Sequence analyses of the deduced amino acid sequence demonstrated that the MCA2590-encoded protein shared significant, but restricted, sequence simi-larity to the bacterial di-heme cytoch
Trang 1of Methylococcus capsulatus (Bath)
A member of a novel group of the bacterial di-heme
cytochrome c peroxidase family of proteins
Odd A Karlsen1, Louise Kindingstad1, Solveig M Angelska˚r1, Live J Bruseth1, Daniel Straume1, Pa˚l Puntervoll2, Anne Fjellbirkeland1, Johan R Lillehaug1and Harald B Jensen1
1 Department of Molecular Biology, University of Bergen, Norway
2 Computational Biology Unit, Bergen Centre for Computational Science, Norway
Copper plays a very significant role in the physiology
of the methanotrophic bacterium Methylococcus
capsul-atus(Bath) The availability of this metal ion regulates
expression of the two forms of the methane-oxidizing
enzyme methane monooxygenase (MMO) the
bacter-ium possesses and formation of an extensive
intracyto-plasmic membrane network [1–4] When copper is
scarce, at a low copper-to-biomass ratio, a soluble cyto-plasmic MMO (sMMO) is responsible for the oxidation
of methane At high copper-to-biomass ratios the parti-culate MMO (pMMO) is expressed and there is no detectable sMMO expression Furthermore, copper also influences the expression of at least two of the four
M capsulatusformaldehyde dehydrogenases [5–7]
Keywords
cell surface exposed; copper regulated;
cytochrome c peroxidase; methanotrophs;
Methylococcus capsulatus
Correspondence
O A Karlsen, Department of Molecular
Biology, University of Bergen, HIB,
Thormøhlensgt 55, 5020 Bergen, Norway
Fax: +47 555 89683
Tel: +47 555 84372
E-mail: odd.karlsen@mbi.uib.no
(Received 27 July 2005, revised 7 October
2005, accepted 17 October 2005)
doi:10.1111/j.1742-4658.2005.05020.x
Genomic sequencing of the methanotrophic bacterium, Methylococcus cap-sulatus (Bath), revealed an open reading frame (MCA2590) immediately upstream of the previously described mopE gene (MCA2589) Sequence analyses of the deduced amino acid sequence demonstrated that the MCA2590-encoded protein shared significant, but restricted, sequence simi-larity to the bacterial di-heme cytochrome c peroxidase (BCCP) family of proteins Two putative C-type heme-binding motifs were predicted, and confirmed by positive heme staining Immunospecific recognition and bioti-nylation of whole cells combined with MS analyses confirmed expression
of MCA2590 in M capsulatus as a protein noncovalently associated with the cellular surface of the bacterium exposed to the cell exterior Similar to MopE, expression of MCA2590 is regulated by the bioavailability of per and is most abundant in M capsulatus cultures grown under low cop-per conditions, thus indicating an important physiological role under these growth conditions MCA2590 is distinguished from previously character-ized members of the BCCP family by containing a much longer primary sequence that generates an increased distance between the two heme-bind-ing motifs in its primary sequence Furthermore, the surface localization of MCA2590 is in contrast to the periplasmic location of the reported BCCP members Based on our experimental and bioinformatical analyses, we suggest that MCA2590 is a member of a novel group of bacterial di-heme cytochrome c peroxidases not previously characterized
Abbreviations
BCCP, bacterial cytochrome c peroxidase; CCP, cytochrome c peroxidase; ECL, enhanced chemiluminescence; MADH, methylamine dehydrogenase; MALDI, matrix-assisted laser desorption ionization; MauG, methylamine-utilizing protein G; MeDH, methanol
dehydrogenase; MMO, methane monooxygenase; Mop, M capsulatus outer membrane protein; NMS, nitrate mineral salt; ORF, open reading frame; pMMO, particulate methane monooxygenase; SACCP, surface-associated cytochrome c peroxidase; sMMO, soluble methane monooxygenase.
