(Received for publication, November 25, 1996, and in revised form, January 14, 1997)
From the A cell wall component of a smooth variant of
Gordona hydrophobica 1775/15 was isolated and purified, and
its structure was determined by various chemical methods, including
chemical synthesis of part structures, Edman degradation, gas
chromatography/mass spectrometry analysis, matrix-assisted laser
desorption ionization-post-source decay (MALDI-PSD) tandem mass
spectrometry, and 1H and 13C NMR using
one- and two-dimensional, homo- and heteronuclear correlated
spectroscopy. The cell wall component was found to be a
(mono-) glycosylated peptidolipid (GPL) consisting of a
tridecapeptide interlinked by a In contrast, rough variants of G. hydrophobica 1775/15 lack
these peptidolipids or synthesize them to a much lesser extent indicating that gordonin contributes significantly to the
physicochemical character of the cell surface.
Biofiltration is the method of choice for the treatment of waste
gases with high volumes and low concentrations of organic pollutants.
This technology requires low investment and operation costs and
guarantees high reliability. Although biofiltration is already widely
used, knowledge about the biology underlying this technology is
lacking. Only a few studies have so far been conducted on the
microorganisms involved in the process of exhaust-air treatment.
Recently, Bendinger et al. (1) isolated coryneform bacteria
from a biofilter that was loaded with the waste gas from an
animal-rendering plant. On the basis of chemotaxonomic differentiation they were able to identify strains of the genus
Corynebacterium, Gordona,
Mycobacterium, and Arthrobacter. Some biofilter
isolates of Gordona hydrophobica (2) showed colony
morphologies, which gave rise to different physicochemical properties
of their cell surface. The rough variants possess an extremely
hydrophobic cell surface due to a hydrophobic mycolic acid layer. In
contrast, the smooth variants exhibit a hydrophilic to moderate
hydrophobic surface (3), although they are also surrounded by a
hydrophobic mycolic acid layer. Obviously, the smooth ones incorporate
into their cell wall an additional surface component, which exposes its
hydrophilic part toward the medium. The observation that rough colonies
of Gordona only gave rise to rough colonies whereas smooth colonies gave rise to smooth as well as rough ones when grown on
complex media indicated that the rough variants lost the ability to
cover their extremely hydrophobic surface with a more hydrophilic compound.
A similar change in colony morphology was reported for mycobacterial
species that are closely related to the genus Gordona. Camphausen et al. (4) found a trehalose containing
lipooligosaccharide to be responsible for the observed change in
Mycobacterium paratuberculosis. Fregnan et al.
(5) showed that smooth variants of an unclassified scotochromogenic
Mycobacterium possessed a mycoside D that is absent in the
rough variants. However, Barrow and Brennan (6) pointed out that
mycoside D is likely to be a mixture of polar and apolar C mycoside
glycopeptidolipids (GPLs).1 Belisle and
Brennan (7) showed that the smooth variants of Mycobacterium
kansasii possess trehalose-containing lipooligosaccharides, whereas the rough variants were devoid of such surface components.
Such a change of morphology within the nocardioform actinomycetes has
so far only been described for mycobacteria and not for other members
of this taxon. Therefore, it was of interest to establish whether the
morphological change observed with G. hydrophobica was due
to the loss of mycosides or lipooligosaccharides or whether a different
cell wall component is responsible for the more hydrophilic cell
surface. Our studies revealed that the smooth variant of G. hydrophobica carries an additional glycosylated peptidolipid,
which we named gordonin. Such peptidolipids have been described as
surfactants or antibiotics (8-10), but so far not as constituents of
bacterial cell walls.
Bacterial Strains
Gordona bronchialis DSM 43247T and
Gordona rubropertinctus DSM 43197T were obtained
from the Deutsche Sammlung für Mikroorganismen und Zellkulturen
GmbH (DSM), Braunschweig, Germany. The isolation and classification of
G. hydrophobica DSM 44015T has been published
previously (1, 2). A detailed description of chemotaxonomical and
physiological markers of G. hydrophobica DSM
44015Tr (=1610/1b), G. hydrophobica DSM
44015Ts (=1610/1a), G. hydrophobica 1775/15r and
G. hydrophobica 1775/15s has been published (1) (s = smooth variants, r = rough variants).
Purification of Gordonin
G. hydrophobica 1775/15s was cultivated in 2 liters
of brain-heart infusion broth (Difco) at 30 °C (pH 7.4) for 3-4
days. The cells were pelleted by centrifugation (3000 × g, 30 min), washed twice with deionized water, and
lyophilized.
