Tài liệu Báo cáo khoa học: Experimental proof for a signal peptidase I like activity in Mycoplasma pneumoniae, but absence of a gene encoding a conserved bacterial type I SPase pdf

Experimental proof for a signal peptidase I like activity
in Mycoplasma pneumoniae, but absence of a gene
encoding a conserved bacterial type I SPase
Ina Catrein, Richard Herrmann, Armin Bosserhoff and Thomas Ruppert
Zentrum fu
¨
r Molekulare Biologie Heidelberg, Universita
¨
t Heidelberg, Germany
Mycoplasma pneumoniae is a human pathogenic bac-
terium [1,2], characterized by a small genome of 816
kbp [3], the lack of a bacterial cell wall and a parasitic
lifestyle [4].
Some species of the genus mycoplasma, e.g. Myco-
plasma genitalium, Mycoplasma gallisepticum and
Mycoplasma pneumoniae exhibit a flask-like shape,
which is believed to be formed and maintained by a
cytoskeleton-like structure [5–10]. This flask-like shape
is caused by the attachment organelle, an asymmetric
extension of the cell composed of an assembly of
unique proteins [11]. M. pneumoniae interacts with its
host cell by adhering with the attachment organelle to
specific receptors. This interaction takes place only if
the P1 protein, the bacterial main adhesin, is inserted
correctly into the attachment organelle [12]. The
proper insertion depends, among others, on the pro-
teins P40 and P90. Absence of these proteins causes a
random insertion of the P1 protein and a cytadher-
ence-negative phenotype [13]. P1 is encoded by
MPN141 and both, P40 and P90 by MPN142. These
genes are organized, together with MPN140 which

probably encodes a phosphoesterase [14], in the P1
operon [15]. In the original publication these genes
were called ORF4 (MPN140), ORF5 (MPN141) and
ORF6 (MPN142) [15]. The names in brackets are the
gene names according to a recent reannotation [15,16].
MPN142 codes for a protein with a molecular mass of
130 kDa, which, however, has been never identified as
single protein of the expected molecular mass. Instead,
two proteins with molecular masses of about 40 kDa
(P40) and 90 kDa (P90) had been found in SDS ⁄
PAGE and Western blotting experiments [17]. An
enzyme responsible for the processing of the proposed
130-kDa protein has not yet been identified. P40
derives from the N-terminal and P90 from the C-ter-
minal part of the predicted 130-kDa precursor protein.
It was proposed that P40 and P90 are identical with
the proteins B and C missing in certain avirulent
mutants [18,19], but so far this has not been proven
Keywords
chemical assisted fragmentation (CAF);
mass spectrometry; protein modification;
signal peptidase
Correspondence
T. Ruppert, Zentrum fu
¨
r Molekulare Biologie
Heidelberg, Universita
¨
t Heidelberg, Im
Neuenheimer Feld 282, 69120 Heidelberg,

Germany
Fax: +49 6221 545891
Tel: +49 6221 546895
E-mail:
(Received 15 December 2004, revised
11 March 2005, accepted 7 April 2005)
doi:10.1111/j.1742-4658.2005.04710.x
Although the annotation of the complete genome sequence of Mycoplasma
pneumoniae did not reveal a bacterial type I signal peptidase (SPase I) we
showed experimentally that such an activity must exist in this bacterium, by
determining the N-terminus of the N-terminal gene product P40 of MPN142,
formerly called ORF6 gene. Combining mass spectrometry with a method
for sulfonating specifically the free amino terminal group of proteins, the
cleavage site for a typical signal peptide was located between amino acids 25
and 26 of the P40 precursor protein. The experimental results were in agree-
ment with the cleavage site predicted by computational methods providing
experimental confirmation for these theoretical analyses.
Abbreviations
CID, collision induced fragmentation; SPase I, signal peptidase I.
2892 FEBS Journal 272 (2005) 2892–2900 ª 2005 FEBS
experimentally. The P1 protein can be cross-linked
with P40 and P90 under in vivo conditions, indicating
that they form a larger protein complex embedded into
the cytoplasmic membrane [20,21].
To get insight into the function of this protein com-
plex, the subunits had to be characterized in detail.
The N-terminal end of P90 was determined by Edman
degradation. This protein begins with an arginine at
amino-acid position 455 of the proposed precursor
[22]. The predicted molecular mass of P40 is therefore

