In vivo studies with an unmarked deletion strain, DnirF, showed that this gene is essential for cd1 assembly and consequently for denitrification, which was restored when the DnirF strain
Trang 1its essential role in d1 heme biogenesis
Shilpa Bali1, Martin J Warren2and Stuart J Ferguson1
1 Department of Biochemistry, University of Oxford, UK
2 Department of Biosciences, University of Kent, Canterbury, UK
Introduction
Denitrification is a four-step transformation of nitrate
to dinitrogen gas by various species of bacteria under
anaerobic conditions [1,2] These four steps are
cataly-sed by complex metalloenzymes and involve stepwise
conversion of nitrate to nitrite, nitrite to nitric oxide,
nitric oxide to nitrous oxide and finally reduction of
nitrous oxide to nitrogen In the denitrification
path-way, nitrite reduction is the key step, as it is the point
of divergence from assimilatory nitrogen metabolism in
which nitrite is reduced to ammonium [2,3] There are
two types of respiratory nitrite reductase involved in
denitrification: one is copper-containing nitrite
reduc-tase (NirK), which is prevalent in, but not exclusive to,
alphaproteobacteria, the other being cytochrome cd1
(NirS), which prevails in betaproteobacteria [4]
Cytochrome cd1 nitrite reductase is a homodimeric
periplasmic enzyme with each subunit containing a
covalently attached c heme and noncovalently attached
d1 heme, bound in a beta-propeller domain, as pros-thetic groups [5,6] Heme d1, which forms the active cen-tre for the one electron reduction of nitrite to nitric oxide, has a unique structure The structure of this mod-ified heme, a dioxoisobacteriochlorin to be more spe-cific, has been known for more than two decades [7,8], but quite how it is biosynthesized by denitrifying bacte-ria under anaerobic conditions is not understood Anal-ysis of insertional mutagenesis and complementation work in Pseudomonas aeruginosa, Pseudomonas fluores-cens, Paracoccus denitrificans and Pseudomonas stutzeri have shown that a set of several contiguous genes that always follows the structural gene, nirS, for cytochrome
cd1, is necessary for the biogenesis of the d1 cofactor [9–13] In P denitrificans and closely related Para-coccus pantotrophus, these genes are cotranscribed as
Keywords
cytochrome cd1; d1heme biosynthesis;
denitrification; nitrite reductase;
Paracoccus pantotrophus; tetrapyrrole
Correspondence
S J Ferguson, Department of
Biochemistry, University of Oxford, South
Parks Road, Oxford OX1 3QU, UK
Fax: +44 1865 613201
Tel: +44 1865 613299
E-mail: stuart.ferguson@bioch.ox.ac.uk
(Received 24 June 2010, revised 27 August
2010, accepted 1 October 2010)
doi:10.1111/j.1742-4658.2010.07899.x
The cytochrome cd1nitrite reductase from Paracoccus pantotrophus catalyses the one electron reduction of nitrite to nitric oxide using two heme cofactors The site of nitrite reduction is the d1heme, which is synthesized under anaer-obic conditions by using nirECFD-LGHJN gene products In vivo studies with an unmarked deletion strain, DnirF, showed that this gene is essential for cd1 assembly and consequently for denitrification, which was restored when the DnirF strain was complemented with wild-type, plasmid-borne, nirF Removal of a signal sequence and deletion of a conserved N-terminal Gly-rich motif from the NirF coded on a plasmid resulted in loss of in vivo NirF activity We demonstrate here that the product of the nirF gene is a periplasmic protein and, hence, must be involved in a late stage of the cofac-tor biosynthesis In vitro studies with purified NirF established that it could bind d1heme It is concluded that His41 of NirF, which aligns with His200
of the d1 heme domain of cd1, is essential both for this binding and for the production of d1heme; replacement of His41 by Ala, Cys, Lys and Met all gave nonfunctional proteins Potential functions of NirF are discussed
Abbreviation
LB, Luria–Bertani.