2 g of lyophilized cells were extracted twice with 60 ml of
chloroform/methanol (2:1, by volume) and once with chloroform/methanol (1:1, by volume). 40 ml of chloroform and 40 ml of 0.3% NaCl were added to the combined extracts. After gentle shaking the phases were
separated by centrifugation (3000 × g, 20 min). The
organic phase was evaporated to dryness on a rotary evaporator, and the pellet was redissolved in 50 ml of chloroform. This fraction was applied to a DEAE-cellulose column (20 × 2 cm) that was
equilibrated with chloroform. The column was washed with chloroform,
and gordonin was eluted with chloroform/methanol (9:1, by volume). The
eluate was evaporated to dryness by rotary evaporation, and the pellet was redissolved in 30 ml of chloroform/methanol/glacial acetic acid/water (186:34:5:3). It was further purified by column
chromatography (50 × 1 cm) on silica gel 60 (Merck, Darmstadt,
Germany). Gordonin was eluted by a linear gradient of
chloroform/methanol/glacial acetic acid/water (186:34:5:3) as solvent A
and chloroform/methanol/glacial acetic acid/water (90:40:15:9) as
solvent B. The gordonin-containing fractions were collected and dried
by rotary evaporation.
Analytical Thin Layer Chromatography
2-5 µg of a sample were dissolved in an appropriate solvent
and spotted on a silica 60-plate (Merck). Plates were developed in
chloroform/glacial acetic acid/methanol/water (80:15:12:4). After
drying, lipids were detected by wetting the plate with 20% sulfuric
acid in ethanol and heating (120 °C) for 2-5 min. Sugar residues
were detected by spraying the plate with 0.1% orcinol in 40% sulfuric
acid followed by heating at 120 °C for 15 min. For detection of
amino groups, plates were sprayed with ninhydrin solution (Riedel de
Haen, Seelze, Germany) and heated at 100 °C for 10 min.
GC/MS Analysis
An aliquot (2 mg) of gordonin was methanolyzed with either 0.5 M HCl/MeOH (65 °C, 45 min) or 2 M HCl in
methanol (85 °C, 16 h) followed by per-acetylation. Gas
chromatography/mass spectrometry (GC/MS) of resulting fragments of
gordonin was carried out with a Hewlett-Packard model 5985 instrument.
GLC was performed with a Varian model 3700 chromatograph equipped with
a capillary column of SPB-5 using a temperature gradient 150-320 °C
at 5 °C/min. GLC-MS was carried out with a Hewlett-Packard model
5989 instrument equipped with a capillary column of HP-1 under the same
chromatographic conditions as in GLC. Electron impact spectra were
recorded at 70 keV, and chemical ionization was done using ammonia as
reactant gas.
Ring Opening of Gordonin by Mild Transesterification
1 mg of gordonin was dissolved in 500 µl of 100 mM
sodium methylate and incubated at 37 °C for 16 h. After
neutralization with 2 M HCl in methanol the solvent was
evaporated under reduced pressure, and the saponified gordonin was
dissolved in chloroform. The precipitated NaCl was discarded.
Fatty Acid Analysis
An aliquot (100 µg) of gordonin was methanolyzed with 2 M HCl in methanol (85 °C, 16 h). GC/MS of the
resulting fragments was carried out with a Hewlett-Packard model 5890 series II gas chromatograph equipped with a capillary column of HP-5
(0.25 mm × 30 m) and a Hewlett-Packard model 5972 MSD. The
temperature gradient was 120-300 °C at 5 °C/min. Electron impact
spectra were recorded at 70 keV. Fatty acid methyl esters were
identified by comparison of retention times and mass spectra using
standards from Sigma (Germany).
Amino Acid Analysis
Gordonin was hydrolyzed in a gas-phase hydrolyzer with 6 M HCl at 150 °C for 1-4 h. The amino acid composition
was analyzed with a ABI type 421 amino acid analyzer (Applied
Biosystems, Foster, CA).
