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Applied and Environmental Microbiology, July 2003, p . 3777-3783, Vol . 69, No . 7
Construction of an Expression System for Site-Directed Mutagenesis of the Lantibiotic Mersacidin
Christiane Szekat,1 Ralph W . Jack,2, Dirk Skutlarek,3 Harald Färber,3 and Gabriele Bierbaum1*
Institut
für Medizinische Mikrobiologie und Immunologie der
Universität Bonn,1
Hygiene-Institut der
Universität Bonn, D-53105 Bonn,3
Institut für
Organische Chemie der Universität Tübingen, D-72076
Tübingen, Germany2
Received 26 December 2002/
Accepted 31 March 2003
The
lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an
antimicrobial peptide of 20 amino acids which is produced by
Bacillus sp . strain HIL Y-85,54728 . Mersacidin inhibits
bacterial cell wall biosynthesis by binding to the precursor molecule
lipid II . The structural gene of mersacidin (mrsA) and the
genes for the enzymes of the biosynthesis pathway, dedicated
transporters, producer self-protection proteins, and regulatory factors
are organized in a biosynthetic gene cluster . For
site-directed mutagenesis of lantibiotics, the engineered genes must be
expressed in an expression system that contains all of the factors
necessary for biosynthesis, export, and producer self-protection . In
order to express engineered mersacidin peptides, a system in which the
engineered gene replaces the wild-type gene on the chromosome was
constructed . To test the expression system, three mutants were
constructed . In S16I mersacidin, the didehydroalanine residue (Dha) at
position 16 was replaced with the Ile residue found in the closely
related lantibiotic actagardine . S16I mersacidin was produced only in
small amounts . The purified peptide had markedly reduced antimicrobial
activity, indicating an essential role for Dha16 in biosynthesis and
biological activity of mersacidin . Similarly, Glu17, which is thought
to be an essential structure in mersacidin, was exchanged for alanine.
E17A mersacidin was obtained in good yields but also showed markedly
reduced activity, thus confirming the importance of the carboxylic acid
function at position 17 in the biological activity of mersacidin.
Finally, the exchange of an aromatic for an aliphatic hydrophobic
residue at position 3 resulted in the mutant peptide F3L mersacidin;
this peptide showed only moderately reduced
activity .
Mersacidin belongs to a group of antibacterial peptides which are
designated lantibiotics because they contain the rare amino acids
lanthionine and/or methyllanthionine
(8) . In contrast to
peptide antibiotics produced by nonribosomal biosynthesis, lantibiotic
precursors are encoded by structural genes and the rare amino acids are
synthesized by posttranslational modification of the prepeptides . The
various enzymes of the biosynthetic pathway, dedicated product
transporters, producer self-protection proteins, and regulatory factors
are encoded in biosynthetic gene clusters, which are found either on
the chromosome or on mobile elements such as plasmids or transposons
(25) . Recently it has
been shown that the biosynthetic gene cluster of mersacidin is located
on the chromosome of the producer strain Bacillus sp . strain
HIL Y-85,54728
(1) .
Since
lantibiotics are gene-encoded peptides, the exchange of single amino
acids by site-directed mutagenesis is possible . However, in the case of
lantibiotics it is not sufficient to simply express a single engineered
gene in an Escherichia coli expression system, since the
modification machinery is indispensable for the biosynthesis of the
lanthionines and other modified amino acids . In addition, lantibiotics
are bacteriocins, and therefore the producer strain has to be protected
against its own product by the self-protection (or immunity) factors.
Systems that meet these requirements have been successfully constructed
for the expression of the lantibiotics subtilin
(20), nisin
(11,
12,
17,
19), Pep5
(4), epidermin
(22), and mutacin II
(9) .
