268
HEINEMANN ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 1. (A) Alaremycin (5-acetamido-4-oxo-5-hexenoic acid). (B) Enzyme reaction of PBGS. PBGS catalyzes the first common step in
tetrapyrrole biosynthesis involving the asymmetric condensation of two 5-aminolevulinic acid molecules to form the monopyrrolic porphobilinogen.
(C) Alaremycin 2 (5-acetamido-4-oxo-5-hexanoic acid).
recorded. The MIC was determined as the lowest alaremycin concentration that
inhibited growth for 5 h.
Expression vectors for recombinant P. aeruginosa and Methanosarcina barkeri
PBGS genes. M. barkeri hemB was amplified from chromosomal DNA by PCR
Many organisms, including bacteria, produce dedicated an-
timicrobial compounds to ward off infections or competing
microbes. Streptomyces sp. A012304 hence produces the com-
pound alaremycin, thereby preventing growth in neighboring
microorganisms. Strategies to counteract the effect of alaremy-
cin on Streptomyces sp. A012304 itself have so far not been
described. We have investigated the molecular basis for the
antimicrobial activity of alaremycin by analyzing its activity
against Escherichia coli, Pseudomonas aeruginosa, Bacillus sub-
tilis, Bacillus megaterium, Streptomyces coelicolor, and Strepto-
myces avermitilis. As PBGS is the molecular target of alaremy-
cin, we studied the impact of the antibiotic on recombinant
Mg2ϩ-stimulated P. aeruginosa PGBS and Zn2ϩ-dependent
Methanosarcina barkeri PBGS. Furthermore, alaremycin was
cocrystallized with the PBGS from P. aeruginosa, and its was
structure solved at a resolution of 1.75 Å to describe its mode
of PBGS inactivation at the atomic level.
with primers 5Ј-CCGGAATTCCGGATGTTTCCAGATGTCAGGTTAAG-3Ј
and 5Ј-CCGCTCGAGCGGTTACTTCAACATGCGGGCAGC-3Ј, which con-
tained EcoRI and XhoI sites, respectively. The resulting 975-bp PCR product
was digested with EcoRI and XhoI and inserted into pET32a (Novagen, Madi-
son, WI) to create pET32aMbhemB. Vector pGEXhemB, which contained P.
aeruginosa PBGS was kindly provided by N. Frankenberg-Dinkel (University of
Bochum, Bochum, Germany) (10).
Protein production and purification. Recombinant P. aeruginosa PBGS was
produced and purified as described previously (10). Protein integrity was analyzed by
mass spectrometry and Western blot analysis. Recombinant M. barkeri PBGS was
produced by using E. coli strain BL21(DE3)RIL (Stratagene, Heidelberg, Germany)
in LB medium at 37°C and 180 rpm. At an optical density at 578 nm of 0.7, protein
production was induced by 150 M isopropyl-1-thio-ß-D-galactopyranoside (IPTG).
Cells were cultivated overnight at 17°C and 150 rpm, harvested, washed with buffer
A (50 mM Tris HCl, pH 8.5, 300 mM NaCl, 10 mM ZnCl2), and resuspended in a
minimal volume of buffer A. After cell disruption by sonication (HD 2070; Bandelin)
and centrifugation, (100,000 ϫ g for 45 min), the soluble fraction was applied to
Protino Ni-IDA agarose (Machery-Nagel, Du¨ren, Germany) and washed and the
PBGS was eluted with 300 mM imidazole in buffer A. Further purification steps
involved anion-exchange chromatography on a DEAE-Sepharose column (PBGS
was eluted with 200 mM NaCl in buffer A) and gel permeation chromatography on
a 30-ml Superdex 200 HR 10/30 column (0.5 ml/min; GE Healthcare) in buffer A.
The overall yield was ϳ9 mg of M. barkeri PBGS per liter of culture.
Enzyme activity assay. PBGS activity was quantified by a modified Ehrlich’s
test, based on the reaction between the product PBG with 4-(dimethylamino)-
benzaldehyde (10, 15). The Michaelis-Menten constant (Km), the maximal
velocity (Vmax), and the catalytic constant (kcat) were determined by measuring the
constant rate of PBG formation for 0 to 10 mM ALA and iteratively optimized
Lineweaver-Burk plots by using the SigmaPlot (version 8.0) and Enzyme Kinetics
(version 1.1) programs. The catalytic efficiency (kcat) was obtained by dividing
Vmax by the enzyme concentration. The activity of PBGS in cell extracts was
determined by adjusting protein concentrations to an A280 of 5 with buffer K1
(100 mM bis-Tris-propane, pH 8.5). As a control, cell extracts were inactivated
by heating to 95°C for 10 min, which resulted in negligible background activity.