Absolute Configuration Analysis of Amino Acids
100 nmol gordonin were hydrolyzed as described above. The
separation of enantiomeric amino acids was achieved after
derivatization with
N Synthesis of Gordonin Part Structures as GC/MS and NMR Reference
Compounds
For the synthesis of
[(R)-3-hydroxytetradecanamido]-D-valine methyl
ester (2), 24 mg (0.1 mmol) of optically pure
(R)-3-hydroxytetradecanoic acid (14:0(3-OH)) were dissolved
in 6 ml of 1,2-dimethoxyethane (Fluka), and 10 mg (0.9 mmol) of
N-hydroxysuccinimide (Aldrich) and 29 mg of
N,N (R)-3-O-[2-N-(Acetamido)-D-valinoxy]-tetradecanoic
acid methyl ester (3) was synthesized starting from 15 mg of
D-Val (0.1 mmol) that were N-acetylated as
described (12). To the resulting
2-N-(acetamido)-D-valine, 1 ml of
trifluoroacetic acid anhydride was added, and the reaction was kept at
40 °C for 24 h. After removal of trifluoroacetic acid anhydride
under a stream of nitrogen, 5 mg (0.02 mmol) of
(R)-3-hydroxytetradecanoic acid methyl ester were added
together with 0.55 ml of pyridine containing catalytic amounts of
2,6-dimethylaminopyridine. The reaction was allowed to proceed for
16 h at 85 °C. GC/MS analysis revealed that the product
obtained was
(R)-3-O-[2-N-(trifluoroacetamido)-D-valinoxy]-tetradecanoic acid methyl ester from which the N-trifluoroacetamido
residue was removed by treatment with 0.25 ml of methanol/water (4:1, by volume) at room temperature. After the product was dried in vacuo, it was dissolved in 1 ml of pyridine/acetanhydride (2:1, by
volume) thus yielding the desired
(R)-3-O-[2-N-(acetamido)-D-valinoxy]-tetradecanoic acid methyl ester (3), which was purified on a small silica gel column as mentioned above (yield 3 mg, 0.01 mmol, 50%).
Amino Acid Sequence Analysis by Edman Degradation
Gordonin was partially hydrolyzed with 2 M HCl
(90 °C, 2-4 h) or with 12 M HCl (37 °C, 4 days). The
hydrolysates were purified by C18 reversed-phase HPLC. The
main peaks (monitored at 220 nm) were collected and analyzed with a
peptide sequencer ABI 476A (Applied Biosystems, Foster, CA). Since
peptides resulting from the hydrolysis were short and hydrophobic, an
arylamine-modified membrane (Sequelon AA; Millipore Corp., Bedford, MA)
was used to covalently bind the carboxyl-terminal amino acid.
Amino Acid Sequence Analysis by GC/MS
Partial hydrolysis of gordonin was carried out after
saponification as described above. Preparation of trifluoroacetylated peptide esters was performed as described elsewhere (13). The resulting
trifluoroacetyl peptide methyl esters were identified by performing gas
chromatography-mass spectrometry using a Hewlett-Packard model 5890 series II gas chromatograph equipped with a 5% phenyl methyl silicone
capillary column (0.25 mm by 30 m) and a model 5972 mass selective
detector. Helium was used as carrier gas; the injection volume was 5 µl, and the injector temperature was 250 °C. Split was set at
1:37. The initial column temperature was 70 °C, which was held for 2 min and than increased to 300 °C at a rate of 5 °C/min. The gas
chromatography-mass spectrometry transfer line temperature was
280 °C.
Time-of-Flight-Secondary Ion Mass Spectrometry (TOF-SIMS)
The TOF-SIMS determinations were carried out with a TOF II
instrument developed at the University of Münster (14). It
consists of a primary ion source with a pulsed 90° deflector, a
reflectron TOF analyzer, and a single ion counting registration system.
The primary ions generated by the ion source bombard the surface as short mass separated ion packets (0.8 ns, 10 keV Ar+) in an
area of about 70 nm diameter. The generated secondary ions are
accelerated to 1.8 keV, mass-separated in a field free flight path, and
then detected with an ion electron-photon conversion system. For the
insulating surfaces, the instrument is equipped with a charge
compensation system, which consists of an electron flood gun producing
low energy electrons. For TOF-SIMS determinations gordonin was
dissolved in chloroform/methanol/glacial acetic acid/water (80:12:15:4)
at a concentration of 0.1 mg/ml and deposited on a clean silver
support.
MALDI and MALDI-PSD Mass Spectrometry
MALDI specimens were prepared by dissolving 50-200 pmol of the
analyte in 10 µl of chloroform/glacial acetic acid/methanol/water (80:15:12:4). To this solution 10 µl of acetone saturated with 2,5-dihydroxybenzoic acid was added. A 5-10-µl aliquot of this mixture was pipetted on the surface of a slightly preheated sample holder, on which the solvent evaporated within a few seconds. Transfer
of the sample into the instrument was performed as quickly as
possible.
The TOF-mass spectrometer is a reflectron-type instrument employing a
gridded two-stage reflectron at the end of a first field drift path of
204 cm in length. Its second field drift path (reflectron to detector)
extends over 175 cm. Standard acceleration voltage in this instrument
is 10 kV. For ion detection a 75-mm diameter dual microchannel plate is
employed. The ion source in the instrument is a coaxial optical/ion
optical device which simultaneously serves for laser focusing, sample
imaging, and optical beam collimation, all in a common axis strictly
perpendicular to the surface plane of the sample.