Trang 2We have previously described the surface-associated
protein, MopE, from which an N-terminally truncated
form, MopE*, is released into the culture medium
[8,9] Furthermore, it was recently shown that MopE
responds to changes in copper concentration during
growth, and was most abundant when copper was
lim-ited, indicating an important physiological role at low
copper-to-biomass ratios [10] Genomic sequencing of
M capsulatus revealed an open reading frame (ORF,
denoted MCA2590) immediately upstream of the mopE
gene [11] In this study, we show that the
MCA2590-encoded protein is expressed by M capsulatus, and,
similarly to MopE, is located on the cellular surface of
the bacterium Furthermore, expression of MCA2590
also responded to the availability of copper, and was
abundant when copper was scarce in the growth
med-ium The MCA2590 primary sequence shares
signifi-cant but restricted sequence similarity to the bacterial
di-heme cytochrome c peroxidase (BCCP) family of
proteins and contains two conserved heme-binding
motifs We also demonstrate that the MCA2590
pro-tein contains C-type heme; this was in line with our
predictions from the primary sequence Extensive
bio-informatical analyses suggest that MCA2590 is a
member of a novel group of the BCCP family
Results
Sequence analyses
An ORF of 2322 nucleotides (MCA2590) was
predic-ted upstream and in the same orientation as mopE
in the M capsulatus genome (Fig 1A) [11] Twenty-six
nucleotides separated the predicted stop codon for the
putative protein MCA2590 and the translation start
site of MopE Sequence analyses including the
MCA2590, mopE and upstream nucleotides of
MCA2590,revealed a candidate promoter region 5¢ of
the potential start codon of the putative MCA2590
protein (Fig 1B) In addition, a transcription
termin-ation site consisting of a GC-rich palindrome sequence
followed by an AT-dominant stretch of nucleotides
was found downstream of the mopE gene [9] The lack
of significant predictions of promoter and terminator
regions immediate 5¢ of mopE indicates an MCA2590 ⁄
mopE operon Furthermore, two putative ribosomal
binding sites could be predicted upstream of both the
MCA2590 and the mopE gene (Fig 1) Our attempts
to detect transcripts overlapping the MCA2590⁄ mopE
transition have so far been unsuccessful
The putative MCA2590-encoded protein consists of
773 amino acids (Fig 1B) Bioinformatical analyses
using signalp and psort predicted a leader peptide
with a putative cleavage site between Ala41 and His42 (Fig 1B) N-Terminal processing would lead to a mature protein of 732 amino acids with a theoretical molecular mass of 78 kDa A search in the PROSITE database of protein families and domains [12] with MCA2590 revealed two regions matching the c-type cytochrome superfamily profiles (PS51007) Both regions contain the c-type cytochrome motif (CxxCH; Fig 1B), suggesting that MCA2590 binds two hemes Sequence similarity searches (BLASTp) revealed signi-ficant similarity to hypothetical proteins of Photobacte-rium profundum (UniProt: Q6LQ47), Pseudomonas fluorescens (GenBank: ZP_00262397.1), and Nostoc punctiforme (GenBank: ZP_00109402.2), with expect (E)-values of 1e)116, 1e)98 and 1e)69, respectively However, neither confirmation of expression nor any functions have been assigned to these putative pro-teins Also proteins with known functions were revealed in the BLASTp searches, e.g the cyto-chrome c peroxidase (CCP) of Nitrosomonas europaea (UniProt: P55929, E¼ 4e)5) and the methylamine util-ization protein MauG of Paracoccus denitrificans (UniProt: Q51658, E ¼ 4e)4) Both these proteins are members of the BCCP family of proteins [13] (pfam 03150), but are representatives of two functionally dis-tinct subsets of the BCCP family [14] To further explore the relationship between MCA2590 and the CCP and MauG proteins, members of each group were collected by performing several BLASTp searches (see Experimental procedures) Pairwise comparisons revealed that the hypothetical MCA2590-related sequences from P profundum, P fluorescens and
N punctiforme are 40–44% identical to the MCA2590,
in contrast to the CCP and MauG sequences that are < 30% identical A multiple sequence alignment including the hypothetical MCA2590-related sequences, and the CCP and MauG sequences, was constructed (alignment of representative members from each group
of sequences are shown in Fig 2) Figure 2 shows that there are conserved segments throughout the alignment and that these segments coincide well with secondary structure elements obtained from the resolved structures of the Pseudomonas aeruginosa and