The activity
spectrum of mersacidin includes several gram-positive bacteria, with
staphylococci among the most sensitive organisms . The peptide acts by
inhibition of bacterial cell wall biosynthesis via interaction with
lipid II, the membrane-bound monomeric cell wall precursor
(6,
7) . Both the crystal and
solution structures of mersacidin have been solved
(24,
28) . Mersacidin is a
compact globular molecule . In methanol as well as in the crystal, the N
terminus folds back in order to interact with the carboxyl group of the
glutamic acid residue at position 17 . Other lantibiotics that resemble
mersacidin with respect to their mode of action are actagardine and
ala(0)-actagardine, which are produced by Actinoplanes
gabardinensis and Actinoplanes liguriae
(23,
30) . Moreover, with the
exception of an exchange of didehydroalanine (Dha) for Ile at position
10 in actagardine and position 16 in mersacidin, the amino acid
sequence of the third ring of mersacidin and the second ring of
actagardine is conserved and has been suggested to constitute the
binding pocket for lipid II
(33) . However the
activity spectrum of actagardine is markedly different from
that of mersacidin; actagardine is most active against
streptococci and displays only low activity against
staphylococcal species .
Here we describe the development of an
expression system that enables the production of engineered mersacidin
peptides, as well as the properties of the first mersacidin
variants .
Bacterial strains and
plasmids.
The strains and
plasmids used for construction of the expression system are described
in Table
1 . The E . coli strains used for mutagenesis were grown on
Luria-Bertani agar, Staphylococcus carnosus was maintained on
blood agar or tryptone soy agar, and Bacillus strains were
cultured on nutrient broth or tryptone soy agar, always in the presence
of the appropriate antibiotics . All strains were stored at
-70°C as glycerol
cultures .
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TABLE 1 . Bacterial
strains and plasmids used in this study
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Site-directed
mutagenesis.
Site-directed
mutagenesis was performed with a commercial phagemid system (Altered
Sites in vitro mutagenesis system; Serva, Heidelberg, Germany)
according to the instructions of the manufacturer . The 1.14-kb
EcoRI-KpnI fragment containing the structural gene of
mersacidin (mrsA)
(3) and the 5'
part of mrsR1 (1)
was cloned into the multiple cloning site of the pALTER-1 vector
provided in the mutagenesis kit . The mutagenic oligonucleotides
employed for site-directed mutagenesis are shown in Table
2 . All mutated genes were confirmed by sequencing on an A.L.F . express
sequencer (Pharmacia Biotech, Freiburg, Germany) or by Sequiserve
(Vaterstetten, Germany) .
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TABLE 2 . Primers
used in this study
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DNA was prepared by using Qiagen genomic
tips, Qiaprep spin miniprep kits, or Qiagen plasmid minikits according
to the recommendations of the supplier (Qiagen, Hilden, Germany).
pTV0/pTV0[MCS] or pTV0/pTV0[MCS] recombinant
plasmids carrying engineered mrsA genes were purified from 50
ml of S . carnosus culture by using the HiSpeed
plasmid midikit (Qiagen) . General protocols were used for cloning
strategies and enzymatic DNA modification
(26) . Digested DNA
fragments were eluted from agarose gels with the QIAquick gel
extraction kit (Qiagen) .
The Bacillus sp . and
Staphylococcus carnosus TM300 were transformed by protoplast
transformation (13,
14), while E.
coli strains were transformed by electroporation . Clones were
screened by plasmid preparations or, in the case of
Bacillus, by PCR (Table
2) performed with AGS Gold
DNA polymerase (Hybaid-AGS, Heidelberg, Germany) in a PCRExpress
thermocycler (Hybaid) with genomic DNA as a template . For competitive
nucleotide priming, a pair of Mut and Umut oligonucleotide primers was
synthesized for each mutation . These primers served as backward
primers, with the 3' terminus annealing to the base which had
been chosen for mutagenesis . Therefore, the sequences of the Mut and
Umut primers differed only by a single base located at the 3'
end . In case of the Mut primer, this base was complementary to the
mutation, whereas the Umut primer was complementary to the wild-type
sequence . The two primers were included into two separate PCRs . The
forward primer (primer 5') was added to both reaction mixtures
and annealed to the sequence downstream of mrsE that is
present only on the chromosome and not on the plasmid . Since
Taq polymerase does not possess proofreading activity, the
enzyme did not work efficiently with the primer whose 3' end
was mismatched . Therefore, only the primer whose 3' end paired
correctly with that copy of the mrsA gene which was located
directly downstream of mrsE yielded a PCR
product .
Purification of engineered
peptides.
