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Journal of Bacteriology, November 1999, p. 7131-7135, Vol. 181, No. 22
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Inactivation of the Tricarboxylic Acid Cycle
Aconitase Gene from Streptomyces viridochromogenes
Tü494 Impairs Morphological and Physiological
Differentiation
D.
Schwartz,*
S.
Kaspar,
G.
Kienzlen,
K.
Muschko, and
W.
Wohlleben
Mikrobiologie/Biotechnologie,
Eberhard-Karls-Universität Tübingen, D-72076
Tübingen, Germany
Received 25 June 1999/Accepted 8 September 1999
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ABSTRACT |
The tricarboxylic acid (TCA) cycle aconitase gene acnA
from Streptomyces viridochromogenes Tü494 was cloned
and analyzed. AcnA catalyzes the isomerization of citrate to isocitrate
in the TCA cycle, as indicated by the ability of acnA to
complement the aconitase-deficient Escherichia coli mutant
JRG3259. An acnA mutant was unable to develop aerial
mycelium and to sporulate, resulting in a bald phenotype. Furthermore,
the mutant did not produce the antibiotic phosphinothricin tripeptide,
demonstrating that AcnA also affects physiological differentiation.
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TEXT |
The structurally identical
antibiotics phosphinothricin tripeptide (PTT) and bialaphos are
produced by Streptomyces viridochromogenes Tü494 and
S. hygroscopicus (1, 9), respectively. They
consist of two molecules of L-alanine and one molecule of
the unusual amino acid phosphinothricin. In both organisms, several
proteins and genes involved in PTT biosynthesis have been characterized (4, 15, 16, 18). By analyzing bialaphos-nonproducing mutants
of S. hygroscopicus, a putative biosynthetic pathway was postulated consisting of at least 13 biosynthetic steps (summarized in
reference 18). In this pathway, the isomerization of
phosphinomethylmalate (step 7) was found to be similar to the aconitase
reaction of the tricarboxylic acid (TCA) cycle and it was speculated
that this step is catalyzed by the TCA cycle-specific enzyme reaction. All aconitases are characterized by a 4Fe-4S cluster at the catalytic site of the enzymes (5). Sequence analyzes of aconitase
genes from several bacteria, plants, and fungi suggest the existence of
two structural forms, called A and B, which differ in protein domain
structure (5). Whereas in type A aconitases domain 4 is
linked at the carboxy-terminal end of the protein, in type B aconitases
domain 4 presents the amino terminus (5). The genes of both
forms have been identified in several gram-negative bacteria, e.g., in
Escherichia coli (5) and Helicobacter
pylori (20). In this paper, we describe the isolation
and characterization of the TCA cycle aconitase gene of S. viridochromogenes. Our results suggest that the aconitase is
involved in the initiation of morphological and physiological
differentiation in S. viridochromogenes.