Trang 2an operon in the following order nirECFD-LGHJN It
has been proved that the biosynthesis of the d1
heme proceeds via a common tetrapyrrole precursor
uroporphyrinogen III, which is transformed into
pre-corrin-2 by S-adenosyl-L-methionine-dependent methyl
transferase, NirE [14,15] In addition, it has been
dem-onstrated that a Paracoccus derivative strain, in which
nirN is replaced with a kanamycin resistance cassette,
still makes holo-cd1, which suggests that this last gene
on the operon is dispensable for d1heme assembly [15]
Also, the nirC gene encodes a periplasmic c type
chrome that may have an electron transfer role in
cyto-chrome cd1activity [16] or maturation [15]
Conflicting evidence exists concerning the subcellular
location of NirF NirF from P pantotrophus shares
54% sequence identity and 72.3% sequence similarity
with the NirF from Ps aeruginosa However, the
pro-tein from Ps aeruginosa lacks the apparent
Sec-depen-dent signal sequence for translocation to the periplasm,
which, in contrast, is readily identified in P
pantotro-phus NirF Information about NirF from the
much-studied Ps aeruginosa has led to the widespread
assumption that NirF is cytoplasmic Accordingly, we
wanted to determine the subcellular location of NirF in
P pantotrophus, which produces larger amounts of cd1
under denitrifying conditions than Ps aeruginosa NirF
also shares 34% sequence similarity with the
beta-pro-peller domain of cd1, indicating a scaffolding role for
an intermediate of heme d1synthesis Roles for NirF in
ferrochelation or dehydrogenase activity have been
pro-posed [10,13] Interestingly, NirF also shows some
simi-larity to a cobalamin decarboxylase, CobT [3,13] The
physiological role of NirF would heavily rely on its
cel-lular location For all these reasons we wanted to
develop an in vivo system to investigate its role by
mak-ing use of the generation and characterization of an
unmarked deletion in nirF The unmarked gene deletion
mutant is expected to lose nitrite respiration and the
capacity to synthesize this tetrapyrrole derivative More
importantly, an unmarked deletion mutant strain
should have denitrification restored on
complementa-tion with plasmid-borne nirF and, therefore, should
provide an in vivo system to further analyse the
physio-logical role of NirF and its variants
Results
Construction of the DnirF strain and its in vivo
nitrite reductase activity
In P pantotrophus, the operon associated with d1heme
biosynthesis has many overlapping genes The nirF
gene overlaps four nucleotides with the preceding nirC
gene and it is also immediately followed by an overlap-ping ORF for nirD-L Previous studies in Ps aerugin-osa demonstrated that a marked mutation in the nirF gene resulted in the formation of inactive nitrite reduc-tase [9] Similar results were also found for an nirF mutant in P stutzeri [10], but in this case a polar effect
of the mutation was not excluded The present study utilized an unmarked deletion in nirF where the entire nirF ORF has been deleted from the chromosome When this unmarked deletion strain of nirF (i.e DnirF), named SBN11, was grown anaerobically in minimal media supplemented with 20 mm nitrate, it converted the entire available nitrate to nitrite within
10 h of growth and lost its nitrite reductase activity, as shown by no consumption of nitrite to yield any gas-eous products The extracellular nitrite concentration peaked at 20 mm and remained there even when the cultures had reached the stationary phase No brown coloration from holo-cytochrome cd1 or gas evolution from nitrogen production was observed in the SBN11 cultures
Reassuringly, the nitrite reductase activity of the DnirF strain was restored within 10 h of anaerobic growth on nitrate-supplemented minimal media, when
it was complemented with a plasmid-borne copy of nirF (Fig 1) Here, the extracellular nitrite concentra-tion reached a maximum value of 14 mm, followed by
a rapid decline This corresponds to a delay in the expression or activation of functional cytochrome cd1, but eventually a complete denitrification pathway was established As shown by the four independent growth results, the extracellular nitrite concentration was a function of cell density, rather than time, for the DnirF strain expressing plasmid-encoded nirF In addition, expression of plasmid-encoded strep II-tagged nirF was demonstrated by western blot analysis with alkaline phosphatase-conjugated strep-tactin antibody (Fig S1) These results confirm the essential role of NirF in d1 heme assembly
Influence of deletion and replacement of the signal sequence on NirF processing and denitrification activity
The derived amino acid sequences of NirF from two different denitrifiers, P pantotrophus and Ps aerugin-osa, differ significantly at the N-termini In P panto-trophus, nirF encodes a ( 42 kDa) protein that has an N-terminal signal sequence suggestive of a location in the periplasm On the other hand, in Ps aeruginosa (PAO1), NirF has no apparent signal sequence and therefore this protein should be located in the cyto-plasm In order to determine whether export of NirF
Trang 3to the periplasm of P pantotrophus is essential for d1
heme formation, and thus for the physiological
func-tion of NirF, we deleted the presumed signal sequence
We also replaced the putative signal sequence of NirF
with the shorter signal sequence of a native periplasmic
protein, NirC, to see whether it could still perform its
physiological function The replacement of the signal
sequence on the NirF coded for on a plasmid had no
effect on nitrite reductase activity as judged by the
res-toration of denitrification when this plasmid was used
to complement the DnirF strain (Fig 2) This result
also ruled out the need for a specific signal sequence for the function of NirF On the other hand, a plasmid carrying an nirF gene lacking the native signal sequence failed to restore denitrification upon attempted complementation of the DnirF strain (Fig 2)
A C-terminally strep II-tagged version of NirF was produced anaerobically from a pEG276-based plasmid
in the DnirF strain using minimal media supplemented with nitrate as the terminal electron acceptor When the cells of this derivative strain producing tagged
22 Time (h) 2 6 10
12 22 24 20
18 16 14 12 10 8 6
4 2 0 GB-17 SBN11 SBN13-1 SBN13-2
Strain
SBN13-3 SBN13-4 SBN14
Fig 1 Restoration of nitrite reduction in a Paracoccus pantotrophus strain carrying an unmarked gene deletion of nirF (DnirF) with plasmid-borne nirF GB17 is the parental wild-type P pantotrophus in which nitrite does not accumulate following reduction of added nitrate; SBN11
is the DnirF strain that does not synthesize d 1 heme and hence cannot turnover nitrite to nitric oxide SBN13 is SBN11 complemented with nirF on pEG276 (four replicas are shown) and SBN14 is a control with the SBN11 strain containing the empty expression vector pEG276 only Replicas of SBN13 indicate that the concentration of extracellular nitrite is dependent on the cell density at any given time The code for the times of analysis is shown on the right.
