Department of Oral Biology, School of
Dentistry, University of Alabama at Birmingham, Birmingham, Alabama
35294
Mutacin II is a ribosomally synthesized peptide lantibiotic
produced by group II Streptococcus mutans. DNA sequencing
has revealed that the mutacin II biosynthetic gene cluster consists of
seven specific open reading frames: a regulator (mutR), the prepromutacin structural gene (mutA), a modifying protein
(mutM), an ABC transporter (mutT), and an
immunity cluster (mutFEG). Transformations of a
non-mutacin-producing strain, S. mutans UA159, and a
mutacin I-producing strain, S. mutans UA140, with
chromosomal DNA from S. mutans T8 with an
aphIII marker inserted upstream of the mutacin II
structural gene yielded transformants producing mutacin II and mutacins
I and II, respectively.
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TEXT |
Many bacteria produce antimicrobial
peptides, termed bacteriocins. One group of bacteriocins is the
lanthionine-containing lantibiotic group. The lantibiotics are
ribosomally synthesized as prepeptides that undergo several
posttranslational modification events (20, 21). The
posttranslational modifications involve dehydration of the hydroxyl
amino acids serine and threonine and addition of sulfur atoms of
cysteines across the formed double bonds, leading to formation of the
thioether amino acids lanthionine and methyllanthionine, respectively.
Cleavage of the leader peptide liberates the active final product.
Mutacin II (4, 17) has been purified from a human isolate,
Streptococcus mutans T8, and characterized (7, 15,
16). Mutacin II, a small thermostable peptide, has a molecular
mass of 3,245 Da and exhibits bactericidal activity over a wide range of pHs against several gram-positive bacteria by inhibiting energy metabolism of sensitive cells (7). Mature mutacin II
contains two lanthionine residues, one methyllanthionine, and one
didehydro amino acid residue (16). Accordingly, mutacin II
is a member of the lantibiotic family of antibiotics (21).
Two genes, mutA and mutM, encoding the
prepromutacin and modification enzyme, respectively, have been cloned,
sequenced, and characterized (29). The deduced primary
sequence of mutacin II showed the presence of 27 amino acids in mature
mutacin II, including three cysteine residues and four hydroxy amino
acids (29). In this study, we provide evidence that all of
the specific genes for mutacin II production are clustered and can be
transferred as a unit. In addition, we identified the rest of the
mutacin II biosynthetic genes in the cluster. Moreover, we constructed a strain capable of producing both mutacin I and mutacin II.
Transformation of non-mutacin-producing S. mutans
UA159.
In order to have a selection marker for the
transformation process, we inserted the kanamycin resistance gene
(aphIII) from Streptococcus faecalis
(27) into the chromosomal DNA of S. mutans T8 at
the HpaI site upstream of the mutacin II structural gene (mutA) by using a plasmid, pCBM6 (5). Plasmid
pCBM6 was constructed in previous structure-activity study
(5). The insert in pCBM6 was linearized by restriction
enzyme XbaI digestion and then transformed into competent
S. mutans T8 by a previously published method
(22). The kanamycin resistance gene (aphIII) was
integrated into the chromosome via the mechanism of double-crossover
recombination. The resultant transformants were mutacin positive and
kanamycin resistant and were designated S. mutans T8kan.
Chromosomal DNA from strain T8kan was isolated with the QIAGEN
genomic-tip DNA isolation kit (QIAGEN, Santa Clarita, Calif.) and used
to transform S. mutans UA159, which is naturally competent
and non-mutacin producing (22). Randomly selected
kanamycin-resistant transformants were tested for mutacin production by
using a deferred-antagonism assay (4, 17). All transformants
tested produced mutacin. As shown in Fig.
1, three randomly picked UA159
transformants, unlike the parental strain UA159, produced mutacin
and inhibited the growth of the indicator strain, Streptococcus
sobrinus OMZ176.
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FIG. 1.
Deferred-antagonism assay of mutacin phenotypes in
selected S. mutans strains. 1, T8 wild type; 2 to 4, UA159
transformants; 5, UA159 wild type; 6, indicator strain S. sobrinus OMZ176. Mutacin production was indicated by the
inhibition zones around the producing strains (strain T8 and UA159
transformants) in the lawn of S. sobrinus OMZ176. The
absence of such an inhibition zone indicated a lack of mutacin
production (parental strain UA159 and the negative control, S. sobrinus OMZ176).
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To further confirm the transfer of the mutacin II biosynthetic gene(s)
into these transformants, Southern blot analysis was carried out with
mutA and mutM as the probe. Chromosomal DNA
isolated from the three randomly picked transformants was digested with the restriction enzyme HindIII. Restriction fragments
were resolved on 0.65% agarose gels, transferred to a nylon
membrane after denaturation and neutralization, and hybridized to the
probe. The results (Fig. 2B) confirmed
transfer of the biosynthetic genes, mutA and
mutM, and a contiguous downstream region by competence
transformation. Comparison of chromosomal DNA restriction patterns of
the transformants and the parental strain UA159 (Fig. 2A) showed that
all transformants were derivatives of the parental strain.
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FIG. 2.
Restriction and Southern blot analysis of S. mutans mutacin-producing and non-mutacin-producing strains and
corresponding transformants. Chromosomal DNA was digested with
HindIII, and the resultant fragments were resolved on a
0.65% agarose gel in Tris-borate-EDTA buffer and photographed under UV
light (A). The gel was transferred to a nylon membrane and probed with
part of the mutacin II biosynthetic operon (mutAM) (B).