47 700. This value, however, differs significantly from
the molecular mass of 36–37 kDa seen in SDS ⁄ PAGE.
This discrepancy could be explained either by abnor-
mal migration of P40 in SDS ⁄ PAGE, or more likely,
by additional processing steps causing the observed
reduction in apparent molecular mass.
A processing step could take place at the N-terminal
region of P40, as a bacterial signal peptide had been
predicted [23–25]. Such signal peptides are normally
cleaved off by a bacterial type I signal peptidase
(SPase). The various type I SPases from Gram-negat-
ive and Gram-positive bacteria show clear differences
concerning gene size, gene copy number and substrate
specificity, despite the substantial sequence similarities
as indicated by six distinct regions with conserved
amino acids [26]. For instance, LepB from Escherichia
coli is 323 amino acids long and exists only as a single
gene copy while the occurrence of multiple type I
SPases within a single species is commonly observed in
Gram-positive bacteria. Bacillus subtilis contains five
chromosomally encoded type I SPases named SipS,
SipT, SipU, SipV and SipW, which are only about 200
amino acids in size [26]. As M. pneumoniae belongs
phylogenetically to the Gram-positive bacteria one
would expect to find a type I SPase similar to the var-
ious sip genes. However, no type I SPase typical for
Gram-positive bacteria or for Gram-negative bacteria
(such as LepB) has been identified in M. pneumoniae,
although a SPase I activity has been shown to be
essential for cell viability in all bacteria analyzed [26].

To test experimentally whether there is a type I
SPase activity in M. pneumoniae, we determined the
N-terminus of P40 as it appears in protein extracts of
M. pneumoniae.
Determination of the N-terminus of P40 by Edman
degradation failed due to the limited amount of start-
ing material. An alternative method is the tryptic
digestion of the purified protein and subsequent ana-
lysis of the derived peptides by mass spectrometry. If
a candidate mass is detected and peptide sequencing
proves, that there is no trypsin cleavage site at the
amino terminus of this peptide, as read from the gene
sequence, then this peptide is taken as the N-terminal
peptide of this protein [27]. This is, however, not an
exact proof, as such a cleavage may also result from
proteolytic contaminants of the trypsin being used like
chymotrypsin or due to pseudotrypsin formed from
trypsin by autolysis [28]. These possibilities can be
excluded, when the amino terminus of the intact pro-
tein is specifically labeled before tryptic digestion.
Liminga and colleagues [29] introduced an N-
hydroxysuccinimide ester of 3-sulfonic-propionic acid
(CAF reagent) to modify peptides after tryptic diges-
tion for enhanced peptide sequencing using matrix-
assisted laser desorption ⁄ ionization time of flight mass
spectrometry (MALDI-TOF-MS) with chemical assis-
ted fragmentation (CAF) [30]. We modified this
method in a way so that only the N-terminus of the
mature P40 was labeled with the CAF reagent. After
tryptic digestion, the N-terminal sulfonated peptide of

P40 could be identified unambiguously.
Results
To analyze in more detail the protein complex
formed by the proteins P1, P40 and P90, it is a pre-
requisite to know the primary structure of the sub-
units. To get sufficient material for the identification
of the amino terminus, P40 was enriched from cell
extracts of M. pneumoniae M129 by immunoprecipi-
tation with a polyclonal antiserum directed against a
P40 fragment corresponding to the sequence from
residue 66 to residue 223 [17]. The final SDS ⁄ poly-
acrylamide gel, which was the source of P40 for the
N-terminal sequence analysis, is shown (Fig. 1). The
indicated Coomassie blue stained protein band was
recognized by antibodies directed against P40 in
western blotting experiments (data not shown). The
P40 band was clearly separated from other proteins,
but the amount of P40 was not sufficient for sequen-
cing by Edman degradation. Therefore, we used mass
spectrometric analysis of an in-gel digest of P40. To
prove that the candidate peptide was the N-terminal
peptide of P40, we labeled the N-terminus of P40
within the gel piece before tryptic digestion by
a method described for peptide sequencing by
MALDI-TOF MS after chemical assisted fragmenta-
tion (CAF) [30]: during the first step of the labeling
reaction the e-amino groups of lysine are specifically
converted to homoarginine. In a second step, the free
amino terminal groups of the peptides are sulfonated
by the CAF reagent (Fig. 2).