N europaea CCPs Strikingly, residues of both the heme-binding sites (CxxCH), in addition to the amino acids coordinating the calcium ion present in the interface domain of the solved CCPs are positionally conserved in MCA2590 It is also evident that the MCA2590 and the hypothetical MCA2590-related sequences, owing to the near double length, introduce large gaps in the CCP and MauG sequences when sequence similarities are aligned Importantly, almost all of these gaps were introduced between secondary
Trang 3structure elements, leaving b sheets and a-helical
struc-tures intact It is also interesting to notice that
MCA2590 and the hypothetical MCA2590-like
sequences share significant sequence similarity in some
of the long segments that remained unaligned to the
CCPs and MauGs, indicating a common secondary
structure⁄ fold for the hypothetical MCA2590-related
sequences in these regions To examine the similarity
between the MCA2590 and the BCCP sequences more
closely, the multiple sequence alignment (Fig 2) was
visualized in the context of the N europaea CCP
struc-ture (Fig 3) This analysis showed that positions that
are identical or display a high degree of similarity
between the MCA2590 and the CCP sequences are located in the core of the CCP structure (Fig 3A) Furthermore, all of the additional insertions of the extended MCA2590 sequence are introduced in loop regions on the surface of the structure, thereby not interrupting secondary structure elements in the
N europaea CCP (Fig 3B) The MCA2590 insertions were mainly dispersed around the entire surface Taken together, these observations strongly suggest that the MCA2590 and hypothetical MCA2590-related sequences are homologous to the BCCP family of pro-teins, and form a separate group with a similar fold and core structure However, the members of this new
A
B
Fig 1 Genomic orientation (A), nucleotide and amino acid sequence (B) of MCA2590 (A) MCA2590 is oriented in the M capsula-tus genome immediate upstream of the mopE gene separated by 26 nucleotides (B) Amino acids are indicated below the nucleo-tide sequence The underlined promoter region was predicted using the Neural Net-work Promoter Prediction (NNPP) and was estimated to have a probability of 0.97 The previously predicted termination loop is indi-cated by black arrows [9] The two putative ribosomal binding sites predicted upstream
of MCA2590 and mopE, respectively, are enlarged in the nucleotide sequence The signal peptide predicted by SIGNALP is col-oured red in the MCA2590 amino acid sequence The two putative C-type heme-binding sites revealed by SCANPROSITE are shown in yellow The amino acid sequence used for construction of the anti-MCA2590 serum is blue The peptides of MCA2590 that were revealed in the MALDI MS ⁄ MS analysis (Table 1) are boxed.
Trang 4Fig.
Trang 5group will contain longer loops and possibly additional
secondary structure elements outside the CCP-similar
core
A similarity search against the translated GenBank
nucleotide database (tBLASTn) revealed significant
similarity between MCA2590 and an unannotated
ORF of Methylomicrobium album (GenBank: U74385 –
nucleotides 3279–4205; E-value 1e)67) [15]
Interest-ingly, this ORF is located immediately downstream of
the corA gene, which encodes the only protein in the
databases that shows significant sequence similarity to
MopE [9,15] The deduced amino acid sequence of the
M album U74385 ORF was added to the multiple
sequence alignment (Fig 2), revealing that it is 50%
identical to the N-terminal half of the MCA2590
pro-tein Because the fully sequenced U74385 ORF is not
available in the databases, it remains to be elucidated
if the sequence similarity of MCA2590 and the
M albumU74385 ORF extends even further
Localization and identification of the mature
MCA2590 protein
Enriched fractions of M capsulatus-soluble proteins,
inner membrane proteins and outer membrane proteins
were obtained as described previously [8] The resulting
fractions were assessed by SDS⁄ PAGE, demonstrating
the presence of the large subunit of the methanol
dehy-drogenase (MeDH) in the soluble fraction and the
outer membrane proteins MopB and MopE in the
A
B
Fig 4 SDS ⁄ PAGE (A) and protein immunoblot analyses (B) of pro-teins obtained during the fractionation of M capsulatus Samples
of each step during the fractionation procedure were collected and comparable samples (10 lL of each 1 mL fraction) were analysed (A) Coomassie Brilliant Blue (CBB) R-250 stained 10% polyacryl-amide gel (A, B) Lane 1, whole cells (W); lane 2, soluble fraction (S); lane 3, total membrane fraction (TM); lane 4, Triton X-100 soluble membranes (enriched inner membrane fraction, IM); lane 5, Triton X-100 insoluble membranes (enriched outer membrane fraction, OM) MopE in addition to the OmpA related MopB [38] are indicated (B) Protein immunoblot of (A) using the anti-MCA2590 serum Molecular mass markers are indicated to the left
of both subfigures.
