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FEM.9Microbiology Letters 132 (1995) 9-l 5
High-molecular-mass, iron-repressed cytoplasmic proteiris in
fluorescent Pseudomonas: potential peptide-synthetases for
pyoverdine biosynthesis
Claude Georges, Jean-Marie Meyer *
Luboratoire
de Microbiologic.
Unite’de Recherche Associk
au Centre National de la Recherche Scientifique No. 1481, 28 rue Goethe,
Uniuersite’ Louis-Pasteur,
67ooO Strasbourg, France
Received 24 April 1995; revised 1 June 1995; accepted 3 June 1995
Abstract
High molecular-mass cytoplasmic proteins were detected in iron-starved, pyoverdine-producing Pseudomonas aeruginosa, P. chlororaphis, P. fluorescens, P. pufih, P. apt&a and P. tofuasii.They appeared to be specifically located in the
cytoplasm and thus were termed ‘IRCPs’, for iron-repressed cytoplasmic proteins. A strain-dependent gel electrophoresis
pattern with multiple bands of M, values ranging from 180 to 600 kDa was usually observed for these proteins. Strains
synthesizing pyoverdines differing in their peptide part presented different IRCP gel electrophoresis profiles, whereas strains
synthesizing identical pyoverdines had identical IRCP gel electrophoresis profiles. Some mutants affected in pyoverdine
biosynthesis presented a perturbed IRCP pattern, and no IRCPs were detected in non-fluorescent Pseudomonasstrains either
unable to synthesize siderophores or synthesizing non-peptidic siderophores. The data strongly suggest that the IRCPs could
be related to peptide synthetases involved in the biosynthesis of the peptidic part of pyoverdine-type siderophores.
Keywords: Pseudomonas
spp.; Iron metabolism; Fyoverdine biosynthesis; Peptide synthetases
1. Introduction
Pyoverdines,
the fluorescent pigments and main
siderophores
of fluorescent
Pseudomonas species
[l], share the presence of a peptidic chain in their
structures. More than 20 pyoverdines produced by
different strains of Pseudomonas have been purified
and the structures of most have been determined.
With the exception of two type strains, P. jluorescens ATCC 13525 and P. chlororaphis ATCC
* Corresponding author. Tel.: +33 88 24 41 50; Fax: 88 35 84
84.
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1995
9446, which produce a structurally identical pyoverdine, each fluorescent
Pseudomonas type strain
studied so far synthesizes
a specific pyoverdine:
these differ from one another in the amino acid
composition of their peptidic parts [2]. This diversity
in structure is even found within a single species, as
structurally different pyoverdines have been recognized among P. aeruginosa or P. jluorescens strains
[2-41. The peptide part of pyoverdine is linked to a
quinoleinic chromophore which is apparently always
identical, whatever the producing strain [2]. Depending on the pyoverdine,
the length of the peptide
chain may vary from 6 to 12 aminoacyl residues [2],
with some of them participating in the complexing of
Federation of European MicrobiologicalSocieties. All rights reserved
IO
C. Georges. J.-M. Meyer / FEMS Microbiology
iron, the others being presumably
involved in the
specific recognition of the ferripyoverdine
receptor
[4]. Another special feature of the peptide chain is
the presence of o-amino acids together with L-amino
acids. These amino acids usually form linear chains
with possible internal loops, e.g. the pyoverdines of
P. ueruginosa ATCC 15692 or P. fluorescens strain
12 [2].
The peculiarities
found in the peptide chain of
pyoverdine raises questions about its biosynthesis.
