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RESEARCH LETTER
Effect of flexibility and positive charge of the C-terminal
domain on the activator P14K function for nitrile hydratase
in Pseudomonas putida
Yi Liu1, Wenjing Cui1, Zhongmei Liu1, Youtian Cui1, Yuanyuan Xia1, Michihiko Kobayashi2 &
Zhemin Zhou1
1
Key Laboratory of Industrial Biotechnology, School of Biotechnology, Jiangnan University, Wuxi, China; and 2Institute of Applied Biochemistry,
The University of Tsukuba, Tsukuba, Ibaraki, Japan
Correspondence: Zhemin Zhou, Key
Laboratory of Industrial Biotechnology,
School of Biotechnology, Jiangnan University,
Wuxi 214122, China.
Tel.: +86 510 85325210;
fax: +86 510 85197551;
e-mail: [email protected]
Received 15 December 2013; accepted 31
December 2013. Final version published
online 30 January 2014.
DOI: 10.1111/1574-6968.12376
Abstract
A self-subunit swapping chaperone is crucial for cobalt incorporation into nitrile
hydratase. However, further information about its structural features is not available. The flexibility and positive charge of the C-terminal domain of the selfsubunit swapping chaperone (P14K) of nitrile hydratase from Pseudomonas
putida NRRL-18668 play an important role in cobalt incorporation. C-terminal
domain truncation, alternation of C-terminal domain flexibility through mutant
P14K(G86I), and elimination of the positive charge in the C-terminal domain
sharply affected nitrile hydratase cobalt content and activity. The flexible, positively charged C-terminal domain most likely carries out an external action that
allows a cobalt-free nitrile hydratase to overcome an energetic barrier, resulting
in a cobalt-containing nitrile hydratase.
MICROBIOLOGY LETTERS
Editor: Simon Silver
Keywords
protein expression; nitrile hydratase; enzyme
activation; cobalt incorporation.
Introduction
Nitrile hydratase (NHase; EC4.2.1.84), which is composed
of a- and b-subunits, contains either a nonheme iron
(Fe-NHase; Greene & Richards, 2006) or a noncorrin
cobalt ion (Co-NHase; Kobayashi & Shimizu, 1999) in the
active center and catalyzes the hydration of a nitrile (RCN)
to the corresponding amide (RCONH2). This reaction is
followed by consecutive reactions, amide ? acid ? acylCoA, which are catalyzed by amidase (Kobayashi et al.,
1997) and acyl-CoA synthetase (Noguchi et al., 2003),
respectively. The metal ions in both the Co-NHase and
Fe-NHase are located in the a-subunits, which share a
characteristic metal-binding motif CXLC(SO2H)SC(SOH)
that contains two modified cysteine residues: cysteinesulfinic acid (aCys-SO2H) and cysteine-sulfenic acid
(aCys-SOH; Murakami et al., 2000; Noguchi et al., 2003).
The apoenzyme is likely to be unmodified (Miyanaga
et al., 2004) and a related enzyme is the thiocyanate hydrolase (Katayama et al., 2006). The noncorrinoid cobalt has
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Published by John Wiley & Sons Ltd. All rights reserved
received increasing interest in bioinorganic chemistry and
in biotechnology. Its availability and remarkable chemical
versatility make it an invaluable catalyst in the chemical
industry (Mitra et al., 2008).
The trafficking of metal ions into NHases is mediated
by activator proteins (Okamoto & Eltis, 2011).
