were free of genomic
DNA. Products were separated on 1% agarose gels.
purified from Klebsiella oxytoca M5al under anaerobic conditions and then used to make rabbit antiserum.
Results and discussion
Comparison of nitrogenase activity
Western blotting
Cells were grown under the different conditions.
Samples that were cultured in the N-deficient medium
were taken after testing the nitrogenase activity. The
Fe protein and MoFe protein were detected using
western blotting with Paenibacillus sp. WLY78 as a
positive control. Western blot experiments were
performed as described previously (Wang et al.
2013a). The recombinant E. coli 78–32 was grown
in N2-fixing conditions with 2 mM IPTG containing
kanamycin (50 lM), and cells were collected after
20 h. The cell pellet collected from 1 ml cultures at
OD600 = 1 was dissolved in 50 ll sodium dodecyl
sulfate (SDS) gel-loading buffer; then, proteins contained in the cell pellet were separated by 10% (w/v)
SDS-PAGE and Tris/glycine SDS buffer at pH 8.3.
MoFe protein and Fe protein of nitrogenase were
The nitrogenase activity of the recombined E. coli
78–32 was compared with those of Paenibacillus sp.
WLY78 and the recombined E. coli 78–7 which is a
derivative E. coli JM109 carrying Paenibacillus nif
gene operon under the control of its nif r70-dependent
promoter cloned in vector pHY300PLK (Fig. 1a. As
shown in Fig. 1b, the native Paenibacillus sp. WLY78
has the highest nitrogenase activity. Unexpectably, the
recombined E. coli 78–32 has 25 % lower nitrogenase
activity than E. coli 78–7.
Influence of NH4? on the nitrogenase activity
of the recombined E. coli 78–32
As shown in Fig. 2, the recombinant E. coli 78–32 has
significant nitrogenase activity even in the presence of
200 mM NH4?, suggesting that nif gene transcription
is not regulated by NH4?. Our data are consistent with
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Fig. 2 Influence of NH4? on nitrogenase activity in the
recombinant strain 78–32. Bacterial cells were grown in the
presence of ammonium (at the initial concentrations shown on
the x axis). Error bars indicate the standard deviation observed
from at least two independent experiments (Position Line
100–101)
Biotechnol Lett (2015) 37:1999–2004
Fig. 4 Transcriptional analysis of nifH and nifK genes in the
recombinant E. coli 78–32 grown in different conditions by
using RT-PCR In each case a parallel RT-PCR reaction was
performed to detect the level of 16S rRNA. Lane M, 100 bp
Marker; Lane 1, positive control (WLY78 DNA PCR product);
Lane 2, RT-PCR product of 78–32 (LD medium and 21 % O2);
Lane 3, RT-PCR product of 78–32(0 mM NH4? and 0 % O2);
Lane 4, RT-PCR product of 78–32(100 mM NH4? and 0 % O2);
Lane 5, RT-PCR product of 78–32(0 mM NH4? and 0 % O2);
Lane 6, RT-PCR product of 78–32(0 mM NH4? and 21 % O2);
Lane 7, the negative control (Position Line 111–112)
Effect of N sources and O2 on nif gene
transcription in E. coli 78–32
Fig. 3 Influence of oxygen on nitrogenase activity of the
recombinant strain 78–32. Bacterial cells were grown in the
presence of oxygen (at the initial concentrations shown on the x
axis). Error bars indicate the standard deviation observed from
at least two independent experiments (Position Line 103–104)
the reports that K. oxytoca nif operons were not
regulated by NH4? when expressed in E. coli JM109
under the control of T7 promoter (Wang et al. 2013b).
Influence of O2 on the nitrogenase activity
of the recombined E. coli 78–32
As shown in Fig. 3, the recombinant E. coli 78–32 has
significant nitrogenase activity in absence of O2, while
it has no activity when O2 concentration is higher than
1 %. The data confirm that nitrogenase is very
sensitive to O2.
