A New Pyoverdin from Pseudomonas aureofaciens

A New Pyoverdin from Pseudomonas aureofaciens

A New Pyoverdin from Pseudomonas aureofaciens*
H. B eiderbeck, D. Risse, H. Budzikiewicz, K. Taraz
In stitu t für O rganische Chem ie d er U niversität zu K öln, G reinstr. 4, D-50939 Köln, G erm an y
Z. N aturforsch. 54c, 1 - 5 (1999); received S ep tem b er 9, 1998
P seudom onas aureofaciens, Pyoverdin, S iderophore
From Pseudom onas aureofaciens a new pyoverdin was isolated and its structure was d e te r­
m ined by various spectroscopic m ethods and by p artial degradation.
In tro d u ctio n
Iron, a fte r oxygen, silicon and alum inum , is the
elem ent fo u rth in abundance by weight in the
earth crust. For most living organisms, however, it
is the critical d eterm inant of prim ary production
because o f its low availability. This is due to the
fact th a t Fe3+ is precipitated at physiological pH values as insoluble ferric hydroxide (K s = 10~39).
To overcom e this iron stress, m any bacteria (and
eucariotic organism s as well) excrete siderophores,
i.e. com pounds, th at form highly stable w ater solu­
ble com plexes with Fe3+. The ferri-siderophore is
recognized and taken up by proteins in the ou ter
m em bran e of the bacterial cell. B acteria of the ge­
nus P seudom onas from the so-called fluorescent
group p ro d u ce characteristic siderophores, the pyoverdins (Budzikiewicz, 1993 and 1997). They
have th re e structural elem ents: a peptide chain of
6 - 1 2
am ino acids, the dihydroxyquinoline chro-
m ophore and a side chain which is an acid derived
from the citrate cycle (Teintze et al., 1981, Michels
et al., 1991). The two hydroxy groups of the chrom ophore and tw o am ino acids from the peptide
chain form an octahedral com plex with Fe3+ as
central ion. The coordinating sites in the peptide
chain can be hydroxam ates derived from O rn or
a-hydroxy-carboxylate am ino acids such as ß-hydroxy-A sp or ß-hydroxy-His. A n o th er im portant
purpose of the peptide chain is the recognition of
the iron com plex on the cell surface. M ost Pseu­
dom onas strains produce th eir own pyoverdins
distinguishable by the com position of their peptide
chain. In this com m unication we describe the iso­
lation and structure d eterm ination of the first py­
overdin from P seudom onas aureofaciens.
M aterials and M ethods
Instrum ents
Abbreviations: C om m on am ino acids, 3-letter-code;
A cO H O rn , N 5-acetyl-N 5-hydroxy-O rn; aThr, alio-Thr;
F o O H O rn ,
N 5-form yl-N 5-hydroxy-O rn;
c-O H -O rn,
cycIo-N5-hydroxy-O rn (3-am ino-l-hydroxy-piperidone2); TAP, trifluoroacetyl (am ino acid) O -isopropylester;
GC/M S, gas chrom atograph coupled with a mass spec­
tro m eter; FAB-M S, fast atom b o m bardem ent mass spec­
tro m eter; H V P E , high voltage p aper electrophoresis;
R P -H P L C , reversed phase high perform ance liquid
chrom atography; TLC, thin layer chrom atography;
N M R -techniques: COSY, correlation spectroscopy;
TOCSY , total correlated spectroscopy; H M BC, heteronuclear m ultiple bond correlation; H M Q C , heteronuclear m ultiple quantum coherence; ROESY, rotating
fram e O v erh au ser and exchange spectroscopy.
* P art LX X V I1I of the series „B acterial C onstituents“.
For p a rt L X X V II see Risse et al. (1998).
R ep rin t req u ests to PD Dr. K. Taraz.
Fax: 49-221-470-5057.