Fe-NHases in Rhodococcus, Pseudomonas chlororaphis,
and Rhodococcus require activators for functional expression (Nishiyama et al., 1991; Hashimoto et al., 1994;
Nojiri et al., 1999). A proposed metal-binding motif,
CXCC, in the NHase activator of Rhodococcus sp. N-771
has been identified, and the activators for Fe-type
NHases have been shown to act as metallochaperones
(Lu et al., 2003). The gene organizations are quite different among the Co-NHases, and their cobalt incorporation mechanisms are also different. One type of
Co-NHases has the gene order <b-subunit> <a-subunit>
<activator>, such as the NHases from Rhodococcus rhodochrous J1 (Zhou et al., 2008). The second type has
the gene order <a-subunit> <activator> <b-subunit>,
FEMS Microbiol Lett 352 (2014) 38–44
39
Flexibility and positive charge of P14K
such as the NHase from Rhodococcus jostii RHA1
(Okamoto et al., 2010). The third type has the gene
order <a-subunit> <b-subunit> <activator>, such as the
NHase from Pseudomonas putida NRRL-18668 (Liu
et al., 2013). Although the activator of the NHase from
R. jostii strain acts as a metallochaperone for cobalt
incorporation (Okamoto et al., 2010), cobalt incorporation into the NHases from R. rhodochrous J1 and
P. putida NRRL-18668 is dependent on a novel mode
of post-translational maturation called self-subunit swapping. The activator protein exists as a complex with the
a-subunit of NHase, and cobalt incorporation involves
swapping the cobalt-free a-subunit of the cobalt-free
NHase with the cobalt-containing a-subunit of the complex (Zhou et al., 2008; Liu et al., 2012). Self-subunit
swapping is quite different from the known general
mechanisms of metallocenter biosynthesis thus far and
reveals the unexpected behavior of a protein in a protein complex (Zhou et al., 2008). Various other
Co-NHases and an NHase family enzyme, thiocyanate
hydrolase, most likely also maturate through selfsubunit swapping (Zhou et al., 2010). The self-subunit
swapping chaperones that exhibit a surprising protein
function are recognized as self-subunit swapping chaperones in contrast with other metallochaperones involved
in metallocenter biosynthesis and molecular chaperones
in protein folding (Zhou et al., 2008). In addition, selfsubunit swapping chaperones also are crucial for the
post-translational cysteine oxidation in the NHase active
center (Zhou et al., 2008). However, there is no further
information on the structural features of self-subunit
swapping chaperones, and the relationship between their
structural features and functions remains unclear.
In the present study, we investigated the relationship
between the structural features and the function of P14K
in P. putida NRRL-18668. We found that the flexibility
and positive charge of the C-terminal domain (C-domain)
of P14K underlay its function of cobalt incorporation
during NHase activation.
Materials and methods
Bacterial strains and plasmids
P14K is barely detectable in sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). However, N-terminal addition of a strep tag, an artificial
peptide used for the purification and detection of
recombinant fusion proteins, can enhance the expression of P14K (Wu et al., 1997; Liu et al., 2013). Therefore, pET-AB-strepP (Liu et al., 2013), a plasmid
containing the a- and b-subunit genes (AB) and a
strep-tagged P14K gene, was used for the expression of
FEMS Microbiol Lett 352 (2014) 38–44
the NHase and P14K, and used as a template for mutagenesis or truncation. Escherichia coli JM109 was the
host for the cloning work. Escherichia coli BL21 (DE3)
was used as the host strain for gene expression.
Corresponding primers for the mutants were shown in
Supporting Information Table S1.
Expression and purification of enzymes
Escherichia coli BL21 (DE3) transformants containing the
recombinant plasmids were grown at 37 °C in TB medium (12 g L 1 tryptone, 24 g L 1 yeast extract, 4 g L 1
glycerol, 17 mM KH2PO4, and 72 mM K2HPO4) contain(50 mg L 1)
and
kanamycin
ing
CoCl2.6H2O
1
(50 lg mL ) until the A600 nm reached 0.8. Isopropyl
b-D-thiogalactopyranoside was added to a final concentration of 0.4 mM, and then, the cells were incubated at
24 °C for 16 h.
All purification steps were performed at 4 °C, and the
procedures were conducted with an AKTA purifier (GE
Healthcare UK Ltd.). Potassium phosphate buffer
(10 mM, pH 7.5) containing 0.5 mM dithiothreitol
(DTT) was used in the purification steps. NHase and
P14K were purified as described previously (Liu et al.,
2013).
Enzyme assay
The NHase activity was assayed in a reaction mixture
comprising 10 mM potassium phosphate (pH 7.5),
20 mM 3-cyanopyridine as a substrate, and 0.1 lg
enzyme in a total volume of 500 lL. The reaction was
carried out at 20 °C for 20 min and stopped by the addition of 0.5 mL acetonitrile. One unit of NHase activity
was defined as the amount of enzyme that catalyzed the
release of 1 lmol of nicotinamide per min at 20 °C
(Zhou et al., 2008).
