(Received for publication, July 7, 1995)
From the
The post-translationally modified, antimicrobial peptide nisin
is secreted by strains of Lactococcus lactis that contain the
chromosomally located nisin biosynthetic gene cluster nisABTCIPRKFEG. When a 4-base pair deletion is introduced into
the structural nisA gene (nisA), transcription
of
nisA is abolished. Transcription of the
nisA gene is restored by adding subinhibitory amounts of nisin, nisin
mutants, or nisin analogs to the culture medium, but not by the
unmodified precursor peptide or by several other antimicrobial
peptides. Upon disruption of the nisK gene, which encodes a
putative sensor protein that belongs to the class of two-component
regulators, transcription of
nisA was no longer inducible
by nisin. Fusion of a nisA promoter fragment to the
promoterless reporter gene gusA resulted in expression of gusA in L. lactis NZ9800 (
nisA) only
upon induction with nisin species. The expression level of gusA was directly related to the amount of inducer that was added
extracellularly. These results provide insight into a new mechanism of
autoregulation through signal transduction in prokaryotes and
demonstrate that antimicrobial peptides can exert a second function as
signaling molecules.
Nisin is an antimicrobial peptide (1, 2, 3) widely used in the food industry as
a safe and natural preservative. The ribosomally synthesized precursor
peptide undergoes extensive post-translational modification, which
includes dehydration of serine and threonine residues and the formation
of thioether bridges called (-methyl)lanthionines, resulting in
five ring structures named A, B, C, D, and E (Fig. 1B).
Peptides containing these characteristic modified residues are named
lantibiotics(4) . Eleven genes organized in a cluster have been
implicated to be involved in the complex biosynthesis of nisin, i.e. nisABTCIPRKFEG (Fig. 1A)(5, 6, 7, 8, 9, 10, 11) .
Of these genes, nisA encodes the nisin A precursor peptide of
57 amino acid residues; nisB and nisC encode putative
enzymes involved in the post-translational modification reactions
(based on homology to genes found exclusively in other lantibiotic gene
clusters); nisT encodes a putative transport protein of the
ABC translocator family that is probably involved in the extrusion of
modified precursor nisin(7, 9) ; nisP encodes
an extracellular subtilisin-like protease involved in precursor
processing (8) ; nisI encodes a lipoprotein involved
in the producer self-protection against nisin(9) ; and nisFEG encodes putative transporter proteins that have also
been implied in immunity (11) . A schematic representation of
the post-translational events yielding mature nisin A is shown in Fig. 1B. Nisin Z is a natural variant of nisin A that
contains an asparagine residue at position 27 instead of the histidine
residue found in nisin A(12) . Both nisin A- and nisin
Z-producing strains are common in nature, and both structural genes (nisA and nisZ) have been
cloned(5, 6, 13) .
Figure 1: A, organization of the nisin gene cluster. Established (nisAIPRKFEG) and putative (nisBCT) functions of the gene products have been indicated. P denotes a mapped promoter, and IR denotes an extensive inverted repeat sequence that could act as a rho-independent terminator(7) . B, schematic outline of the biosynthesis of nisin A. Rings are labeled A-E. Asterisks indicate residues that will be modified. The black arrow indicates processing of the N-terminal Met residue, while the small white arrow indicates processing of the leader peptide by the action of NisP(8) . Dha, dehydroalanine; Dhb, dehydrobutyrine.
The proteins encoded by nisR(8) and nisK(10) have shown to be involved in the regulation of nisin biosynthesis(8, 10) . NisR is a response regulator, and NisK is a sensor histidine kinase which belong to the class of two-component regulatory systems (14, 15, 16) . When the genes nisABTCIR are present on a multicopy plasmid, production of fully modified precursor nisin is observed, indicating that overexpression of nisR alone is sufficient to activate transcription of nisA and obviously also of the biosynthetic genes downstream by partially reading through an inverted repeat sequence (Fig. 1A)(8) . This observation is similar to the regulation of expression of iep and degU genes in Bacillus subtilis, where overexpression of the response regulator activates transcription of the target genes(17) , and to the case of overexpression of epiQ, which encodes a response regulator involved in the biosynthesis of the lantibiotic epidermin(18) . When only the genes nisABTCI are present on a multicopy plasmid (pNZ9000) in Lactococcus lactis MG1614, no transcription of nisA is observed(9) . Two gene products have been identified for the regulation of the biosynthesis of the related lantibiotic subtilin(19) , which also belong to the class of two-component regulators, i.e. SpaR, the response regulator, and SpaK, the sensor histidine kinase(20, 21) . Upon disruption of either of these genes, subtilin production was abolished, indicating the involvement of these gene products in subtilin biosynthesis(20) . The regulation was shown to be growth-phase dependent, but an inducing signal was not identified(20, 21) .