22
SBN20 (ΔnirF + NirF (no signal sequence))
20 18 16 14 12 10 8 6
4 2 0
2.2 2 1.8 1.6 1.4 1.2
0.8 0.6 0.4 0.2 0 1
Time (h)
20
SBN28 (ΔnirF + NirF (NirC signal sequence))
1.8 1.6 1.4 1.2
0.8 A600
0.6 0.4 0.2 0 1
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Time (h)
20
Fig 2 A periplasmic targeting sequence is essential for Paracoccus pantotrophus NirF function Growth plots and time courses of nitrite appearance and disappearance for the SBN11 (DnirF) strain complemented with a plasmid coding for NirF from which the putative periplas-mic targeting sequence had been deleted (to give SBN20) Also shown is the effect of providing the DnirF strain with a plasmid coding for NirF where its native signal sequence had been replaced by the proven periplasmic targeting sequence of NirC (to give SBN28) Cell density was determined at A 600 and is depicted by grey diamonds, whereas the extracellular nitrite concentration was determined using a colorimet-ric method (for more details, see Experimental procedures) and is shown by black triangles The data shown here are the average of four different experiments.
Trang 4NirF were fractionated and run on the SDS⁄ PAGE
for western analysis by using the alkaline phosphatase
conjugate of strep-tactin antibody, we found that both
membrane and cytoplasmic fractions were free of NirF
protein and it was present only in the periplasmic
frac-tion (Fig 3) These results, together with the outcome
of the complementation analysis, prove that NirF is a
periplasmic protein in P pantotrophus
Influence of variations of conserved residues on
the in vivo activity of NirF
Interestingly, like NirN, NirF shares sequence
similar-ity with the C-terminal d1 heme-containing domain of
cytochrome cd1 Strikingly, the axial ligand of iron in
d1heme in P pantotrophus cd1(His200) is conserved in
NirF (His41); this conservation applies to all other
NirF sequences known in the database (Fig S2)
How-ever, the other catalytic site histidines (His 345 and
His388) of NirS are not conserved in NirF
Restoration of denitrification upon complementation
of the unmarked nirF deletion strain of P pantotrophus
with plasmid-borne nirF provided a good in vivo
sys-tem for testing the molecular basis for the NirF
activ-ity (Fig 4) Replacement of the aforementioned His41
with Ala completely abolished the in vivo nitrite
reduc-tase activity as seen by the accumulation of large
amounts of extracellular nitrite in the DnirF strain complemented with nirF (H41A), when growing under denitrifying conditions (Fig 5) We were also curious whether denitrification could be rescued to any extent
if this His were replaced with some of the other heme iron-binding residues, such as Met, Cys or Lys All plasmids bearing nirF with this residue changed to any
of these three potential heme ligands failed to rescue denitrification in the DnirF strain (Fig 5) These results indicate that His41 is important for NirF function
It has been reported that NirF shows 21% sequence similarity to the first 100 amino acids of NirE [13] There is also a highly conserved N-terminal Gly-rich (GXGX2GX7G) motif in all NirF sequences, which is suggestive of a binding to a nucleotide-containing cofactor This motif has also been found in several other dehydrogenases involved in tetrapyrrole biosyn-thesis pathways, including CysGA and SirC in the siroheme biogenesis pathway [17] Furthermore, when
a pairwise alignment of NirF was performed with Met8P (a bifunctional dehydrogenase-ferrochelatase from Saccharomyces cerevisiae), we found that the two proteins had 24% sequence similarity A crystal structure of Met8P has shown that this protein has
an aspartate residue (Asp141), which is important for both chelatase and dehydrogenase function [17]; interestingly, this aspartate, Asp129, is also conserved
98
kDa
le
riplasm Membr ane Cytoplasm kDa
le
riplasm Membr ane Cytoplasm
62
49
38
28
17
14
98 62 49
38
28
17 14
Fig 3 Distribution of NirF between different cell fractions of P pantotrophus (A) Western blot assay with the different fractions of the cells expressing plasmid-borne and strep-tagged NirF from SBN13 strain, using an alkaline phosphatase conjugate of strep-tactin antibody (for more information see Experimental procedures) NirF ( 42 kDa) with a C-terminal strep II tag could be found in the total cell lysate and the periplasmic fraction, but was absent from the membrane and cytoplasmic fractions (B) The same cell fractions as shown in (A) when sub-jected to SDS⁄ PAGE analysis and stained with Coomassie blue for proteins.