Analytical methods
The UV-Visible spectra were obtained with a U-0080D
spectrophotometer (Hitachi, Tokyo, Japan) at room temperature. The enzymes were dialyzed against 10 mM
potassium phosphate (pH 7.5), and 1.0 mg mL 1 samples
were prepared.
Homology modeling of P14K
The MODELLER 9.7 (Eswar et al., 2007) software was used
for the structure prediction. Stereochemical analysis of
the structures was performed using the PROCHECK
software (http://nihserver.mbi.ucla.edu/SAVS/), and the
final models that displayed good geometry (with < 1%
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Published by John Wiley & Sons Ltd. All rights reserved
40
Y. Liu et al.
of residues in the disallowed region) were used in this
study.
Quantum chemical calculations of the
transition state
All calculations were carried out using the MOPAC2012
program. The energetic profile of the reaction barrier was
mapped out by carrying out conventional self-consistent
field calculations (http://OpenMOPAC.net).
Results and discussion
The simulation of structure fluctuations
of P14K
To investigate the structural features of P14K, molecular
dynamic simulations were carried out. P14K and other
self-subunit swapping chaperones exhibit meaningful
sequence similarity to the NHase b-subunits (Zhou et al.,
2010). Therefore, P14K was modeled using the N-terminal domain of b-subunit of NHase as the template as
reported previously (Cameron et al., 2005). The average
RMSF (root-mean-square fluctuation) per residue for the
backbone atoms was calculated using a fast simulation of
protein structure fluctuations online server CABS-flex
(http://biocomp.chem.uw.edu.pl/CABSflex/). On the basis
of the mean RMSF, residues L85-A144 of the C-domain
are considered to be flexible (Fig. 1a).
The C-domain of P14K is important for its
function
To investigate the effect of C-domain on the function of
P14K, a mutant gene AB-strepP(DC) was designed, in
which the residues L85-A144 (60 amino acids) of the
C-domain of P14K were truncated, and constructed the
pET-AB-strepP(DC) plasmid. The transformant harboring
pET-AB-strepP(DC) was used for NHase expression
(Fig. 1b). The NHase encoded by the gene AB-strepP(DC)
was purified (Fig. 1c) and used to compare with the
wild-type NHase encoded by gene AB-strepP. The activity
of the NHase encoded by the gene AB-strepP(DC) was
only 3% that of the wild-type NHase (Table 1). This
result demonstrated that the C-domain plays an important role in the function of P14K.
The effect of the flexibility of the C-domain on
P14K function
To investigate how the C-domain affects the function of
P14K, we aligned several self-subunit swapping chaperones (Fig. 2). Gly86 conserved in all of these self-subunit
swapping chaperones. Further, we analyzed the modeled
structure of P14K, the C-domain appears to be a long
loop despite containing two short a-helixes, and there are
three Glys (Gly86, Gly90, and Gly91) at its start point
(Fig. 3a). According to the general relationships between
the Gly, loop structure, and flexibility (Epand et al., 1986;
(a)
(b)
(c)
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Published by John Wiley & Sons Ltd. All rights reserved
Fig. 1. Flexibility analysis of P14K and SDSPAGE analysis of NHases and P14K-containing
complexes [a(P14K)2]. (a) The average
calculated RMSF (root-mean-square
fluctuation) per residue for the backbone
atoms. The dash ellipse surrounds the
C-domain. (b) SDS-PAGE analysis of lane (1)
MW markers and the cell extracts of the
transformants carrying (2) pET-AB-strepP, (3)
pET-AB-strepP(DC), (4) pET-AB-strepP(C-G86I),
(5) pET-AB-strepP(C-sixH), (6) pET-AB-strepP
(C-R96A), (7) pET-AB-strepP(C-12positive).
(c) SDS-PAGE analysis of lane (1) MW markers
and purified NHases from the transformants
carrying (2) pET-AB-strepP, (3) pET-AB-strepP
(DC), (4) pET-AB-strepP(C-G86I), (5) pETAB-strepP(C-sixH), (6) pET-AB-strepP(C-R96A),
(7) pET-AB-strepP(C-12positive), and the a
(P14K)2 from the transformants carrying (8)
pET-AB-strepP, (9) pET-AB-strepP(C-G86I),
(10) pET-AB-strepP(C-12positive).
