Cyanobacterial phytochrome A has the canonical cysteine residue, by which covalent chromophore attach-ment is accomplished in the same manner as in plant phytochromes; how-ever, its para
Trang 1The cyanobacterium Fremyella diplosiphon incorporates biliverdin
as a genuine, functional chromophore
Benjamin Quest1,*, Thomas Hu¨bschmann2, Shivani Sharda1, Nicole Tandeau de Marsac3and Wolfgang Ga¨rtner1
1 Max-Planck-Institute for Bioinorganic Chemistry, Mu¨lheim, Germany
2 Institute for Biology, Humboldt-University, Berlin, Germany
3 Unite´ des Cyanobacteries, De´partement de Microbiologie, Institut Pasteur (URA-CNRS 2172), Paris, France
Keywords
bacteriophytochrome; biliverdin IXa;
photoreceptor; phycocyanobilin;
two-component signal transduction
Correspondence
W Ga¨rtner, Max-Planck-Institute for
Bioinorganic Chemistry, Stiftstr 34–36,
D-45470 Mu¨lheim, Germany
Fax: +49 208306 3951
Tel: +49 208306 3693
E-mail: gaertner@mpi-muelheim.mpg.de
*Present address
Institut de Biologie Structurale, Jean Pierre
Ebel (UMR5075 CNRS-CEA-UJF), Grenoble,
France
(Received 5 October 2006, revised 17
January 2007, accepted 20 February 2007)
doi:10.1111/j.1742-4658.2007.05751.x
Bacteriophytochromes constitute a light-sensing subgroup of sensory kin-ases with a chromophore-binding motif in the N-terminal half and a C-ter-minally located histidine kinase activity The cyanobacterium Fremyella diplosiphon (also designated Calothrix sp.) expresses two sequentially very similar bacteriophytochromes, cyanobacterial phytochrome A (CphA) and cyanobacterial phytochrome B (CphB) Cyanobacterial phytochrome A has the canonical cysteine residue, by which covalent chromophore attach-ment is accomplished in the same manner as in plant phytochromes; how-ever, its paralog cyanobacterial phytochrome B carries a leucine residue at that position On the basis of in vitro experiments that showed, for both cyanobacterial phytochrome A and cyanobacterial phytochrome B, light-induced autophosphorylation and phosphate transfer to their cognate response regulator proteins RcpA and RcpB [Hu¨bschmann T, Jorissen HJMM, Bo¨rner T, Ga¨rtner W & deMarsac NT (2001) Eur J Biochem 268, 3383–3389], we aimed at the identification of a chromophore that is incor-porated in vivo into cyanobacterial phytochrome B within the cyanobacte-rial cell The approach was based on the introduction of a copy of cphB into the cyanobacterium via triparental conjugation The His-tagged puri-fied, recombinant protein (CphBcy) showed photoreversible absorption bands similar to those of plant and bacterial phytochromes, but with remarkably red-shifted maxima [kmax 700 and 748 nm, red-absorbing (Pr) and far red-absorbing (Pfr) forms of phytochrome, respectively] A com-parison of the absorption maxima with those of the heterologously gener-ated apoprotein, assembled with phycocyanobilin (kmax 686 and 734 nm)
or with biliverdin IXa (kmax 700 and 750 ± 2 nm), shows biliverdin IXa
to be a genuine chromophore The kinase activity of CphBcy and phos-photransfer to its cognate response regulator was found to be strictly
Pr-dependent As an N-terminally located cysteine was found as an alternative covalent binding site for several bacteriophytochrome photore-ceptors that bind biliverdin and lack the canonical cysteine residue (e.g Agrobacterium tumefaciens and Deinococcus radiodurans), this correspond-ing residue in heterologously expressed cyanobacterial phytochrome B was mutated into a serine (C24S); however, there was no change in its spectral
Abbreviations
BphP, bacteriophytochrome photoreceptor; BV, biliverdin; Cph, cyanobacterial phytochrome; CphBcy, CphB from homologous expression in Fremyella diplosiphon SF 33; PCB, phycocyanobilin; PEB, phycoerythrobilin; PFB, phytochromobilin.