Fig 3 The sequence similarity between MCA2590 and the CCP proteins visualized on the N europaea CCP structure Bound ligands are shown in green; the heme groups are shown as sticks, and the Ca2+ion is shown as a sphere (A) The surface of the structure is shown in transparent white Residues that are identical between the MCA2590 and CCP sequences in the alignment are shown in red, and similar residues are shown from yellow to red with increasing similarity (B) A cartoon representation of the structure Positions in the structure where gaps were introduced in the alignment are marked with blue spheres The N- and C-termini are marked.
Trang 6Triton X-100 insoluble fraction (enriched outer
mem-brane fraction) (Fig 4A) When protein immunoblots
of the enriched fractions were probed with a constructed
antibody to a short peptide derived from the MCA2590
sequence, an anti-MCA2590 immunoreactive
polypep-tide was found to cofractionate with the outer
mem-brane from cells grown at a low copper-to-biomass ratio
(Fig 4B) The polypeptide migrated with a relative
molecular mass of 80 kDa, which is close to the mass
predicted for the mature MCA2590 (78 kDa)
To further establish the cellular localization of
MCA2590, M capsulatus whole cells were treated with
a high ionic strength buffer to extract proteins
associ-ated with the cell surface Treatment of whole cells
with high concentrations of NaCl disrupts
electro-static⁄ ionic bonds between biomembranes and
pro-teins, thus leaving surface-associated polypeptides in
the resulting supernatant upon centrifugation [16,17]
The NaCl extraction was performed in two steps
with increasing NaCl concentrations Analyses of the
resulting fractions by SDS⁄ PAGE displayed a complex
crude cell-extract protein pattern in the untreated cells
and in cells treated with 0.5 and 1 m NaCl (Fig 5A,
lanes 1, 4 and 6) Importantly, the large 60 kDa
sub-unit of the MeDH was seen only in these fractions
MeDH is a periplasmic protein [18], hence indicating
that the cells remained undisrupted during the
extrac-tion procedure and thereby avoiding leakage of
pro-teins from the periplasm Protein immunoblot using
antibodies raised against MCA2590 revealed this
protein in the 0.5 m NaCl-extracted fraction (Fig 5B,
lane 3) This observation suggests a specific association
of MCA2590 to the outer membrane and that it is
exposed to the cell exterior As expected and in line
with previous reports, a similar extraction behavior
was observed for MopE [9] (Fig 5B) The integral
outer membrane OmpA-related MopB protein was not
extracted from the outer membrane by the NaCl
treat-ment, demonstrating the selectivity of the extraction
procedure (Fig 5B)
To ensure that the localization of MCA2590 is at
the external surface and not at the periplasmic
inter-face, we performed two additional experiments
M capsulatuswhole cells were immobilized on a
nitro-cellulose membrane and treated with the
anti-MCA2590 serum (Fig 6A) The anti-MCA2590-specific
staining obtained is consistent with SACCP being
sur-face exposed As another demonstration of sursur-face
exposure, we labelled intact cells grown in flask
cul-tures with biotin (Fig 6B) Biotin is too large to
penet-rate the outer membrane and will only label
polypeptides that have a lysine-containing sequence
accessible at the cell surface The biotinylated cells
B
Fig 5 SDS ⁄ PAGE and protein immunoblot analyses of protein frac-tions obtained during the NaCl extraction of surface proteins (A) 10% polyacrylamide gel stained with CBB R-250 The periplasmic methanol dehydrogenase (MeDH) and the surface associated MopE are indicated (A, B) Lane 1, cells resuspended in 5 mL buffer with low ionic strength (20 m M Tris ⁄ HCl) (10 lL sample applied); lane 2,
20 m M Tris ⁄ HCl wash (20 lL sample applied); lane 3, 0.5 M NaCl extract (20 lL sample applied); lane 4, whole cells treated with 0.5 M NaCl (10 lL sample applied); lane 5, 1 M NaCl extract (20 lL sample applied); lane 6, whole cells treated with 1 M NaCl (10 lL sample applied) (B) Protein immunoblots of (A) using anti-MCA2590, anti-MopE and anti-MopB sera, respectively Molecular mass markers are indicated to the left of (A) and (B) (C) Concentra-ted (selective centrifugation, Amicon 10 kDa cut-off) 0.5 M NaCl extracted fraction separated by SDS ⁄ PAGE and stained with CBB R-250 The polypeptide migrating with a corresponding molecular mass to the anti-MCA2590 immunogenic band is indicated.