Subsequently, the protein was digested by trypsin
and the supernatant was analyzed by ESI-QTOF MS
(Fig. 3) in the positive ion mode, to get high quality
fragmentation pattern for peptide sequencing. Fifteen
I. Catrein et al. Signal peptidase I activity in M. pneumoniae
FEBS Journal 272 (2005) 2892–2900 ª 2005 FEBS 2893
signals of various m ⁄ z-values from the peptide mass
fingerprint were subjected to collision induced frag-
mentation (CID). P40 was identified (probability
based Mowse score of 276) by sequence tags of six
different peptides covering the N-terminal region
(position 26–203) of P40 (Table 1). The fourfold
charged signal with m ⁄ z ¼ 850.2 (rmm: 3396.8) corre-
lated with the mass of the peptide 26–55 (calculated
rmm: 3260.8), if an increase in the molecular
mass by 136 Da, the mass of the sulfonic acid modi-
fication, is taken into account. The identity of this
peptide was confirmed by collision induced fragmen-
tation (CID). The complex fragmentation pattern of
the fourfold protonated peptide shows single, double
and triple charged fragment ions, which can be easily
distinguished from the isotopic pattern. After
deconvolution, the peptide was identified by the
almost complete series of y-fragments and a long
series of b-fragments (Fig. 4). From the difference of
the precursor mass to the y-28 fragment and from
the b1 fragment, which both represent the N-terminal
part of the peptide, it is evident that the N-terminal
amino acid is asparagine (position 26), but increased
in mass by 136 Da. Because this modification is only

possible at the free a amino group of asparagine,
this amino acid must represent the N-terminal amino
acid of the protein. Interestingly, this peptide can
acquire up to four positive charges even when there
are only two basic residues present in this sequence.
In the peptide mass fingerprint (Fig. 3, inset) the
unmodified peptide (m ⁄ z ¼ 816.18) is also detected,
but the signal intensity is only about 20% of that
of the modified peptide. Therefore, labeling of P40
within the gel by the CAF reagent occurred in a very
efficient way and allowed the identification of the
new N-terminus after cleavage of the signal sequence
(Fig. 5).
Remarkably, the sulfonated peptide 26–55 also
appeared with high intensity as sodium and a potas-
sium adduct ion, respectively (Fig. 3). These salt
adducts were most presumably bound by the strong
negative charge of the sulfonate group. The same
observation was found only for one additional peptide
Fig. 3. Peptide mass fingerprint of P40. The tryptic digest of P40
was analyzed by ESI-QTOF MS. Multiple charged peptide ions cor-
responding to P40 are indicated by arrows. The inset shows the
m ⁄ z region of the fourfold charged ions of the N-terminal peptide
(position 26–54). 816.2 (+ 4) and 850.2 (+ 4) represent the unmodi-
fied and the sulfonated peptide, respectively. Two additional, four-
fold charged peptides with m ⁄ z ¼ 855.67 (rmm: 3418.7) and with
m ⁄ z ¼ 859.66 (rmm: 3434.6) represent the sodium and potassium
adduct of the sulfonated peptide 26–55 as evident from the frag-
mentation spectra (data not shown).
Fig. 2. Labeling of the N-terminus of a protein by the CAF reagent.