Trang 2Sensing of light quality is of paramount importance
for all photosynthetic organisms Higher and lower
plants employ phytochromes [1] for determining the
quality, quantity and direction of light in the
long-wavelength range The recent finding of
phytochrome-like chromoproteins in phototrophic [2] and even
heterotrophic [3,4] bacteria has extended the
occur-rence and utilization of this efficient photoreceptor
sys-tem into the prokaryotic phylum Besides the sequence
similarities to plant phytochromes in the N-terminal
half, many of the bacteriophytochrome photoreceptors
(BphPs) so far identified exhibit a histidine kinase
activity in their C-terminal half Typically, the
BphP-encoding genes form an operon together with genes
encoding their cognate response regulators, thus
add-ing light as a trigger to the bacterial two-component
signal transduction [5,6] However, the finding of
pro-karyotic phytochromes has not only extended the
vari-ation in protein sequences, but has also shown a
greater variety in the chromophores employed in these
photoreceptors, and also in the type of chromophore
binding The first BphP identified, cyanobacterial
phyto-chrome (Cph) 1 from Synechocystis PCC6803, is
fur-nished in vivo with phycocyanobilin (PCB), and in vitro
is able to bind the open-chain tetrapyrroles
phytochro-mobilin (PFB), PCB and phycoerythrobilin (PEB) in a
covalent manner via a thioether linkage to a cysteine
residue, identical to that found in plant phytochromes
Also, it undergoes light-induced reactions similar to
those undergone by the phytochromes [7,8] Homologs
of Cph1 have been found in a number of other
cyano-bacteria [9], e.g Calothrix PCC7601 [10] and Anabaena
PCC7120, and also in proteobacteria such as Deinococcus
radiodurans, Pseudomonas aeruginosa [3,11] and
Agro-bacterium tumefaciens [12–14] Interestingly, these
proteobacterial BphPs all lack the plant
phytochrome-specific cysteine in the chromophore-binding domain,
and make use of another cysteine residue, located at
the N-terminal end within the first 30 amino acids, to
bind covalently biliverdin (BV) IXa as chromophore
This unusual type of binding was confirmed by a
recently presented three-dimensional structure of the
GAF-PHY domain of D radiodurans [15]
Evidence for a light-modulated phosphorelay
between histidine kinase and a response regulator was
first found for Cph1 [16], and this was also
demonstra-ted for heterologously expressed CphA and CphB, after
they had been assembled with tetrapyrrole chromo-phores They undergo red⁄ far-red light-modulated auto-phosphorylation in a similar fashion to Cph1, and perform remarkably selective transphosphorylation to their cognate response regulators, RcpA and RcpB [17] The finding of two bacteriophytochromes in one organism, Calothrix PCC7601 [9,10], caused some con-fusion in our understanding of this novel group of prokaryotic photoreceptors, in particular because only CphA carries the canonical cysteine residue that, ana-logously to plant phytochromes, accomplishes covalent binding of the tetrapyrrole chromophore by formation
of a thioether linkage CphB, instead, has a leucine at that position (Fig 1C) This exchange (leucine instead
of cysteine) apparently prevents CphB from covalently binding the chromophore (in the phytochrome-typical fashion; Fig 1A); however, incubation of CphB with tetrapyrrole chromophores, e.g PCB, generated photo-chemically active chromoproteins reminiscent of the plant phytochromes, but with slightly red-shifted absorption maxima [10] Even more surprisingly, the replacement of only that particular leucine residue of CphB by a cysteine yielded a covalently binding BphP with spectral properties very similar to those of its paralog, CphA [10]
Like the above-mentioned BphPs, CphB also has
a cysteine at position 24, which would be able to bind BV However, we showed recently that in chromophore competition experiments, heterologously expressed apo-CphB binds BV very tightly and with preference over PCB, but could be expelled from the binding site upon extended incubation with PCB [18] The various chromophores that have been identified in prokaryote phytochromes and the different types of binding or incorporation led us to investigate which chromophore is incorporated in vivo The homologous expression of Cph1 [19] has revealed that Synechocystis furnishes this protein with PCB, and this most prob-ably also holds true for the closely related CphA from Calothrix Up to now, however, no BphP lacking the canonical cysteine as the chromophore attachment site has been homologously expressed in its genuine host organism Here, we describe the first expression and isolation of such a bacteriophytochrome, CphB, in Calothrix PCC7601, its photochemistry, and its light-induced kinase activity The homologous expression was of particular interest, as Calothrix synthesizes large
properties On the other hand, the mutation of His267, which is located directly after the canonical cysteine, into alanine (H267A), caused com-plete loss of the capability of cyanobacterial phytochrome B to form a chromoprotein
Trang 3amounts of PCB (and PEB) for its light-harvesting
complexes, in which process BV appears as only a
transient intermediate at much lower concentrations
Results
Homologous expression of cphB
The cyanobacterium Calothrix PCC7601 expresses two
bacteriophytochromes, CphA and CphB [9] Whereas
CphA binds bilin chromophores (PCB and PFB) in a
covalent, phytochrome-typical manner via a thioether
linkage with a cysteine residue of the protein, this
essential amino acid is replaced in CphB by a leucine
In vitro, heterologously expressed apo-CphB is able to form photochemically active complexes with PCB and also with BV IXa [28] As both of these tetrapyrroles, and also PEB, are present in the cyanobacterium, it was of interest to determine which chromophore is added to the apoprotein by the cyanobacterial cell The cphB gene was furnished with an oligonucleotide providing a His6-tag at its 5¢-end, and was cloned under the control of the tac promoter (plasmid pPL9b; see Experimental procedures) The plasmid pPL9b was transferred to Fremyella diplosiphon SF33, by triparen-tal conjugation The cyanobacteria were grown in a
12 L fermenter, and yielded, after 5 days, 15 g of cell pellet (wet weight), from which 3 mg of CphBcy could be purified via affinity chromatography, followed
by a gel filtration step
When purified, CphBcy was subjected to Zn-gel elec-trophoresis The protein showed a strong fluorescence
in the molecular mass range of the holoprotein (molecular mass 87 kDa; Fig 2, right panel) The comigration of the chromophore-induced fluorescence
is evidence for a covalently bound tetrapyrrole The heterologously expressed apo-CphB, assembled with PCB, does not show the Zn-induced fluorescence after the purification (data not shown) The lower affinity of apo-CphB for PCB has been formerly observed during affinity purification of CphB–PCB adducts [28] and recently confirmed by competition experiments [18] The purified holoprotein showed an absorption band
at 700 nm that could be converted by irradiation into
an even further red-shifted absorption band at 748 nm (Fig 2) This photochemistry could be repeated several times without any loss of absorption A comparison of
Fig 2 Absorption and absorption difference spectra (Pr) P fr ) of CphBcy from homologous expression in F diplosiphon SF33 Inset: Comparison of CphBcy with CphB from heterologous expression, assembled with BV Coomassie-stained Scha¨gger-PAGE and Zn-gel
of CphBcy are also shown.