For purification
of S16I mersacidin, 400 ml of culture supernatant were applied to a
35-ml XAD Serdolite AD2 (Serva) column, the column was washed with
water-50% methanol containing 50 mM potassium phosphate
buffer (pH 7), and the peptide was eluted with acetonitrile containing
0.1% trifluoroacetic acid . The eluate was concentrated by
evaporation and applied in several runs to a POROS R2 10 column
(Applied Biosystems, Lincoln, Calif.) at 3 ml/min and eluted with the
following gradient (eluent A, water-0.1% trifluoroacetic
acid; eluent B, acetonitrile-0.1% trifluoroacetic acid):
0 min, 10% B; 3 min, 10% B; 15 min, 30% B; 23 min,
40% B; and 25 min, 100% B . The adsorption of the eluate
was monitored at 220 nm . Since S16I mersacidin could not be identified
directly by mass spectrometry (MS) of the crude lyophilized fractions,
the fractions were analyzed by high-pressure liquid chromatography
(HPLC)-MS . Lyophilized fractions were dissolved in 30%
acetonitrile, and a 20-µl aliquot was separated on a 100- by
1-mm column of Gromsil ODS-5ST (Grom Analytik+HPLC, Herrenberg,
Germany) in a continuous gradient of 10 to 90% acetonitrile
containing 0.1% trifluoroacetic acid over 30 min at a constant
flow rate of 60 µl/min . Eluant was fed directly into a VG
Quattro II triple-quadropole mass spectrometer (MicroMass) fitted with
an electrospray ionization interface, using air as the carrier gas . The
mass spectrometer was operated in positive mode, and data were
collected and analyzed with the software provided by the manufacturer
(MassLynx) . The fractions containing S16I mersacidin were
rechromatographed in order to obtain sufficient material for the
determination of MICs . After lyophilization, the mass of the peptide
was analyzed by MS as described above .
E17A mersacidin and F3L
mersacidin were purified from 100 ml of culture supernatant, which was
sterilized by filtration, lyophilized, dissolved in about 30 ml of
30% acetonitrile-0.1% trifluoroacetic acid, and
applied in several runs to a POROS R2 20 column . The mass was analyzed
with an API 2000 Triple Quad MS (Applied Biosystems) .
For the
agar diffusion assay, the culture supernatant was sterilized by
filtration, and aliquots were pipetted into wells in a blood agar plate
that had been overlaid with soft agar containing Micrococcus
luteus as indicator strain .
Determination of the MICs of
mersacidin and engineered mersacidin peptides was performed by use of a
microtiter plate assay with half-concentrated Mueller-Hinton broth and
an inoculum of 105 cells of M . luteus per well,
which was read after 16 h of incubation at
37°C .
Construction
of an expression system for variant mersacidin peptides.
For expression of mutated peptides, we
constructed a system that relies on replacement of the wild-type gene
by the engineered gene on the chromosome . To this end, the
EcoRV fragment carrying the engineered mrsA gene was
ligated into the temperature-sensitive vector pTV0 or its derivative
pTV0[MCS], both of which carry a chloramphenicol resistance
gene, and was subcloned in S . carnosus at 30°C . The
recombinant plasmid was then transformed into the wild-type producer
strain (Fig.
1A), which was also cultured at 30°C . Since pTV0 and
pTV0[MCS] replicate at a very low copy number, the colonies
obtained on regeneration agar were screened by PCR with primers pTV01
and pTV02 (Table 2; Fig.
1) . Subsequently, colonies
which had integrated the plasmid into their chromosome were selected by
a temperature shift to 45°C and growth on agar containing
chloramphenicol . These colonies carried two copies of mrsA,
the wild-type gene and the mutagenized gene, which are separated by the
vector . It has to be kept in mind that the mutagenized mrsA
gene differs from the wild-type gene by only a single or double base
exchange, i.e., a point mutation . Therefore, two different kinds of
mutant strains could be expected from the integration of the plasmid
into the chromosome of the producer strain by a single-crossover event.
When the crossover event takes place upstream of the point mutation,
the mutant gene will be located downstream of mrsE, as shown
in Fig . 1B . On the other
hand, the wild-type gene will remain downstream of mrsE when
the crossover event takes place downstream of the point mutation, as
shown in Fig . 1C . The
locations of the mutant and wild-type genes were checked by competitive
nucleotide priming with Taq polymerase . However, these clones
did not show any mersacidin production, irrespective of orientation of
the two mrsA genes . The reason for this phenomenon was not
investigated, but it might be the result of a polar effect caused by
the insertion of the plasmid into the biosynthetic gene
cluster .