While the structure and function of two-component regulators have been studied in great detail(14, 15, 16) , the nature of the inducing signal has remained unclear in many cases. It is demonstrated here that fully modified nisin can induce the transcription of its own structural gene as well as of the downstream genes by limited read-through, via signal transduction, by acting as the extracellular signal for the sensor histidine kinase NisK.
The nisA promoter
region including part of the nisA gene was isolated as a
1442-bp BglII-Ecl136II fragment from plasmid
pNZ9000(8) . This fragment was cloned into pNZ273, containing
the promoterless gusA gene(24) , which had been
digested with BglII and ScaI, generating plasmid
pNZ8003. Part of the upstream promoter region was deleted by digesting
pNZ8003 with BglII and Tth111I. These sites were made
blunt by Klenow polymerase and ligated, generating plasmid pNZ8008,
which eventually contained a 312-bp nisA promoter fragment in
front of the gusA gene. Another part of the nisA promoter region, including the full nisA gene and the
first part of the nisB gene, was isolated as a 1904-bp BglII-MunI fragment from plasmid pNZ9000. This
fragment was cloned into pNZ273(24) , which had been digested
with BglII and EcoRI, generating plasmid pNZ8002. A
1442-bp BglII-Ecl136II promoter fragment was deleted
in pNZ8002, generating pNZ8002, by making the BglII site
blunt with Klenow polymerase and subsequent ligation to the Ecl136II site. All constructs were initially made in E.
coli MC1061(26) . Plasmids pNZ8008, pNZ8002, and
pNZ
8002 were used to transform L. lactis NZ9700 and L. lactis NZ9800(9) , and transformants were obtained
by selecting for resistance to chloramphenicol.
The nisB gene was disrupted by introducing a 162-bp in-frame deletion into
the middle of the gene. This was accomplished by cloning a 4.4-kilobase
pair BglII-EcoRI fragment, containing nisB and surrounding regions from the nisin gene cluster, into a BamHI-EcoRI-digested pUC19 vector, which harbored an
additional erythromycin resistance marker, as has been described
previously(9) . The deletion was made by removing an internal HpaI fragment from the nisB gene and subsequent
ligation. The resulting plasmid was named pNZ9135 and was used for
transformation of L. lactis NZ9700. Following transformation,
erythromycin-resistant colonies were obtained that had integrated the
plasmid by recombination of the plasmid with one of the flanking
regions of the deleted fragment. After growing for 200 generations in
the absence of erythromycin and plating, a colony was obtained that was
sensitive to erythromycin. This had apparently been caused by a second
recombination event involving the flanking region on the other side of
the deletion than the side of the first recombination event, resulting
in the replacement of nisB with nisB on the
chromosome. The configuration of the desired construct was confirmed by
polymerase chain reaction analysis of the nisB region with the
deletion and by Southern analysis of BglII-digested
chromosomal DNA. The desired strain was called NZ9700
nisB.
Northern blotting showed that in
strain NZ9800, the transcript of nisA was absent, but
after adding small amounts of nisin A to the culture medium at an A
of 0.5, nisA transcripts appeared
again (Fig. 2). Interestingly, the amount of these transcripts
was dependent on the amount of nisin A added (Fig. 2, lanes
3-7). Several other related peptides were able to induce
transcription, such as nisin Z and various nisin Z mutants, i.e. T2S nisin Z, S5T nisin Z(25) , M17W nisin Z, S3T nisin Z,
and sl-nisin Z, a fully modified nisin Z species that has the subtilin
leader peptide still attached(30) . However, the last two
species were >100-fold less effective inducers compared with nisin Z
(data not shown). In contrast, the T2S and M17W nisin Z mutants were
more potent inducers than nisin Z. These findings demonstrate that the
modified lantibiotic part plays an important role in the induction
process. Interestingly, several less related peptides evoked no
restoration of transcription, i.e. the unmodified synthetic
nisin A precursor of 57 amino acid residues (Fig. 1B and Fig. 2, lane 12), the 56% homologous
lantibiotic subtilin (19) , the lantibiotic lacticin
481(31, 32) , the lantibiotic Pep5 (Fig. 2, lane 13)(33) , and the antimicrobial peptide
lactococcin A (34) (data not shown for subtilin, lacticin 481,
and lactococcin A).
