Tài liệu Báo cáo khoa học: Direct identification of hydrophobins and their processing in Trichoderma using intact-cell MALDI-TOF MS docx

Intact-cell MALDI-TOF mass spectra of mycelia and spores of Trichoderma strains.. This characteristic feature of hydrophobins could therefore be used as a diagnostic tool to identify hyd

in Trichoderma using intact-cell MALDI-TOF MS

Torsten Neuhof1, Ralf Dieckmann1,*, Irina S Druzhinina2, Christian P Kubicek2,

Tiina Nakari-Seta¨la¨3, Merja Penttila¨3 and Hans von Do¨hren1

1 TU Berlin, Institut fu¨r Chemie, FG Biochemie und Molekulare Biologie, Berlin, Germany

2 FB Gentechnik und Angewandte Biochemie, Institut fu¨r Verfahrenstechnik, Umwelttechnik und Technische Biowissenschaften, TU Wien, Vienna, Austria

3 VTT Technical Research Centre of Finland, Espoo, Finland

Hydrophobins are small proteins thought to be

ubi-quitous in filamentous fungi They are usually present

on the outer surfaces of cell walls of hyphae and

coni-dia Here, they mediate interactions between the

fun-gus and the environment, such as surface recognition

during pathogenic interactions with plants, insects or

other fungi, and also in symbiosis The size of

hydro-phobins ranges from approximately 75 to 400 amino

acid residues; they contain eight positionally conserved

cysteine residues, and can be divided into two classes

according to their hydropathy profiles and spacing

between the conserved cysteines [1]

The anamorphic fungal genus Trichoderma (Hypocre-ales, Ascomycota) contains cosmopolitan soil-borne fungi with economic importance as biocontrol agents and producers of beneficial metabolites and enzymes In addition, Trichoderma spp have recently been reported

to occur as endophytes, eliciting positive plant responses against potential pathogens [2] Hydrophobins are likely

to play a role in this process, and a hydrophobin gene has in fact recently been isolated that leads to overpro-duction of hydrophobins during endophytic interactions between Trichoderma asperellum and cucumber roots [3] However, hydrophobins may also be involved in the

Keywords

fungal biomarker; hydrophobin; intact-cell

MS; MALDI-TOF MS; Trichoderma

Correspondence

H von Do¨hren, TU Berlin, Institut fu¨r

Chemie, FG Biochemie und Molekulare

Biologie, Franklinstr 29, 10587 Berlin,

Germany

Fax: +49 30 314 24783

Tel: +49 30 314 22697

E-mail: Doehren@chem.tu-berlin.de

*Present address

AnagnosTec, Gesellschaft fu¨r Analytische

Biochemie und Diagnostik mbH,

Potsdam-Golm, Germany

(Received 19 September 2006, revised 27

November 2006, accepted 6 December

2006)

doi:10.1111/j.1742-4658.2007.05636.x

Intact-cell MS (ICMS) was applied for the direct detection of hydropho-bins in various species and strains of Hypocrea⁄ Trichoderma In both myce-lia and spores, dominating peaks were identified as hydrophobins by detecting mass shifts of 8 Da of reduced and unreduced forms, the analysis

of knockout mutants, and comparison with protein databases

Strain-speci-fic processing was observed in the case of Hypocrea jecorina (anamorph Trichoderma reesei) An analysis of 32 strains comprising 29 different spe-cies of Trichoderma and Hypocrea showed hydrophobin patterns that were specific at both at the species and isolate (subspecies) levels The method therefore permits rapid and direct detection of hydrophobin class II com-positions and may also provide a means to identify Trichoderma (and other fungal) species and strains from microgram amounts of biomass without prior cultivation

Abbreviations

HFB, hydrophobin; ICMS, intact-cell MALDI-TOF MS.