The acceleration stage is a split device (2/6 mm) with the option to
apply delayed extraction conditions. This means that desorbed ions are
initially (for 50-500 µs) allowed to expand against a slightly
retarding field before the accelerating field is turned on (fast HV
switch). If the delay time and the ratio of field strength in the two
acceleration stages are properly chosen (settings dependent on the mass
range of interest), so-called velocity focusing conditions can be
fulfilled, resulting in a dramatic improvement of mass resolution. In
the present investigation delay times between the desorption pulse and
the onset of the extraction field of 230-440 µs were chosen, and a
ratio of 0.47 for the field strength in the first and second
acceleration stages, respectively, was found to perform best with
respect to mass resolution of the quasimolecular ion species. Typical
values obtained under these conditions were 2500-4000 M/dM (FWHM). The
TOF-mass spectrometer was equipped with a so-called precursor ion
selector. This is an electrostatically switched ion gate located at a
distance of about 220 mm from the sample. It allows a precursor ion
selection with a mass selectivity of about 60 M/dM and is a mandatory
device for post-source decay fragment ion mass analysis in
nonhomogenous samples.
PSD fragment ion spectra were sequentially recorded over 12-15 mass
windows by incremental reduction of the reflectron voltages. In each
spectral window 30-100 single-shot spectra were averaged depending on
sample conditions. For signal recording, the output of the detector was
fed to a 300-MHz digital oscilloscope (Le Croy 9450A Chestnut Ridge,
NY) and digitized at a sampling rate of either 10 or 5 ns. Signal
averaging and processing were carried out using a 486 PC with Ulysses
software (version 7.31, Chips at Work, Bonn, Germany). A subroutine
provides for easy PSD ion mass assignment without the need to calibrate
the instrument by PSD reference standards (for further details see Ref.
15). Accuracy and precision of PSD mass assignments reach ± 0.2-0.3 atomic mass units in the mass range of m/z
500-1000. Compilation (stitching) of a set of PSD mass windows was
performed by a subroutine contained in the MALDI-PSD Peptide Sequencer
software (version 3.2, copyright by Frank Lützenkirchen, ILM,
Düsseldorf, Germany).
NMR Spectroscopy
1H and 13C NMR spectra were obtained
with a Bruker AM-360 spectrometer for solutions in
Me2SO-d6 containing 2%
D2O at room temperature referenced to dimethyl sulfoxide (H
2.49, C 39.5). For the detection of NH protons and correlated signals,
additional one-dimensional and two-dimensional 1H spectra
were run containing 2% water (by volume) under the same conditions.
Standard Bruker software was used for two-dimensional COSY, relayed
COSY, homo- and heteronuclear correlated experiments.
Identification, Isolation, and Purification of Gordonin
A systematic analysis of complex lipids in G. hydrophobica 1775/15s and G. hydrophobica 1775/15r
according to the method of Dobson et al. (16) revealed that
an additional compound was present in the fraction of the most polar
lipids of the smooth variants compared with that of the rough ones. The
less polar lipids were identical between smooth and rough variants
(data not shown). In TLC analysis this compound showed an
Rf value of 0.23 (Fig. 1). In
contrast to the rough variants, all investigated smooth ones synthesize
this compound. The ninhydrin reaction was negative, whereas the
reaction with orcinol was positive. This indicated that the lipid
contains no free amino group but instead carries a sugar moiety. We
therefore tentatively concluded that the isolated compound is a
glycolipid, which was named gordonin. For structural analysis we used
the glycolipid of the strain 1775/15s, which was confirmed to be
G. hydrophobica by DNA-DNA hybridization with G. hydrophobica DSM 44015T and that was the object of
further physiological investigations.2
Structural Analysis of Gordonin
After weak methanolysis and subsequent peracetylation of the
purified gordonin, glucose could be detected as peracetylated methyl
glycoside by GC/MS analysis to be the only sugar component (Table
I). Strong methanolysis released glucose, serine, and two fatty acids. Two further fragments, in which the valine was bound
to two different fatty acids, could be identified, but it was not
possible to decide whether the amino acid was amide- or ester-linked to
the fatty acid.
Calculated molecular weight of gordonin and its homologue
Abteilung Mikrobiologie,
-hydroxylated fatty acid
(3-hydroxyeicosanoic acid, 20:0 (3-OH)) to form a cyclic lactone ring
structure. The main fraction of GPL, for which we propose the name
gordonin, was identified as
3-hydroxyeicosanoyl-L-seryl-L-phenylalanyl-L-seryl-L-seryl-D-alanyl-L-(O-
-D-glucopyranosyl)-threonyl-glycyl-D-leucyl-L-valyl-L-seryl-L-phenylalanyl-glycyl-L-valyl lactone. The other GPLs constitute structural variations within the nature of the
-hydroxylated fatty acid (20:0(3-OH)
versus 22:1(3-OH)) in a ratio of about 1:0.9 as well as
within one amino acid (D-Leu versus
L-Phe) in about 30%. Sequence information was obtained in
part by Edman degradation as well as gas chromatography/mass spectrometry analysis of di- and tripeptide fragments. However, the
complete amino acid sequence could only be established by MALDI-PSD
from the linear molecule, i.e. after ring opening of the
lactone.