Identification and characterization of the TCA cycle aconitase from
S. viridochromogenes.
During the analysis of PTT
biosynthesis in S. viridochromogenes, the PTT biosynthetic
gene pmi (phosphinomethylmalate isomerase gene) was
identified, whose deduced gene product has 48% overall identity to
type A aconitases from E. coli and Legionella
pneumophila (14a). A 2-kb internal
EcoRI/SacI fragment of this gene was used to
isolate the aconitase gene of the TCA cycle by Southern hybridization against a 
phage library of S. viridochromogenes
(hybridization at 68°C using a DIG-DNA labeling kit [Boehringer];
stringent washing step with 0.5× SSC [1× SSC is 0.15 M NaCl plus
0.015 M sodium citrate]-0.1% sodium dodecyl sulfate). Approximately
6,500 phage clones were examined, and 33 hybridizing clones were
isolated. One phage clone (-ACN2) was characterized that carries a
1.6-kb hybridizing SacI DNA fragment which is not found in
the pmi locus of the PTT biosynthetic gene cluster. The
hybridizing SacI fragment and adjacent overlapping DNA
fragments of -ACN2 were subcloned in vector pUC18, and the
nucleotide sequence of a 4.3-kb DNA fragment was determined. The
analysis of this sequence by the codon usage program of Staden and
McLachlan (17) led to the identification of a single
complete open reading frame on one strand and an incomplete open
reading frame on the opposite strand named acnA and
murA', respectively. murA is located upstream of
acnA (Fig. 1) and encodes a
protein with similarity to MurA (UDP-N-acetylglucosamine
enolpyruvoyl transferase) from different bacteria. By cloning of a
0.7-kb PvuII/BglII fragment in the promoter probe
vectors pIJ486 and pIJ487, divergent DNA regions capable of promoting
transcription initiation were identified on this fragment (Fig. 1). By
homology search, the deduced AcnA protein with 931 amino acids clearly
resembled type A aconitases from bacteria, plants, and fungi, such as
AcnA of E. coli (55% overall identity) and Bacillus
subtilis (50% overall identity). The highest similarity was found
to the aconitases of S. coelicolor (17a) and
Mycobacterium avium (10) with identities of 89 and 66%, respectively. Conserved amino acid residues involved in the
formation of the 4Fe-4S cluster typical of this kind of enzyme
(3) were identified in the deduced AcnA amino acid sequence (Fig. 2).
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FIG. 1.
Genetic localization and gene insertion mutagenesis of
the aconitase gene acnA. The genetic organization of a
4.3-kb DNA fragment of -ACN2 carrying the acnA gene is
shown. acnA, gene encoding the TCA aconitase AcnA;
murA', 3' terminus of a murA-like gene from
S. viridochromogenes. Restriction sites used in subcloning
experiments are marked, and regions with promoter activity are
indicated by arrows. The kanamycin resistance cassette
(aphII gene from transposon Tn5) and insertion
sites used for inactivation of the acnA gene are shown.
Restriction sites destroyed by insertion of the cassette are bracketed.
The 0.7-kb PvuII/BglII fragment used in promoter
probe experiments is marked.
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FIG. 2.
Alignment of conserved regions of aconitases and iron
regulatory proteins, including AcnA and Pmi from S. viridochromogenes. The regions shown are located in domain 3 (amino acids 639 to 700) and domain 4 (amino acids 821 to 880) of
aconitases. Conserved amino acid residues are marked by inverse
letters. Corresponding to the structure of the pig heart aconitase
(3),  and  , respectively, indicate amino acids and
cysteine residues which are structurally conserved and involved in the
formation of the 4Fe-4S cluster at the catalytic site. AcnA_S.vir.,
AcnA from S. viridochromogenes; Pmi_S.vir.,
phosphinomethylmalate isomerase from S. viridochromogenes;
AcnA_Mycav, AcnA from M. avium; AcnA_E.coli; AcnA from
E. coli; CitB_B.sub., aconitase CitB from B. subtilis; AcnA_A.thal., AcnA from Arabidopsis thaliana;
Human_IRP1, human iron regulatory protein 1; Human_IRP2, human iron
regulatory protein 2; Acn_Por.he., porcine heart mitochondrial
aconitase.
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In order to verify the sequence data, an aconitase-deficient
E. coli mutant (
6) was used for complementation
experiments
with the
acnA gene. The
E. coli
mutant JRG3259 is characterized
by the inactivation of aconitase genes
acnA and
acnB (
6). The
mutant is
L-glutamate auxotrophic on glucose-containing minimal
medium. A 3.4-kb
SphI/
Acc65I DNA fragment
containing the native
acnA gene of
S. viridochromogenes was subcloned downstream of
the
lacZ
promoter, resulting in plasmid pDS91.
E. coli JRG3259
was
transformed with pDS91 and pDS89 (expression vector used as
a control).