Because of their short length, unusual amino acid
composition and possible internal cyclic structures,
these peptides appear to be structurally closely related to peptidic antibiotics of microbial origin such
as the gramicidins or tyrocidine produced by Bacillus spp. [5], actinomycin from Streptomyces spp. [6],
or syringomycin and syringotoxin produced by strains
of P. syringae [7,8]. Thus, the pyoverdine peptides
may be synthesized through a multi-enzyme thiotemplate mechanism
involving peptide synthetases as
demonstrated
for the antibiotics
[5], rather than
through a classical ribosomal pathway. Moreover, a
multi-enzyme
system catalysing the biosynthesis of
the cyclic
hexapeptide
ferrichrome,
a fungal
siderophore, has already been characterized [9], as
have some amino acid-activating enzymatic activities
in Azotobacter, which suggest the involvement of a
non-ribosomal
pathway for the biosynthesis of the
pyoverdine-like
azotobactin
siderophore
[IO]. Finally, the recently published nucleotide sequence of
gene pcdD involved in pyoverdine biosynthesis in
P. ueruginosu demonstrated clear similarities with a
range of bacterial and fungal antibiotic peptide synthetase genes [ 1 I], again strongly suggesting a nonribosomal way of biosynthesis for pyoverdine. In this
paper, we report on the search for such pyoverdinerelated peptide-synthetases
in fluorescent
Pseudomonas spp., undertaken
by looking for high M,
soluble proteins, a general feature of antibiotic-related peptide synthetases [5].
2. Materials and methods
2. I. Bacterial strains and growth conditions
The fluorescent
Pseudomonas
strains
used
throughout this work were mainly collection strains
Leirers 132 (1995) 9- 15
as P. ueruginosu
ATCC 15692, P.
ATCC 27853, P. putidu ATCC 12633,
P. fluorescens ATCC 13525, P. fluorescens
ATCC
17400, P. fluorescens CCM 2798, P. chlororaphis
ATCC 9446, and P. tolaasii NCPPB 2192. P. fluorescens strain 12, P. f7uorescens strain ii, and P.
aptata strain 4a were from the laboratory collection
of H. Budzikiewicz (University of Kiiln, Germany)
and 10 P. aeruginosa clinical isolates (strains Pa3 to
Pal2), with pyoverdines previously characterized [3],
were from G. Wauters (St. Luc Hospital, Brussels,
Pseudomonas
included
Belgium). Non-fluorescent
P. fragi ATCC 4973, P. stutzeri ATCC 17588 and
P. diminuta CIP 7129. The P. ueruginosu pyoverdine-deficient
mutants
PA06601,
PA06606,
PA06609,
PA06622,
PA06624
and their parent
strain PA06049,
a methionine-auxotroph
of P.
ueruginosu ATCC 15692 (PA01 strain), have been
described elsewhere [ 121. Iron-starved cells were obtained from 40-h cultures grown in succinate medium
[3] at 25°C with shaking (200 rpm). Iron-replete cells
were obtained from cultures grown under identical
conditions in succinate medium supplemented, after
sterilization, with 100 PM FeCl,. The PA0 mutants
were grown in 1 mM methionine-supplemented
succinate media as required [ 121.
referenced
ueruginosa
2.2. Detection of the IRCPs
Bacterial protein extracts from stationary phase
cells were prepared and partially purified by using a
procedure described for the purification of antibiotic
peptide synthetases [6], consisting of disruption of
the cells using a French press or by sonication in 0.1
M phosphate buffer (pH 7.0). Partial purification of
the soluble proteins was then obtained by a treatment
with polymine P (O.l%, final concentration),
followed by an overnight 50% saturation ammonium
sulfate precipitation
at 4°C. The resulting protein
precipitate was solubilized in 0.125 M Tris HCl
buffer (pH 6.8) and, after protein quantification
by
the Lowry method [13], subjected to SDS-PAGE
according to Laemmli [14] with 5% polyacrylamide
gels. Detection of the protein bands was performed
with the conventional Coomassie brilliant blue R-250
reagent or by more sensitive procedures, i.e. a silver
staining [15] or a modified Coomassie brilliant blue
staining [ 161. A selective purification of the periplas-
C. Georges, J.-M. Meyer/ FEMS Microbiology Letters 132 (1995) 9-15
mic proteins of P. aeruginosa ATCC 15692 was
done according to [17].