Trang 5in NirF sequences from P pantotrophus and other
den-itrifiers (Fig S3) When plasmids carrying the variation
in NirF of Asp129 to Ala or to Gln were used to
complement the DnirF strain, they showed the same
phenotype as when complementation was done with
wild-type NirF There were no growth defects and
nei-ther of these strains showed a large accumulation of
extracellular nitrite during the log phase, when grown
on 20 mm nitrate under denitrifying conditions
(Fig 5) This result demonstrated that Asp129 of NirF
could not be essential for any function similar to that
in Asp141 of Met8P
The idea of NirF being a dehydrogenase is appealing
because of the presence of a putative
nucleotide-bind-ing motif in the N-terminal of the protein sequence
and also because there is a need for oxidation in the d1
heme biosynthetic pathway, for example, oxidation of
C17 propionate to give an acrylate side chain This
type of step would normally require FAD-based
chem-istry Another potential dehydrogenation is NAD⁄
NADP-dependent oxidation of precorrin-2 to
sirohy-drochlorin that might be a shared intermediate in the
d1 heme and cobalamin biosynthesis pathway Some,
but not all, flavoproteins have tightly bound flavin
when overexpressed and thus are yellow on extraction
However, no such coloration was observed for the NirF when it was overproduced in either Escherichia coli or in P pantotrophus under either aerobic or anaerobic conditions We also did not observe any interaction between the purified NirF and a range of nucleotide-containing cofactors by using a variety of biophysical methods (data not shown) Nonetheless,
we still decided to test the effect of the deletion of the entire GXGX2GX7G motif on the in vivo NirF and nitrite reductase activity Although deletion of the entire Gly-rich region resulted in an inactive NirF, analysis of variant NirF species with one or more of the individual Gly residues changed to Ala did not result in loss of NirF function Thus, we conclude that although a significant stretch of the N-terminus is important for the formation of a functional protein,
we have no evidence that this functionality relates to the Gly residues; thus, the important residues may lie elsewhere within this N-terminal region
Purification and in vitro characterization of NirF and its variants
NirF was recombinantly produced in E coli and puri-fied from the periplasmic fraction to near homogeneity
22
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2.2
1.2 1.7
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SBN13 (ΔnirF + NirF)
SBN11 (ΔnirF )
A600
A600
A600
A600
Time (h)
0 5 10 15 20 25
Time (h)
0 5 10 15 20 25
Time (h)
0 5 10 15 20 25
Time (h)
Fig 4 Time courses of nitrite accumulation and consumption in Paracoccus pantotro-phus strains Starter cultures were grown aerobically in LB with shaking before inocu-lation of mineral salt medium containing
20 m M nitrate in a 1% v ⁄ v dilution and appropriate antibiotics These cultures were incubated without shaking at 37 C Growth
of wild-type P pantotrophus GB17 strain is shown in the upper left panel and of the kanamycin insertion mutant nirF strain (SBN03) in the upper right panel Growth of SBN11 (DnirF) is shown in the bottom left panel and of SBN13 (DnirF) containing pEG276-nirF in the bottom right panel Cell density was determined at A 600 and is depicted by grey diamonds, whereas the extracellular nitrite concentration was deter-mined using a colorimetric method and is shown by black triangles The data shown here are the averages of four different experiments.