A
B
C
Fig 1 (A) Covalent attachment of PCB to a cysteine residue of an
apophytochrome The photochemistry of phytochromes (Z fi E
photoisomerization of the methine bridge between rings C and D) is
also indicated The protonated state of the chromophore in the
pro-tein-bound form has been demonstrated by resonance Raman
spectroscopy [33] (B) Structures of tetrapyrrole compounds
(in nonprotein-bound form) serving as chromophores in
phyto-chromes (PFB, phytochromobilin; BV, biliverdin IXa) Note that
(a) BV has one additional double bond in the A-ring as compared
to the other chromophores, and (b) one double bond ) of the
3¢-ethylidene substituent ) is lost upon covalent attachment to the
protein via thioether linkage formation in PCB and PFB (C)
Sequence comparison of representative bacteriophytochromes in
the region of the chromophore-binding site (Cph1 from
Synechocys-tis PCC6803; CphA and CphB from Calothrix PCC7601; AphA ⁄ B
from Anabaena PCC7120; Bph1 from Deinococcus radiodurans; and
BphP from Pseudomonas aeruginosa) Sequences of Arabidopsis
thaliana PhyA and PhyB have been added to demonstrate the
simi-larity to plant phytochromes The arrowhead indicates the position
of covalent binding in the case of a cysteine residue being present.
Trang 4these absorption maxima with heterologously
exp-ressed apo-CphB, incubated with either PCB or
BV IXa, gave practically complete agreement with the
absorption of the BV IXa adduct [BV IXa adduct,
kmax¼ 702 nm and 754 nm, red-absorbing (Pr) and far
red-absorbing (Pfr) form of phytochrome, respectively;
PCB adduct: kmax¼ 686 nm and 734 nm, Pr and Pfr]
As also seen for heterologously expressed CphB [18],
the photochemically generated Pfr form of CphBcy
exhibited remarkable thermal stability when kept in
darkness at ambient temperature A fraction of only
25% of CphBcy-Pfr converted back to Pr within
2 days Excess PCB added to CphBcy–BV adduct did
not alter its spectroscopic properties (data not shown)
Identification of CphBcy by HPLC and MS
CphBcy was unambiguously identified by MALDI-TOF
MS after SDS⁄ PAGE, excision of the band from the
gel, and tryptic digestion The peptide mixture of a
tryptic digest of purified CphBcy represented 95% of
all theoretically predicted peptides Among these, the
peptide spanning the putative chromophore-binding
site (positions 262–277 with the conserved histidine at
position 267) could also be identified in the MS
analysis; however, no peptide with a bound chromo-phore was detected When the tryptic digestion mixture was subjected to LC-MS analysis, the inspection of the
LC trace (chromatographic separation precedes MS identification) revealed three peaks with similarly strong absorptions at kmax¼ 370 and 680 nm (elution times 17.03 min and, for the double peak, 17.72 min), indicat-ive of the presence of a peptide with a bound chromo-phore (Fig 3) None of these peaks matched the retention time of a free chromophore control sample Subsequently, one of these peaks was identified by
MS analysis as the above-mentioned putative chromo-peptide, although without its chromophore We fur-thermore observed that the free chromophore is not detected by MS analysis; thus, we conclude that the bound⁄ incorporated BV IXa molecule remains attached to the peptide during the tryptic digest and the
LC, but is apparently lost during the conditions of MS analysis
Light-induced autophosphorylation and transphosphorylation of CphBcy
As previously reported for the heterologously expressed CphB [17], the homologously expressed CphBcy was
19.13 100
%
0 100
%
0
250 300 350 400 450 500 550 600 650 700 750 800
nm
15.00 CphBcy peptide at 17.750 min diode array spectrum
280 375
688
15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00
Time (min)
680 nm
Fig 3 MS identification of chromopeptide
from CphBcy after tryptic digestion and
HPLC separation.