A
B
Fig 6 blot and biotinylation analyses of whole cells (A) Dot-blot analyses of M capsulatus cells (0.5 · 10 8
) immobilized onto nitrocellulose membranes (1) Cells treated with the anti-MCA2590 serum (2) Negative control; cells treated exclusively with secon-dary HRP-conjugated antibody (B) Protein immunoblott of biotin-labelled M capsulatus cells (1) Biotin-biotin-labelled proteins visualized
by treatment with streptavidine biotinylated HRP complex (2) The same protein immunoblot as (1) but treated with the anti-MCA2590 serum.
Trang 7were analyzed with protein immunoblotting using the
anti-MCA2590 serum, followed by removal of bound
antibodies and staining of all biotinylated proteins
using streptavidin-biotinylated horseradish peroxidase
complex on the very same membrane By using
mark-ers on the nitrocellulose membrane we observed an
exact comigration of a biotinylated protein with the
immunoreactive MCA2590 band, strongly suggesting
that MCA2590 has been biotinylated The major
peri-plasmic protein, MeDH, was not labelled by biotin,
demonstrating that only surface-exposed proteins were
labelled during the procedure
A 0.5 m NaCl-extracted fraction of M capsulatus
whole cells was concentrated by selective centrifugation
(Amicon, 10 kDa cut-off) and the polypeptide
migra-ting with an apparent molecular mass of 80 kDa in the
SDS⁄ PAGE analysis of the concentrate was excised
from the gel and subjected to MS analyses (Fig 5C)
MALDI MS⁄ MS analyses revealed three sequenced
peptides (Table 1), which identified the excised
poly-peptide as the gene product of MCA2590 Based on
these findings and our bioinformatical analyses, we
have named the MCA2590-encoded
protein-surface-associated cytochrome c peroxidase (SACCP)
Expression
The expression of MopE is influenced by the
avail-ability of copper [10] Thus, it was of great interest
to explore if MCA2590 and mopE were
concomit-antly expressed if organized in a single
transcrip-tional unit M capsulatus was grown at high and
low copper-to-biomass ratios in batch cultures (0, 0.8
and 5 lm copper included in the growth medium)
and expression was analysed by protein immunoblots
using the anti-MCA2590 serum (Fig 7) The subunits
of pMMO (Fig 7A), as well as detection of sMMO
activity [19], were used as markers for
copper-to-bio-mass ratios in M capsulatus cultures The protein
immunoblots revealed that the expression of SACCP
was altered by the different copper concentrations,
SACCP being most strongly expressed in cells grown
in low copper media (Fig 7B) Specific PCR
prim-ers were designed for MCA2590, and differential expression in cells cultured under high- or low-copper conditions was verified by RT-PCR analyses (Fig 7C)
Detection of C-type heme Because of the sequence similarity of SACCP to mem-bers of the BCCP family of proteins and the predic-tion of heme-binding motifs in the primary sequence,
it was of interest to assay for the presence of C-type heme in SACCP However, because our SACCP pre-paration contains other proteins that may also express C-type heme-specific peroxidase activity, we designed
an assay that would distinguish SACCP from other proteins with similar activity NaCl-extracted proteins separated by SDS⁄ PAGE were transferred to a nitro-cellulose membrane and directly stained for heme using enhanced chemiluminescence (ECL) This method takes advantage of the intrinsic peroxidase activity of the covalently bound heme group of dena-tured c-type cytochromes, and should, in principle, detect any hemeprotein that retains heme after such treatment as described above [20,21] The activity measurement of the NaCl extract resulted in four
Table 1 MALDI MS ⁄ MS analyses of anti-MCA2590 immunogenic
band The localization of the identified peptides in the MCA2590
amino acid sequence is shown in Fig 1.