Trang 6in a single affinity chromatography step, with a yield
of 3.0 mg protein per litre of culture All the NirF
variants were also produced and purified in a similar
manner with a yield ranging from 1.5 to 4.0 mgÆL)1of
culture SDS⁄ PAGE gels for all proteins are shown in
Fig S4 Size exclusion chromatography demonstrated
that NirF is monomeric Dynamic light scattering
showed that the protein was well folded with a
hydro-dynamic radius fitting with the molecular weight of the
mature protein CD of the protein in potassium
phos-phate buffer at pH 7.5 displayed a predominantly
beta-sheet structure (data not shown) This is
consis-tent with the sequence similarity of this protein with the C-terminal beta-propeller domain of NirS (cyto-chrome cd1) that houses d1 heme MS confirmed the molecular mass of the protein to be 41.937 kDa, which
is expected after processing and cleavage of the signal peptide Surprisingly, a D129A mutant of NirF failed
to give any soluble protein when overexpressed in
E coli, although this variant rescued denitrification when it complemented the Paracoccus DnirF strain under denitrifying conditions This observation sug-gests that the conserved Asp129 is important for fold-ing of the recombinant protein
SBN23 (ΔnirF + NirF (H41M))
SBN19 (ΔnirF + NirF (H41A))
SBN21 (ΔnirF + NirF (H41K)) SBN22 (ΔNirF + NirF (H41C))
SBN25 (ΔnirF + NirF (D129Q))
SBN24 (ΔnirF + NirF (D129A))
22 20 18 16 12 14 10 8 6 4 2 0
10 20 30
2.2 2 1.8 1.4 1.2 1.6
1 0.8 0.6 0.4 0.2 0
Time (h)
22 20 18 16 12 14 10 8 6 4 2 0
5 10 15 20 25
2.2 2 1.8 1.4 1.2 1.6
1 0.8 0.6 0.4 0.2 0
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Time (h)
0 5 5
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15 15
25 Time (h)
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A600
A600
A600
Time (h)
Fig 5 His41 is essential for
Paracoc-cus pantotrophus NirF, but Asp129 is
dis-pensable Growth plots and time courses of
nitrite appearance and disappearance for
P pantotrophus SBN11 (DnirF) strain
com-plemented with plasmid carrying a gene
coding for NirF(H41A) (upper left),
NirF(H41M) (upper right), NirF(H41K) (middle
left), NirF(H41C) (middle right), NirF(D129A)
(lower left) and NirF(D129Q) (lower right).
Cell density was determined at A 600 and is
depicted by grey diamonds, whereas the
extracellular nitrite concentration was
deter-mined using a colorimetric method and is
shown by black triangles The data shown
here are the averages of four different
experiments.
Trang 7In vitro binding of d1heme to NirF
As explained above, there are sequence similarities
(Fig S2) between NirF and the d1 heme-binding
domain of cytochrome cd1 Therefore, we tested for
the binding of d1 heme to purified NirF The addition
of d1heme to the NirF resulted in the appearance of a
distinctive visible absorption spectrum (Fig 6) The d1
heme peak shifted from 681 to 630 nm Considering
that NirF is colourless and has no absorbance in the
UV–visible region, this shift of 50 nm in the spectrum
is due to extreme changes in the d1heme environment
This binding of d1heme with NirF was stoichiometric,
i.e 1 mol of heme was taken up by 1 mol of NirF
(concentrations of heme and protein were calculated
by using the extinction coefficient mentioned in the
experimental section and the standard Bradford assay,
respectively) To test whether the binding of d1 heme
to NirF was specific, and thus physiologically
signifi-cant, we added heme to NirF and found that there
were no shifts in the visible absorption spectrum and
thus no interaction It is already known for other
peri-plasmic proteins that the d1 heme-binding region is
very sensitive to proton concentration and prefers a
lower pH for d1 heme addition, consistent with the
periplasm probably having a pH lower than 7 [15]
Similarly, the process of d1 heme addition to NirF was
also pH dependent At relatively high pH values (8 or
higher) the spectral change described above for adding
d1 to apo-protein, did not occur; however, when the
pH was lowered to neutral pH the uptake of d1 heme
proceeded
As NirF could have at least two other interacting
partners in the periplasm for the d1 heme assembly,
namely NirC and NirN, we wanted to test if the com-plex of NirF.d1could transfer d1 heme to NirN, which was recently also shown to bind d1 heme [15] This binding would be difficult to judge, as the NirN.d1 heme complex shows a peak at 627 nm Unfortunately,
we could not observe any significant peak shifts when NirN was added to the NirF.d1heme complex in slight molar excess under anaerobic conditions (data not shown) His200 of P pantotrophus NirS is conserved between NirF and NirS; it is the His residue that in NirS is the proximal axial ligand to the d1 heme Replacement of an equivalent His, His41, in NirF by Ala abolished binding of the heme to the protein Known distal heme-binding residues, such as Met, Lys