-(2,4-dinitro-5-fluorophenyl)-L-alaninamide.
Derivatization was performed as described (11). Amino acid derivatives
were separated at 50 °C by HPLC on a C18 reversed-phase
column (Hewlett-Packard ODS Hypersil, 5 µm, 250 × 4 mm) fitted
with a LiChrospher®100 RP-18 guard column (5 µm, 4 × 4 mm). The separation was achieved by a linear gradient
of 50 mM triethylaminophosphate, pH 2.9 (buffer A), and
acetonitrile (buffer B). The gradient increased from 15 to 50% buffer
B in 35 min. The flow rate was 500 µl/min and amino acid derivatives
were detected at 340 nm. Peaks were recorded and quantified with the
Hewlett-Packard 3D Chem Station software.
-dicyclohexylcarbodiimide (0.14 mmol, Sigma) were added
to the solution. The reaction was allowed to proceed for 36 h at
4 °C, and precipitating material was removed by filtration. The
solution was evaporated to dryness under a stream of nitrogen, and the
product was dissolved in 1 ml of pyridine. 10 mg of
D-valine methyl ester (freshly prepared from
D-valine (Sigma) and diazomethane in ether) were added to
the solution, and the reaction was allowed to proceed for 1 h at
room temperature. The product (2) was applied to a small
silica gel column (0.5 × 5 cm, Kiesel gel 60, 230-400 mesh,
Merck) and stepwise eluted with ether/hexane (10:90, by volume),
chloroform, chloroform/methanol (95:5 and 90:10, by volume). The
product (2) eluted in the chloroform fraction (yield 3 mg,
0.01 mmol, 10%) and was further characterized by GC/MS and NMR
spectroscopy.
Fig. 1.
Identification of an additional glycolipid
synthesized by smooth colony-forming variants of Gordona
biofilter isolates. Analytical TLC was carried out as described
under "Materials and Methods." The most polar fractions of the
following strains have been analyzed: lane 1, Gordona
bronchialis DSM 43247T; lane 2, G. rubropertinctus DSM 43197T; lane 3, G. hydrophobica DSM 44015Ts; lane 4, G. hydrophobica DSM 44015Tr; lane 5, G. hydrophobica 1775/15s; lane 6, G. hydrophobica 1775/15r (s = smooth variants, r = rough variants).
[View Larger Version of this Image (81K GIF file)]
Residue
Mr
Number of
residues
Molecular weight
Serine
105
4
420
420
Glycine
75
2
150
150
Threonine
119
1
119
119
Alanine
89
1
89
89
Valine
117
2
234
234
Leucine
131
1
131
131
Phenylalanine
165
2
330
330
Glucose
180
1
180
180
FA
C20-3OH
328
1
328
FA C22:1-30H
354
1
354
Sum
1981
2007
H2O
subtracted
15
270
270
Calculated molecular
weight
1711
1737
After strong hydrolysis and subsequent amino acid analysis the
amino acids alanine, leucine, threonine, glycine, valine,
phenylalanine, and serine could be identified in a molar ratio of
1:1:1:2:2:2:4, respectively (Table I). The determination of the
absolute configuration of the amino acids was achieved by separation of
diastereomeric N-(2,4-dinitro-5-fluorophenyl)-L-alaninamide-amino
acid derivatives by HPLC (data not shown) and showed that only leucine
and alanine were present in the D configuration.
The fatty acids were isolated, methylated, and analyzed by GC/MS. Two main peaks were detected. One of them belonged to a saturated 3-OH fatty acid since its basal peak at m/z 103 originated from [CH(OH)CH2COOCH3]+. The other one was an unsaturated 3-OH fatty acid with a significant peak at m/z 103 and a basal peak at m/z 55 due to [H3C(CH)2CH2]+. The parental peaks of the methyl esters were difficult to detect by this ionization method and were only observed for the unsaturated fatty acid, but their molecular weight could be obtained from the peaks at m/z = M-50, which are of significant height in the case of 3-OH fatty acids (17). The data indicated that the fatty acid fraction was a mixture of two components, 20:0 (3-OH) and 22:1 (3-OH) (Table I).