In a first reaction lysine residues are specifically converted to
homoarginine by O-methylisourea. Then, the free a-amino group at
the N-terminus is sulfonated by the CAF reagent.
12 3
kDa
250
150
100
75
50
37
25
15
Fig. 1. Enrichment of P40 for mass spectrometric analysis. The pro-
teins were separated by SDS ⁄ PAGE (12.5%). Lane 1, molecular
mass marker; lane 2, proteins (1.5 lg) enriched by immunoprecipita-
tion; lane 3, total cell extract of M. pneumoniae (7 lg). The gel was
stained with colloidal Coomassie blue. The protein band, which was
cut out for mass spectrometric analysis is indicated by an arrow.
Signal peptidase I activity in M. pneumoniae I. Catrein et al.
2894 FEBS Journal 272 (2005) 2892–2900 ª 2005 FEBS
with m ⁄ z of 436.3, threefold charged. It turned out
that this peptide 71–81 (VGDTKLVALVR) contained
a lysine in the middle of the sequence, also modified
by sulfonation (Fig. 6).
Other amino acids were hardly, if at all, influenced
by this two step labeling procedure. The oxidation of
tryptophan or the conversion of asparagine to aspartic
acid, as observed for peptide 146–152 (ATWVFER)
and peptide 204–226 (VNGVAQDTVHFGSGQESSW

NSQR), respectively, was also found during normal
sample preparation [28].
Discussion
Protein identification is greatly facilitated by the
growing number of genome sequences. To get further
insight into the function of a protein, additional infor-
mation about post-translational modifications, like
Table 1. The tryptic digest of P40 was analyzed by ESI-QTOF MS. Peptide ions of the indicated m ⁄ z-value were fragmented by CID. Amino
acid sequences were calculated from the fragment ions (letters in bold) or taken from the P40 sequence (letters in italics).
m ⁄ z-value Rel. mol. mass Sequence (position) Remarks
850.2 3396.6 N(+136)TYLLQDHNTLTPYTPFTTPXDGGXDVVR (26–54) Sulfonated at N (position 26)
855.7 3418.6 N(+136)TYLLQDHNTLTPYTPFTTPXDGGXDVVR (26–54) Sulfonated at N (position 26), Na + adduct
859.7 3434.6 N(+136)TYLLQDHNTLTPYTPFTTPXDGGXDVVR (26–54) Sulfonated at N (position 26), K + adduct
436.3 1305.9 VGDTK(+136)XVAXVR (70–80) Sulfonated at K (position 74)
653.9 1305.7 VGDTK(+136)XVAXVR (70–80) Sulfonated at K (position 74)
443.6 1327.7 VGDTK(+136)XVAXVR (70–80) Sulfonated at K (position 74), Na + adduct
448.9 1343.6 VGDTK(+136)XVAXVR (70–80) Sulfonated at K (position 74), K + adduct
444.2 1329.7 VGDTKXVAXVR (70–80) Unknown modification (+ 160 Da)
663.8 1325.6 RVGDTKXVAXVR (69–80)
454.7 907.4 ATWVFER (146–152)
462.7 923.4 ATW(ox)VFER (146–152) Oxidized W
470.7 939.4 ATW(2 · ox)VFER (146–152) 2 · oxidized W
799.5 2395.5 TLQDLXVEQPVTPYTPNAGLAR (182–203)
831.1 2490.2 VDGVAQDTVHFGSGQESSWNSQR (204–226) N (position 205) hydrolysed to D
623.3 2489.2 VNGVAQDTVHFGSGQESSWNSQR (204–226)
Fig. 4. Identification of the N-terminus of P40. A fourfold charged peptide (m ⁄ z ¼ 850.18) with a molecular mass of 3396.4 Da was fragmen-
ted by collision-induced fragmentation, because the calculated mass of peptide 26–54 (rmm: 3260.7) fits well if a CAF modification is
assumed (+136 Da). The fragmentation spectrum showed dominant double and triple charged fragment ions. To reduce the complexity, the
spectrum was deconvoluted and the peptide sequence was obtained from the single charged y-fragment ions (…) and the b-fragment ions
(– –). Calculated from the b1 fragment and the y 28 fragment, the N-terminal amino acid consists of asparagine modified by the CAF reagent