or Cys [18,19], were substituted for His41 in NirF No changes in the visible spectra were observed when all three variants, NirF(H41K), NirF(H41M) and NirF(H41C), were added individually to the d1 heme
in slight molar excess There was no equivalent peak at
630 nm, which was observed for the NirF.d1 complex These results, when taken together with in vivo comple-mentation analysis of NirF(H41) variants, suggest that interaction of NirF with d1 heme is very specific for His41 This His41 residue must play a part in both structural and functional roles of NirF
Discussion
On the basis of several criteria, including cell fraction-ation and the consequences of either deleting the putative signal sequence or replacing it by a proven signal sequence from nirC, it can be concluded that NirF is a periplasmic protein in P pantotrophus This has an important implication as the only other known
d1 biogenesis proteins with a periplasmic location are NirC and NirN, both of which are not essential for
d1 heme synthesis [15,16] It follows that, unless there are other unrecognized d1 biogenesis proteins, then NirF must catalyse the last step(s) in d1 heme synthesis
The nature of these synthesis steps is conjectural at this stage, but the NirF.d1 heme complex might reflect
a product complex The failure of the D129A mutation
to prevent d1 heme biogenesis suggests that the activity
of NirF cannot be similar to that of Met8P activity where a comparable mutation is inhibitory A puzzle
is that some NirF sequences, notably for two strains
of Ps aeruginosa, PA7 and PAO1, but also that in Magnetospirillum magneticum, do not have any readily recognizable, i.e N-terminal positive charges, central hydrophobic core (or h-region) of seven to 15 amino acid residues, followed by a peptidase recognized
‘c-region’ [20], signal sequences These sequences are in
0.21
0.18
0.15
0.12
0.09
0.06
0.03
0
Wavelength (nm)
Fig 6 Visible absorption spectra of oxidized d 1 heme, 0.060 m M ,
before ( ) and after the addition of NirF ( ) in slight molar
excess The flat trace at the bottom is the visible absorption
spectrum of NirF at 0.041 m M All spectra were taken in 50 m M
phosphate buffer, pH 7, at room temperature.
Trang 8contrast to many other sequences for NirF proteins
where the signal sequence is readily recognizable It is
possible that the function of NirF can be realized in
the cytoplasm of Ps aeruginosa, with the resulting d1
heme then being translocated to the periplasm In the
case of P pantotrophus it would be the substrate for
NirF that is translocated In either case the transport
process is enigmatic as none of the Nir proteins codes
for a transmembrane protein that could be a candidate
for moving d1 heme, or a precursor, across the
mem-brane Alternatively, as suggested by Suzuki et al [21],
NirF in some organisms might be periplasmic but with
an N-terminal transmembrane helix anchoring the
pro-tein to the membrane However, our bioinformatics
analysis of the N-terminal sequences for NirF for
Ps aeruginosaand M magneticum does not agree with
this suggestion A function of NirF related to binding
nucleotide in a putative Rossman fold now appears
unlikely, as the putative Gly of such a fold are not
essential This result also correlates well with the
export of NirF via the sec system; a periplasmic
pro-tein with a bound nucleotide would be exported via
the Tat system in a folded conformation along with
the cofactor
Heme d1 differs from other tetrapyrrole derivatives
in that it is a dioxoisobacteriochlorin, as opposed to
porphyrin, characterized by the presence of two oxo
groups at C3 and C8 and methyl groups at positions
C2 and C7 [7,22] Also, its synthesis is mediated via a
separate branch of the tetrapyrrole biosynthetic
path-way from uroporphyrinogen III [14,15,23] Recently
we showed that methylation at C2 and C7 is catalysed
by NirE to give another tetrapyrrole intermediate
pre-corrin-2 [14,15] Further modifications would include:
(a) decarboxylations of the acetate side chain at
posi-tions C12 and C18, (b) dehydrogenation of the C17
side chain to give an acrylate moiety, (c) introduction
of oxo groups at positions C3 and C8, (d)
ferrochela-tion and (f) transport to the periplasm Not only the
enzymes and chemistry of all these steps are unknown,
but even the order in which the modifications occur
remains mostly unknown Our result that NirF is a
periplasmic enzyme indicates that this protein catalyses
the chemistry required for the last stages of d1 heme
biosynthesis However, defining the substrate for NirF
will not be an easy task Possibilities include the d1
heme lacking iron and⁄ or with the side chain