FEMS Microbiol Lett 352 (2014) 38–44
41
Flexibility and positive charge of P14K
Nilmeier et al., 2011), we considered that the C-domain
possesses certain flexibility.
Protein function is intimately linked to protein flexibility, and any interaction between a protein and another
molecule requires the protein to be able to change its
conformation. Proteins rely on flexibility to respond to
Table 1. Activity of the purified NHases co-expressed with the P14K
mutants in Escherichia coli
Plasmid (NHase)
NHase activity,
U mg 1 protein
pET-AB-strepP
*pET-AB-strepP(DC)
*pET-AB-strepP(C-G86I)
*pET-AB-strepP(C-sixH)
*pET-AB-strepP(C-R96A)
*pET-AB-strepP(C-K101A)
*pET-AB-strepP(C-K127A)
*pET-AB-strepP(C-K129A)
*pET-AB-strepP(C-R134A)
*pET-AB-strepP(C-K138A)
*pET-AB-strepP(C-12positive)
439
12
42
310
422
420
406
410
411
416
20
3.6
1.2
6.0
5.3
1.6
1.4
3.6
4.3
7.1
3.7
4.9
The values represent the means SD for at least triplicate independent experiments. pET-AB-strepP contains the a- and b-subunit genes
(AB) and a strep-tagged P14K gene (strepP). pET-AB-strepP(DC) contains AB genes and a mutant strep-tagged P14K gene (strepP(DC)) in
which the residues L85-A144 (60 amino acids) were truncated. pETAB-strepP(C-sixH) contains AB genes and a mutant strep-tagged P14K
gene (strepP(C-sixH)) in which six histidines (H104, H109, H111,
H117, H128, and H130) were substituted with neutral Alanines. pETAB-strepP(C-12positive) contains AB genes and a mutant strep-tagged
P14K gene (strepP(C-12positive)) in which 12 positive amino acids
(H104, H109, H111, H117, H128, H130, R96, K101, K127, K129,
R134, and K138) were substituted with neutral alanines.
*The character C means the C-domain of P14K.
environmental changes; a perturbation that changes the
flexibility of a protein may potentially interfere with its
function (Teilum et al., 2011). We modeled a structure of
mutated P14K, in which Gly86 was substituted by Ile; a
large side-chain amino acid such as Ile would likely be an
obstacle for the conformational flexibility of the
C-domain. The average RMSF per residue was calculated
using the online server CABS-flex (Fig. 1a). Compared to
the wild-type P14K, the flexibility of the C-domain of
P14K(G86I) was decreased, while that of the rest of the
protein was similar. Subsequently, we designed the
mutant gene AB-strepP(C-G86I) and constructed the plasmid pET-AB-strepP(C-G86I). The transformant harboring
pET-AB-strepP(C-G86I) was then used for NHase expression (Fig. 1b). The NHase encoded by the gene AB-strepP
(C-G86I) was purified (Fig. 1c) and used to compare with
the wild-type NHase. The activity of the NHase encoded
by gene AB-strepP(C-G86I) was only 10% that of the
wild-type NHase (Table 1). These findings demonstrated
that the flexibility of the C-domain might be an important factor for P14K function.
The positively charged amino acids of the
C-domain might be crucial for P14K function
NHase is a metalloprotein, and P14K is essential for
cobalt incorporation into NHase (Liu et al., 2012). In
addition, the C-domain of P14K plays an important role
in P14K function (Table 1). These findings indicate that
there may be some functional amino acid residues related
to cobalt binding in this domain. Six histidine residues
(H104, H109, H111, H117, H128, and H130) in the
C-domain (Fig. 2) were proposed to be involved in cobalt
binding because histidine is considered to have a high
Fig. 2. Protein sequence alignment of selfsubunit swapping chaperones. PP P14K,
RAPc8 P14K, J1 E, and J1 G indicate the selfsubunit swapping chaperones of NHases in