(+136 Da). Most of the unlabelled fragments in the spectrum are y- and b-ions after neutral loss of H
2
O from the side chains of aspartic acid
and threonine.
I. Catrein et al. Signal peptidase I activity in M. pneumoniae
FEBS Journal 272 (2005) 2892–2900 ª 2005 FEBS 2895
proteolytic processing steps, are necessary. Edman de-
gradation is a widely used technique for determining
the N-terminus of a protein. This method, however, is
not only limited by its inability to deal with amino ter-
minal modified proteins, but also by its poor sensitivity
compared to mass spectrometry. On the other hand,
mass spectrometry is still not very efficient for analyz-
ing whole proteins despite the progress made during
the last years. Such an analysis normally begins
with the proteolytic degradation of a protein, followed
by the analysis of the cleavage products. It is, how-
ever, difficult to prove which of the identified peptide
represents the amino terminus of the protein. To over-
come this problem, we labeled the amino terminus of
the protein after electrophoretic separation within the
excised gel piece.
As shown, the N-terminal amino group of P40 could
be sulfonated by the CAF reagent. After tryptic diges-
tion, the N-terminal sulfonated peptide 26–54 was
identified by collision induced dissociation of the four-
fold protonated peptide ion. The charge state of this
peptide is unexpected: a peptide containing two basic
amino acids (arginine and histidine) and a blocked
amino terminus should not acquire more than two

positive charges in positive ion mode. On the other
hand, the maximal charge state of a peptide can be
extended with increasing length which may be due to
additional protonation of the peptide backbone in the
gas phase under electrospray conditions (T Ruppert,
unpublished results). Due to the multiple charged
peptide ions formed by electrospray ionization the
fragmentation pattern of the N-terminal sulfonated
peptide differed significantly from the fragmentation
pattern of similar modified peptides using post source
decay (PSD) in MALDI-TOF mass spectrometry,
where b-ions are not observed [31]. The b1 to b5 frag-
ments containing the sulfonated N-terminus of the
fourfold protonated peptide were detected as positively
charged fragment ions. This indicated that the strong
acid group at the N-terminus was not deprotonated as
assumed for MALDI-PSD. Therefore, the N-terminal
labeling is not only visible from y-ion series but also
by the b-ion series. The mass of the b1 fragment cor-
responded to an asparagine, increasing in mass by
136 Da. Detection of the b1 fragment ion indicates
that an amide bond, formed after the reaction the
sulfonation reagent, is present at its N-terminus
instead of a free amino group [32]. Therefore, the
asparagine in position 26 was labeled within the intact
protein at its a-amino group and represents the amino
terminal amino acid of P40. The efficiency of this
reaction was quite high, as judged from the signal
intensities of the corresponding N-terminal modified
peptide, compared to that of the unmodified peptide

(Fig. 3).
In the peptide mass fingerprint, the N-terminal sulfo-
nated peptide 26–54 was detected not only in its pro-
tonated form, but also as a sodium and potassium
adduct ion (Fig. 3). Interestingly, after collision
induced dissociation of these peptide ions, only y-frag-
ments were observed. The sodium and potassium ions,
respectively, were most likely bound at the deproto-
Fig. 5. SIGNAL P predicted and experimentally verified cleavage site
for P40. The cleavage site for the P40 precursor protein was
predicted by
SIGNAL P (Gram-positive network) to be located
between amino acid 25 (alanine) and 26 (asparagine). The values of
the C- (output from cleavage site networks), S- (output from signal
peptide network) and Y-scores (combined cleavage site score) are
shown for each position in the sequence. The data were generated
by feeding the first 50 amino acids of the gene products of
MPN142 to the publicly available web server http://www.
cbs.dtu.dk/services/SignalP/. For more details see [24,25]. The
experimentally defined N-terminus of the mature P40 agreed with
this prediction (black arrow).
Fig. 6. Fragmentation spectrum of the sulfonated peptide 70–80.
The doubly charged peptide ion with m ⁄ z ¼ 653.9 showed intense
sodium and potassium adducts as observed for the sulfonated
N-terminal peptide 26–54. From the fragmentation pattern (y-frag-
ments are indicated by arrow) the peptide was identified as
70-VGDTKLVALVR-80 containing a lysine in the middle of the
sequence, which is probably modified by sulfonation (+136 Da).
Signal peptidase I activity in M. pneumoniae I. Catrein et al.
2896 FEBS Journal 272 (2005) 2892–2900 ª 2005 FEBS