E-m ail: aco98@ pop.rrz.uni-koeln.de
0939-5075/99/0100-0001 $ 06.00
U V /V IS
spectroscopy:
H IT A C H I
200,
L A M B D A 7 (both P erkin-E lm er, Ü berlingen), 1
mg of l a in 2 0 ml w ater, 2 0 ml 0 . 1 m acetic acid
pH 2.7 and 20 ml 0.066 m phosphate buffer pH 7.
M ass spectrom etry: FAB-M S: HSQ-30 (Finnigan-M AT, B rem en) with FA B-gun from IonTech
Ltd. (Teddington, G B ), FAB-gas: Xe, and VARIA N M AT 731 (Finnigan-M A T, B rem en) with the
sam e FA B-gun, FAB-gas: Xe; sam ple preparation
by adsorption on Sep-Pak RP-18, washing with
H 2 0 , desorption with C H 3 0 H /H 20 1:1 and dry­
ing i.v.
NM R: A M 300 (B ruker, R heinstetten), ( ‘H: 300
M Hz, 13C 75,5 M H z); 15 mg of la were dissolved
in 0.5 ml D 2 0 , purified on Biogel P2 with 0.1 m
acetic acid as solvent; the acetate anions w ere re ­
placed on a D E A E -colum n by C1“ , H 20 as
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2
H. B eiderbeck et al. ■A New Pyoverdin from P seudom onas aureofaciens
D-aThr (Thri)
0H
L-Thr<Th«)
\
«-(
- C i»
hA
?
PH D-Sen
NHR
OH
D-Ser2
c-OH-Orn
1a: R = CO-CH -CH -CONH
2
2
2
1b: R = CO-CH2 -CH2 -COOH
Fig. 1. S tructure of the pyoverdin from P. aureofaciens.
solvent. C hem ical shifts are given relative to H D O
as internal standard ( 8 = 4.8 ppm ).
C hrom atography: G C -colum n Chirasil-L-Val
(22 m x 0.22 m m ) (C hrom pack, F rankfurt/M .);
colum n chrom atography: X A D Type 4 (Serva,
H eidelberg), Biogel P2 (B ioR ad, H ercules, CA,
U S A ), Sephadex G-25, C M -Sephadex C-25 and
D E A E -S ep h ad ex C-25 (Pharm acia, U ppsala, S),
SepPak RP-18 (W aters, M ilford, G B ), TLC -plates:
Silicagel 60 F 2 5 4 and H P T L C -F ertig p latten C H IR
(M erck, D arm stadt).
H V PE : C am ag H V E System 60600, pap er
MN261 (Cam ag, M uttenz, CH ); sam ples w ere dis­
solved in 0.1 m acetic acid (p H 2.7) and 0.1 m
CH 3CO O N H 4 (pH 6.5), with glucose and desferal
as standards (Poppe et al., 1987).
Growth conditions, isolation and characterisation
o f the pyoverdin
The strain of P. aureofaciens was o b tain ed from
the Institute for M icrobiology, U niversity B onn. It
was grown at 23 °C in a m edium with gluconate as
C -source (per 1: 13 g N a-D -gluconate, 4 g K H 2 P 0 4,
5 g (N H 4 ) 2 S 0 4, 0.5 g M g S 0 4 -7H 2 0 ) for 92 h.