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To study the impact of environmental factors on the
transcription of the nif cluster, RT-PCR was performed
using RNA isolated from the recombinant E. coli 78–32
under different culture conditions. The nifH and nifK
were used as markers to monitor the transcription of the
nif cluster, and 16S rDNA was a control. As shown in
Fig. 4, the nifH and nifK products were amplified from
RNA extracted from the recombinant E. coli 78–32
cells grown in different conditions with Paenibacillus
sp. WLY78 as a positive control. The nifH and nifK
were expressed in both N2-fixing conditions and nonN2-fixing conditions. The same result was observed for
organic N compounds as the N source (Fig. 4, lane 2).
The data indicate that NH4?, organic N compounds,
and O2 did not inhibit the transcription of nif genes in
the recombinant E. coli 78–32.
Translation of structural nitrogenase proteins
(NifH, NifD and NifK) of the recombined E. coli
78–32
To investigate that the transcribed nif mRNA products
are efficiently translated in E. coli 78–32, the amounts
Biotechnol Lett (2015) 37:1999–2004
2003
Fig. 5 Comparison of the protein expression levels of nif genes
between the E. coli 78–32 and E. coli 78–7 by using Western
blot whole cell proteins were subjected to SDS-PAGE followed
by Western blotting using anti-Fe protein and anti-MoFe protein
from K. oxyctoa in different condition. Lane 1, the positive
control (Paenibacillus sp. WLY78 in N2-fixing condition); Lane
2, the negative control (Paenibacillus sp. WLY78 in non-N2fixing condition with LD medium and 21 % O2); Lane 3, E. coli
78–32 grown in N2-fixing condition; Lane 4, E. coli 78–32
grown in LD medium with 21 % O2; Lane 5, the negative
control (only pET-28b vector in N2-fixing condition); Lane 6,
E. coli 78–7 grown in N2-fixing condition; Lane 7, E. coli 78–7
grown in LD medium with 21 % O2; Lane 8, the negative
control (only pHY300PLK vector in N2-fixing condition)
(Position Line 123–124)
of the Fe protein and MoFe protein were examined by
western blotting analysis. We demonstrate that Fe
protein (NifH, 27.7 kDa) and MoFe proteins (NifD,
54 kDa; NifK, 57.4 kDa) in the native Paenibacillus
sp. WLY78 were only synthesized in N2-fixing
condition (Fig. 5. Lanes 1, 2), while the same sizes
of Fe protein and MoFe proteins were translated in
E. coli 78–32 under both the N2-fixing and non-N2fixing conditions (Fig. 5 lanes 3 and 4). Notably, the
intensities of the bands corresponding to MoFe protein
and Fe protein from E. coli 78–32 are similar to those
from the native Paenibacillus sp. WLY78, whether it
was cultured in N2-fixing or non-N2-fixing conditions.
These results demonstrated that the translation of Nif
proteins in E. coli 78-32 is not regulated by a high
concentration of NH4?, O2, or organic N compounds.
Although Fe and MoFe proteins were significantly
formed in the recombinant E. coli 78–32 under both
conditions, nitrogenase activity exhibited only under
N2-fixing conditions. The results support that N2
fixation is a complex process and a high concentration
of O2 under non-N2-fixing conditions led to the loss of
nitrogenase activity since almost all nitrogenases are
very sensitive to O2.