A
B
C
Fig 7 Copper regulation analyses of MCA2590 (SACCP) (A) SDS ⁄ PAGE analyses of M capsulatus cells grown at 5 (lane 1), 0.8 (lane 2), and 0 l M copper included in the growth medium, respectively The 10% polyacrylamide gel was stained with CBB R-250 The PmoA, PmoB and PmoC components of the particulate methane monooxygenase are indicated (observed in lane 1 and 2) sMMO activity in the cell cultures are shown by either (+) or (–) below the lanes (B) Protein immunoblot of (A) using anti-MCA2590 and anti-MopE serum, respectively Molecular mass markers are indicated to the left of both (A) and (B) (C) RT-PCR analyses of the MCA2590, mopE and mopB transcripts prepared from the same cultures as used in (A) and (B).
Trang 8distinct bands, all of which could be traced to
corres-ponding bands on a parallel CBB-stained
polyacryl-amide gel (Fig 8) Most importantly, a distinct band
of molecular mass corresponding to the migration of
SACCP was identified, suggesting that SACCP
contained bound heme A protein immunoblot using
antibodies directed against SACCP on the same
nitro-cellulose membrane showed that the
peroxidase-activ-ity-stained band and the SACCP immunogenic band
colocalized (Fig 8) Using MS, The two additional
high molecular mass bands, which also stained positive
for heme, were identified as the proteins encoded by
the genes MCA0421 and MCA0423, annotated as a
cytochrome c553o family protein and cytochrome c553o,
respectively (data not shown) [11,22]
Discussion
In this study, we show that the predicted ORF
(MCA2590) upstream of the mopE gene in M
capsula-tus encodes a C-type heme protein (SACCP) whose
expression is strongly increased in bacteria cultured at
low-copper concentrations The protein is located on
the surface of the bacterium and is noncovalently
attached to the outer membrane The extracellular
localization is in accordance with the prediction of a
signal peptide in a primary translation product
Bioinformatical analyses revealed that SACCP
shares characteristics with previously described
mem-bers of the BCCP family of proteins by both having
significant sequence similarity and containing two
con-served C-type heme-binding motifs [13] This family
constitutes the bacterial di-heme cytochrome c
peroxid-ases Similar to other CCPs, they reduce hydrogen per-oxide to water using C-type heme as cofactor The BCCP family also includes the MauG proteins, whose similarity to di-heme CCPs has previously been recog-nized [23,24] However, the MauG subset is distinct from the CCPs, and MauGs are proposed to function
in the oxidation of methylamine in facultative methylo-trophs [23,24] Importantly, SACCP significantly differ from the BCCP family of proteins in its much longer amino acid sequence The regions of similarities to the CCPs and MauGs are therefore restricted, and in mul-tiple alignments distributed as conserved segments throughout the SACCP amino acid sequence As in the BCCPs, two putative heme-binding motifs were found conserved N- and C-terminally of the SACCP sequence, indicating the binding of heme-groups anal-ogous to the low- and high-potential heme present in both CCPs and in the MauG proteins Furthermore, the regions in SACCP with highest sequence similarity
to the BCCP family of proteins coincided nicely with the known secondary structure elements of both the
P aeruginosa and N europaea CCPs [25], indicating a native fold of SACCP which resembles the structure of CCPs This supposition was substantially supported
by structural visualization of the multiple alignments between the SACCP sequence and its close homo-logues, and the BCCP sequences (Fig 3) These analy-ses showed that the additional SACCP-specific amino acid sequences were all located on the protein surface, and in-loop regions not interrupting secondary struc-ture elements Furthermore, the majority of the con-served residues were buried amino acids, thereby maintaining the structural integrity of an N europaea CCP-similar fold of SACCP, and thus bringing the hemes in close proximity to each other in contrast to increased interheme distance found in the primary sequence We also showed that SACCP stained posit-ively for peroxidase activity typical of the covalently bound heme groups of denatured c-type cytochromes [20,21] The C-type heme-associated peroxidase activity
is in accordance with the presence of heme-binding motifs in the primary sequence Interestingly, in the course of this study, two other surface-associated proteins that also stained positive for C-type heme were identified Both of these proteins have previously been described as multi-c-heme cytochromes (cyto-chrome c553o and cytochrome c553o family protein) of
M capsulatus, and the respective genes are clustered in the genome [11,22] We have now provided novel evi-dence for these proteins being noncovalently attached
to the cellular surface It is interesting to notice that 57 putative c-type cytochrome proteins are annotated in the M capsulatus genome, of which five are members
Fig 8 Detection of C-type heme in MCA2590 (SACCP) (A–C) Lane
1, 0.5 M NaCl extract from M capsulatus; lane 2, bovine serum
albumin (A) 10% CBB R-250 stained polyacrylamide gel obtained
from SDS ⁄ PAGE analysis (B) Samples corresponding to (A)
trans-ferred to a nitrocellulose membrane and directly stained for C-type
heme peroxidase activity (C) Protein immunoblot of a membrane
corresponding to (A) using the anti-MCA2590 serum Molecular
mass markers are indicated to the left of (A).