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FIG . 1 . Construction
of clones that are produced by integration and subsequent excision of a
plasmid carrying an engineered mrsA gene and the N terminus of
mrsR1 (white open reading frames, thick line) into the
chromosome (black open reading frames, thin line) . The mutation is
marked by an asterisk . (A) Wild type . (B and C) The plasmid
(thick line, cat resistance gene) has been integrated into the
chromosome, and the crossover took place either upstream of the
mutation (B) or downstream of the mutation (C) . (D)
The plasmid has been excised by the second homologous recombination
step, which took place either downstream (B) or upstream
(C) of the mutation in mrsA, resulting in an
exchange of the wild-type mrsA gene for the engineered gene.
The primers are shown by arrows: P1, primer 5'; P2, NPTV0; P3,
Mut; P4, Umut; P5, PTV01; P6,
PTV02.
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In order to obtain production of engineered peptides, we
cultured the clones for about 100 generations in the absence of
chloramphenicol and screened for colonies that had lost the
chloramphenicol resistance by a second crossover but had kept the
engineered mrsA gene (Fig.
1D) . Colonies that had not
grown on agar containing chloramphenicol after 2 days of incubation
were screened by PCR with primers specific for the vector (PTV01 and
PTV02), primers which anneal on the chromosome upstream and downstream
of the EcoRV fragment containing mrsA (NPTV02 and
primer 5'), and the specific Mut and Umut primers (Fig.
1) . When the PCR indicated
that the colony had integrated the engineered gene correctly into the
biosynthetic gene cluster and had lost pTV0[MCS], the peptide
was produced and purified as described above . The purified peptide was
checked by MS for the correct
modification .
Identification of amino acid
residues essential for activity of mersacidin.
The first peptide to be produced by the
method described above was S16I mersacidin, which contains the Ile
residue that is found in the related lantibiotic actagardine instead of
the Dha residue present in mersacidin (Fig.
2) . Since no antibacterial activity and no mersacidin peak were detected in
the supernatant of the correct clone after 72 h of culture,
the peptide either was inactive, was produced only in small
amounts, or was not synthesized at all . Loss of production has been
described for some engineered nisin, epidermin, and Pep5 peptides
(18) . Therefore, the
culture supernatant was fractionated on XAD and POROS R2 columns and
the fractions eluting between 31 and 37% acetonitrile were
screened by HPLC-MS (Fig.
3) . The peptide with the correct mass of 1,869 Da was detected at
36% acetonitrile and thereafter was purified from 400 ml of
culture supernatant with a yield lower than 0.5 mg/liter . The
determination of the MIC showed that the activity of S16I mersacidin
against M . luteus was nearly 1,000-fold lower than that of the
wild-type peptide (mersacidin MIC, 0.1 mg/liter; S16I mersacidin MIC,
87.5 mg/liter) . The antibacterial activities of HPLC fractions of
culture supernatant were checked against 25 Streptococcus
strains (belonging to Lancefield groups A, G, and B), two enterococci,
and Micrococcus flavus, but no inhibition zones could be
discerned on Mueller-Hinton agar plates . It was also inactive in a
broth microdilution test against a clinical Streptococcus
pyogenes isolate (I-12662) with good sensitivity to wild-type
mersacidin (mersacidin MIC, 0.78
mg/liter; S16I mersacidin MIC, >25 mg/liter) .
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FIG . 2 . (A)
Structure of mersacidin
(8) and exchanges that
were introduced by site-directed mutagenesis . (B) Structure
of the closely related lantibiotic actagardine
(34).
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FIG . 3 . HPLC-electrospray
ionization-MS of S16I mersacidin . (A) Total ion chromatogram
obtained after separation of the fraction containing S16I mersacidin.
(B) Extracted chromatogram for peaks with masses in the range
of 1869.0 to 1872.5 Da . (C) Mass determined for the peak
detected at 21.28 min (expected mass for S16I
mersacidin, 1,869.32
Da).