Trang 5subjected to light-induced autophosphorylation and
phosphotransfer to its cognate response regulator
RcpB Phosphorylation of CphBcy, after it had been
converted into the Pror the Pfrform and then
incuba-ted in the dark with ATP, took place relatively slowly
and was complete after 40–60 min, when the
auto-phosphorylation reached a plateau (Fig 4, left) The
kinase activity of the Pr form of CphBcy was clearly
stronger than the activity of the Pfr form, which
reached, maximally, 20% of that of the Prform
Tak-ing into account the maximal conversion of Pr into Pfr
of 70%, due to the partial overlap of their
absorp-tion spectra, it is concluded that the Pr form is
selec-tively active in CphBcy, and that the residual kinase
activity of the Pfrform can be ascribed to the amount
of Prleft in the irradiation mixture When the response
regulator RcpB was added to the maximally
phosphory-lated CphBcy, a nearly immediate phosphate transfer
took place (Fig 4, right) The transphosphorylation
reaction driven from the Pr form of CphBcy was twice
as high as the transphosphorylation driven from the
Pfrform of CphBcy, indicating that both the
autophos-phorylation and the transphosautophos-phorylation reactions
are Pr-dependent processes
Heterologously expressed mutated CphB
The recently identified BphPs from A tumefaciens,
Agp1 [4,7] and from D radiodurans, DrBphP [3,11],
were reported to carry BV as chromophore, covalently
bound to an N-terminally located cysteine (position 20
in Agp1, and position 24 in DrBphP) We investigated
a putative role of this amino acid in the chromophore-binding capacity of CphB, which also carries an N-ter-minally located cysteine mutated into a serine (C24S) This in vitro expressed mutated protein, when incuba-ted with BV, showed identical absorption properties to the wild-type CphBm (spectra not shown), indicating that this position is of no importance for the forma-tion of the CphB chromoprotein
Inspection of phytochrome sequences reveals, besides the presence of a chromophore-binding cys-teine residue in the GAF domain, a highly conserved histidine, directly after the canonical chromophore-binding position This residue, H267 in CphB, was mutated into alanine (H267A) After the addition of PCB to the heterologously expressed, purified apopro-tein CphB-H267A, no photochemically active bacterio-phytochrome was obtained (Fig 5) However, a slightly visible shoulder at around 700 nm indicates a very weak interaction of PCB with CphBm-H267A (Fig 5, inset) Upon assembly with BV in the dark, the broad unstructured absorption band of free chro-mophore (BV and t0 in Fig 6) slowly converted into
a structured Pr-like absorption band around 700 nm within 36 min This absorption was lost upon red light illumination (Fig 6), and could not be restored either by illumination with far-red light or by pro-longed incubation in the dark (data not shown) Thus, we conclude that the histidine residue is of utmost importance for chromophore incorporation and the maintenance of the spectral properties of CphB
Fig 5 CphBm H267A assembled with a 2.5 molar excess of PCB and PCB control sample Inset: Zoom of the region 625–775 nm The arrow highlights the observed shoulder of the initial dark-assembled PCB adduct.
A
B
Fig 4 Autophosphorylation of CphBcy and transphosphorylation to
its cognate response regulator RcpB (A) Kinetics of
autophosphory-lation (left) and the corresponding blot (right) The arrow indicates
the addition of RcpB to the autophosphorylation reaction (B)
Kinet-ics of transphosphorylation from CphBcy to RcpB (left) and the
cor-responding blot (right).