nated sulfonic acid group and lost during fragmenta-
tion, which causes neutralization of the b-fragments.
The formation of sodium and potassium adducts was
possibly a general property of the sulfonic acid group,
which may help to select such peptides in a complex
mixture after electrospray ionization. In fact, we detec-
ted an additional peptide in this tryptic digest, showing
such salt adducts (Fig. 3). This peptide contained the
sulfonic acid group in the middle of the sequence
bound to a lysine residue, which was not converted to
homoarginine due to incomplete guanidation. Other
amino acids were hardly, if at all, affected by these
two reactions. Two amino acid modifications were
detected: tryptophan 248 was oxidized to a minor
extent and asparagine 205 was, to some extent, con-
verted to aspartic acid.
We conclude that sulfonation with the CAF reagent
is a simple and efficient procedure to label the amino
terminus of a protein after gel electrophoresis. After in-
gel digestion, the identification of a peptide containing
this label at the amino terminus by mass spectrometry
proves that it represents the amino terminus of the pro-
tein. Using this method, we showed that asparagine 26
represents the amino terminus of P40. Knowing, that
the first amino acids of the processed P40 and P90 is
the asparagine at position 26 and the arginine at posi-
tion 455 we calculated for P40 a molecular mass of
44.873 kDa. As the molecular mass of P40 in protein
extracts, measured by SDS ⁄ PAGE is about 36 kDa,
there is an obvious difference in molecular mass of

about 9 kDa between the calculated and the actually
observed masses (Fig. 7). It seems reasonable to
assume, that additional proteolytic cleavage took place.
Without further information, we can presently not
decide which were the true intermediates in this pro-
cess. There could be a premature P40 with an extended
C-terminal region, but a premature P90 with an exten-
ded N-terminal region can not be excluded (Fig. 7).
The best way to analyze this precursor-product rela-
tionship would be through puls-chase experiments
with radioactively labeled amino acids. This approach,
however, is hampered by the lack of a defined minimal
medium for M. pneumoniae.
Several predictive methods have been published
[23,24], which facilitate the identification of prokaryotic
signal peptides. A recent study [23] of bacterial and
archaeal proteomes proposed that the fraction of puta-
tive exported or secreted proteins ranges from 8%
(Methanococcus jannaschii) to 37% (M. pneumoniae).
This means that in M. pneumoniae 254 from 688 predic-
ted proteins contain a signal peptide. Establishing and
improving such prediction methods depend on the
availability of experimental data. Although Nielsen
et al. [24] explicitly excluded members of the cell wall-
less mollicutes from their analysis of signal peptides,
applying their program signal p [25,33] precisely pre-
dicted the cleavage site for P40 (Fig. 5). This result was
confirmed by the program exprot [23]. Both methods
failed to confirm the cleavage site of the main adhesine
P1 of M. pneumoniae. This is the only other M. pneu-

moniae protein, of which the N-terminus of the mature
protein was experimentally determined. For this pro-
tein, the predicted cleavage site [23,24] between amino
acids 27 and 28 disagreed with the experimental data,
which showed that the processed P1 protein begins with
amino acid 70 of the precursor protein [25,34,35].
Although a multivariate data analysis indicated that
signal peptides of the mollicutes differ significantly from
E. coli and Gram-positive bacteria [36], a signal peptide
of 70 amino acids is not in agreement with this multi-
variate data analysis. Therefore, the simplest explan-
ation would be that the N-terminus of the mature P1 is
generated by an additional processing step.
The most interesting question concerns the signal
peptidase activity observed in M. pneumoniae [26],
because a conserved bacterial type I signal peptidase
has not been found in annotations of complete genome
sequences, neither in M. pneumoniae [3,16] nor in the
phylogenetically closely related Mycoplasma genitalium
[37]. They were, however, found in Mycoplasma galli-
septicum [38] and Mycoplasma pulmonis [39]. In con-
trast, the type II signal peptidase, that is specific for
signal peptides from lipoproteins of the murein lipo-
protein type of E. coli [40], occurs in all mollicute spe-
cies sequenced so far. As the results of our analysis of
the gene products of MPN142 proved that a SPase I
like activity is present in M. pneumoniae, a correspond-
ing, hitherto unidentified gene, has to code for it. In
the course of the re-annotation of the M. pneumoniae
genome sequence the two genes MPN032 and