Pseudomonas putida NRRL-18668, Bacillus
RAPc8, and Rhodococcus rhodochrous J1 (the
two self-subunit swapping chaperones for low
and high molecular mass NHases are
represented by E and G), respectively. Closed
points indicate Gly86 and Arg96, and asterisks
indicate the 12 positively charged amino acids
(H104, H109, H111, H117, H128, H130, R96,
K101, K127, K129, R134, and K138),
respectively, in the C-domain of P14K of
P. putida NRRL-18668.
FEMS Microbiol Lett 352 (2014) 38–44
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Published by John Wiley & Sons Ltd. All rights reserved
42
Y. Liu et al.
(a)
(b)
Fig. 3. The structure of P14K obtained by homology modeling and
the surface electrostatic potentials prediction. (a) The a-helical regions
of the 60 amino acids in C-domain are shown as cylinder. The
residues displaying side chain represent Gly86, Gly90, and Gly91,
respectively. The dashed curve represents the surface of the
C-domain of P14K. (b) The surface electrostatic potentials of P14K
and the cobalt-free a-subunit. I, the surface electrostatic potentials of
P14K; II, the surface electrostatic potentials of the cobalt-free
a-subunit. The positive charges and negative charges are shown in
blue and red, respectively.
affinity for metals. We designed a gene AB-strepP(C-sixH)
in which the six His residues were substituted with Ala
and constructed the plasmid pET-AB-strepP(C-sixH). The
transformant harboring pET-AB-strepP(C-sixH) was used
for the mutant NHase expression (Fig. 1b), and the
NHase encoded by gene AB-strepP(C-sixH) was purified
(Fig. 1c) and used to compare with the wild-type NHase.
70% activity of the wild-type NHase still remained in this
mutant (Table 1), indicating that these His residues only
partially participate in P14K function. In addition, residue
Arg96, which is conserved among the self-subunit swapping chaperones (Fig. 2), was also changed to investigate
any effect on P14K function. The purified mutant NHase
encoded by AB-strepP(C-R96A) exhibited the same level
of activity as the wild-type NHase (Table 1), showing that
the Arg96 residue does not affect P14K function.
As there are no other conservative amino acids in the
C-domain besides above referred Gly86 and Arg96
(Fig. 2), molecular electrostatic potentials (MEP) were
used for further analysis of the C-domain. P14K has been
confirmed to form a complex with the a-subunit of
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Published by John Wiley & Sons Ltd. All rights reserved
NHase (Liu et al., 2012). Therefore, we modeled the MEP
of both P14K and the a-subunit using the Adaptive Poisson-Boltzmann Solver (APBS; Baker et al., 2001) implemented in PyMOL (Fig. 3b). The flexible C-domain
forms a positively charged region on the surface of P14K,
similar to a positively charged stick, while the active center in the a-subunit forms a negatively charged region,
indicating that the flexible C-domain might effectively
access the a-subunit via the molecular electrostatic
pulling forces between the positively charged region in
C-domain and the negatively charged active center in the
a-subunit. Following to this speculation, other five positively charged amino acids (K101, K127, K129, R134, and
K138) in the C-domain (Fig. 2) were substituted by Ala,
respectively, and each mutant NHase was purified. Each
mutation had little influence on NHase activity (Table 1).
However, when all the 12 positive amino acids (H104,
H109, H111, H117, H128, H130, R96, K101, K127, K129,
R134, and K138) in the C-domain (Fig. 2) were substituted with neutral Ala, the activity of this purified mutant
NHase (encoded by AB-strepP(C-12positive)) decreased to
c. 5% that of the wild-type NHase (Table 1). These findings demonstrated that the total positively charged environment of C-domain plays an important role in P14K
function.