mechanism of mycoparasitism as well as the

coloniza-tion of decaying wood

Our information about the roles of hydrophobins

in the physiology of Trichoderma, as well as in other

fungi, is mostly derived from reversed genetics Little

is known about the occurrence and processing of the

individual hydrophobins on the fungal surface As a

method for the rapid detection of hydrophobins

from a large number of small samples, we have

investigated the potential of intact-cell MALDI-TOF

MS (ICMS) Initial applications of ICMS on

fila-mentous fungi have demonstrated characteristic sets

of ions for strain identification in mycelia [4–8] and

spores [9–12] We will show here that class II

hydro-phobins account for the main characteristic peaks of

Trichoderma, as the intact-cell extraction procedure

employing a solvent mixture of acetonitrile and

methanol is suitable for dissolving these cell wall

constituents

Results

Identification of new Trichoderma hydrophobins

by EST search

In order to determine which hydrophobins could

potentially be detected in Hypocrea jecorina, we first

mined its genome database for members of class II

hydrophobins Besides the well-known HFB1 and

HFB2, the HFB3 hydrophobin has been identified by

cloning the corresponding gene [13] and further

char-acterization of the protein [14] We here identified

HFB4, HFB5, and HFB6 In addition, two

hydropho-bin-encloding EST sequences were retrieved from the TrichEST database (http://www.trichoderma.org): one encoding an ortholog of HFB3 from T longibra-chiatum (L22T11P141R12690, L14T53P137R01628, L22T11P138R12431, and L22T11P137R12300), and the other one encoding an ortholog of HFB1 of

T atroviride (L12T11P119R10608) Their sequence relationships and putative processing sites are illustra-ted in the alignment given in Fig 1

ICMS analysis of Trichoderma Several strains of Trichoderma were studied initially to examine the effectiveness of ICMS as an analytical method for distinguishing different species of

Trichoder-ma A rapid analytical procedure based on ICMS was established in order to characterize the low-molecular-weight proteometric (up to 20 000 Da) and peptidomet-ric (up to 2000 Da) profiles at the same time Thirty-two Trichoderma strains belonging to various species were subcultivated on agar plates at an incubation tem-perature of 25C and analyzed without further pre-treatments as described in Experimental procedures Vegetative mycelia or spores were transferred from the biomass growing on agar plates directly to the MALDI sample plate and mixed with an acidic matrix in an organic solvent mixture An estimated 106cells were used per spot Data obtained from triplicate samples grown for different times confirmed that the MS analy-ses were reproducible with respect to the characteristic biomarkers obtained Figure 2 shows typical MALDI-TOF mass spectra of four strains of H koningii, T long-ibrachiatum, H virens and T atroviride The spectra

Fig 1 Alignment of the six hydrophobin protein sequences of H jecorina Protein accession numbers and first amino acid of the protein after predicted signal peptide cleavage are: HFB1 (P52754; Q17), HFB2 (S62621; A16), HFB3 (trire.GWV1.31.87.1, scaffold 31: 136 622–

136 948; A17), HFB4 (estExt_fgenesh1_pg.C_50116, scaffold 5: 390 006–390 493; D25), HFB5 (trire.GWV1.11.179.1, scaffold 11: 162 998–

163 354; A17), HFB6 (trire.GWV1.3.266.1, scaffold 3: 1 189 586–1 190 177; no predicted signal peptide).

showed a characteristic set of mass peaks in the range

of 5–10 kDa, typically including two dominating peaks

at approximately m⁄ z 7000 As mycelia and spores

largely remained intact, and the extraction solution

contained acetonitrile and methanol, the well-known

hydrophobins were suspected to be the signal source,

and some were assigned by database analysis

Identification of the class II hydrophobins

produced by H jecorina

In order to identify the already known hydrophobin

peaks and to validate the method described here, we

first performed a detailed analysis of H jecorina¼

T reesei QM 9414 To date, two major hydrophobin

(HFB) proteins have been characterized in detail: the

97-residue HFB1-precursor with a molecular mass of

9874.32 Da is processed to a 75 amino acid peptide with a molecular mass of 7540.58 Da, which is further reduced by disulfide bond formation to 7532.58 Da [15] The 86 residue HFB2 precursor with a mass of 8766.28 Da is processed to a 71 amino acid peptide with a calculated molecular mass of 7196.42 kDa, and further reduced by disulfide formation to 7188.42 Da [15] Both hydrophobins were detected as [MH]+ sig-nals of the oxidized forms (Hfb1, m⁄ z 7533; Hfb2,

m⁄ z 7189) A minor peak of m ⁄ z 7041 presumably cor-responds to the processed Hfb2 lacking the terminal Phe (7041.24 Da) (Fig 3)

The same peaks were observed in the spectra obtained from isolated reference substances of HFB1 and HFB2 proteins (Fig 3C) A second minor peak of

m⁄ z 7229 correlates with oxidized HFB2 cleaved at Ala13 lacking the N-terminal Phe This tentative

corre-Fig 2 Intact-cell MALDI-TOF mass spectra

of mycelia and spores of Trichoderma

strains The masses 7347 and 7494 of

T atroviride spores correlate with two

proc-essed products of the spore hydrophobin

SRH1 [16] cleaved at the N-terminal

MQFSI-VALFATGALA site and the C-terminal Phe,

respectively.