T hen 20 ml of 5% iro n (III) citrate solution was
added p er 1 of the culture and the pH -value was
adjusted to 5.5 with 6 m hydrochloric acid. Cells
w ere rem oved by tangential filtration (TH LF-Sys-
tem , M illipore, Bedford, M A, U SA ). The brow n
filtrate was passed through a X A D -colum n. A fte r
w ashing with deionized w ater the pyoverdin frac­
tion was eluted with m ethanol/w ater 1 : 1 (v/v) and
dried i.v. The residue was chrom atographed on B i­
ogel P2 with 0.2 m pyridinium acetate as solvent,
the fraction absorbing at 405 nm was collected and
dried i.v. The residue was applied to a C M -Sepha­
dex colum n and desorbed with 0 . 2 m pyridinium
acetate. Two fractions ( la and lb ) w ere obtained,
the la tte r being probably an artefact derived from
l a by hydrolysis (Schäfer et al., 1991). The frac­
tions w ere rechrom atographed with 0 . 0 2 m pyri­
dinium acetate. The substances w ere dried i.v. and
their purity was checked by TLC with m ethanol/
H 2 0 /a c e tic acid 70:30:0.005 (v:v:v) as solvent. For
m ass spectrom etry and N M R -spectroscopy the
ferri-pyoverdins were decom plexed with 8 -hydroxyquinoline/citrate and rechrom atographed as d e ­
scribed before (B riskot et al., 1986).
Q ualitative analysis of the am ino acids was p e r­
form ed by T A P-derivatisation and GC-M S as d e ­
scribed earlier (Jacques et al., 1995). P artial hy­
drolysis: To determ ine the position of L-Thr and
D-aThr in the peptide chain 1 mg of the pyoverdin
la was hydrolyzed with 1 ml 3 m formic acid for
10 min at 90 °C. The solution was dried i.v., FA B MS of the residue showed that the peptide chain
H. B eiderbeck et al. ■A New Pyoverdin from P seudom onas aureofaciens
was hydrolyzed at the am ide bond betw een G ly 1
and T h r 1 (see Fig. 1). The m aterial was subse­
quently dansylated and hydrolyzed again with 1 ml
of 6 m HC1 at 90 °C for 8 h (Risse et al., 1998).
D ansyl-derivatives of the am ino acids w ere ad ­
sorbed on SepPak cartridges and fu rth er investi­
gated by TLC on chiral plates with m eth an o l/H 2 0 /
acetonitrile 50:40:20, 0.05 m K H 2 P 0 4 as solvent.
The aqueous eluate of the SepPak cartridges was
dried i.v. and TA P-derivatized.
R esults and Discussion
The UV/Vis spectra are typical for pyoverdins:
ferri-la: 399 nm at pH 5.3; desferri-la: 363 nm at
pH 3.2 und 399 at pH 7.1 (B riskot et al., 1986).
The m olecular mass as d eterm ined by FAB-M S
was 1277 u for la and 1278 u for lb . A fte r total
hydrolysis and T A P-derivatisation l - G 1 u , Gly, d O rn, D-Ser, L-Thr, D-aThr could be observed. The
electrophoretic m obility of the free pyoverdin la
is +0.6 (at pH 2.7) and +0.41 (at p H 6.5).
Partial hydrolysis resulted in a cleavage of the
peptide chain betw een G ly 1 and T h r 1 (s. Fig. 1).
The m aterial was dansylated, com pletely hy­
drolyzed and dried i.v. The dansylated am ino acids
contained only dansyl-D-aThr. T h erefore T h r 1 is d aThr. A fter T A P-derivatisation the free am ino
acids showed a L-Thr/D-aThr ratio of 1.5. T h ere­
3
fore the T hr closer to the C-term inus (T hr2) is l Thr. The fact that D-aThr was observed in the
TA P-derivatives is probably due to incom plete
dansylation of the partial hydrolysate.
N M R -spectroscopy
The pyoverdin from P. aureofaciens contains
several bifunctional am ino acids (Thr, Ser, G in
and O rn). It had to be shown in which way these
am ino acids w ere inserted in the peptide chain.
F u rth erm o re sequence-specific signals should be
o btained in the R O ESY -experim ent. The data
w ere assem bled by a variety of tw o-dim ensional
experim ents: H ,H -C O SY shows V -coupling of HC-C-H , w hereas 4J- and higher couplings could be
observed by TOCSY. C arbon shifts w ere obtained
by H M Q C and H M B C for the carbonyl carbon
atoms. For a m ore detailed description of the ex­
perim ents see Evans (1995), Braun et al. (1996)
and Schaffner et al. (1996).