MPN294 were proposed as possible candidates enco-
Fig. 7. Schematic model for processing of the gene product of
MPN 142. Cleavage into P40 and P90 takes place after amino acid
454. The N-terminus of the mature P40 starts with amino acid 26.
The molecular mass of about 36 000 Da of P40, as determined by
SDS ⁄ PAGE would correspond to a protein reaching from amino
acid 26–365 (P40
340
). According to this scheme a premature P40
(amino acid 26–454) could be an intermediate as well as a pre-
mature P90 (amino acid 366–1218).
I. Catrein et al. Signal peptidase I activity in M. pneumoniae
FEBS Journal 272 (2005) 2892–2900 ª 2005 FEBS 2897
ding a SPase I like activity [16]. Using recombinant
P40 preprotein or suitable synthetic peptide sub-
strates [26] subfractions of protein extracts from
M. pneumoniae can now be assayed for signal pepti-
dase I activity facilitating the identification of the
corresponding gene ⁄ protein.
Experimental procedures
Bacteria
M. pneumoniae reference strains M129 (ATCC 29342; sub-
type 1; broth passage no. 31) and FH (ATCC 15531; sub-
type 2 [41]; broth passage no. 5) were cultivated as
described previously [42] and stored at )80 °C. For sample
preparation, M. pneumoniae cells were grown adherently at
37 °C in 100 mL of modified Hayflick medium [43] in
150 cm
2
cell culture flasks (Greiner, Flacht, Germany).

Enrichment of IgG
The IgG fraction of 2 mL rabbit anti-FP130K-1 serum (no.
42328 [17]), was precipitated with Na
2
SO
4
by mixing the
serum with 2 mL phosphate buffer (0.1 m KH
2
PO
4
, 0.1 m
Na
2
HPO
4
, pH 7.4) and adding 4 mL of a Na
2
SO
4
solution
(34%, w ⁄ v). The suspension was incubated for 5 min at
20 °C and then centrifuged at 20 °C for 5 min at 12 000 g.
The supernatant was discarded, the pellet dissolved in 2 mL
phosphate buffer and the Na
2
SO
4
precipitation repeated
twice. The final pellet was dissolved in 2 mL phosphate buf-

fer. The concentration of this suspension was determined
by reading the absorbance at 280 nm and using an extinc-
tion coefficient of 1.4 for a 1 mgÆmL
)1
solution of IgG. We
obtained from 2 mL of serum about 10 mg protein, mainly
IgG as revealed by SDS ⁄ PAGE. The IgG-enriched fraction
retained the ability to recognize P40 in western blotting
experiments (I. Catrein, unpublished results). SDS ⁄ PAGE
and western blotting were performed as published recently
[44].
Cross-linking of IgG to magnetic beads
The enriched IgG fraction (10 mg) was bound to 2 mL sus-
pension of magnetic beads, which carried recombinant Pro-
tein A covalently attached (DynabeadsÒ Protein A, Dynal
Biotech, Oslo, Norway) following the instructions of the
manufacturer.
Isolation of P40 from cell extracts
M. pneumoniae M129 from 10 cell culture flasks (150 cm
2
,
100 mL modified Hayflick medium) were collected, washed
twice with phosphate buffer and the pellet suspended in
2 mL lysis buffer (500 mm NaCl, 50 mm Tris ⁄ HCl pH 7.5,
0.5% Triton X-100 and the protease inhibitor cocktail com-
plete, EDTA-free (according to the manufactures recom-
mendation; Roche, Basel, Switzerland). The bacteria were
sonicated with a Branson sonifier for 8 · 15 s at 4 °C with
intervals of 1 min and the suspension separated in pellet
and supernatant by centrifugation (60 min, 75 000 g)ina