Isolation and Characterization of Part Structures of Gordonin GC/MS Analysis of Val-20:0(3-OH) After Strong MethanolysisUsing strong methanolysis and subsequent peracetylation, GC/MS analysis revealed that valine was attached to 20:0 (3-OH) as a major compound. It was expected that ester-bound valine would not have resisted the strong methanolysis conditions applied (2 M HCl/MeOH, 85 °C, 2 h). As judged by GLC/MS analysis it was difficult to decide whether valine is ester- or amide-linked to the 20:0 (3-OH), since both derivatives exhibit identical molecular weights. To solve this problem, the peracetylated derivatives [(R)-3-hydroxy-tetradecanamido]-D-valine methyl ester (2) and [(R)-3-O-[2-N-(acetamido)-D-valinoxy]-tetradecanoic acid methyl ester (3) have been synthesized whereby the (R)-3-hydroxymyristic acid (14:0 (3-OH)) was used instead of 3-hydroxyeicosanoic acid (20:0 (3-OH)), since it was the only (R)-configurated 3-hydroxylated fatty acid available.
As compared with GC/MS analysis, compounds 2 and
3 displayed in the electron impact-mass spectrum
characteristic fragment ions for either the amide- or the ester-linked
valine to the -hydroxylated fatty acid (compounds 2 and
3, Figs. 2, a and b).
Comparing the electron impact-mass spectra of 2 and
3 with that of the compound isolated and derivatized in a
similar way from gordonin, it was unequivocally proven that valine is
ester linked to the
-hydroxy group of the
-hydroxylated fatty
acid (Fig. 2c). Therefore, the valine residue in the intact
gordonin is either amino-terminally blocked or gordonin is a cyclic
depsipeptide, because it is ninhydrin-negative.
Amino Acid Sequence Analysis
To determine the amino acid
sequence of gordonin, the compound was partially hydrolyzed. After
separation on C18 reversed-phase HPLC, resulting fragments
were analyzed by Edman degradation. The separation pattern of partially
hydrolyzed peptides (a-f) and their amino acid sequences
are shown in Fig. 3. Edman degradation of the other
fragments has been unsuccessful because of amino-terminal blocking.
Hydrolysis, trifluoracetylation, and GC/MS analysis revealed that
besides the monomeric amino acids, dimeric fragments being composed of
Thr-Gly, Gly-Val, Val-Ser, Leu-Val, Ser-Phe, Phe-Ser, and Phe-Val and
also a trimeric Ser-Ser-Ala fragment could be found (Fig.
4).
Determination of Molecular Weight by TOF-SIMS
The molecular weight of gordonin was determined by TOF-SIMS. This
analysis further revealed that gordonin is not homogeneous (Fig.
5). Besides two main masses with m/z 1711 and
1737, additional masses with m/z 1745, 1755, and 1771 were
detected. The assignment of the signals shown in Fig. 5 could be
confirmed by thin layer chromatography and thin layer
chromatography-SIMS (18) of gordonin. The main molecule (gordonin) with
m/z 1711 could only be explained when 15 mol of water per
molecule of gordonin are released (Table I). The same holds true for
the gordonin homologue (m/z 1737) containing the 22:1 (3-OH)
fatty acid. This argues strongly in favor of a cyclic structure.
MALDI and MALDI-PSD Analysis
To obtain the amino acid sequence of gordonin, MALDI and MALDI-PSD
mass analysis were employed. This could be best achieved after mild
alkaline treatment of the cyclic gordonin molecule and its homologues
with sodium methylate and subsequent methylation, by which the cyclic
lactone ring was transferred to the linear methyl ester, and the
glucose was released from the molecule. The derivatives, although
complex in structure, were suitable for MALDI and MALDI-PSD
time-of-flight mass analysis. The MALDI spectra contained a large
variety of parent ions up to the expected mass range, where abundant
parent ion signals could be recorded at 1254 and 1286 atomic mass
units, respectively. However, both precursors did not match the
expected molecular masses based on the amino acid analysis by 18
atomic mass units.
On a 1020 atomic mass unit parent ion, a MALDI-PSD fragment, an ion
analysis was performed (Fig. 6), which unambiguously
confirmed (by sets of consecutive bn and ym
signals) the presence of two carboxyl-terminal sequence
ladders: ... GLVSFGV-OCH3 and ...
GFVSFGV-OCH3. Since the mass increment of 83 atomic mass
units between y7 and y8 cannot represent
any standard amino acid, a dehydrothreonine (101 18 atomic mass
units) was suggested for this position. This assumption was further
corroborated by the
18 atomic mass unit differences between the
calculated and measured masses of the full stretch precursor ions.
Alkaline treatment of gordonin causes obviously detachment of glucose
by
-elimination. This observation supports the notion that the
Glc residue was attached to Thr and not to any other of the putative
hydroxyl groups in the four serine residues (compare also NMR analysis
below).