Trang 6The identification of phytochrome-like photoreceptors
(BphPs) in many bacterial and cyanobacterial species
has not only extended the occurrence of bilin-based
light perception into the prokaryote kingdom, but
has also shed light on the many stimuli of the
two-component signal transduction pathways The finding
of genes encoding BphPs with strong sequence
simi-larities to phytochromes, but without the covalently
binding cysteine in the GAF domain [3], raised the
question of whether a different type of chromophore
and⁄ or a different binding mechanism occurred in
these proteins Whereas for D radiodurans and
A tumefaciens, covalent attachment of BV via its
3¢-position has been confirmed, CphB from Calothrix
appears to be an exception to all other phytochromes
described so far Although it shows all the features
of an interaction with BV, i.e the lack of the
canon-ical cysteine and the presence of an alternative
cys-teine residue within the first 30 amino acids at its
N-terminus, noncovalent binding was proposed on
the basis of competition between PCB and BV [18]
In fact, the data on the C24S mutant, presented here,
demonstrate that the overall shape of the protein
cavity is already sufficient to incorporate a tetrapyrrole
and to allow photochemistry Because, for
heterolo-gously expressed CphB, binding of both PCB and
BV IXa has been reported [10], the nature of the
native chromophore incorporated by the
cyanobacte-rial cell was addressed in this work Up to now,
BphPs employing BV IXa as a chromophore have
been exclusively found in bacteria with a heme
oxyg-enase gene as the only enzyme of chromophore
syn-thesis, as reported for Bph1 from D radiodurans
[11] and Agp1 from A tumefaciens [12] Thus, the
identification of the native CphB chromophore was
of particular interest, as Calothrix generates PCB (as
a reduction product of BV IXa) in large quantities to equip cells with their light antennae, the phycobili-somes
The expression⁄ purification of CphB from F diplo-siphon SF33 yielded a chromoprotein (CphBcy) with spectral properties virtually indistinguishable from those of the heterologously expressed, BV IXa-assem-bled protein Although the peptide covering the chro-mophore-binding region lost the chromophore during the MS analysis, the assignment of BV IXa as a chromophore is straightforward The first line of evi-dence arises from the detection of a chromopeptide
in the HPLC separation that matches the spectral properties of a peptide-attached tetrapyrrole (in addi-tion, the MS analysis of this peak revealed the expec-ted molecular mass for the peptide containing the putative chromophore-binding site; the finding of more than one peptide with chromophore absorption might be due to incomplete digestion or mechanical cleavage of peptide bonds) The second line of evi-dence arises from the high affinity of CphB for BV
In contrast to the CphBm–PCB adduct, which relea-ses the chromophore during purification, no loss of chromophore was detected for CphBcy; however, chromophore exchange (BV versus PCB and vice versa) has been shown to be possible [18] Further-more, the tight interaction between BV IXa and apo-CphBcy, allowing Zn-mediated fluorescence (which is not seen with the CphB–PCB adduct), and the stabil-ity of the BV adduct against an excess of PCB, are both indicative of the fact that PCB cannot be the chromophore of CphBcy Accordingly, we recently showed that BV is able to actively replace PCB in the binding pocket of CphB [18] As an additional argument, the absorption properties of CphBcy match the spectra of the heterologously expressed protein assembled with BV, not only in the position of the absorption maxima, but also in the shape of the Pr and Pfr forms Slight differences in the positions of the absorption maxima of homologously versus heterologously expressed BphPs like those observed with CphBcy (maximum variations of 2 nm were observed) were also reported for Cph1 from Synechocystis [19]
In particular, the positions of the absorption max-ima provide a clear indication of the type of chromo-phore–protein interaction, when we take into account that covalent binding of a 3-ethylidene-substituted tetrapyrrole (such as PCB) leads to the loss of one double bond (3–3¢) due to the formation of a thio-ether linkage to the protein This effect has recently
Fig 6 Assembly kinetics and subsequent red light illumination of
CphBm H267A assembled with a 2.5 molar excess of BV and BV
control sample.
Trang 7been demonstrated by comparing the absorption
max-ima of the noncovalently bound PCB adduct of CphB
(kmax: 686 and 734 nm for Pr and Pfr, respectively
[10]) with those of the L266C mutant of CphB that
binds PCB covalently (kmax: 656 and 702 nm), and
with those of the BV IXa adduct of CphB (702 and
754 nm) This mutation (L266C), which enables
cova-lent binding of PCB with removal of the 3–3¢ double
bond in the chromophore, is sufficient to convert the
binding mode of CphB into that of Cph1 or CphA
(cf kmax of CphA: 663 and 710 nm for Pr and Pfr,
respectively) Moreover, the spectral shift of the