Figure 2:
Northern blot prepared using nisA as a probe of RNA from several uninduced lactococcal cultures and
cultures induced with different amounts of nisin A or unmodified
precursor nisin A or with the lantibiotic Pep5. Lane 1, NZ9700
(nisin A producer); lane 2, MG1614; lanes 3-7,
NZ9800 with nisin A (0, 1, 2.5, 10, and 50 ng/ml, respectively); lanes 8 and 9, NZ9800 nisK with nisin A
(0 and 2.5 ng/ml, respectively); lanes 10 and 11,
NZ9700
nisB with nisin A (0 and 2.5 ng/ml, respectively); lane 12, NZ9800 with unmodified precursor nisin A (1000
ng/ml); lane 13, NZ9800 with Pep5 (1000
ng/ml).
Figure 3:
Dose
response of purified (mutant) nisins as inducers of gusA expression in L. lactis strain NZ9800 harboring pNZ8008.
Nisin species were as follows: , T2S nisin Z;
, M17W
nisin Z;
, wild-type nisin Z;
, S3T nisin Z;
, I1W
nisin Z. Standard errors were <20% for each given value. A.U., arbitrary units.
In further experiments, the nisin-producing strain NZ9700 with either plasmid pNZ273 (containing the promoterless gusA gene) or pNZ8008 (containing the nisA promoter fragment followed by the gusA gene) was used in an agar diffusion assay (8) to determine the amount of nisin produced. Fifty times lower nisin production and severely reduced immunity were observed when plasmid pNZ8008 was present compared with the situation where pNZ273 was present. This can be explained by titration of the response regulator NisR by the multicopy presence of the nisA promoter region containing the putative NisR-binding site.
Figure 4: Model for nisin biosynthesis and regulation. In Step 1, NisK senses the presence of nisin in the medium and autophosphorylates. In Step 2, the phosphate group is transferred to NisR, which acts as a transcriptional activator, followed by mRNA synthesis and ribosomal synthesis of unmodified precursor nisin and of biosynthetic proteins. In Step 3, the precursor is modified by the putative enzymes NisB and NisC(7, 9) . In Step 4, the fully modified precursor peptide is translocated across the membrane by the putative ABC transporter NisT(7, 9) . In Step 5, fully modified precursor nisin is extracellularly processed by NisP(8) , resulting in the release of active nisin. NisI(9) , together with NisF, NisE, and NisG(11) , protects the cell from the bacteriocidal action of nisin by a thus far unknown mechanism.
Mutants of nisin or precursors of nisin that have the leader peptide attached to the mature lantibiotic (second molecule shown in Fig. 1B) can also act as inducers, whereas other antimicrobial peptides are incapable of induction. The presence of the modified residues is of crucial importance for induction capacity, especially those present in the N-terminal part of nisin. To our knowledge, this is the only report of peptides that can induce transcription of their own structural gene via signal transduction. Interestingly, a recent report on syndecan biosynthesis in mice, which plays a role in wound repair, describes the role of the antimicrobial peptide PR39 in induction of syndecan gene transcription(44) , although the amount of inducer needed (0.5 mM) is at least a factor of 10,000 higher than for nisin (30 pM). This suggests that the role of antimicrobial peptides in nature might be broader than just the antagonistic action because in some cases these peptides can also act as signals for transcription activation of their own structural gene or of other genes. There may be several evolutionary reasons for the autoregulation of nisin gene transcription via signal transduction, e.g. (i) to save energy by control of the integrity of the gene cluster since any dysfunctional biosynthetic gene will abolish inducer formation and thus expression of biosynthetic genes; (ii) to raise immunity levels in response to high nisin production by neighboring cells, in other words, to amplify the response to environmental signals; or (iii) to promote cell to cell communication that allows the production of antimicrobial peptides in high quantities in a concerted action, thereby decreasing the chance of resistance development in target organisms.