The !H - and 1 3 C -data of l a are assem bled in
Tables I and II. The signals of the chrom ophore
and the succinic acid am ide side chain correspond
to those of other pyoverdins published before
(B riskot et al., 1989, G eisen et al., 1992). The
chem ical shifts of the ß-protons of the Ser and Thr
indicate th at the O H -groups are free; otherw ise
their chem ical shifts should be shifted dow nfield
Fig. 2. Sequence-specific N O E s as observed in the R O E SY -spectrum .
4
H. B eiderbeck et al. ■A New Pyoverdin from P seudom onas aureofaciens
Table I. ‘H -N M R -D ata of la.
2'
Suca
C hr
Ser 1
Ser 2
A cO rn
G ly 2
Gly 1
T hr 1
T hr 2
Gin
cO H O rn
3'
2
nh
2.78
2.73
9.81
1
2a
2b
3a
3b
4H N +
5.68
2.49
2.69
3.39
3.69
8.81
HN
a
ß
Y
Ö
acetyl
9.60
8.47
8.83
8.62
8.41
8.30
8.58
8.79
8.66
4.52
4.46
4.44
3.98
3.98
4.32
4.34
4.38
4.45
3.93
3.88
1.61/1.79
1.45
3.35
2.00/2.05
4.14
4.25
2.16
1.86/2.04
1.23
1.21
2.35
2.00
3.63
Table II. 13C -N M R -D A ta of la .
c o (r12'
177.5
31.7
30.7
178.9
C hr
CO
1
2
3
4a
5
171.4
58.2
23.1
36.2
150.6
118.7
6
6
7
8
9
10
10
115.3
144.8
152.5
101.4
133.0
Ö
acetyl
S er 1
S er 2
A c O rn
G ly 2
G ly 1
Thr 1
Thr 2
G in
c O H O rn
a
C O N H 2 (4 ')
140.3
116.1
CO
a
ß
Y
172.4
172.4
174.9
172.5
172.7
173.2
173.4
174.9
167.6
56.7
57.1
54.4
43.6
43.7
60.7
60.2
54.4
51.6
62.3
61.7
29.2
23.4
6 8 .0
2 0 .0
68.4
27.7
27.7
2 0 .0
32.2
2 1 .2
6
7
10
7.93
7.18
6.97
experim ent. It follows that the structure of the
new pyoverdin is la .
Suca
3'
5H N
48.0
Conclusion
a
175.1/20.3
178.9
52.7
by approxim ately 0.5 ppm (Risse et al., 1998). G lu
found in the am ino acid analysis is derived from
G in as shown by the chemical shift of the carbonyl
carbon at 174.9 ppm . The carbonyl group of the
free acid has a chemical shift of approxim ately
182.2 ppm (G eisen et al., 1992). The data for the
two O rn show that one of them is cO H O rn at the
C -term inus and the oth er one bears a hydroxam ate end group, the nitrogen being acetylated and
hydroxylated (A cO H O rn ).
From the m olecular mass and the N M R -data it
follows th at every am ino acid besides Gin is p re ­
sent twice in the peptide chain. Figure 2 shows the
sequence specific signals obtained in the ROESY-
l a is the first pyoverdin obtained from a Pseu­
dom onas aureofaciens culture. P. aureofaciens was
originally classified as biovar E of P. fluorescens,
but was sep arated as the m ost notable producer
of phenazine pigm ents (R öm er et a l, 1981), and
lateron com bined with P. chlororaphis, the original
biovar D (Palleroni, 1992), pigm ented by phenazine-l-carbam ide (chlororaphin). Several p hen­
azine derivatives w ere also obtained from cultures
of a P. fluorescens strain, especially when grown
under iron deficiency in the presence of rather
high concentrations of B e2+ (Taraz et al., 1990).