Fig 3 Intact-cell MALDI-TOF mass spectra

of H jecorina strain QM 9414 (D), and the

mutant strains QM 9414 Dhfb1 (B) and

QM 9414 Dhfb1Dhfb2 (A) HFB I and HFB II

indicate the processed hydrophobins.

Whereas the HFB I peak is missing in the

disruption mutant, the proportions of the

processed HFB2 hydrophobins have been

shifted The double knockout (A) has no

sig-nificant mass peaks in this region As a

con-trol spectrum, the purified hydrophobins are

shown in (C).

lation of mass data is achieved by the calculation of

successive deletions of terminal amino acid residues of

all six hydrophobins providing this single match

A rapid means of identifying peaks corresponding to

hydrophobins in MALDI-TOF mass spectra was

elab-orated, making use of the fact that hydrophobins

con-tain eight cysteines forming four disulfide bonds

Reduction of the four disulfides with dithiothreitol

increased the masses of all peaks corresponding to

hydrophobins by 8 Da Thus, the masses of processed

HFB1 and HFB2 show shifts from m⁄ z 7533 to 7541 and from m⁄ z 7189 to 7197, respectively (supplementary Fig S1) This characteristic feature of hydrophobins could therefore be used as a diagnostic tool to identify hydrophobin peaks in intact-cell MALDI-TOF spectra

In order to prove that the peaks described above originate from HFB1 and HFB2, hfb1D and hfb1⁄ hfb2DD strains were also analyzed to confirm the peak assignments: The double mutant hfb1–⁄ hfb2– did not show HFB1 and HFB2 signals (Fig 3A), whereas

Fig 4 Intact-cell MALDI-TOF spectra of mycelia (A, C, E) and sporulating mycelia (B, D, F) of three strains of H jecorina grown on malt agar The masses displayed have an error of about 0.1%, so peaks of 7232 (A), 7237 (E) and 7234 (F) represent similar peptides Strain 618 mycelia (A) show a variety of peaks, in contrast to strains 665 and 937, shown in (C) and (E) However, there are only few similarities: 7232 and 7237 in (A) and (E), or 7509 and 7514 in (A) and (C) An obvious shift is the appearance of higher mass peaks upon sporulation, presum-ably related to the only large hydrophobin of H jecorina.

in the knockout mutant hfbI–, only the respective mass

peak was missing (Fig 3B)

Deviating post-translational processing

of hydrophobins in H jecorina strains

To investigate strain diversity with respect to

meta-bolite production and low-mass proteomics by ICMS,

three phylogenetically described isolates of H jecorina

were studied As shown in Fig 4, spectra of mycelia

and sporulating mycelia directly taken from the

plates after 1 or 3 days, respectively, differ in peak

compositions and intensities Surprisingly, all spectra differ with respect to strain QM 9414 Strain CPK 618 mycelia show a prominent signal of

m⁄ z 7232 (Fig 4A), which disappears in the sporula-tion process, with new signals of m⁄ z 8859, 8802 and

7521 appearing (Fig 4B) To obtain a preliminary correlation of observed masses with hydrophobin data, we again calculated from the available sequence data sets of masses for each hydrophobin, succes-sively subtracting terminal residues and introducing disulfide bonds The m⁄ z 7232 peptide could thus be tentatively assigned to the hydrophobin HFB3 in the

Fig 4 (Continued).

oxidized form, with cleavage of the N-terminal

pep-tide at Ala31 and the C-terminal dipeptide at

Pro102 The same mass is also observed in strain

CPK 937 (Fig 4E,F) as a prominent signal in

myce-lia that almost disappears in the sporulation process

Peaks of m⁄ z 8858 and 8863 were also observed in

sporulating strains CPK 618 and CPK 665,

respect-ively (Fig 4B,D) These correspond in mass to HFB5

with an N-terminal cleavage at Ala8 including

oxida-tion (8861 Da)