3. Results and discussion
3.1. Detection of high M,, iron-regulated cytoplasmic proteins in P. aeruginosa ATCC 15692
When the soluble proteins of the pyoverdine producer P. aeruginosa ATCC 15692 (PA01 strain)
were analysed by SDS-PAGE (5% polyacrylamide),
high M, proteins were observed (Fig. 1). Depending
on the bacterial growth conditions and on the amount
of protein loaded on the gel, five protein bands
ranging from 185 to 550 kDa were stained by the
Coomassie brilliant blue reagent. These bands were
not visible when gels were loaded with less than 100
pg protein. They were detectable and increased in
intensity at higher protein concentrations, appearing
PA0
PA0
P.chl.
P.chl.
P. n.
P.rl.
15692
15692 9446
9446
13525
13525
P.SWw.. P.SlUlz.
17588
17588
kDa
II
as pronounced bands when gels were loaded with
400 Fg protein per lane. As shown in Fig. 1, these
bands were observed in bacteria grown under iron
starvation (succinate medium), but were never visible in cell extracts obtained from bacteria grown in a
100 PM iron-enriched succinate medium. For intermediate iron concentrations in growth medium, the
high M, proteins were visible at roughly the same
intensity over the range of O-4 PM added iron and
not seen at higher iron concentrations. It should be
emphasized that the O-4 PM iron concentration
range in succinate growth medium allows the production, by P. aeruginosa ATCC 15692, of pyoverdine and the synthesis of a bacterial outer membrane
protein acting as ferri-pyoverdine receptor [18]. For
higher iron concentrations, all these biosyntheses
(high molecular mass proteins, siderophore, outer
membrane receptor) were severely repressed. Thus,
the soluble high molecular mass proteins appeared to
be under the same iron control as the siderophore
and the ferri-siderophore receptor. Analysis of the
periplasmic proteins of P. aeruginosa ATCC 15692
did not reveal any high M, proteins (data not shown).
Thus, the large proteins should be located in the
cytoplasm only. For convenience, we propose to
name these high M, proteins ‘IRCPs’, for iron-repressed cytoplasmic proteins, by analogy with the
term ‘IROMPs’ which is commonly used to designate iron-regulated outer membrane proteins expected to act as ferri-siderophore receptors.
3.2. lRCPs in other Pseudomonas strains
-
w
Fig. 1. Iron-repressed cytoplasmic proteins (IRCPs) in Pseudomonas. Bacterial extracts (400 pg protein) of P. aeruginosa
ATCC 15692 (PA0 15692). P. chlororuphis ATCC 9446 (P.
chlor. 9446). P. fruorescens ATCC 13525 (P. fl. 13525) and P.
stutzeri ATCC 17588 (P. stutz. 17588) grown in succinate medium
( -1, or in 100 PM FeCl,-supplemented succinate medium ( + ),
were analysed by SDS-PAGE (5% polyactylamide) according to
Laemmli [14]. The electrophomtic mobility of high molecular
protein markers (Pharmacia) with size in kDa am indicated on the
right side. Electrophoresis conditions were maintained during a
supplementary hour after the dye had reached the bottom of the
gel.
Initially determined for P. aeruginosa ATCC
15692, the 400 pg protein load for SDS-PAGE was
convenient to observe well formed IRCP bands for a
majority of other pyoverdine producer Pseudomonas
strains, as illustrated in Fig. 1 for P. chlororuphis
ATCC 9446 (lane 3) and P. jluorescens ATCC
13525 (lane 51, and also seen for P. aptata 4a, P.
j7uorescens ATCC 17400, P. jluorescens CCM 2798,
P. jluorescens 12, P. jluorescens ii (data not shown).
In some cases, however, as in P. aeruginosu ATCC
27853 and P. putida ATCC 12633, these proteins
appeared as diffuse or very faint bands, even with a
400 pg protein load. Higher amounts of proteins did
not result in a better visualization of these bands and
disturbed the electrophoresis pattern. The use of
12
C. Geoqes, J.-M. Meyer/ FEMS MicrobiologyLetters132 11995) 9-15
more sensitive methods for gel protein detection, i.e.