The only o th er species within the fluorescent
P seudom onas group that produces a phenazine
derivative is P. aeruginosa characterized by the betaine pyocyanin (responsible for the color of the
blue-green pus from which the bacterium had its
original nam e “Bacillus p yo cyaneus”) derived
from l-hydroxy-phenazine-5-oxide. Pyocyanin has
probably nothing to do with the iron transport. P.
fluorescens Y4, how ever, produces under iron defi­
ciency no pyoverdins, but rath er phenazine deriva­
tives containing the structural elem ent 2,3-dihydroxybenzoic acid, the constituent of catecholate
siderophores (Taraz et al., 1991). In the iron defi­
cient culture of the P. aureofaciens strain under
investigation no phenazines were observed. To our
know ledge, o th e r phenazine producers were not
H. B eiderbeck et al. ■A New Pyoverdin from P seudom onas aureofaciens
investigated in view o f th eir ability to use pyoverdins as siderophores.
Pyoverdins have been used before to establish
relationships betw een Pseudom onas spp. (Budzikiewicz et al., 1997). It seem ed, therefore, of in ter­
est to com pare the am ino acid sequence of la with
those of o th e r pyoverdins (Kilz et al., 1998). The
m ost frequently en co u n tered p attern am ongst P.
fluorescens strains show s at the N -term inus a small
n eutral am ino acid (A la, Ser) followed by Lys. The
N -term inus D-Ser-D -A cO H O rn was encountered
so far only once in a pyoverdin (which shows sim­
ilarities with l a insofar as it also has only neutral
am ino acids b etw een A cO H O rn and the C-terminal cO H O rn ) from a P. fluorescens (B arelm ann,
B raun S., K alinow ski H .-O . and B erger S. (1996), 100
and M ore Basic N M R Experim ents. V C H , W einheim.
B riskot G., Taraz K. and Budzikiewicz H. (1986), Siderop hore vom Pyoverdin-T yp aus Pseudom onas aerugi­
nosa. Z. N aturforsch. 41c, 497-506.
B riskot G., T araz K. and Budzikiewicz H. (1989), Pyoverdine-type sid ero p h o res from Pseudom onas aerugi­
nosa. Liebigs A nn. chem . 375, 375-384.
B udzikiewicz H. (1993), Secondary m etabolites from flu­
orescent pseudom onads. FEM S M icrobiol. Rev. 104,
2 0 9 -228.
B udzikiewicz H. (1997), S iderophores of fluorescent
pseudom onads. Z. N aturforsch. 52c, 41 3 -4 2 0 .
B udzikiewicz H., Kilz S., Taraz K. and M eyer J.-M.
(1997), Identical pyoverdines from Pseudom onas flu ­
orescens 9AW and Pseudom onas putida 9BW. Z. Naturforsch. 52c, 7 2 1 -7 2 8 .
Evans, J. N. S. (1995), B iom olecular N M R Spectroscopy.
O xford Univ. Press, O xford.
G eisen K., Taraz K. and Budzikiewicz H . (1992), N eue
Sideropore des Pyoverdin-Typs aus P seudom onas flu ­
orescens. M onatsh. C hem . 123, 151-178.
Jacques Ph., O ngena M., Gwose I., Seinsche D.,
Schröder H., D elfosse Ph., T honart Ph., Taraz K. and
Budzikiewicz H. (1995), Structure and ch aracterisa­
tion of isopyoverdin from P seudom onas putida BTP1
and its relation to the biogenetic pathw ay leading to
pyoverdins. Z. N aturforsch. 50c, 6 2 2 -629.
Kilz S., Lenz Ch.. Fuchs R. and Budzikiewicz H. (1998),
A fast screening m eth o d for the identification of sid­
ero p h o res from fluorescent P seudom onas spp. by li­
quid chrom atography/electrospray mass spectrom etry.