Comparison of the mycelial compositions of strains

CPK 665 (Fig 4C) and CPK 937 (Fig 4E) shows four

major peaks, all differing in mass, but none of them

corresponds to the QM 9414 strain (Fig 3) The main

peak of CPK 665, m⁄ z 7147, correlates with HFB1

with four disulfide bonds, cleaved N-terminally at

Arg22 and at the C-terminal tetrapeptide In

QM 9414, HFB1I is not processed C-terminally The

m⁄ z 6999 peak, the second prominent peak of strain

CPK 665, can be assigned to Hfb2 C-terminally

cleaved at Lys66 and oxidized This peak is not present

in any other strain investigated

Although they are rather speculative, the interpreted

masses agree with verified cleavage sites observed for

HFB1 and HFB2 and known sites for signal

peptidas-es and Kex2-type peptidaspeptidas-es (Table 1) Verification of

these assessments by tryptic digestion and sequencing

is in progress

Hydrophobin patterns in other T atroviride and

T longibrachiatum strains

T atroviride

A hydrophobin gene (srh1) encoding a class II

hydro-phobin with phylogenetic similarity to H jecorina

HFB2 (I S Druzhinina and C P Kubicek,

unpub-lished results) has been found in T atroviride (therein

named ‘T harzianum’ [16]) The main components of

the sporulating mycelia of the same strain (T

atrovi-rideP1) could indeed be assigned to this hydrophobin,

assuming similar post-translational processing as for

the H jecorina HFB2 (Fig 2, top spectrum) The

peaks at m⁄ z 7499 and 7352 correspond to the

proc-essed spore hydrophobin SRH1 with the cleaved signal

sequence MQFSIVALFATGALA and an additional

C-terminal Phe cleavage, respectively, including loss of

8 Da for the disulfide bonds A minor peak at

m⁄ z 7741 could be tentatively correlated with the

SRH1 hydrophobin with N-terminal cleavage of

MQFSIVA, C-terminal processing following the two

Glu residues of AAAQGTF, and four disulfide bonds

Interestingly, these peaks could not be detected in

vegetative mycelia of T atroviride P1 (Fig 2), which

displayed a similar peak pattern, but with slightly dif-ferent masses of 7181, 7339 and 7739 Da A database search in TrichoEST for the presence of other T atro-viride hydrophobins led to the identification of an HFB1-like protein, which, after N-terminal processing (MKFFTAAALFAAVAIA), C-terminal processing (AVGA) and disulfide bond formation, has a mass of

7743 Da

T longibrachiatum The main mass peak of T longibrachiatum of m⁄ z 7242 was assigned to an HFB3-type of hydrophobin identified

by searching the TrichoEST database The 10 235 Da precursor peptide would have been cleaved at the unique Arg site MQFLAVAALLFTAAFAAPSSEAHGLRRR, comprising 3 Arg residues in sequence (underlined), and this would have been followed by the formation of four disulfide bonds, leading to a mass of 7241 Da

ICMS of class II hydrophobins can distinguish Trichoderma strains at the subspecies level The results described above for H jecorina show that even under carefully controlled culture conditions, different strains of this taxon displayed different MS fingerprints Because of the ease and speed of the analysis, we investigated whether the hydrophobin class II pattern of Trichoderma could be used in strain diagnosis at the species level To this end, we selected isolates for 29 different species of Hypo-crea⁄ Trichoderma and compared their hydrophobin molecular masses Table 2 shows that, indeed, all of the species tested exhibited a unique combination of peaks with unique molecular masses It is therefore interesting to note that even phylogenetically closely related species (such as T hamatum and T asperel-lum, or T harzianum and T fulvum [17], or T fas-ciculatum and T strictipile, which were recently revised to be actually the same species [18]), could be clearly separated This is in accordance with the data

on H jecorina shown above, and implies that hydro-phobin fingerprints can in fact distinguish isolates at the subspecies level All spectra are compiled in sup-plementary Fig S2

Discussion

Hydrophobin patterns Genome sequencing of filamentous fungi has revealed the presence of multiple hydrophobin genes in filamen-tous fungi We here report the the sequences of four

new type II hydrophobins for H jecorina, in addition

to the known HFB1 and HFB2 Likewise, we

identi-fied new hydrophobins in T atroviride in addition to

the known sporulation-specific one, and in T

longibra-chiatum Direct MS analysis of mycelia in differing

physiologic states provides evidence for differential

expression of these genes in relation to the

morpho-logic state However, there is no clear match of the

observed mass peaks to the predicted propeptides

expected to originate from cleavage of signal peptides

Instead, further processing has been observed, as has been demonstrated before from N-terminal sequence analysis of H jecorina HFB1 and HFB2 Hydrophobin patterns even suggest strain-specific multiple cleavages

of propepides

Hydrophobin processing The export of most type II hydrophobins involves both signal peptide cleavage and maturation of the

propep-Table 1 Confirmed and predicted hydrophobin cleavage sites.

