(Received for publication, June 15, 1995; and in revised form, July 11, 1995)
From the
Biosynthesis of lantibiotics such as nisin and subtilin involves
post-translational modifications, including dehydration of serines and
threonines, formation of thioether cross-linkages, translocation,
cleavage of a leader sequence, and release into the medium. We have
studied the cellular machinery that performs the modifications by
constructing and expressing nisin-subtilin chimeric prepeptides in a
strain of Bacillus subtilis 168 that possesses all of the
cellular machinery for making subtilin except for the presubtilin gene.
The chimeras consisted of a normal subtilin leader region
(S), fused to nisin-subtilin chimeric structural regions,
one of which was
S
-Nis
-Sub
, in
which the N-terminal portion of the structural region was derived from
nisin, and the C-terminal portion derived from subtilin. This chimera
was accurately and efficiently converted to the corresponding mature
lantibiotic, as established by reverse phase high performance liquid
chromatography profiles, proton NMR spectroscopy, mass spectral
analysis, and biological activity. A succinylated form of the chimera
was also produced. Another chimera was in the reverse sense, with
subtilin sequence at the N terminus and nisin sequence at the C
terminus of the structural region
(S
-Sub
-Nis
). It
was processed into a heterogeneous mixture of products, none of which
had the characteristics of a correctly processed polypeptide, but did
contain a minor component that was active, with a specific activity
that considerably exceeded nisin itself. These results, together with
results published earlier, establish that processing requires specific
recognition between the prelantibiotic peptide and the processing
machinery, and in order for the processing to occur correctly, there
must be an appropriate combination of the N-terminal part of the leader
region and the C-terminal part of the structural region of the
prepeptide.
Nisin (produced by Lactococcus lactis) and subtilin
(produced by Bacillus subtilis) are the most thoroughly
studied examples of lantibiotics, which are ribosomally synthesized
antimicrobial peptides that are characterized by the presence of
unusual lanthio and dehydro residues. Their structures are shown in Fig. 1. Their biosynthesis involves several post-translational
modifications: dehydration of serines and threonines, formation of
thioether cross-linkages between dehydro residues and cysteines,
translocation, removal of a leader sequence, and release of the mature
antimicrobial peptide into the extracellular medium (reviewed in (1, 2, 3) ). Gene-encoded antimicrobial
peptides constitute a family of natural products, whose known members
are expanding rapidly in number and diversity and are produced by many
kinds of organisms, ranging from bacteria to eukaryotes, including
mammals(1, 4, 5, 6) . Their ubiquity
among widely diverged organisms implies that they have had an
opportunity to explore many strategies for achieving their
antimicrobial properties, some of which are quite different from the
mechanisms of classical antibiotics such as penicillin. They may
therefore be able to supplement the arsenal of therapeutic
antimicrobial agents that has been depleted as a result of the
evolution of resistance among microbes. An advantage that is unique to
gene-encoded antimicrobial peptides is that their structures can be
readily manipulated by mutagenesis, which provides a facile means for
constructing and producing the large numbers of structural analogs
needed for structure-function studies and rational design. Whereas this
advantage is shared by all gene-encoded antimicrobial peptides, the
lantibiotics are unique in possessing the unusual dehydro and lanthio
residues, which are absent from
magainins(7, 8, 9) ,
defensins(10, 11, 12, 13) , or
cecropins(14, 15) . This means that the lantibiotics
offer chemical, physical, and hence biological properties that are not
attainable by polypeptides that lack these residues. For example, the
dehydro residues (Dha ()and Dhb) are electrophiles, whereas
none of the ordinary amino acids is electrophilic. The thioether
cross-linkages are more resistant to breakage than the disulfide bridge
and can better survive reducing conditions and extremes of pH and
temperature(16) .
Figure 1:
Structures of
nisin and subtilin as determined by Gross and
co-workers(38, 39, 40) . Aba,
aminobutyric acid; Dha, dehydroalanine; Dhb,
dehydrobutyrine (-methyldehydroalanine); Ala-S-Ala,
lanthionine; Aba-S-Ala,
-methyllanthionine. The unusual
amino acids are introduced by posttranslational modifications as
described in the text.