L266C mutant is only observed when PCB is used
for the in vitro assembly, and not in the case of BV,
which retains practically unchanged absorption
max-ima, irrespective of whether the wild-type or the
mutated apo-protein is used [18] Also, in BV-binding
proteins a double bond (3¢)3¢ of the vinyl group) is
lost during covalent bond formation, and this should
also lead to a less red-shifted absorption than
observed This unexpected result, however, is
pro-posed to be due to a rearrangement of double bonds
in BV upon covalent binding, in accordance with a
more detailed inspection of the D radiodurans BphP
crystal structure (K Forest, personal communication)
Such a rearrangement (Fig 7) changes the
hybridiza-tion of C2 into sp3 in accordance with the electron
density of the crystal structure, and converts the
A-ring of bound BV into a PCB-like structure, now
with an ethylidene substituent In addition, this type
of binding is reversible, explaining the observation
that a bound BV can be expelled from the binding
site by an excess of PCB [18]
The ability of CphB to incorporate BV noncovalently
(C24S mutant) places it between these two classes of
phytochromes, and the mutation demonstrates that
C24 is not necessary for the formation of the
photo-receptor complex, as has been shown by us for other
phytochromes [28] An inspection of the
three-dimen-sional structure of D radiodurans does not reveal any
other appropriately located cysteine that would allow a
similar conformation of a bound chromophore The
observed preferred binding of BV to CphB, although
PCB is synthesized in the cyanobacterial cell in large
quantities, is an interesting ability of Calothrix that
allows adjustment of the spectral sensitivity through the use of two related photoreceptors This selection,
of course, can only be employed on the basis of an additional photoreceptor (CphB) that binds BV and provides a bathochromically shifted absorption It should be noted that although the demonstration of light-induced phosphorylation of both phytochromes from Calothrix, which differ in their absorption max-ima by 50 nm, represents a simple color discrimin-ation system, there is, as yet, no evidence for a physiologic role
The loss of chromophore incorporation upon muta-tion of H267 reveals a very important role for this residue for both PCB-binding and BV-binding phytochromes Inspection of the crystal structure of
D radiodurans phytochrome indicates interactions with the chromophore (via hydrogen bonding to the pro-pionate group of ring C), which can be assumed to have
a stabilizing effect on the extended conformation that the chromophore adopts in the binding site (in contrast
to the helically coiled conformation in organic solvents [29]) This mutation has been reported to prevent chro-mophore binding in Bph1 [3], in oat phytochrome [30], and in CphB (this study) and CphA (B Quest, unpub-lished results) In addition, this histidine might also be important for the assembly process itself, as has been demonstrated by addition of imidazole to the incuba-tion mixture [18] On the other hand, recent studies have shown that in Cph1 also, a glutamine residue at that position is sufficient for the spectral properties of this cyanobacterial phytochrome [31]; these authors demonstrate, moreover, that a major contribution of this histidine (260 in Cph1) is the stabilization of the protonated state of the chromophore (H260Q shows strong pH dependence in its absorption properties) The homologously expressed protein showed an even more pronounced phosphorylation capability origin-ating from the Pr form than the heterologously expressed protein, and reacted in a precise manner with its cognate response regulator, as shown by the crystal structures of the response regulators RcpA and RcpB [32] The physiologic relevance of this signal transduction pathway, which leads to phosphorylation
of the response regulator under conditions of either continuous far-red light irradiation or continuous
Fig 7 Proposed double bond rearrange-ment during covalent binding of BV Note that this binding is reversible.
Trang 8darkness after assembly of CphB in the dark, will be
the subject of future work
Experimental procedures
Cyanobacterial strains and culture media
Fremyella diplosiphonstrain SF33 is a
hormogonium-defici-ent mutant of F diplosiphon UTEX 481 (also designated
Calothrix PCC7601) [20,21] that grows as short filaments
and is easier to use for genetic studies than the wild-type
strain Cyanobacteria were routinely maintained in liquid
medium BG-11 [20] or on solid medium GN (BG-11
med-ium containing 0.38 mm Na2CO3), at 30C under a
photo-synthetic photon flux density of 6–7 lEÆm)2Æs)1 for
conditions without a gas supply, and 20–35 lEÆm)2Æs)1 for
growth with a gas supply provided by Sylvania (Osram,
Munich, Germany) GRO-Lux 18 W fluorescent lamps
Cloning
cphBwas amplified from previously described constructs [10]
using the following primers: oBQ35, 5¢-CATATGACGAA
TTGCCATCGCGAACC-3¢; and oBQ36, 5¢-GGATCCTTA
TTTGACCTCCTGCAATGTGAAATAG-3¢ (restriction sites
are underlined, and start and stop codons are given in bold)