Trang 9of the cytochrome c553o family and four are annotated
as putative cytochrome c peroxidases [11] In this
study, three of these have been localized to the cellular
surface
As shown previously for MopE [10], the expression
of SACCP was found regulated by the copper
concen-tration in the growth medium and was found most
abundant in cells grown in copper-depleted medium
RT-PCR analyses revealed that this regulation takes
place at the transcriptional level The concomitant
regulation of SACCP and MopE is also in accordance
with the possibility that the MCA2590 and mopE genes
constitute an operon, but evidence of this assumption
has not yet been provided Furthermore, a potential
promoter was predicted with high significance both by
BPROM and NNPP immediate 5¢ of MCA2590 Very
interestingly, the following nucleotide sequence was
identified within the predicted promoter region:
5¢-TTGAGN(5)ATCGA-3¢ This nucleotide sequence
closely resembles the consensus binding site of the
transcriptional factor Fnr, 5¢-TTGATN(4)ATCAA-3¢,
introducing only two mismatched nucleotides
FNR-type regulators are known to regulate aerobic⁄
anaer-obic-dependent gene expression in c-proteobacteria,
and the gene product of the fnrA gene in Pseudomonas
stutzeri controls the expression of cythochrome
cbb3-type terminal oxidase, cytochrome c peroxidase and
the oxygen-independent coproporphyrinogen III
oxidase [26] M capsulatus harbours one copy of a
Fnr-type transcriptional regulator (MCA2120) [11]
However, further studies are necessary to elucidate the
regulatory mechanisms for MCA2590 and mopE
The biological function of SACCP remains to be
elucidated However, upregulation of SACCP in
M capsulatuswhen grown at a low copper-to-biomass
ratio indicates an important physiological role under
these growth conditions Furthermore, the concomitant
expression, in addition to both the genomic and
cellu-lar colocalization, of SACCP and MopE, indicates that
these proteins may cooperate and have linked
func-tions In general, CCPs are thought to play a
protect-ive role in the periplasm by reducing peroxides
generated in oxidative metabolism [27] MauG seems
to have a more specific function, as demonstrated in
P denitrificans, by being involved in the maturation of
the tryptophan tryptophylquinone cofactor of
methyl-amine dehydrogenase (MADH) [14,28] This enzyme is
responsible for the oxidative deamination of
methyl-amine when facultative methylotrophs are grown on
methylamine as the sole source for carbon and energy
[24] The strong indications of SACCP having a core
structure that resembles the CCPs may point toward
similar enzymatic mechanisms However, the increased
size and cellular localization of SACCP open the possi-bility of other physiological functions distinct from what has been described for CCPs and MauGs
In conclusion, we have described a novel C-type heme protein located to the cellular surface of the methanotrophic bacterium M capsulatus This protein shares characteristics with the members of the BCCP family, but separates itself from the described CCPs and MauGs in both amino acid sequence and cellular location Significant sequence similarity to three hypo-thetical proteins found in the prokaryotes P profun-dum, P fluorescens and N punctiforme, respectively, was observed, in addition to a partial sequenced ORF
of the methanotroph, M album Based on the amino acid sequence, experimental and bioinformatical data,
we propose that SACCP belongs to a novel class of the bacterial di-heme cytochrome c peroxidases Inter-estingly, the M album ORF was found in close prox-imity to the CorA encoding gene (corA) which shares sequence similarity to MopE of M capsulatus It remains to be seen if this colocalization is a unique feature of methanotrophs However, the colocalization may indicate a linked and unique function of these proteins in bacteria possessing both genes
Experimental procedures
Growth of Methylococcus capsulatus (Bath)
described previously [29] Most analyses were performed using cells grown at a low copper-to-biomass ratio in
‘copper-free’ medium (no copper added) The cultures were screened for soluble methane monooxygenase (sMMO) activity by the naphthalene assay described by Brusseau