A concern when making mutants of
lantibiotics is the effect of the mutations on the post-translational
modification process, because a mutation that disrupts processing makes
the biosynthesis of the corresponding mature lantibiotic peptide
impossible. All known lantibiotic prepeptides contain an N-terminal
region that is cleaved during maturation, and for the Type A
lantibiotics (e.g. nisin, subtilin, and epidermin), this
leader region is highly conserved (17) , and its participation
in the orchestration of post-translational modification and secretion
has been proposed(17, 18) . Certain mutations in the
leader region of the nisin prepeptide have rendered the cell incapable
of nisin production(19) , whereas many mutations in the
structural region of several lantibiotics that do not disrupt
processing have been reported (e.g.(20) and (21) ). When the complete nisin prepeptide consisting of the
nisin leader region and the nisin structural region
(N-Nis
) was expressed in a
subtilin-producing cell, no nisin-related peptide products were
detectable(22, 23) . However, when a chimera
consisting of the subtilin leader region and the nisin structural
region (S
-Nis
) was expressed in a
subtilin-producing cell, an inactive nisin-like peptide was produced in
which the leader region had been correctly cleaved and contained a full
complement of unusual amino acids(22) . The lack of activity
was attributed to the formation of incorrect thioether
cross-linkages(22) . Similarly, when a prepeptide consisting of
a subtilin leader region and a nisin structural region was expressed in
a nisin-producing cell, the nisin structural region contained the
unusual amino acids, but the leader was not cleaved(24) . It
has also been reported that expression of a prepeptide consisting of
the nisin structural region fused to a subtilin-nisin chimeric leader
region,
S
-N
-Nis
,
forms active nisin when expressed in a subtilin-producing
cell(23) .
These results imply that the subtilin processing machinery in B. subtilis is not capable of recognizing the nisin prepeptide (which is ordinarily expressed in Lactococcus lactis) and converting it to nisin. However, the subtilin machinery will perform the modification reactions on the nisin structural peptide if it is attached to a subtilin leader region, although the modifications seem to be misdirected so that active nisin is not produced. Finally, the subtilin machinery will produce active nisin if the leader region is an appropriate combination of subtilin leader and nisin leader sequences. In this work, we explore the contribution of the structural region and its relationship to the leader region by the construction and expression of nisin-subtilin chimeras, which contain chimeric nisin-subtilin structural regions fused to the subtilin leader region. We have discovered that chimeras in which the C-terminal portion of the structural region correspond to subtilin are processed correctly and give active products, whereas those in which the C-terminal portion of the structural region corresponds to nisin produces a heterogeneous mixture of products, most of which, but not all, are inactive.
Inspection of the structures of nisin and subtilin in Fig. 1reveals that the number and locations of the thioether
rings and the Dha residues are conserved. Each has one Dhb residue, but
its position is not conserved. The N-terminal region is relatively
conserved, except for three non-conservative differences out of the
first 11 residues. Nisin has isoleucine at position 1, whereas subtilin
has a bulky aromatic tryptophan. Subtilin has a positively charged
lysine at position 2, whereas nisin has an unusual Dhb residue.
Finally, subtilin has a negatively charged glutamate at position 4,
whereas nisin has a neutral aliphatic isoleucine. In previous work, we
changed the Glu of subtilin to the Ile
of
nisin, and obtained a mutant with enhanced chemical stability and
activity(21) . We wondered what would happen if we changed the
other two residues as well, to give a subtilin analog with a nisin-like
N terminus. This analog would have only hydrophobic residues at the N
terminus, as well as a fourth dehydro residue at a location that is
unfamiliar to the subtilin processing machinery, and if it were unable
to process it properly, the entire processing pathway could abort.
Since the subtilin machinery cannot process the
S
-Nis
prepeptide to an active product (22) , it is difficult to predict how the machinery would
handle the S
-Nis
-Sub
prepeptide. We therefore constructed and expressed this
prepeptide and examined its products.
Figure 2:
Strategy for construction of
Nisin-Subtilin chimeras. Mutagenesis was performed in the plasmid
pSMcat, which is a cassette-mutagenesis plasmid that contains
a copy of the subtilin structural gene upstream from a cat gene(21) . When this plasmid is transformed into the B. subtilis 168 host LH45ermS and selected on
chloramphenicol, the subtilin gene is integrated into the chromosomal
subtilin (spa) operon (21) at the site from which the
natural subtilin gene has been deleted. The sequence of the
Nis
-Sub
chimera and the
nucleotide sequence that encodes it is shown (top), in which
the 32-residue mature Nis
-Sub
sequence is numbered. Immediately below are the mutagenic
oligonucleotides used to construct this sequence. The sequence of the
S
-Nis
chimera and the
oligonucleotides used to produce it are at the bottom.