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E17A
mersacidin was constructed in order to assess the importance of the
glutamic acid residue at position 17 for the antimicrobial activity of
mersacidin . Culture supernatant of the E17A mersacidin producer did not
show antimicrobial activity against M . luteus . However, the
peptide could be identified by double detection at 220 and 266 nm
during HPLC runs, since due to the NH-CH=CH-S structure in the
C terminus of mersacidin, the extinction at 266 nm is about half of
that at 220 nm . E17A mersacidin was produced in greater amounts than
S16I mersacidin, and the purification yielded 0.41 mg of pure peptide
from 100 ml of culture supernatant . The peptide with the correct mass
(1,767 Da) (Fig.
4A) was inactive against M . luteus, with an MIC that was higher
than 175 mg/liter (wild-type mersacidin MIC, 0.195 mg/liter) . The yield
of purified F3L mersacidin was 3.4 mg/liter of culture . The MIC of this
peptide with the correct mass of 1,791 Da (Fig.
4B) against M.
luteus was 12.5 mg/liter (wild-type mersacidin MIC, 0.195
mg/liter) .
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FIG . 4 . MS
of E17A mersacidin (expected mass, 1767.2 Da) (A) and F3L
mersacidin (expected mass, 1791.2 Da)
(B).
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For successful
production of a mutagenized lantibiotic, the peptide must be expressed
and correctly modified to contain the rare amino acids,
e.g., methyllanthionine, didehydroalanine, and
S-aminovinylmethylcysteine for mersacidin . The modification
reactions are catalyzed by the modifying enzymes MrsM (dehydration and
thioether formation) and MrsD (oxidative decarboxylation of Cys20)
(1) . In the study
presented here, a gene replacement strategy was successfully
employed for production of mutated mersacidin peptides . Compared to
a complementation approach, it is advantageous in that the
relative gene dosage of the structural gene versus the
modification-export machinery (MrsD, MrsM, and MrsT) is kept at the
wild-type level . Similarly, in the case of nisin yields were also
higher when a gene replacement system was employed than when a
complementation approach was used
(11) . In the
present study, the rates of production of the engineered
mersacidin peptides varied, but all three peptides were produced at
lower rates than wild-type mersacidin, where yields of 5 to 10 mg/liter
are expected . The yield was especially low for S16I mersacidin; in this
peptide, the serine residue at position 16 had been exchanged for
isoleucine, thereby preventing the modification reaction of Ser16 to
dehydroalanine . It is possible that this exchange and the hydrophobic
side chain of the Ile residue introduced interfere with the dehydration
of other residues and/or thioether formation . Therefore, the lower
production rates, especially of S16I mersacidin, may be caused by
inhibition of the enzymes of the modification machinery . Similar
observations have also been described for other lantibiotics,
especially after exchange of hydroxy amino acids; e.g., T20A Pep5 was
not completely modified and was produced only at low rates
(5) . Furthermore, some
engineered peptides that had lost hydroxy amino acids (S3N epidermin
and T23A nisin A) were not produced at all
(18,
22) . Incomplete
dehydration, indicated by the presence of peptides with molecular
masses corresponding to hydrated variants (having an additional 18 Da),
as found with the expression system for engineering of the lantibiotic
Pep5 (4), was not seen in
this system, and incompletely modified peptides were not detected.
However, Bacillus is a potent producer of extracellular
proteases, and one function of the ring structures in lantibiotics is
stabilization of the peptides against the activity of proteases
(5) . An incompletely
modified mersacidin peptide, lacking the stabilizing ring structures,
would most probably be quickly degraded by one of the numerous
extracellular proteases of the producer strain . This phenomenon may
also contribute to the low yields of S16I mersacidin .
The
exchanges S16I and E17A are situated in the third ring of mersacidin,
which is conserved between mersacidin and the lantibiotic actagardine
from A . gabardinensis (Fig.