et al [19] to ensure that a low copper-to-biomass ratio was
MCA2590, cells grown at high copper-to-biomass ratios were included in the experiment (0.8 and 5 lm copper in the growth medium) Batch cultures of M capsulatus were
harvesting
Fractionation of cells
Cells were harvested by centrifugation at 5000 g for
10 min Enriched fractions of the soluble proteins, inner membrane proteins and outer membrane proteins were obtained as described by Fjellbirkeland et al [8] 20 mm
throughout the fractionation procedure
Trang 10Extraction of cell-surface proteins
5000 g for 10 min, and resuspended in a small volume of
suspen-sion was centrifuged (as described above), and the
NaCl) to disrupt noncovalent bonds between
surface-asso-ciated proteins and components of the outer membrane
[16,17] Cells were incubated with rotation in this buffer
centrifugation, and the resulting supernatant contained
the 0.5 m NaCl-extracted proteins Treated cells were
pH 7.4, thereby increasing the ionic strength of the
extraction buffer Cells were thereafter incubated with
rotation and harvested as outlined above, resulting in the
1 m NaCl extracted protein fraction Cells were
centrifugation
Biotinylation of cell-surface proteins
Biotinylation of whole cells was performed with EZ-Link
Sulfo-NHS-biotin according to the manufacturer’s
instruc-tions (Pierce, Rockford, IL) Biotinylated proteins were
visualized on nitrocellulose membranes by treatment with
streptavidin-biotinylated horseradish peroxidase complex as
described by the manufacturer (Amersham Biosciences,
Little Chalfont, UK)
SDS⁄ PAGE and Western blotting
[30] using 10% (w⁄ v) running gels and 3% (w ⁄ v) stacking
gels Protein immunoblotting was carried out as described
previously [8] Rabbit polyclonal peptide-specific antibodies
against MCA2590 were produced by Sigma Genosys The
immunogen correspond to the amino acids 742–755 of the
MCA2590 primary sequence (Fig 1B) The specificity of
the produced antibody was confirmed by indirect ELISA
Horseradish peroxidase-linked anti-rabbit sera were
pur-chased from Bio-Rad (Hercules, CA) Protein immunoblots
that were incubated with anti-MopE or anti-MopB sera
were developed using the colour reagent HRP CDR
(Bio-Rad) Anti-MCA2590-treated membranes were developed
using ECL (Amersham Biosciences)
Detection of C-type heme
nitrocellulose membrane by electroblotting and directly
stained for C-type heme with the ECL assay as described previously [20,21] The ECL reagent was supplied from Amersham Biosciences
MS identification of proteins
MS analyses were performed at the PROBE facility at the University of Bergen, Norway
Isolation of total RNA
pH 8.0) and submerged directly into liquid nitrogen and
treatment were carried out using the QIAGEN RNeasy Mini Kit as described in the supplied ‘RNAprotect Bacteria Reagent Handbook’ with an additional DNase treatment on the column The RNA was dissolved in 50 lL of RNase-free
RNA quality and quantity was determined with the Agilent
2100 Bioanalyser and the 2100 expert software
RT-PCR
First-strand synthesis was carried out with 1 lg of total
(Invitrogen, Carlsbad, CA) was used for first strand synthe-sis according to the manufacturer’s protocol RNasin (Promega, Madison, WI) was used in the mixture to inhibit any RNAse activity The cDNA synthesis was carried out
primers used in the synthesis were 40 oligomers calculated
to anneal to all ORFs in the M capsulatus genome The primers were defined using the program ‘Genome directed primers’ as described by Talaat et al [31]
PCR was performed according to standard procedures [32] The reverse transcription reaction (1 lL) was
DNA Polymerase (Finnzymes), in a 25-lL reaction volume with 0.2 mm dNTP, 0.5 lm of each specific primer, 1.5 mm
protein B, rplB, and M capsulatus outer membrane protein
B, mopB, were amplified The amounts of PCR products were determined using the Fuji FLA-2000 phosphoimager, and the amount of template of first-strand cDNA was corrected to give an equal amount of amplified PCR prod-ucts for each ‘housekeeping’ gene The differential expres-sion of the sapE and mopE transcripts was determined using the primers: sapE–F sapE–R and mopE–F mopE–R (Table 2)