Reactions without additional ALA were used to determine the concentration of
cellular PBG in the cell extracts.
MATERIALS AND METHODS
Materials. ALA was purchased from Merck (Darmstadt, Germany), por-
phobilinogen was purchased from Porphyrin Products (Logan, UT), and
other chemicals were purchased from Sigma-Aldrich (Hamburg, Germany).
Oligonucleotides were purchased from Metabion (Planegg-Martinsried, Ger-
many), Protino Ni-IDA resin was purchased from Machery-Nagel (Du¨ren,
Germany), and ethyl methanesulfonate was purchased from ABCR
(Karlsruhe, Germany).
Bacterial strains and growth conditions. To determine the antibacterial effect
of alaremycin, P. aeruginosa PAO1 was grown in AB minimal medium; Bacillus
subtilis JH642, S. coelicolor (DMS 40233), and S. avermitilis (DSM 46492) were
grown in Spizizen minimal medium; and E. coli CSA1 was grown in M9 minimal
medium containing 20 M hemin (20). B. megaterium (DSM 319) was grown in
MOPSO [3-(N-morpholino)-2-hydroxypropanesulfonic acid] minimal medium.
To quantify the activity of PBGS in cell extracts, S. avermitilis was cultured in 2%
oat meal, pH 7.2, at 30°C and 200 rpm for 4 days. S. coelicolor was grown in 0.4%
glucose–0.4% yeast extract–1% malt extract, pH 7.2, at 30°C and 200 rpm for 4
days. Streptomyces sp. A012304 was grown in seed medium (4% glucose, 1% dry
bouillon, 0.3% soybean meal, 0.3% CaCO3, pH 7.0) at 30°C and 200 rpm for 2
days. All other bacterial strains were grown in LB medium at 37°C for 20 h.
Isolation of alaremycin. For alaremycin production, Streptomyces sp. A012304
was cultivated in production medium containing 6% dextrin, 2% yeast extract,
0.3% NaCl, 0.3% CaCO3, 0.1% dry bouillon, and 0.1% K2HPO4, pH 7.0, at 30°C
and 200 rpm for 4 days. Alaremycin was isolated as described previously (2). It
was shown to be at least 98% pure, as analyzed by nuclear magnetic resonance
(NMR) spectroscopy (2).
Determination of inhibition constants. P. aeruginosa PBGS at 2 g/ml and M.
barkeri PBGS at 25 g/ml were diluted to a final absorbance of 5 at
with
280
kinetic buffer K1 (100 mM bis-Tris-propane, pH 8.5). ALA (40 mM) and alare-
mycin solutions were prepared in buffer K1. For P. aeruginosa PBGS, 10 mM
MgCl2 was added, and for M. barkeri PBGS, 10 mM ZnCl2 was added. Protein,
buffer, and alaremycin (0 to 10 mM) were mixed and incubated at 37°C for 10
min. Longer incubation times did not affect enzyme inhibition. Substrate (5 mM)
was added to start the reaction. The PBGS-catalyzed reaction was stopped at
times of between 1 and 60 min by adding equivalent volumes of stop reagent
(50% trichloroacetic acid, 100 mM HgCl2) to the reaction mixture. After cen-
trifugation (at 5,000 ϫ g for 3 min), the supernatant was treated with an equiv-
alent amount of Ehrlich’s reagent (0.4 g 4-dimethylamino benzaldehyde in 10 ml
acetic acid and 10 ml HClO4). After 15 min of incubation at room temperature,
the product was quantified by measurement of the absorbance at 555 nm (ε ϭ
60,200 MϪ1 cmϪ1). The alaremycin concentration that inhibited the enzyme
Determination of MICs. MICs were determined by microdilution techniques:
twofold dilutions (1 to 100 mg/liter) of alaremycin in double-distilled water were
placed in microtiter plates; and 108 cells/ml exponentially growing B. subtilis, B.
megaterium, P. aeruginosa, S. coelicolor, S. avermitilis, E. coli DH10b, and E. coli
CSA1 were added. E. coli strain CSA1 was incubated in the presence and the
absence of 50 g/ml hemin. For all strains, at least four independent curves of
growth in the presence of different alaremycin concentrations over 24 h were