satu-rated, but accessing these putative substrates is not
trivial An alternative approach would be to seek
accu-mulation of the substrate of NirF in a mutant that
lacks NirF; this too is not trivial as the DnirF strain
does not accumulate readily detectable amounts of an
intermediate of d1synthesis
Experimental procedures
DNA manipulations DNA manipulations were performed by standard methods Primers were synthesized by Sigma–Genosys (Haverhill, UK) Amplifications by PCR using KOD DNA polymerase (from Thermococcus kodakaraensis) were according to sup-plier’s instructions (Novagen, now Merck Biosciences, Not-tingham, UK) All constructs generated by PCR were confirmed to be correct by sequencing All the primers used
in this study are shown in Table S1
Construction of bacterial strains Initially, an unmarked deletion was generated in nirF in a wild-type GB17 P pantotrophus strain This was performed
in a two-step process First, the 5¢ and 3¢ flanking regions of nirF were cloned and the kanR cassette inserted between them This was cloned into pJQ200ks (gentamicin-resistant), which is incapable of replication in P pantotrophus Chro-mosomal integrants in which double crossover events had replaced the nirF ORF with the kanamycin-resistance cas-sette, but lost the pJQ200ks backbone, were selected as kanamycin-resistant gentamicin-sensitive strains Correct integration of the cassette was confirmed by PCR screening Second, the deletion was made unmarked using a con-struct in which the nirF flanks were cloned into the pRVS2 vector (gentamicin resistance), which was modified from pRVS1 [24] This vector is also incapable of replication in
P pantotrophus Single crossover events were selected via resistance to streptomycin (P pantotrophus), gentamicin (pRVS2) and kanamycin (nirF::kanR) This strain was then screened for a second crossover event in which the kanR cassette was removed via homologous recombination This strain was selected essentially as described in [24] and iden-tified by the growth of kanamycin-sensitive white colonies
in the presence of 200 mgÆmL)1X-gal (5-bromo-4-chloro-3-indolyl-b-galactoside) Putative strains were confirmed to be correct by PCR screening (D nirF) Full details of the con-struct generation and strategy employed can be found in supporting information (Doc S1, S2 and S3)
Cloning of P pantotrophus nirF and nirF variants The nirF ORF was amplified from P pantotrophus genomic DNA using SB5 and SB6, digested with NcoI and XhoI and ligated into NcoI⁄ XhoI-digested pET22b (for overex-pression in E coli) A C-terminal strep II tag was intro-duced in the pET22b-based construct by inverse PCR using primers SB45 and SB46, and by self-ligating the purified PCR product after phosphorylation with T4 PNK The native P pantotrophus signal sequence of nirF was removed
by inverse PCR with SB62 and SB63 to generate a new construct that had the PelB signal sequence in frame with
Trang 9downstream nirF, for recombinant production of NirF in
E coli The internal EcoRI site within the nirF gene was
silently mutated using the primers SB30 and SB31 and the
product of this PCR was used to amplify EcoRI and
Hin-dIII flanked nirF to clone into EcoRI⁄ HindIII digested
pEG276 (for expression in P pantotrophus strains) [25]
Inverse PCR was used to generate a number of mutations
using the following primer combinations on both the
pET22b- and pEG276-based clones: H41A – SB82 and
SB83, H41K – SB114 and SB115, H41C – SB116 and SB117,
H41M – SB118 and SB119, D129A – SB101 and SB102,
D129Q – SB103 and SB104 The Gly-rich region in the
N-terminus of the nirF was deleted by inverse PCR using
primers SB110 and SB111 Similarly, the native signal
sequence of the nirF was deleted from the pEG276-based
plasmid using the primers SB112 and SB113 A hybrid NirF
was made by introduction of the NirC signal sequence in
front of NirF by two sequential inverse PCRs using primers
SB121, SB122, SB123 and SB124 The mutants generated in
this study are detailed in Table 1
Bacterial strains, plasmids and growth conditions
The bacterial strains and plasmids used in this study are
detailed in Table 1 Paracoccus pantotrophus strains were
grown in Luria–Bertani (LB) medium or in a defined
mini-mal medium [26] supplemented with 20 mm succinate as a
carbon and energy source Overnight aerobic growth was
achieved in 5 mL growth medium in 50 mL universals, which
were incubated in a shaker at 250 rpm at 37C Anaerobic
growth was conducted in 600 mL growth medium in
com-pletely filled bottles, with stationary incubation at 37C
For anaerobic growth, cultures were supplemented with
20 mm sodium nitrate Anaerobic cultures were inoculated
with 1% v⁄ v freshly grown aerobic overnight culture in LB