Figure 3:
HPLC chromatography of the
Nis-Sub
chimera. The
Nis
-Sub
chimera constructed
as shown in Fig. 2was expressed, isolated, and subjected to
HPLC chromatography. Samples were collected at 1-min intervals and
assayed for activity using the halo assay. The major peak contained the
only activity. A portion of this peak sample was subjected to
SDS-polyacrylamide gel electrophoresis and silver-stained. The stained
gel is shown in a panel beside the peak (sample, left lane,
size standard, right lane). Standards were 2.5-kDa myoglobin
fragment (F3), 6.2-kDa myoglobin fragment (F2), and
8.1-kDa myoglobin fragment (F1). The expected mass of the
Nis
-Sub
chimera is 3186 Da,
which is consistent with the position of the band in the sample
lane.
Figure 4:
Resolution of the
Nis-Sub
chimera into two
forms. The Nis
-Sub
chimera
constructed as shown in Fig. 2was expressed and isolated as for Fig. 3, except that the cells were grown for a longer time into
the stationary phase, resulting in the appearance of a new peak on the
HPLC column (inset), with the original peak (Early
Peak) and the new peak (Late Peak). These peaks were
separated by only 1 min, so were chromatographed using a shallower
gradient (35-60% acetonitrile over 45 min, center),
whereupon the early peak and late peak were separated by 4 min. The
results of halo assays are shown above the center HPLC
profile, with arrows indicating the positions in the profile
from which the samples used for halo assays had been
derived.
Figure 5:
NMR
and mass spectra of the early peak and late peak. The early peak and
late peak are those as defined in the HPLC elution profiles in Fig. 4. The mass spectrum of the early peak is shown in panel A, with a calculated mass of 3185.98 Da that is
identical to the expected value of 3186 Da for the mature
Nis-Sub
chimera. The mass
spectrum of the the purified late peak is shown in panel B,
with its calculated mass of 3286.78 Da exactly corresponding to the 100
Da increase expected from addition of a succinyl group to the mature
Nis
-Sub
chimera. The NMR
spectrum of the early peak is shown in panel D. Identification
of the Dhb
, Dha
, Dhb
, and
Dha
peaks was by correlation with NMR spectra obtained
previously for nisin (33) and subtilin(21) . The NMR
spectrum of a mixture of the early peak and late peak is shown in panel C. Resonances shifted by the presence of the succinyl
group are identified by asterisks (Dhb
*, Dha
*).
Figure 6:
Stability of the
Nis-Sub
chimera during a
72-day incubation. The NMR spectrum of the
Nis
-Sub
chimera was obtained
after incubation in aqueous solution after 0, 34, and 72 days. The
sample was a mixture of both the early peak and the late peak, as
defined in Fig. 4and Fig. 5, and shows the expected
resonances of Dhb
, Dhb
*, Dha
,
Dha
*, Dhb
, and Dha
,. There was no
significant change in these resonances during the 72-day incubation
period.
We conclude from these results
that the Dha residue is subject to profound changes in its
chemical reactivity, ranging from the most reactive state observed in
natural subtilin, to the least reactive state observed in the
Nis
-Sub
chimera; with
E4I-subtilin being in between. Somewhat surprisingly, the biological
activity displayed by these structural variants varies inversely with
the reactivity, with the unstable subtilin having the lowest activity,
and the highly stable Nis
-Sub
chimera displaying the greatest activity. The fact that the
chemical reactivity of Dha
varies inversely with biological
activity argues that role of the Dha
residue in the
antimicrobial mechanism is not related to its chemical reactivity in a
simple fashion, and that other factors, such as the specificity imposed
by the peptide sequence surrounding the dehydro residue, are also
important.
Figure 7:
HPLC
profiles and mass spectra of the
Sub-Nis
chimera. The HPLC
profile of the Sub
-Nis
chimera is shown in the center. The halo assays of
samples from the profile are shown at the bottom, with the arrows showing were the samples were derived from the profile.