A 20-ml volume of M9 minimal medium (
14) (with or
without 20 mM
L-glutamate) in a 100-ml Erlenmeyer flask containing
50 µg of kanamycin ml
1, 15 µg of tetracycline
ml
1, and 40 µg of chloramphenicol ml
1 was
inoculated with 0.2 ml of an overnight culture of plasmid-carrying
strain JRG3259 grown in Luria-Bertani medium. Incubation was performed
for 21 h at 37°C and 180 rpm. Induction of the
lacZ
promoter was
done with 1 mM
isopropyl-
-
D-thiogalactopyranoside (IPTG). At
different
times, growth was measured by determination of the optical
density at
580 nm. Under aerobic conditions, the
S. viridochromogenes acnA gene was able to partially complement the mutant, as
indicated
by an increase in the growth rate compared to that of the
nonsupplemented
mutant JRG3259 carrying the expression vector only
(Fig.
3).
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FIG. 3.
Complementation of aconitase-negative E. coli
mutant JRG3259. A culture of JRG3259 with acnA expression
plasmid pDS91 was incubated in M9 minimal medium with and without
supplementation with L-glutamate (L-glu) for 21 h. As
a control, JRG3259 with vector pDS89 was incubated under the same
conditions. At different times, growth was measured by determination of
the optical density (OD) at 580 nm.
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Analysis of the physiological role of AcnA.
In order to
examine the function of the AcnA aconitase, the acnA gene
was inactivated. Inactivation was carried out with the nonreplicative
plasmid pGK1, which carried an acnA gene fragment disrupted
by insertion of the aphII gene (Table
1). The transcription of the inserted
resistance gene aphII was oriented in the same direction as
that of the acnA gene (Fig. 1). E. coli plasmids used for transformation of S. viridochromogenes were
isolated from the methylase-negative strain E. coli ET12567
(11). Wild-type S. viridochromogenes was
transformed by polyethylene glycol-mediated transformation by using
this plasmid as described by Schwartz et al. (15). In
Southern hybridization experiments, kanamycin-resistant transformants
were identified showing a double-crossover event between the
chromosomal copy of the acnA and the mutated fragment of
pGK1. In contrast to the wild type, the acnA mutant (ACOA) was not able to develop aerial mycelium and to sporulate on yeast-malt (YM) medium (bald phenotype). The same phenotype was observed for the
wild type when the competitive aconitase inhibitor sodium fluoroacetate
(0.5% final concentration) was added to the YM medium, indicating that
the bald phenotype resulted from the inactivation of acnA.
In order to exclude the possibility that the bald phenotype of ACOA is
caused by a decreased pH value due to the excretion of organic acids,
filter disks soaked with Tris/HCl buffer (0.1 M, pH 8.0) were placed on
ACOA mycelium. Thereby, no aerial mycelium formation around the disks
was observed. Transformation of ACOA with plasmid pDS92 (Table 1)
restored the ability of the mutant to develop aerial mycelium, to
sporulate, and to product PTT. The genetic complementation results
indicate that the phenotype of the insertion mutation in
acnA is not a result of polar effects on genes located
downstream.
ACOA was also unable to produce the secondary metabolite PTT,
suggesting a defect in physiological differentiation. Addition
of
L-glutamate (5 to 25 mM) to the YM medium partially
restored
the ability of ACOA to develop white aerial mycelium but not
the
formation of green spores. The formation of aerial mycelium of
the
ACOA mutant growing on medium supplemented with
L-glutamate
was further examined by electron microscopy as described by Tillotson
et al. (
19). On YM medium without
L-glutamate,
substrate mycelium
with short hyphae was observed (Fig.
4B). Addition of
L-glutamate
at increasing concentrations resulted in the formation of longer
aerial
hyphae (Fig.
4C and D), as examined for the wild type (Fig.
4A). The
formation of rough spores, characteristic of wild-type
S. viridochromogenes (Fig.
4A), was not observed. However, on
YM
medium with 25 mM
L-glutamate, unusual structural elements
could be detected at the end of the hyphae, probably representing
deformed spores or aerial hyphae (Fig.
4D).
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FIG. 4.
Scanning raster electron microscopy of wild-type
S. viridochromogenes and the mutant ACOA. Aerial mycelium of
wild-type S. viridochromogenes and mutant ACOA grown on YM
medium with and without supplementation with L-glutamate
for 72 h was examined by raster electron microscopy. Panels: A,
wild-type S. viridochromogenes grown on YM; B, ACOA grown on
YM medium; C, ACOA grown on YM medium-12.5 mM L-glutamate;
D, ACOA grown on YM medium-25 mM L-glutamate.