a silver staining [15] or a modified Coomassie blue
staining [16], likewise did not result in better visualization. Nonetheless, the general conclusion of these
studies was that all the pyoverdine-producing
strains
analysed synthesized
IRCPs. Usually they all responded to iron supplementation
as P. aeruginosu
ATCC 15692 did, with no high M, protein bands
detectable for cells grown in presence of 100 PM
added iron, as shown in Fig. 1 for P. aeruginosa
ATCC 15692 (lane 2) and P. fluorescens ATCC
13525 (lane 6). P. chlororuphis ATCC 9446 was the
only strain which showed a high M, protein, with a
molecular mass different from the two major IRCPs
detected in this strain (370 kDa and 530-480 kDa,
respectively), specific for iron-fed cells (Fig. 1, lane
4). In this respect, P. chlororaphis behaved the same
cytoas P. syringae, which produces iron-inducible
plasmic proteins related to syringotoxin
and syringomycin synthesis [7,8]. For all the pyoverdineproducing strains analysed, IRCP biosynthesis correlated well with pyoverdine biosynthesis
and both
syntheses appeared to be under the same iron regulation.
Additional evidence for a relationship
between
IRCPs and pyoverdines came from analyses of the
cytoplasmic protein content of several pyoverdinedeficient mutants of P. aeruginosa PA06049,
a
methionine auxotroph of P. aeruginosu ATCC 15692
(PA01 strain). The mutants analysed belonged to the
two mutation groups recognized previously [ 121, harbouring mutations at the 23-min region of the bacterial chromosome (35 min in a former map, strains
PA06601 and PA06622),
or at the 47-49 min region (65-70 min) for strains PA06606, PA06609
and PA06624. As shown in Fig. 2, PA06601 presented an IRCP profile which lacked the two 550and 480-kDa upper bands and showed only a faint
band at 250 kDa when compared to the parent strain
PA06049; the two other IRCPs (290 and 185 kDa)
were apparently not affected. However, PA06622,
which belongs to the same mutation
group as
PA06601, presented the same IRCP profile as the
parent strain (data not shown), suggesting for this
strain a mutational event not related to the synthesis
of the peptidic part of the pyoverdine
molecule.
Mutants of the second group presented a similar
heterogeneity since PA06606 gave an identical IRCP
PA0
PA0
PA0
PA0
PA0
PA0
PA0
PA0
fxiol
-
6601
+
6ah
-
6606
6609
-
6609
+
6019
-
601’)
+
+
Fig. 2. Iron-repressed cytoplasmic proteins (IRCPs) in the pyoverdine-deficient
mutants PAO6601,
PA06606,
PA06609
their pyoverdine producer parental strain PAO6049.
and in
Electrophore-
sis, bacterial growth conditions. symbols and markers are the
same as in the legend of Fig.
I,
excepted that electrophoresis was
stopped when the blue dye had reached the bottom of the gel, and
that the growth media were supplemented with
1 mM
methionine
according to Hohnadel et al. [ 121.
profile as PA06049
(Fig. 2), whereas PA06609
(Fig. 2) and PA06624 (not shown) were characterized by an almost complete absence of the 250-kDa
IRCP, the other IRCPs being unchanged.
3.3. Correlations between peptidic siderophore-reluted IRCPs and peptide synthetuses
In order to determine if the IRCPs are directly
related to pyoverdine biosynthesis
and if they are
specific to peptidic siderophore-producing
bacteria,
we extended the search of IRCPs to a strain known
to produce a non-peptidic siderophore, i.e. the desferriferrioxamine
E-producer
P. stutzeri ATCC
17588 [ 191, and to wild-type strains which apparently
do not produce siderophores under iron starvation,
such as P. diminuta CIP 7 129 or P. frugi ATCC
4973 [l]. As shown in Fig. 1, lanes 7-8, no IRCPs
were detectable in iron-starved
P. stutzeri ATCC
17588 cell extracts, even at the maximal 400 kg
protein load. The non-siderophore-producing
strains
reacted the same: cells of P. diminutu and P. frugi,
which were iron-deficient as judged by their lower
C. Georges, J.-M. Meyer/ FEMS Microbiology Letters 132 (1995) 9-15
growth yield in succinate medium compared to
growth in iron-supplemented medium, were devoid
of IRCPs, just like P. stutzeri (not shown).