J. Mass. Spectrom ., in press.
M ichels J., B enoni H., B riskot G., Lex J , Schm ickler H.,
Taraz K., Budzikiew icz H.. Korth H. and Pulverer G.
(1991), Isolierung und spectroskopische C h arak te ri­
sierung des Pyoverdin-C hrom ophors sowie seines 5H ydroxy-A nalogen. Z. N aturforsch. 46c, 993-1000.
Palleroni N. J. (1992), “ Introduction to the family of
P seu d o m o n ad aceae” and “ H um an- and anim al-pa­
5
unpublished); D-Ser-D-FoOHOrn is the N -term i­
nus of the pyoverdin from one of the siderovars
of P. aeruginosa (Tappe et al., 1993). It is thus too
early to speculate w hether a second characteristic
pattern may evolve.
Acknow ledgem ents
The research was supported by the E u ro p ean
Com m ission D G X II u nder the project “Cell
factories for the production of bioactive peptides
from Bacillus subtilis and Pseudom onas"’ (Bio4CT95-0176). We wish to thank Dr. L. Pang, Insti­
tut für M icrobiologie, U niversität B onn, for the P.
aureofaciens-strain.
thogenic p seu d o m o n ad s” . The P ro k ary o tes (Balow s
A., Tröger H. G., D w orkin M., H a rd e r W. and H olt
J G., eds.), Springer, New York; vol. 3, ch ap ters 160
and 161.
Poppe K., Taraz K. and Budzikiewicz H. (1987), Pyoverdine type sid ero p h o res from Pseudom onas flu o ­
rescens. T etrah ed ro n 43, 2261-2272.
Risse D., B eiderbeck H., Taraz K.. B udzikiewicz H. and
G ustine D. (1998), C orrugatin, a lipopeptide siderop hore from P seudom onas corrugata. Z. N aturforsch.
53c, 2 9 5 -304.
R öm er A., Scholl H., Budzikiewicz H., K orth H. and
Pulverer G. (1981), Phenazine aus Pseudom onaden.
Z. N aturforsch. 36b, 1037-1046.
Schaffner E. M., H a rtm an n R., Taraz K. and B udzikie­
wicz H. (1996), S tructure elucidation of A zotobactin
87, isolated from A zo to b a cter vinelandii A TC C 12837.
Z. N aturforsch. 51c, 139-150.
Schafer H., T araz K. and Budzikiewicz H. (1991), Z u r
G enese d er am idisch an den C h ro m o p h o r von Pyoverdinen g eb u n d en en D icarbonsäuren. Z. N aturforsch.
46c, 398-406.
T appe R.. Taraz K., Budzikiewicz H.. M eyer J.-M. and
L efevere J F. (1993), S tructure elucidation of a pyov­
erdin pro d u ced by P seudom onas aeruginosa A R C C
27853. J prakt. C hem ie 335, 8 3 -8 7 .
Taraz K., Schaffner E. M., B udzikiewicz H., K orth H.
and P u lverer G. (1990), 2,3,9-T rihydroxyphenazin-lcarbonsäure - ein u n ter B eryllium einw irkung gebil­
detes neues P h enazinderivat aus P seudom onas flu o ­
rescens. Z. N aturforsch. 45b, 55 2 -5 5 6 .
Taraz K., Schaffner E. M., Budzikiewicz H., K orth H.
and P ulverer G. (1991), D ie Bildung hydroxylierter
Phenazine durch P seudom onas fluorescens Y 4 bei
B e2+-Z usatz zum K ulturm edium - ein A b w eh rm e­
chanism us? Z. N aturforsch. 46c, 194-196.
Teintze M.. H ossain M. B., B arnes C. L., L eong J. and
van d er H elm D. (1981), S tru ctu re of ferric pseudobactin, a sid ero p h o re from a plan t grow th p ro m o t­
ing Pseudom onas. B iochem istry 20, 6446-6457.