H jecorina

QM 9414

protease processing, shown by N-terminal sequencing

shown by N-terminal sequencing

predicted C-terminal cleavage

predicted C-terminal cleavage

H jecorina

strains 618 and

937

peptidase and predicted C-terminal cleavage by unknown protease

within signal peptide

H jecorina

strain 665

predicted non-Kex2 sites

peptide site, predicted non-Kex2 site

peptide cleavage

peptide cleavage, predicted C-terminal cleavage

signal peptide cleavage, predicted C-terminal cleavage

peptide cleavage, predicted C-terminal cleavage

cleavage

tides A considerable amount of information is

avail-able on eukaryotic signal peptidase specificities, and

cleavage site predictions can be performed using,

for example, signalp (http://www.cbs.dtu.dk/services/

SignalP/) version 3.0 [19]) Owing to the limited

amount of information available on eukaryotic

pro-teins, an evaluation of version 2 reported a 78%

accu-racy [20] In this study, a selection of verified and

predicted sites for hydrophobins is reported (Table 2)

On the basis of confirmed structural data of H jecorina

HFB1, HFB2 and HFB3, we here predict the presence

and cleavage patterns of additional hydrophobins

These data are based on calculated masses, which need

to be confirmed by other experiments, such as isolation

and sequencing work, or indirect studies employing proteinase knockouts or proteinase inhibition This may indeed be speculative, but provides useful hypo-thetical data based on: (a) selective extraction leading

to a limited set of no more than six small proteins; (b) known cleavage sites of signal peptidases and fungal Kex2-like proteinases; and (c) actual structural studies

of H jecorina HFB1 and HFB2

In H jecorina, maturation of HFB1, HFB2 and HFB3 includes signal protease processing that removes peptides 15 and 16 amino acids in length This is fol-lowed by propeptide cleavage by a non-Kex2 protein-ase in the cprotein-ase of HFB1, cleaving at a monobasic site, and by Kex2 cleavage in the case of HFB3 [21],

Table 2 Biomarker masses of various Hypocrea ⁄ Trichoderma strains detected by ICMS CBS, Centraalbureau voor Schimmelcultures, the Netherlands; DAOM, Eastern Cereal and Oilseed Research Centre, Canada; ATTC, American Type Strain Culture Collection, USA The iden-tity of all strains was verified by sequencing internal transcribed spacer (ITS)1 and ITS2 and (if necessary) the long intron of tef1, and analyz-ing the sequences by TRICHOKEY [28] and TRICHOBLAST [29].

T stromaticum CBS 101875 7093

a T fasciculatum is a synonym of H strictipilis b T flavofuscum is a synonym of H virens c T croceum is a synonym of T polysporum (¼H pachybasioides) d Identified as HFB3 type with processing at the Arg site: MQFLAVAALLFTAAFAAPSSEAHGLRRR e Identified as processed HFB2 (see Fig 2).fIdentified as processed HFB1 (see Fig 2).gIdentified as processed HFB1-type hydrophobin: GPVEVRTGGG-SICPDGLFSNPQCCDTQLLGIIGLGCEVPSQTPRDGADFKNICAKTGDQALCCVLPIAGQDLLCQA h Identified as processed SRH1 (see Fig 1).

i Identified as processed SRH1: LASVSVCPNGLYSNPQCCGANVLGVAALDCHTPRVDVLTGPIFQAVCAAEGGKQPLCCVVPVAGQDLLCEE.