Beckman TL100 ultracentrifuge. The protein concentration
of the 2 mL supernatant was about 25 mgÆmL
)1
as meas-
ured with the Quick Start Bradford Protein Assay (Bio-
Rad, Hercules, CA, USA) using bovine serum albumin as
standard. It contained almost all of the P40 protein as
revealed by western blotting. For the isolation of P40, the
2 mL supernatant were incubated with the prepared mag-
netic beads (see previous paragraph) overnight at 4 °C with
gentle agitation. After magnetic separation, the beads were
washed 6 times with 6 mL of 0.1 m sodium phosphate buf-
fer, pH 8,1. P40 was eluted by adding 540 lL 0.1 m citrate
buffer, pH 2,8 to the beads and incubating the suspension
for 2 min at 20 °C. The beads were again removed by the
magnet and the supernatant containing now the P40 was
neutralized by adding 1 mL of 0.1 m sodium phosphate
buffer, pH 8,1. This protein solution was concentrated with
a spin column with a 10 kDa cut-off (Vivaspin concen-
trator, Vivascience, Hannover, Germany) to a volume of
24 lL. It contained enough P40 for separating the P40
protein by SDS ⁄ PAGE and characterizing it by mass
spectrometry.
Protein determination and mass spectrometry
The Coomassie blue stained band containing P40 was
excised from the gel. After washing, the gel piece was trea-
ted with 30 lL10mm DTT at 60 °C for 10 min and alkyl-
ated with 30 lL40mm iodacetamide at room temperature
for 15 min. The gel piece was again washed with 25 mm
ammonium bicarbonate, shrunk in acetonitrile and incuba-

ted with water for 15 min. After removal of excess water,
100 lLofO-methylisourea hemisulfate (140 mm in 200 mm
sodium bicarbonate, pH 10) was added and incubated over
night at room temperature. The supernatant was removed,
the gel piece washed twice with water and shrunk in aceto-
nitrile on ice. After removal of acetonitrile, 6 mg CAF rea-
gent (chemical assisted fragmentation reagent; kindly
provided by Amersham Biosciences, Freiburg, Germany)
(Fig. 1) dissolved in 60 lL 250 mm sodium bicarbonate,
pH 9.4, was added. After 10 min on ice the Eppendorf tube
was quickly brought to room temperature for additional
5 min. Then, the reaction was stopped by addition of 2 lL
50% hydroxylamine solution. After 1 h the supernatant
was removed and the gel piece washed twice with water,
25 mm ammonium bicarbonate, pH 8.5. After shrinkage in
acetonitrile, the gel piece was incubated with 15 lL25mm
ammonium bicarbonate, pH 8.5 containing 140 ng modified
porcine trypsin (8 ngÆlL
)1
) (Promega, Madison, WI, USA)
for 4 h at 37 °C. Digestion was stopped by formic acid
Signal peptidase I activity in M. pneumoniae I. Catrein et al.
2898 FEBS Journal 272 (2005) 2892–2900 ª 2005 FEBS
(final concentration: 2%) and the sample was stored at
)20 °C. MS analysis was performed on a Q-TOF mass
spectrometer (Applied Biosystems, Darmstadt, Germany)
equipped with a nano-ESI ion source (Protana, Odense,
Denmark) as described previously [27]. If indicated,
MS ⁄ MS spectra were deconvoluted using Bayesian Peptide
Reconstruct (mass tolerance: 0.1 Da; S ⁄ N threshold: 2) pro-

vided with the analyst qs software (Applied Biosystems).
Acknowledgements
We thank E. Pirkl and M. Ellis for skillful technical
assistance, C U. Zimmerman for critically reading the
manuscript, M. T. Saleh for providing the latest results
of a signal peptide analysis of M. pneumoniae and the
Deutsche Forschungsgemeinschaft for financial sup-
port (He 780 ⁄ 12–2 and SFB 382).
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Supplementary material
The following material is available from http://www.
blackwellpublishing.com/products/journals/suppmat/ EJB/
EJB4710/EJB4710sm.htm
Fig. S1. (A) Isolation of P40 from cell extracts, (B)

isolation of P40 from cell extracts as revealed by wes-
tern blot and (C) identification of the N-terminus of
P40.
Table S1. Peak list of the fragmentation spectrum of
the fourfold charged peptide with m/z ¼ 850.18.
Signal peptidase I activity in M. pneumoniae I. Catrein et al.
2900 FEBS Journal 272 (2005) 2892–2900 ª 2005 FEBS