As shown in Fig. 5, the expressed amounts of Fe
protein and MoFe protein from E. coli 78–32 are
higher in non-N2-fixing condition than those in N2fixing condition. The amounts of a subunit and b
subunit of MoFe protein from E. coli 78–32 are similar
in both conditions. However, the expressed amounts of
Fe protein and MoFe protein from E. coli 78–7 are
lower in non-N2-fixing condition than those in N2fixing condition. The amount of the b subunit is much
higher than that of the a subunit in E. coli 78–7 in both
conditions. Although the amounts of the expressed Fe
and MoFe protein in E. coli 78–32 are more similar to
those of Paenibacillus sp. WLY78 than to those of the
recombined strain E. coli 78–7, the N2 fixation ability
of E. coli strain 78–32 is 25 % lower than that of
E. coli 78–7.
Discussion
Nitrogenase is sensitive to O2, and N2 fixation
catalyzed by nitrogenase is an energy-consuming
process. Thus, transcription of nif genes in diazotrophs
is tightly controlled in response to the external O2 and
NH4? concentrations (Dixon and Kahn 2004). Similar
to almost of other diazotrophs, the nitrogenase activity
and nif gene transcription in Paenibacillus sp. WLY78
are regulated by NH4? and O2 (Wang et al. 2013a).
Here, our studies show that nif gene transcription in
E. coli 78–32 is not regulated by NH4? and O2, while
nitrogenase activity is not affected by NH4?, but still
by O2, due to nitrogenase being very sensitive to O2.
Translational analysis showed that Fe protein and
MoFe protein were significantly expressed in the
recombinant E. coli 78–32 in different N sources. The
amounts of the expressed Fe protein and MoFe protein
in medium containing organic source (yeast extract
and Tryptone) or in N2-fixing conditions were larger
than those of in the recombinant E. coli 78–7.
However, the nitrogenase activity of recombinant
E. coli 78-32 is 4% lower than that of the native
Paenibacillus sp. WLY78. Compared to the nif gene
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cluster of K. oxytoca, nifF, J, M, Z, W, T, Y, Q, U, S,
which are involved in biosynthesis of nitrogenase, are
absent in the Paenibacillus nif gene operon. The
functions of these genes might be completed by the
corresponding related genes in Paenibacillus genome.
The absence of these genes in E. coli might be one
reason for low nitrogenase activity. Unexpectably, the
nitrogenase activity of recombinant E. coli 78–32 is
25 % lower than that of the recombinant E. coli 78–7.
Similar results that the low nitrogenase activity in the
recombinant E. coli strains carrying the nif gene
operons of K. oxytoca under the control of T7
promoter in E. coli MG1655 was reported (Temme
et al. 2012). We deduce that massive overproduction
of foreign Nif proteins under the strong T7 promoter
damages the host cell, leading to lower nitrogenase
activity. The nitrogenase activities were improved by
modifying the T7 promoter activities which controlling nif operons and T7 RNA polymerase activities
(Wang et al. 2013b). In addition, we are not sure
whether E. coli strains affect the nitrogenase activity.
In our studies, E. coli BL21 which carries T7
polymerase is used to work with T7 expression vectors
which control the expression of nif gene operons from
Paenibacillus. However, E. coli MG1655 or E. coli
JM109 carrying T7 polymerase was used to work with
T7 promoters which control the expression of nif gene
operons from K. oxytoca (Temme et al. 2012; Wang
et al. 2013b).
Conclusion
The Paenibacillus nif operon composed of nine genes
(nifBHDKENXhesAnifV) under the control of the
bacteriophage T7 promoter enables E. coli BL21 to
synthesize active nitrogenase. The nif gene transcription and translation are not regulated by O2, organic N
compounds (yeast extract and Tryptone) or NH4?. The
nitrogenase activity in E. coli 78–32 is regulated by O2
but not by NH4?. However, the nitrogenase activity in
E. coli 78–32 is 4 % lower than that of the native
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Paenibacillus sp. WLY78. Thus nitrogenase activity
needs to be improved in the future studies.
Acknowledgments This work was supported by Guangdong
Innovative and Entrepreneurial Research Team Program (No.
2013S033) and by the National High Technology Research and
Development Program (‘‘863’’ Program: No. 2013AA10280204) and by College of Life Science of Shanxi Normal University
key project (No. SMYKZ-35).
Supporting information Supplementary Table 1—Primers
used in this study (Position Lane 80-81).
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