The flexibility and positive charge of the
C-domain related to cobalt incorporation
P14K forms a complex a(P14K)2 with the a-subunit of
the NHase, and the incorporation of cobalt into the
NHase of P. putida was confirmed to be dependent on
the a-subunit substitution between the cobalt-containing
a(P14K)2 and the cobalt-free NHase (self-subunit swapping; Liu et al., 2012). P14K acts not only as a chaperone
for self-subunit swapping but also as a metallochaperone
that is crucial for cobalt insertion into the a-subunit
(Zhou et al., 2009; Liu et al., 2012). The low activity of
the mutant NHases [encoded by AB-strepP(C-G86I) and
AB-strepP(C-12positive)] may be caused by low cobalt
content in the a-subunit of a(P14K)2 and then affects the
cobalt content in NHase. Subsequently, the mutant
NHases and the mutant a(P14K)2 [encoded by AB-strepP
(C-G86I) and AB-strepP(C-12positive), respectively] were
purified (Fig. 1c) and used to compare with the wild-type
NHase and the wild-type a(P14K)2. The absorption in
the 300–350 nm region of Co-NHase reflects the S->Co3+
charge transfer (Zhou et al., 2009), and an extra shoulder
in the 300–350 nm is found in cobalt-containing a
(P14K)2 and NHase but not in cobalt-free a(P14K)2 and
NHase (Liu et al., 2012). The extra shoulder in the 300–
350 nm region was only observed in the wild-type a
(P14K)2 and NHase, but not in the two mutant enzymes
FEMS Microbiol Lett 352 (2014) 38–44
43
Flexibility and positive charge of P14K
(a)
(b)
(c)
Fig. 4. UV-Visible absorption spectra of the purified NHase and a
(P14K)2 and quantum chemical and energy calculation. (a) UV-Visible
absorption of the purified a(P14K)2 expressed from the transformants
harboring pET-AB-strepP, pET-AB-strepP(C-G86I), and pET-AB-strepP
(C-12positive), respectively. (b) UV-Visible absorption of the purified
NHase expressed from the transformants harboring pET-AB-strepP,
pET-AB-strepP(C-G86I), and pET-AB-strepP(C-12positive), respectively.
(c) The crystal structure of the NHase in Pseudonocardia thermophila
(PDB: 1UGQ, 95% amino acid identity) was used as the template for
the homology modeling of immature NHase. The crystal structure of
mature NHase is known (PDB: 3QXE). S1, S2, S3, N1, and N2 indicate
the S and N atoms corresponding to cobalt binding in the active
center. The distances and dihedrals among the cobalt-linked atoms of
the active center in the immature NHase are S1-N1 3.617 A, S2-N2
5.033 A, and S1-N2-N1-S3 57.9°, respectively, while those of the
mature NHase are S1-N1 4.075 A, S2-N2 4.358 A and S1-N2-N1-S3
58.7°. TS indicates the transition state of NHase during cobalt
incorporation. The numbers
16851,
16648,
16918, and
203 kcal mol 1 indicate the calculated total energy of the immature,
transition state, and mature NHase and the energy barrier,
respectively.
(Fig. 4a and b), indicating that the cobalt is not successfully incorporated into the two mutant a(P14K)2 and the
corresponding NHases.
In addition, the process of cobalt binding was
simulated through quantum chemical calculations
implemented using MOPAC2012. As the differences
between the conformations of the immature and mature
NHases in the cobalt-linked atoms of the active centers
are insignificant, with < 0.7 A in distance and 0.8° in
dihedral (Fig. 4c), it seems that NHase has the potential
ability for cobalt incorporation itself. However, a high
FEMS Microbiol Lett 352 (2014) 38–44
calculated energetic barrier 203 kcal mol 1 was observed
among the immature, transition state, and mature NHases (Fig. 4c). This finding indicates that the immature
NHase cannot maturate on its own. Therefore, an external action must occur for the immature NHase to overcome the energetic barrier and result in a mature NHase.
The C-domain of P14K most likely performs this external
action because the high flexibility and positive charge permit the C-domain to associate with the active center of
the a-subunit. As a result, the energetic barrier is overcome, resulting in the cobalt-containing mature NHase.
These findings suggest that the flexible, positively charged
C-domain of P14K might play an important role in
cobalt incorporation into NHase.
Self-subunit swapping chaperones are essential for
cobalt incorporation into NHase. However, the direct
experimental evidence for the mechanism of cobalt incorporation is limited. Further study of the disordered
region of P14K would be useful for investigating the
function of the self-subunit swapping chaperones
involved in NHase biosynthesis.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (31070711), the Doctoral Scientific
Research Fund Project of Jiangnan University of China
(JUDCF10011), the General University Doctor Research,
and the Innovation Program of Jiangsu Province of China
(CXZZ11_0475).
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. Oligonucleotide primers used in this study.
FEMS Microbiol Lett 352 (2014) 38–44