2)
(8,
34) and includes the
conserved Glu residue . Actagardine also inhibits cell wall biosynthesis
(29) and is able to
strongly inhibit the binding of mersacidin to M . luteus cells,
indicating that both lantibiotics compete for a similar binding site
(6) . However, actagardine
and its variant Ala(0)-actagardine show a different activity spectrum
from mersacidin (2,
30) . Whereas mersacidin
displays high antibacterial activity against M . luteus and
Staphylococcus aureus, actagardine and its variant show good
activity against streptococci . The three-dimensional
structure of actagardine was elucidated by nuclear magnetic
resonance (33) and seems
to be rather rigid . The only hydrophilic amino acids of actagardine,
Ser2 and Glu11, are part of a putative binding pocket that is formed by
Ser2-Ala1-S-Ala6- -aminobutyric acid 7
(Abu7)-S-Ala12-Glu11 . This structure is located in the part
of actagardine that is conserved compared to mersacidin, and it was
speculated that these residues might form the binding site for the
antibacterial target of mersacidin and actagardine, the cell wall
precursor lipid II (6,
33) . In the conformation
of mersacidin that is adopted in methanol, the N terminus is folded
back into the center of the molecule and the amino group of Ala1
interacts with the carboxylate group of Glu17
(24), and in the crystal
structure an additional interaction with Gly7 was observed
(28) . The polar
interactions of Glu17 and Ala1 or Gly7 varied in their strength in the
six slightly different structures that constitute the hexameric
crystal, indicating a rather loose contact . This observation led to a
speculative model in which the carboxylate group interacts with Ala1 or
Gly7 in the crystal but upon encountering its target shifts to binding
a ligand directly upon lipid II
(28) . Recent nuclear
magnetic resonance experiments have indeed shown that mersacidin is a
flexible molecule and that the three-dimensional structure that is
adopted by mersacidin in methanol-water mixtures changes upon contact
with dodecylphosphocholine micelles and then shifts again
upon addition of lipid II . The first shift is especially strong in
Abu13, Abu15, and Dha16; the second shift is especially pronounced for
Abu13, which seems to act as a hinge region, and Gly7 and Glu17
(16) . From these
structural data, it is most likely that Glu17 is one of the key
residues in mersacidin . The loss of antibacterial activity in E17A
mersacidin demonstrated here confirms this hypothesis . In contrast, the
low antibacterial activity displayed by S16I mersacidin was not
expected from the comparison of the primary structures of mersacidin
and actagardine . However, these results indicate that in mersacidin,
either Dha16 is either essential for the interaction with lipid II or
the presence of the bulky Ile residue in position 16 influences the
position of the neighboring essential residue Glu17 . Obviously, the
primary structure of actagardine cannot simply be superimposed on
mersacidin even if the amino acid sequence of both rings is otherwise
conserved . In contrast, the comparatively better activity of
F3L mersacidin shows that exchanges seem to be more
compatible with antibacterial activity in the two N-terminal rings of
mersacidin .
The mode of action of mersacidin and actagardine is
not covered by any other antibiotics, since both substances interact
with a novel target site that is present only on lipid II and do not
bind to lipid I or intracellular peptidoglycan precursors
(6) . Although ramoplanin
also binds to lipid II, this lipoglycodepsipeptide recognizes lipid I
and UDP-N-acetylmuramylpentapeptide as well
(10), and vancomycin and
teicoplanin target the terminal
D-Ala-D-Ala residues of the peptide side
chain of lipid II . The promising activities of the lantibiotics against
Staphylococcus and Streptococcus make them lead
structures for the design of novel antibiotics against these
problematic pathogens . Chemical derivatives of actagardine have been
synthesized (21,
30), but chemical
modification is restricted to the C and N termini and the hydroxyl
group of Ser5 . In mersacidin, only the N terminus and the Glu residue
represent reactive groups . The development of a system for
site-directed mutagenesis of mersacidin is an important step towards
the design of novel antibiotics, since it allows targeted exchange of
amino acids in the structure of mersacidin .
This work was supported by
the Bundesministerium für Bildung, Wissenschaft,
Forschung und Technologie (grant 01KI9705), the Deutsche
Forschungsgemeinschaft (grants Bi 504/1-2 and Bi 504/1-3), and the
BONFOR program of the Medizinische Einrichtungen, Universität
Bonn .
We are grateful to Aventis Pharma GmbH (Frankfurt, Germany)
for providing mersacidin and the mersacidin producer strain . We thank
S . Schmitz for expert technical
assistance .
* Corresponding
author . Mailing address: Institut für Medizinische Mikrobiologie
und Immunologie, Sigmund-Freud-Str . 25, D-53105 Bonn, Germany . Phone:
49-228-2879103 . Fax: 49-228-2874808 . E-mail:
bierbaum{at}mibi03.meb.uni-bonn.de .
Present
address: Department of Microbiology, University of Otago,
Dunedin, New Zealand .
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