whereas HFB2 is not cleaved further in the N-teminal

region Mass spectra provide evidence for a C-terminal

Phe cleavage for HFB2

The ICMS spectra presented here provide evidence

for similar cleavage patterns of hydrophobins HFBIII

of H jecorina and T longibrachiatum, and SRH1 and

HFB1 of T atroviride, involving signal peptides,

Kex2-type processing, and C-terminal amino acid cleavage

(Table 2) In addition, the recorded masses provide

evidence for alternative processing reactions One

reac-tion concerns alternative signal peptide cleavage sites

at Ala13 for H jecorina HFB2 and at Ala7 for T

atr-ovirideSRH1 The predicted sites are compatible with

the specificity profiles of signal peptidases, whereas

their length is less than the average 15–40 residues

The second type of reaction is the cleavage of

C-ter-minal peptides at Ala sites, e.g at sites + 2 from the

last conserved Cys, at Lys-Ala positions, at Glu-Ala

positions, or at Thr-Ala positions These predictions

need confirmation by further analysis

Comparing ICMS of bacteria and fungi

ICMS provides a rapid means for distinguishing

bac-teria, spores, viruses and fungi [4–12] It has the

advantages of being very rapid, using small samples

(subcolony amounts) and requiring minimal sample

preparation Bacterial intact-cell MALDI-TOF spectra

in the range 2–20 kDa are dominated by a set of

ri-bosomal proteins as highly abundant intracellular

constituents [22] This set of about 10–30 defined

masses permits the identification at the species and

subspecies⁄ strain level We here show that in

filamen-tous fungi, hydrophobins are the dominating protein

masses, whereas ribosomal proteins have not been

identified This result can be ascribed to the unique

solubility properties of these hydrophobic proteins

Although there are several hydrophobin genes

pre-sent, only a some of these might be expressed under

differing physiologic conditions Thus, it has been

reported that the expression of hydrophobins is

dependent on the morphologic state of T atroviride

(erroneously described as T harzianum [16]) It has

also been shown that expression and localization of

specific hydrophobins in Cladosporium fulvum is

dependent on the stages of the plant infection

pro-cess, the hydrophobins being either retained on

coni-dia and aerial structures or being excreted [23] In

Magnaporthe grisea, it has been demonstrated that

the formation of disulfide linkages is required for

secretion and cell wall localization [24]

Indeed, we have shown here that sporulating and

nonsporulating mycelia of several species differ in

hydrophobin composition Unexpectedly, the patterns observed indicate diverse cleavage reactions of the respective prepropeptides These patterns are unlikely

to be proteolytic artefacts of extraction, as proteases are unlikely to be active in methanol⁄ acetonitrile mix-tures As MALDI-TOF MS involves an especially gen-tle ionization process, cleavages of peptide bonds are generally not observed Differences in hydrophobin processing are thus interpreted as being dependent on the presence and concentrations of specific proteinases acting on the respective propeptides

Hydrophobins as biomarkers for ICMS

of filamentous fungi Hydrophobins are proposed to be suitable ICMS bio-markers for the following reasons: (a) fungi contain a set of hydrophobin genes, generally with developmen-tally regulated expression; (b) the hydrophobic pep-tides can be selectively dissolved and rapidly analyzed

by MALDI-TOF MS; (c) hydrophobin patterns are diverse, due to post-translational processing; (d) the presence of the characteristic four disulfide bonds can

be easily demonstrated by reduction; (e) owing to their fairly small size, sequence information for the hydro-phobins can be obtained by MS; (f) the respective genes are accessible by standard PCR methods; and (g) the variability of hydrophobins is fairly significant, and exceeds the similarities of other biomarkers pro-posed, e.g ubiquitins The sensitivity of detection employing the MALDI-TOF technique can be expec-ted to allow the identification of bacterial peptides with 100 microbial cells

Experimental procedures

Reagents and standards

2,5-Dihydroxybenzoic acid from Anagnostec (Potsdam, Germany) was used as the matrix for MALDI-TOF experi-ments Trifluoroacetic acid, ethanol, acetonitrile and meth-anol from Merck (Darmstadt, Germany) were used as solvents The reference hydrophobins HFBI and HFBII were purified by two-phase separation and RP-HPLC puri-fication as described previously [25]

Microbial strains and cultivations

The strains of H jecorina used were: the wild-type strains

QM 6a, TUB F-1038 (CPK 618), TUB F-733 (CPK 665) and CBS 498.97 (CPK 937) (all described in [26]); the cellu-lase moderately overproducing mutant strain QM 9414 (ATTC 26921) [27]; and QM 9414 Dhfb1 (VTT D-99724