A comparison
of the IRCP profiles of
pyoverdine-producing
Pseudomonas strains as a
function of the structure of the pyoverdine they
produce disclosed siderophore-specific profiles of
IRCPs. As shown in Fig. 1, strains which are known
to produce pyoverdines, differing by their peptide
chain, presented a different number of IRCPs: four
major IRCP bands were detectable for P. aenrginosa
ATCC 15692, whereas P. C~~OFOFC-@~S ATCC 9446
only showed two major IRCPs. Other examples are
found in Table 1, where both the number of IRCPs
13
recognized for each strain and the peptidic structures
of the respective pyoverdines are given. Conversely,
two strains producing the same pyoverdine presented
identical IRCP profiles. This is illustrated in Fig. 1
for P. chlororaphis ATCC 9446 and P. jluorescens
ATCC 13525. The same conclusion held when comparing IRCP profiles of several P. aeruginosa isolates belonging to an identical pyoverdine group:
strains Pa5, Pa& Pa9, Pal0 and Pall, which have
been shown to elaborate identical pyoverdines and
pyoverdine uptake systems as P. aeruginosa ATCC
15692 [3], presented exactly the same IRCP profile
as their type-strain (data not shown), whereas strains
Pa3, Pa4 and Pa7, belonging to the pyoverdine group
Table 1
Pyoverdines and IRCPs in various fluorescent pseudomonas species
Stitl
Numberof Number Apparentmolecularmass
a.a.resfdues OflRBs
OffRCPs &Da+
. _
sequenceof thepyoveldinepepddea
P. aer~@~sa Pa6
(Chr)-Srt-Dab_FooHom-Gln-GlprFoOHaCp-CIy
c
6
4
520,xlo. 465.330
P. optatasmin 4a
(Chr)-dir-L~Tbr_SIl-AcOHOm-cOHOHOm
6
2
430.400
P. uemginosa ATCC 27853 (au)Scc_FooHorn-OrGl~~-~r~OHOm
7
3
550,470.3od
P. ch&nw&&ATCC 9446 (~~-L~-Cly-FoOfIOnr-L~~HOrnSerC
7
3
530.480.180
P. jluonwcenr ATCC! 13525 (Cln)-gCr-LJlcly-F-L~~HOm~J
7
3
SM. 480.180
P. wngines~ ATCC 15692 (au)Srr-Arlr-SrC-OHOm-~~OHOm-Thr-Thrl
a
5
550.480,290.250,185f
9
4
520,510,4MJ380
P.j7uorescensATCC17400 (Chr)-Ala-Lys-GIy-GIy-O~pGln-D~er-~8-~HOm
8
P. jhwesrenr CCM 2798
(Chr)Scr-~~Gly~r~~A~-Gly-~-Gly~OHOm
9
4
530.sclo.330,240
P. tohsii NCPPB 2192
(~)_Src-Lurser-Src_Tar-~-~HOm-~r~~~
10
5
550.510.460.400.360
P.j78lOrescenssuain 12
(chr>src-Lys-Cb_FoOHOm~~-Gl~-LfbF
11
4
480.440.380.310
P.jlmescens
(Chr)-~-~~lyly-cly-O~~-~-A~-~-~-~~Orn
12
5
600,470.440,230,210
strainii
a See [2,20] for sources. BAmino acids are underlined. Abbreviations:Chr, chtomophore; OHOm, 6N-hydroxy Om; cOHOm, cycle-OHOm;
OHAsp, threo-Shydroxy Asp; aThr, alloThr; FdAc,Bu)OHOm, 6N-formyl (ace@, Phydroxy butyryl) OHOm, Dab, diaminobutyric acid.
h Average values obtained from two to ten independent experiments, depending on the strain (see also footnote 0.
’ Structure determined for the pyoverdine of strain P. aeruginosa R [2]. The pyoverdine of strain Pa6 has an identical structure (R. Tappe,
personal communication).
d Values obtained for strains P. aeruginosa Pa3, Pa4 and Pa7 which produce the same pyoverdine as P. aeruginosa ATCC 27853 [3].
e H. Budzikiewicz and K. Taraz, personal communication.