The PCR product was then cloned in vector pET28a(+)
(Novagen⁄ Merck, Darmstadt, Germany) between the NdeI
and BamHI sites located downstream of the nucleotide
sequence providing the N-terminal His-tag sequence The T7
promoter, present in the vector, was replaced by a tac
pro-moter that was amplified from vector pGEX-4T-1
(Pharma-cia Biotech⁄ GE Freiburg, Germany) using the following
primers: oBQ60, 5¢-GGGCCCTGCACGGTGCACCAA
TGC-3¢; and oBQ61, 5¢-CCATGGATACTGTTTCCTGTG
TGA-3¢ The resulting PCR fragment was cloned between
the ApaI and NcoI sites, thereby removing half of the 5¢
nuc-leotide sequence of the lacI repressor gene of pET28a(+)
After removal of the BamHI site of this construct by
diges-tion, Klenow fill-in and religadiges-tion, the DNA fragment
carry-ing the tac promoter, the cloned gene and the T7 terminator
was subcloned into the single BamHI site of the vector
pPL2.8 using the following primers: oBQ88, 5¢-CGGGA
TCCTGCACGGTGCACCAATGCTTC-3¢; and oBQ89,
5¢-ACGGATCCAAAAAACCCCTCAAGACCCG-3¢ pPL2.8
is a derivative of pPL2.7 [22], generated by EcoRI digestion,
Klenow fill-in, and religation The resulting construct was
termed pPL9b CphBm was amplified from genomic DNA
from PCC7601 using primers oBQ146 (5¢-TATACCATGG
GCTTAAGTCCTGAAAATTCTCCAG-3¢) and oBQ147
(5¢-AAACTCGAGCCGGCCCTCAATTTTGACCTCCTGC
AATGTGAAATAGAACG-3¢), and cloned between the
NcoI and XhoI sites into pET28a(+), providing a His-tag in
the C-terminus of the recombinant protein
Generation of site-directed mutations The C24S and H267A mutations were generated with the QuickChange site-directed mutagenesis kit (Stratagene-Europe, Amsterdam, the Netherlands), according to the instructions of the manufacturer Generation of the C24S mutant was performed with the following primers: CphBm C24S-sen, 5¢-GAGGTGGACTTGACGAATTCAGATCGCG AACCAATTCAC-3¢, and CphBm C24S-antisense 5¢-GTGAA TTGGTTCGCGATCTGAATTCGTCAAGTCCACCTC-3¢ The primers used for H267A were: oBQ144-2, 5¢-CACT CGGTACTCCGCAGCGTTTCGCCGTTARCCATTGAA TATTTGCACAATATGG-3¢ (R ¼ purine); and oBQ145-2, 5¢-CCATATTGTGCAAATATTCAATGGYTAACGGCG AAACGCTGCGGAGTACCGAGTG-3¢ (Y ¼ pyrimidine) The differences from the wild-type sequence are indicated The mutations were identified and verified by sequencing
Conjugal transfer of DNA to cyanobacteria DNA was transferred to F diplosiphon cells by means of a triparental conjugation system as previously described [23], with minor changes The cargo strain containing the plasmid
of interest was Epicurian coli XL1 blue MR (Stratagene-Europe); the conjugal strain, bearing the RP4 plasmid [24] necessary for conjugal transfer, was Escherichia coli J53 Mil-lipore HATF nitrocellulose filters were used for conjugation, and the cell mixtures were spotted in different cyanobac-teria⁄ conjugal strain ⁄ cargo strain ratios The filters were incubated under a photosynthetic photon flux density of 6–7 lEÆm)2Æs)1 for 48 h on GN plates supplemented with 5% (v⁄ v) LB medium, and subsequently transferred to GN plates containing 25 lgÆmL)1neomycin
Homologous expression Cyanobacterial cells carrying plasmid pPL9b were grown
in BG-11 medium [20] supplemented with 25 lgÆmL)1 neo-mycin Phosphate buffer (5 mm, pH 7.4) was added to the fermenter cultivation (Braun, Melsungen, Germany,
880 137⁄ 1, culture volume of 12 L) All cyanobacterial cul-tures were incubated at 30C under a photosynthetic pho-ton flux density between 20 and 35 lEÆm)2Æs)1 (GROLUX F18W⁄ GRO fluorescent white light tubes, Osram) For the homologous expression of CphB in F diplosiphon (CphBcy), the fermenter was inoculated with a 1 L precul-ture (D750 0.8) The doubling time was approximately
20 h Cells were harvested at a D750of around 0.8 by cen-trifugation (6000 g, 10 min, 4C, Avanti centrifuge with JA10 rotor, Beckman-Coulter, Fullerton, CA, USA) Prior
to cell breakage by a French Pressure Cell (Aminco,
1100 lbÆin)2) in NaCl⁄ Tris buffer (supplied with protease inhibitor cocktail, EDTA-free, Boehringer, Mannheim, Germany), the cyanobacterial cells were washed several
Trang 9times with BG-11 to remove residual E coli cells from the
conjugation mixture
Heterologous expression
The heterologous expression of CphBm and RcpB in
BL21DE3 RIL (Stratagene-Europe) was carried out as
pre-viously described [18] In brief, the expression was carried
out in TB medium at 18–20C for 16–20 h after induction
with 0.4 mm isopropyl-thio-b-d-galactoside The amount of
soluble photoactive CphBm was thereby increased 30-fold
in comparison to the expression system described previously
[10] Typical yields reached approximately 6 mg of purified
protein per liter of culture
Protein purification
After cell breakage, cellular debris was removed by
ultracen-trifugation (150 000 g, 1 h, 4C, LX80 XP centrifuge with
Ti60 rotor, Beckman-Coulter) For cyanobacterial
prepara-tions, one spatula of streptomycin sulfate was added to the
supernatant prior to the centrifugation to remove
chloro-phyll-containing microvesicles Centrifugation with
strepto-mycin sulfate was repeated up to three times Holo-CphBcy
and Apo-CphBm were purified with Ni–nitrilotriacetic acid