[28]) and QM 9414 Dhfb1Dhfb2 (VTT D-99725 [25]) Other

Trichoderma spp strains used in this study are listed in

Table 1

Hypocrea jecorinaQM 9414 and its mutants were

cultiva-ted in liquid cultures on microtiter plates (200 lL volume)

for 4 days in buffered minimal medium [15] complemented

with 3% glucose and 0.2% peptone The other strains of

H jecorina, as well as strains of other Trichoderma spp.,

were cultivated on malt extract agar (3%) at 25C

Extraction and preparation of mycelia

for MALDI-TOF analysis

A few micrograms of fungal mycelia were suspended in

acetonitrile⁄ methanol ⁄ water (1 : 1 : 1), and 1 lL of the

sus-pension was directly spotted onto target wells of a

100-posi-tion sample plate and immediately mixed with 1 lL of

matrix solution [10 mgÆmL)1 2,5-dihydroxybenzoic acid in

acetonitrile⁄ methanol ⁄ water (1 : 1 : 1) and 0.3%

trifluoro-acetic acid] The sample matrix mixture was allowed to air

dry prior to analysis Alternatively, freeze-dried mycelium

obtained from shaken cultures or fungi grown on plates

was homogenized in 60% ethanol and centrifuged at

13 000 g using a Beckman Microfuge 11 (Beckman Coulter,

Unterscheissheim, Germany) One microliter of the protein

solution was spotted on a MALDI target plate and mixed

with matrix

Reduction of disulfide bonds

For reduction of proteins containing disulfide bonds, cells

were suspended in 60% methanol, vortexed, and

centri-fuged at 13 000 g using a Beckman Microfuge 11, and the

supernatant was concentrated to dryness The residual was

redissolved in 50 mm Tris⁄ HCl (pH 8) and 1 mm

dithio-threitol and incubated for 1 h at room temperature

Microbial characterization by MALDI-TOF MS

analysis

MS measurements were performed on a VOYAGER

DE-PRO TOF mass spectrometer from Applied Biosystems

(Foster City, CA, USA) Mass spectra were acquired in

lin-ear delayed extraction mode using an acceleration voltage

of 20 kV and a low mass gate of 1500 Da For desorption

of the components, a nitrogen laser beam (k¼ 337 nm)

was focused on the template The laser power was set to

just above the threshold of ionization Spectra for

individ-ual specimens were compiled, and results were averaged

from at least 100 shots taken across the width of the

speci-men for m⁄ z values of 2000–20 000 In the linear mode, the

1000 p.p.m (0.1%) Calibration was done with the H

jeco-rina HFB1 protein with the 7533 Da calculated average

atomic mass All masses determined correspond to average atomic masses

MS analysis of low-molecular-mass peptides

Measurements were performed in the delayed extraction mode, allowing the determination of monoisotopic mass values A low mass gate of 800 Da improved the measure-ment by filtering out the most intensive matrix ions The mass spectrometer was used in the positive ion detection and reflector mode

Database search and alignments ) H jecorina hydrophobins

The H jecorina genome (http://gsphere.lanl.gov/trire1/ trire1.home.html) was screened for hydrophobin-encoding genes by using the tblastn (protein versus translated nuc-leotide) program We used the hydrophobin class II protein sequences of other fungal species as queries to search the

H jecorinagenome Then, all putative hydrophobins, inclu-ding the newly identified hydrophobin from H jecorina, were used to identify further proteins with similar domains, and finally all hypothetical proteins encoding hydrophobins from the annotated genomes of the Broad Institute (http:// www.broad.mit.edu/), Neurospora crassa, Gibberella zeae (Fusarium graminearum) and Magnaporthe griseae, were also used

Calculations of monoisotopic molecular masses of hydro-phobins was performed with the expasy proteomics server (http://www.expasy.org/) or the peptide mass calculator (http://rna.rega.kuleuven.ac.be/masspec/pepcalc.htm) To correlate observed mass peak data, masses of various N- and C-terminally processed and oxidized peptides were calculated and compared

Acknowledgements

This work was supported by a fellowship from the Deut-sche Forschungsgemeinschaft (Do270⁄ 10) and by the Fifth Framework program (Quality of Life and Man-agement of Living Resources; Project EUROFUNG 2; QLK3-1999-00729) of the European Community The

T reesei genome sequencing project was funded by the Department of Energy The authors thank M

Salohei-mo for helpful comments and discussions

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