The only activity in the profile appeared at the position of the small
peak appearing at 16 min. The mass spectrum at the left was
obtained for the material in the large peak indicated by the arrow. The mass spectrum at the right was obtained
for the material in the small peak (which was active) indicated by the
other arrow.
Our ability to incorporate the unusual dehydro and lanthio
type amino acids into lantibiotic analogs and non-lantibiotic
polypeptides depends on the ability of the lantibiotic processing
machinery to cope with foreign precursor sequences. Our working
hypothesis is that the leader region is primarily responsible for
engaging the prepeptide with the processing machinery, and, once
engaged, serines and threonines are dehydrated with little regard for
the sequence in which they reside. Cysteines then react with particular
dehydro partners in accordance with the forces of folding and
conformation that exist within the polypeptide in a manner that is
reminiscent of the specific selection of disulfide bond partners in
polypeptides such as ribonuclease A and insulin(34) . Although
the results presented here do not prove this hypothesis, they are
consistent with it and therefore support it. There are now several
known instances in which prelantibiotic peptides undergo processing
reactions, but give rise to inactive products. These are summarized in Table 1. Examples are the S-Nis
chimera, which produces a processed (22) but inactive (22, 23) product when expressed in a cell that
possesses the subtilin machinery, and the
S
-Sub
-Nis
chimera (this paper), which produces a heterogeneous mixture of
products that are mainly inactive, although at least one active form is
produced. This means that although N
-Nis
is an authentic lantibiotic precursor, the subtilin machinery
seems incapable of coping with it, and its gene products have not been
detected(22, 23) . However, if you place the subtilin
leader in front of the nisin structural region to give
S
-Nis
, a processed, but inactive,
product is produced by the subtilin machinery (22) . From this,
we conclude that the subtilin leader is competent in engaging the
processing machinery, but there is something about the conformational
and folding interactions between the leader and structural region in
the S
-Nis
construct that causes some
of the processing reactions, perhaps the partner selection in thioether
formation, to malfunction. The fact that the
S
-N
-Nis
construct is processed properly to give active nisin (23) argues that critical conformational interactions are
restored when an appropriate N-terminal sequence element from the nisin
leader region is combined with a C-terminal sequence element of the
subtilin leader. However, this combination of leader sequence elements
must be appropriately complemented by the structural region. Whereas
the S
-Nis
-Sub
construct is processed correctly (this work), the
S
-Sub
-Nis
construct is not. Moreover, the processing reactions for the
latter construct really run amok, giving a complex mixture of mainly
inactive products. Surprisingly, at least one component in this mixture
was active. Since none of the components had the mass of a correctly
processed product, this activity must be due to an incorrectly
processed component, and its specific activity was at least 4-fold and
as much as 35-fold higher than nisin itself. We would like to know more
about what is responsible for such high activity, because it may
provide insight about how to design lantibiotics that are dramatically
more effective than the natural forms. We conclude from these results
that correct processing of the prelantibiotic peptide requires specific
conformational communication between the N-terminal portion of the
leader region and the C-terminal portion of the structural region.
These results also provide new insight about the relationship
between the structure of lantibiotics and their chemical properties and
biological activity. Subtilin and nisin are highly disparate in their
chemical stability and specific activity, with nisin being superior to
subtilin in both categories. The
Nis-Sub
chimera has the
superior properties of nisin, showing that the 3 residues that differ
in the N-terminal regions of nisin and subtilin are primarily
responsible for the disparity between nisin and subtilin. We note that
Nis
-Sub
has a very
hydrophobic N-terminal region, which may facilitate insertion of the
lantibiotic into the membrane, which is its target of
action(26, 35, 36, 37) . However,
another possible explanation for its elevated activity is the presence
of a second dehydro residue (Dhb) at position 2 in the
Nis
-Sub
chimera. One might
expect that the Dhb
would have a dramatic effect on the
antimicrobial properties of the chimera, since it is so close to the
critical Dha
and might cooperate in reacting with its
microbial target; however, if it does, there could be no more than a
2-fold effect. This illustrates a frustrating aspect of our knowledge
about lantibiotics. The ubiquitous occurrence of the unusual residues
among the many known lantibiotics argues that they are conserved
because they have important functions. Except for the critical role of
Dha
in inhibition of spore outgrowth, functions that
clearly justify this ubiquitous occurrence have yet to be identified.
This suggests that there are some very important attributes of
lantibiotics that we know nothing about.