Subsequent analysis of the full prompt precursor ion spectra (data not
shown) revealed that the missing amino-terminal stretch had the
following sequences SFSSAT(18)GL ... and
SFSSAT(
18)GF... . Based on these sets of complementary
information and in full agreement with the sequence information derived
from partial hydrolysis (see above), Sequence 1 as
determined by MALDI and MALDI-PSD-MS could be compiled as shown.
NMR Spectrometric Analysis
In the 1H NMR spectrum of gordonin (Fig.
7) one characteristic doublet for the anomeric proton of
a hexose (H-1, 4.22 ppm) was present at high field with a high
coupling constant (J1,2 7.2 Hz) characteristic
for
-anomeric-linked pyranoses. Although the other signals from the
glucose were not resolved completely, they could be assigned following
the contour plot in the two-dimensional COSY spectrum to be a
-glycosidically linked Glc residue. Adding traces of water to the
sample, NH signals of the amino acids were detected thus providing
further help for the assignment of the various characteristic H-2
proton signals of the 13 amino acids. Therefore, the 13C
NMR spectrum as well as the distortionless-enhancement by
polarization-transfer (DEPT) spectrum (Fig. 8,
a and b) could be completely
assigned by the help of the 13C,1H COSY.
The 13C NMR spectrum contained 14 carbonyl carbons,
including those for 13 amino acids and 1 for a fatty acid (Table
II). Characteristic signals for a -linked terminal
pyranosidic glucose residue were also identified. From the low field
shift of the C-3Thr signal (
72.73 versus
67.1 ppm; Ref. 19) it was deduced that Glc was linked to C-3 of
Thr.
|
The linkages of 20:0 (3-OH) in the cyclic gordonin were investigated with the help of two synthetic reference compounds [(R)-3-hydroxytetradecanoamide]-D-valine methyl ester (2) and [2-N-(acetamido)-D-valinamido]-(R)-3-hydroxytetradecanoic acid methyl ester (3). The 13C NMR of the synthetic compound 3 was very similar to that of gordonin (1) (Table III) but different to that of compound 2 (data not shown). These data support the result of the GC/MS analysis and are also in good agreement with the data from the MALDI-PSD analysis of the linear molecule (see above).
|
In conclusion, chemical analysis, MALDI-TOF-mass spectrometry, and NMR
spectroscopy revealed that gordonin is a cyclic tridecapeptidolipid carrying a fatty acid (20:0 (3-OH)) as part of the ring structure and a
pyranosidic Glc in -anomeric linkage attached to Thr (1). Besides gordonin (1) (Fig. 9), at least three
additional GPL molecules were identified being structurally related to
gordonin. These molecules carry either L-Phe instead of
D-Leu or 22:1 (3-OH) instead of 20:0 (3-OH).
The plasma membrane of mycobacteria and related actinomycetes like Rhodococcus, Corynebacterium, Nocardia, and Gordona is not very different from that of the plasma membrane of other bacteria, but the cell envelope is very distinct (20-23). The peptidoglycan of mycobacteria and other Nocardiaceae is linked to characteristic arabinogalactan polysaccharides (24), which are esterified with high molecular weight mycolic acids. A range of complex free lipids are associated with the mycolic acid matrix in the mycobacterial cell envelope. These complex free lipids are composed of very apolar lipids like dimycocerosates of the phtiocerol family up to polar glycopeptidolipids and lipooligosaccharides. All of these free lipids contain fatty acids or long chain alcohols, which are structurally different from those constituting the hydrophobic section of the plasma membrane's amphiphatic polar lipids. These long chain components are thought to intercalate with the mycolic acid layer by hydrophobic interaction resulting in the exposition of the more polar groups of the free lipids to the hydrophilic exterior. Recently, Ortalo-Magné et al. (25) identified the surface-exposed lipids of several mycobacterial species. They isolated the lipids of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium gastri, Mycobacterium smegmatis, and Mycobacterium aurum by gently treating the cells with glass beads thereby demonstrating a selective location of classes of ubiquitous lipids on the surface of mycobacteria. Phosphatidylethanolamine and phosphatidylinositol mannosides were exposed in all species examined, whereas dimycolyl trehalose ("cord factor") was only found at the surface of M. aurum. Monomycolyl trehaloses and triacylglycerides were exposed in M. avium and M. smegmatis but not in other mycobacterial species studied so far. Phenolic glycolipids, dimycocerosates of phtiocerols and lipooligosaccharides were identified at the surface of M. tuberculosis (Canetti), M. kansasii, and M. gastri, whereas glycopeptidolipids were identified in the outermost layer of M. avium and M. smegmatis (25). However, a glycosylated N-acyl peptide on the cell surface of G. hydrophobica has so far not been isolated as a constituent of a cell envelope.