superflow affinity resin (Qiagen, Hilden, Germany) and
sub-sequent gel filtration on a 16⁄ 60 Superdex 200 preparative
grade column (Pharmacia⁄ GE, Freiburg, Germany), using
A¨kta FPLC systems RcpB was purified to homogeneity by
affinity purification on Ni-nitrilotriacetic acid superflow
resins and gel filtration on a 26⁄ 60 Superdex 75 preparative
grade column Proteins were analyzed by SDS⁄ PAGE
fol-lowing the protocol of Scha¨gger & Jagow [25], and stored at
4C in NaCl ⁄ Tris buffer (50 mm Tris ⁄ HCl, pH 8.0, 150 mm
NaCl), including 1 mm dithiothreitol, until further use
Visualization of chromoproteins via
Zn-fluorescence
Zn-gel electrophoresis was performed as previously
des-cribed [26] In brief, 1 mm Zn acetate was added to all
solu-tions of a standard SDS⁄ PAGE The gels were placed on a
UV-transilluminator, and images were recorded with
integ-ration times between 2 and 4 s
Assembly of recombinant chromoproteins,
determination of absorption and difference
absorption spectra, and measurement of Pfr
stability
For the assembly of heterologously expressed CphBm in
the wild-type, mutated or truncated form, with BV or
PCB, the apoprotein was incubated in the dark with a
2.5-fold molar excess of BV or PCB, respectively The
molar extinction coefficients of the chromophores in NaCl⁄ Tris buffer (BV e674¼ 13 000 m)1Æcm)1, PCB e610¼
16 000 m)1Æcm)1) were taken from Lindner et al [27] The fully assembled chromoproteins were subjected to repeated red⁄ far-red illumination Interference filters at 636 ± 9 nm and 730 ± 12 nm were used to generate the Pfrand the Pr forms of the PCB adducts, respectively, and for the BV adducts and for CphBcy, filters at 680 ± 8 nm and
788 ± 11 nm were used The probes were illuminated with the stated light qualities, until no further absorption chan-ges occurred Absorption spectra were recorded with a Shimadzu (Duisburg, Germany) UV-2401 PC spectropho-tometer All samples were measured at 15C For the determination of the thermal stability of the Pfr form of CphBcy, the samples were irradiated with a saturating red light pulse, and the conversion back to Prwas followed by UV-visible spectroscopy Between the successive recordings
of absorption spectra (from 260 to 820 nm), the samples were protected against the measuring light of the spectro-photometer The absence of secondary photochemistry was confirmed by several consecutive measurements
Phosphorylation of CphBcy and transphosphorylation to RcpB Autophosphorylation and phosphotransfer were carried out
as previously described [17] In brief, a single reaction con-tained 3 lg of CphBcy for the autophosphorylation, or
3 lg of CphBcy and 0.75 lg of RcpB for the phosphotrans-fer reactions The reactions were carried out in phospho-transfer buffer containing 50 mm Tris⁄ HCl (pH 7.8),
50 mm KCl, 1 mm dithiothreitol, 0.5 mm MgCl2, 10 lm unlabeled ATP, and 0.2 lm [32P]ATP[cP] (110 TBqÆmmol)1) (Hartmann Analytik, Braunschweig, Germany) The reac-tion volume was 15 lL Reacreac-tions were started by the addi-tion of [32P]ATP[cP], and terminated at given time points
by adding 5 lL of SDS stop buffer (250 mm Tris⁄ HCl,
pH 6.8, 15 mm EDTA, 30% v⁄ v glycerol, 11% w ⁄ v SDS, 10% v⁄ v 2-mercaptoethanol, 0.02% w ⁄ v bromophenol blue) and incubating for 5 min at 50C Phosphotransfer was initiated by adding 15 lL of the autophosphorylation reaction mixture to 0.75 lg of RcpB, dissolved in 3 lL of phosphotransfer buffer without ATP All reactions were performed at room temperature The 32P-labeled products were separated by SDS⁄ PAGE (12.5%, La¨mmli) and trans-ferred to poly(vinylidene difluoride) membranes (Pharma-cia) The membranes were dried and quantified using a GS-525 PhosphorImager (Bio-Rad, Munich, Germany)
MALDI-TOF MS Samples were excised from a Coomassie-stained gel and washed three times alternately in 50% acetonitrile and
50 mm ammonium bicarbonate Destained samples were
Trang 10processed by incubation at 56C in ammonium
bicarbon-ate + 10 mm dithiothreitol for 45 min followed by
incuba-tion in ammonium bicarbonate + 55 mm iodoacetamide
for 30 min and an initial washing cycle For tryptic digests
of CphBcy, protein⁄ protease mixtures (40 : 1, w ⁄ w) were
incubated in NaCl⁄ Tris + 1 mm CaCl2at 37C overnight
Digested samples were analyzed on a Maldi Reflex III
(Bruker-Daltonik, Bremen, Germany)
LC-MS
Digested samples were separated with a Waters (Milford,
MA, USA) Symmetry C18 column (5 lm; 0.32· 150 mm)
on a Waters CAP-LC, supplied with a photodiode array
detector (eluent A, 0.025% v⁄ v trifluoroacetic acid in H2O;
eluent B, 0.02% v⁄ v trifluoroacetic acid in acetonitrile;
gra-dients, 5 min 5% v⁄ v B, from 5% to 45% v ⁄ v B in 25 min,
from 45% to 90% v⁄ v B in 3 min, 7 min 90% B) The
sam-ples were transferred online for ESI-MS-MS analysis
Acknowledgements
We thank Dr Frank Siedler and Bea Scheffer (MPI
Biochemie, Martinsried) for technical assistance in the
MS experiments This work was partially supported by
the SFB533 and by a grant of the Fonds der
Chemis-chen Industrie to B Quest
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