and cell density determined at A600 Antibiotic-resistant
strains were supplemented with antibiotics at the following
concentrations: streptomycin (100 mgÆmL)1), kanamycin
(50 mgÆmL)1), carbenicillin (100 mgÆmL)1) and gentamicin
(20 mgÆmL)1) Growth on solid media used liquid growth
medium supplemented with 1.5% bacteriological agar
Analysis of extracellular nitrite
Cells were pelleted from 1 mL anaerobic culture via
centri-fugation at 14 000 g for 1 min The nitrite concentration in
the medium was estimated colorimetrically using the
method in [27]
Fractionation of P pantotrophus extracts and
western blotting
Paracoccus pantotrophus strains were grown in 2 L cultures
of minimal media supplemented with 20 mm sodium nitrate
and 20 mm sodium succinate and harvested at 6000 g for
20 min Cell pellets were resuspended in 10 mL SET buffer (100 mm Tris⁄ HCl pH 7.5, 3 mm EDTA and 0.5 m sucrose)
to which 1 mgÆmL)1lysozyme, 75 mg DNaseI and 1⁄ 5 of a protease inhibitor tablet were added This suspension was incubated at 37C for 40 min and spun at 26 000 g for
40 min to collect the periplasmic fraction The pellet from the last step was resuspended in 20 mm Tris⁄ HCl pH 7.5, and French-pressed three times at 1000 psi Cell debris and the insoluble fraction were removed by centrifugation at
12 000 g for 30 min The supernatant was centrifuged at
150 000 g for 2 h to collect the membranes, which were resuspended in 5 mL 20 mm Tris⁄ HCl pH 7.5 and stored at )80 C The supernatant from the 150 000 g step was kept
as cytoplasm and stored at)20 C
Paracoccus pantotrophusstrains were grown anaerobically
in 50 mL minimal salt medium supplemented with 20 mm sodium nitrate, to an A600 of 1 before harvesting at
6000 g for 10 min Pellets were resuspended in BugBuster (Novagen, now Merck) at 0.2 g dry pelletÆmL)1 and incu-bated at room temperature with rocking for 30 min Ten millilitre samples of lysate or 3 mL membrane extracts con-taining equal protein concentrations (total 30 mg) were run
on an SDS⁄ PAGE gel for analysis Western blots to detect strep II tags were performed using an alkaline phos-phatase conjugate of strep-tactin antibody (IBA, Go¨ttingen, Germany) according to the manufacturer’s instructions For all SDS⁄ PAGE, the markers used were SeeBlue Plus 2 (Invitrogen, Paisley, UK)
Recombinant production and purification of NirF and its variants
Overexpression was performed in the E coli strain BL21 codonplus (RIPL) (Stratagene, Leicester, UK) All cells expressing protein were grown at 37C in 500 mL volumes
of LB broth in 2 L flasks from overnight starter cultures to
an A600 of 0.6–0.7 and transferred to 16C before induc-tion with 0.2 mm isopropyl thio-b-d-galactoside After fur-ther incubation for 16 h, the cells from the 2 L culture were harvested and resuspended in 6 mL 50 mm Tris⁄ HCl pH 7.5, containing a trace amount of DNaseI and protease inhibitor tablet Periplasmic fractions were obtained by incubating the resuspended cells with 1 mgÆmL)1polymyxin
B sulphate at 4C for 45 min and removing the insoluble material by centrifuging at 15 000 g for 40 min The peri-plasmic fraction was applied to 5 mL of Strep-Tactin-Sepharose (IBA) equilibrated with 50 mm Tris⁄ HCl,
250 mm NaCl (pH 7.5) The column was washed with six column volumes of 50 mm Tris⁄ HCl, 250 mm NaCl (pH 7.5) and the protein was eluted with 50 mm Tris⁄ HCl (pH 7.5), 150 mm NaCl, 2.5 mm desthiobiotin (IBA) according
to the manufacturer’s instructions All the NirF variants were also produced in the same manner The purity of the samples was checked by running SDS⁄ PAGE 10% Bis ⁄ Tris NuPAGE gels (Invitrogen)
Trang 10MS with the purified NirF
For MALDI analysis, the purified protein was desalted
on a C18 column Approximately 10 lm of the purified
solution was premixed with the matrix:
a-cyano-4-hy-droxycinnamic acid (10 mm in 35% aqueous acetonitrile,
0.1% trifluoroacetic acid) at a 1 : 1 ratio and 1 lL of
mixture applied directly to the sample plate The droplet
was air-dried before analysis in the MS MALDI spectra
were obtained in reflectron mode and a nitrogen laser, emitting 337 nm light in a 3 ns pulse, was the ionization source The accelerating voltage in the ion source was
30 kV
Acknowledgements
This work was funded by research grant BBE0229441 from the Biotechnology and Biological Sciences
Table 1 Strains and plasmids used in the present study.
endA1 gyrA96 thi1 relA1 (general cloning vehicle)
Gibco BRL
pEG276-NirF (NirC signal sequence) P pantotrophus nirF (NirC signal sequence) cloned into pEG276 This work pEG276-NirF (no signal sequence) P pantotrophus nirF (no signal sequence) cloned into pEG276 This work pEG276-NirF(D4-17) P pantotrophus nirF(D4-17)cloned into pEG276 where
(D4-17) is a deletion of N-terminal GXGX2GX7G motif
This work