N-Acyl peptides can be structurally divided into two classes: those having a linear peptide moiety like stenothricin (13, 26), fortuitin (27), and amphomyxin (28) and cyclic peptides. The latter group can be further subdivided in several subgroups depending on the functional groups of the molecule responsible for the ring formation such as lactones and lactams. The lactones can be further subdivided depending on the involvement of the hydroxyl group of the fatty acid in the ring formation or not. Finally, also glycosides of cyclic peptidolipids have been found. A comprehensive synopsis of bacterial lipids containing amino acids or peptides linked by amide bonds is given by Asselineau (29).
Gordonin described here was found to be a glycoside of an N-acyltridecapeptide containing a hydroxy fatty acid thus forming a cyclic lactone ring structure. The glucose is bound to L-threonine, similar to one of the four most polar peptidolipids of Rhodococcus erythropolis isolated by Koronelli (30). In two other polar peptidolipids glucose is bound to serine, and the fourth is lacking glucose. Furthermore, these peptidolipids contain 1 mol of a normal chain fatty acid and 1 mol of a mycolic acid residue. In addition to this, Koronelli (30) found a different amino acid pattern compared with gordonin. A cyclic structure was only found for one of the three apolar peptidolipids. The low polarity lipids are N-acyl-L-threonyl-L-valyl-L-valyl-L-leucinolyde (I), N-acyl-L-(O-acyl)-threonyl-L-valyl-L-valyl-L-leucine (II), and N-acyl-L-(O-mycolyl)-threonyl-L-valyl-L-valyl-L-leucine (III). In all cases the N-acyl units and the O-acyl unit in lipid II are moieties of C20, C22, C24, C26, and C28 saturated and monoenoic acids.
Aydin et al. (8) isolated two antimicrobial agents from
Erwinia herbicola called herbicolin A and herbicolin B. Both
compounds have the same fatty acid and amino acid composition with the
following sequence:
dehydro-Abu-L-Thr-D-allo-Thr-D-Leu-Gly-D-Gln-Gly-N-Me-L-allo-Thr-L-Arg (dehydro-Abu is 2,3-dehydro--aminobutyric acid). This peptide is
N-acylated by an 3-hydroxytetradecanoic acid. It is
interesting to note that only herbicolin A carries
D-glucose. This carbohydrate residue is linked in an
-glycosidic bond to the hydroxy group of the 3-hydroxytetradecanoic
acid. The carboxyl-terminal arginine residue forms a lactone ring with
the hydroxy group of L-threonine.
Globomycin and surfactin are well-known N-acyl peptides containing a hydroxy fatty acid as part of a lactone ring (9, 10). Globomycin exhibits antibiotic activity against Gram-negative bacteria affecting cell wall synthesis. Surfactin was isolated and characterized by Arima et al. (10). They found that this bacterial peptidolipid remarkably extents the time necessary for fibrin clot formation by inhibiting the conversion of fibrin monomers to fibrin polymers. In addition, surfactin was found to lower the surface tension of 0.1 M NaHCO3 from 71.6 to 27.0 millinewton/m. An even better surface activity was found for arthrofactin, a lipopeptide biosurfactant isolated from Arthrobacter species strain MIS38 (31). The minimum surface tension of arthrofactin was 24 millinewton/m above the critical micelle concentration.
The amphiphilic character of many peptidolipids give rise to several biological functions of these molecules like hemolytic properties, antibiotic activities, or enzyme-inhibiting properties (29). The amphiphilic nature of gordonin is caused by the 20:0 (3-OH) fatty acid and several hydrophobic amino acids clustered at one side of the proposed structure (Fig. 9), and the glucose and several L-serine residues, which are located on the opposite side. Therefore, gordonin is able to intercalate with the very hydrophobic mycolic acid layer of the cell wall by the hydrophobic tail of the 3-hydroxy fatty acid and to expose the hydrophilic head of the molecule to the exterior to make the cell surface more hydrophilic. This hydrophilic cell surface enables the smooth variants of G. hydrophobica to grow in suspension in liquid culture. In contrast, the rough variants, lacking gordonin, grow in large flocks resulting in anaerobic microenvironments. This was shown by demonstrating that the rough variants in contrast to the smooth ones show nitrate dissimilation despite aerobic culture conditions (32).
It is also conceivable that gordonin is partially released to the environment during growth to dissolve hydrophobic substrates. Therefore, gordonin could act as a surfactant, like surfactin or arthrofactin, and this would allow G. hydrophobica to use pollutants of the animal-rendering plant waste gas as carbon source, which are hardly soluble in water. Currently, we are investigating if gordonin is able to reduce interfacial tension between air and water or water and organic solvents, respectively.
We thank L. Merschel (Institute of Analytical Chemistry, University of Muenster) for performing TOF-SIMS investigations.