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Not only morphological but also physiological differentiation is
affected by supplementation of the mutant with 25 mM
L-glutamate,
as indicated by the reduced amount of PTT
produced (approximately
10% of the wild-type
activity).
In other bacteria, the TCA cycle and aconitases also affect
morphological differentiation. In
B. subtilis, the influence
of
the TCA cycle, especially of aconitase CitB, on sporulation was
examined (
2,
7). Inactivation of
citB resulted in
mutants
which were defective in transcription of the expressed earliest
sporulation genes (
2). The activation of these sporulation
genes is dependent on the activated transcription factor Spo0A.
In a
citB mutant, activation of Spo0A by phosphorylation failed,
indicating that the accumulation of citrate or the subsequent
lack of
TCA intermediates may be responsible for the early sporulation
block.
It can be speculated that a similar mode of action is the
reason for
the inhibition of morphological differentiation in
S. viridochromogenes.
S. viridochromogenes possesses a second aconitase
activity.
Since mutant ACOA was able to grow on glucose minimal
medium (12) with 10 g of glucose per liter, a second
aconitase may be present in S. viridochromogenes. In order
to detect a putative residual aconitase activity, cells of the wild
type and mutant ACOA from comparable growth phases (24 to 96 h)
were examined for aconitase activity. A 150-ml volume of YM medium in a
500-ml Erlenmeyer flask was inoculated with 1.5 ml of homogenized cells of a 2-day (wild type) or a 5-day (ACOA) preculture and incubated in an
orbital shaker (180 rpm) at 30°C. Crude cell extracts were examined
for aconitase activity as described by Kennedy et al. (8).
Compared to the wild type, a residual aconitase activity of
approximately 7% was determined for mutant ACOA (Table
2). The disrupted acnA gene
encodes only a truncated AcnA protein missing domain 4, which has been
shown to be essential for the function of aconitases (5).
Considering the conserved protein structure of aconitases (3,
5), it seems unlikely that the residual activity is caused by
this protein. Up to now, it was not known, whether the activity is due
to a second aconitase or to an unrelated hydratase-dehydratase having a
broad substrate specificity that includes citrate. This protein seems
not to be encoded by the aconitase-like gene pmi of the PTT
biosynthetic gene cluster, as indicated by the ability of an acnA
pmi double mutant to grow on glucose minimal medium
(14a).
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TABLE 2.
Aconitase specific activity in crude cell extracts of
wild-type S. viridochromogenes and acnA mutant
ACOA from different
growth phases
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Nucleotide sequence accession numbers.
The nucleotide
sequences reported here have been assigned accession no. Y17270 and
Y17269 in the EMBL data library. The GenBank accession numbers of the
genes used for alignments are X82841, Z99113, X60293, AF002133, and
J05224, and the Swiss-Prot numbers are P21399 and P48200.
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ACKNOWLEDGMENTS |
This research was supported by the DFG (Graduiertenkolleg
Mikrobiologie) and by the BMBF (ZSP Bioverfahrenstechnik, D 3.2 E). G. Kienzlen was supported by a grant of the Konrad-Adenauer-Stiftung. Part
of this work was financed by a grant of the Fonds der Chemischen Industrie (no. 163607).
We are very grateful to Charles Thompson for communicating results
prior to publication and for helpful suggestions, to John R. Guest for
providing the E. coli aconitase mutant JRG3259, and to
C. F. Bardele and H. Schoeppmann for taking the electron micrographs.
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FOOTNOTES |
*
Corresponding author. Mailing address:
Mikrobiologie/Biotechnologie, Eberhard-Karls-Universität
Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen,
Germany. Phone: 49 7071 29-74638. Fax: 49 7071 29-5979. E-mail:
schwartz{at}molbio.biol.biologie.uni-tuebingen.de.
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Journal of Bacteriology, November 1999, p. 7131-7135, Vol. 181, No. 22
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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