‘Average values with standard deviations (10 independent experiments) were 550 f 37. 480 f 22, 290 f 17, 250 f 22 and 185 f 18,
respectively. Strains P. aeruginosa Pa5, Pa8, Pa9, Pal0 and Pal 1, which produce the same pyoverdine as P. aeruginosa ATCC 15692 [3],
displayed the same IRCP profile.
g Stereochemistry partially determined or unknown.
14
C. George& J.-M.
Meyer/
FEMS Microbiology
P. aeruginusa
ATCC 27853 [3])
presented all the same IRCP gel pattern, with bands
at 550, 470 and 300 kDa. Another strain belonging
to the same group (Pa12) and the type strain itself
gave too faint and diffuse bands on SDS gels to
accurately estimate their apparent molecular mass.
They appeared, however, at positions corresponding
to the cited mass values. Attempts to correlate the
number of IRCPs detected in one strain with the
peptide length of its pyoverdine roughly suggested
that the longer peptide chain, the greater the number
of IRCPs: strains with pyoverdines
having 6 or 7
aminoacyl residues presented the lowest number of
IRCPs (middle average of 3), whereas strains with
pyoverdines
exceeding 7 amino acids had 4 or 5
IRCPs.
Finally, a last correlation between IRCPs, peptidic
siderophores and peptide synthetases concerned the
unusual high IV, observed for both kinds of proteins.
Antibiotic-related
peptide synthetases are characterized by a M, higher than 100 kDa, often reaching
the 200-500-kDa
range [5]. Examples close to our
work concern the production of the antibiotics syringomycin and syringotoxin by the pyoverdine-producing P. syringae pv. syringae. Five proteins ranging from 130 to 470 kDa are involved in syringomycin production [8], whereas two proteins of
M, 470 and 435 kDa were related to syringotoxin
production [7]. Moreover, at least four other large
proteins in the range of 200-400 kDa were described
in that strain and tentatively related to pyoverdine
synthesis since, unlike the antibiotic-related
proteins,
they were visible only in iron-deficient cells [7]. The
IRCP IV, values, as estimated in the present work,
appeared within the same order of magnitude, ranging from 180 to 600 kDa (Table 1). Interestingly, a
molecular size of 1100 kDa has been determined by
gel filtration for ferrichrome synthetase, the only
multi-enzyme
complex involved in a non-ribosomal
synthesis
so far characterized
for a peptidic
siderophore [9].
In conclusion, the presence of high A4, iron-repressed cytoplasmic proteins in all the pyoverdineproducing Pseudomonas strains so far analysed, together with their absence in strains devoid of pyoverdine production, as well as the multiple correlations
between these proteins and pyoverdine structures and
regulation, strongly suggest that IRCPs are peptide
II (type strain
Letters 132 (1995) 9-15
synthetases which are specifically involved in the
biosynthesis of the peptidic chain of pyoverdines. It
should be emphasized that during the completion of
this work the gene pvdD involved in the biosynthesis of pyoverdine in P. aeruginosa OTll (a strain
related to P. aeruginosa ATCC 15692; I.L. Lamont,
personal communication)
has been fully sequenced
[ 111. Although the gene product and its enzymatic
function remain unknown, the multiple similarities of
this gene with peptide synthetase genes strongly
suggests that pvdD indeed codes for a peptide synthetase. The length of the gene (7374 bp) allows a
deduced M, of 273 kDa for the PvdD protein, a size
which is within the range of the IRCP h4, values and
close to the estimated 290 5 17 and 250 + 22 kDa
IRCPs (Table I) found in P. aeruginosa ATCC
15692.
Acknowledgements
We are indebted to H. Kleinkauf and H. von
Dohren for valuable discussions and encouragements
and to I.L. Lamont for providing us with the pvdD
sequence before publication. C.F. Earhart is particularly acknowledged for advice and manuscript improvement, and H. Budzikiewicz,
K. Taraz and R.
Tappe for strains and pyoverdine structures. This
work has been done with the technical assistance of
G. Seyer.
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