Lanes: 1, T8 wild type; 2, UA159 wild type; 3 to 5, UA159
transformants; 6, UA140 wild type, 7 to 9, UA140 transformants. DNA
marker sizes are indicated on the left side of each panel, in
kilobases.
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Biosynthesis of lantibiotics involves complex posttranslational
modifications (12, 20). While Southern blot analysis
indicated the transfer of the mutacin II biosynthetic genes
mutA and mutM and the downstream region into
these transformants, it was not obvious whether the active mutacins
from these transformants were fully modified, as was the wild type from
T8. To verify the correct processing of the mutA gene
product in these transformants, we purified the mutacin from these
transformants, as described previously (15), and analyzed it
by electrospray ionization mass spectroscopy on a PE Sciex API III
biomolecular mass analyzer (15). The results (Fig.
3) showed that the mutacins from the
S. mutans UA159 transformants and S. mutans T8
(15) were identical, indicating that the transferred genetic
determinants were sufficient to catalyze the synthesis of fully
modified lantibiotic molecules.
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FIG. 3.
Electrospray ionization mass spectroscopic analysis of
purified mutacin isolated from S. mutans UA159 transformants
by a previously described method (15). The molecular mass
indicated by multiple charged ions was calculated as 3,245.4 ± 0.6 Da, which is identical to that of mutacin II from S. mutans T8 (15).
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Transformation of mutacin I-producing S. mutans
UA140.
The above results suggested that the entire mutacin
biosynthetic locus was transferred into a non-mutacin-producing strain as a unit via competence transformation. It was not known, however, whether this locus could be also transferred into strains producing other mutacins. A group I strain, S. mutans UA140, which
produces mutacin I (4), was transformed with the chromosomal
DNA from strain T8kan. The resultant kanamycin-resistant transformants inhibited the growth of strain T8, as shown in Fig.
4A, suggesting that these transformants
continued to produce mutacin I. Mutacin II production by these
transformants was indicated by their ability to inhibit the growth of
Staphylococcus aureus MSSA4, a strain susceptible only to
mutacin II, in a deferred-antagonism assay (Fig. 4B). The Southern blot
(Fig. 2B, lanes 7 to 9) indicated the successful transfer of the
mutacin II biosynthetic operon, which was absent in the UA140 wild type
(lane 6). The restriction patterns of the corresponding chromosomal DNA
preparations (Fig. 2A, lanes 6 to 9) confirmed that these transformants
were truly derivatives of UA140.
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FIG. 4.
Deferred-antagonism assay of the mutacin I and II
phenotypes in selected S. mutans strains. (A) When S. mutans T8 was used as the indicator strain, the inhibition zones
around S. mutans UA140 wild type (5) and its transformants
(2 to 4) indicated mutacin I production in these strains, while the
absence of such an inhibition zone around T8 wild type (1) indicated
that mutacin I was not produced by this strain. (B) Mutacin II
production by T8 wild type (1) and UA140 transformants (2 to 4) was
indicated by their ability to inhibit growth of S. aureus
MSSA4.
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Identification and characterization of the mutacin II biosynthetic
gene cluster.
The above-mentioned results indicated that mutacin
II-specific genes are likely to be clustered. To confirm this, up- and downstream fragments of the mutacin II structural gene
(mutA) from S. mutans T8 were cloned by
single-specific-primer PCR chromosomal walking (23, 24). The
cloned fragments were sequenced by automated sequencing (Applied
Biosystems 373A DNA sequencer), and the resultant sequence data were
analyzed by using several programs in the GCG package (Genetics
Computer Group, Madison, Wis.). The entire mutacin II biosynthetic gene
cluster was determined.
This cluster consists of seven genes (Fig.
5) in the order mutRAMTFEG,
based on BLAST similarity search. Southern blot analyses with an
internal portion of each gene as a probe indicated that these seven
genes exist only in mutacin II-producing strains (data not shown). In
addition to the previously identified MutA and MutM (29),
the products of five additional open reading frames (MutR, MutT, MutF,
MutE, and MutG) were identified. The gene upstream of mutA
was designated mutR because it encodes a protein with similarity (25% identity and 49% positives) to a positive
transcriptional regulator, Rgg (25, 26), which regulates
expression of glucosyltransferase G and influences the Spp phenotype of
Streptococcus gordonii Challis. MutR is therefore proposed
to be the regulator for the mutacin biosynthetic operon. Internal
disruption of mutR resulted in mutants that did not produce
any mutacin, due to abolished transcription of mutA (data
not shown). This result further supported the proposed function of MutR
as a regulator. Approximately 130 bp upstream of mutR
resides a putative structural gene with a deduced amino acid sequence
having similarity to transposases (tra) from lactococcal insertion elements (9, 28, 30) but lacking a translation start codon and a ribosomal binding site. PCR analyses indicated that
this transposase does not exist in S. mutans UA140 (data not
shown). A 1.2-kb intergenic region and CTP synthetase (UTP-ammonium ligase) gene follows the tra gene.
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FIG. 5.
Genetic organization of the mutacin II biosynthetic gene
cluster. The cluster consists of seven specific genes in the following
order: regulator gene (mutR), structural gene
(mutA), modifying enzyme gene (mutM), transporter
and leader peptidase gene (mutT), and accessory
self-protection genes (mutFEG). Upstream of mutR
is a silent transposase gene (tra).
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The amino acid sequence deduced downstream of the mutM gene,
MutT, exhibits a high degree of similarity with sequences of ATP-dependent transport proteins, especially LanT (38% identity and
59% positives) of lacticin 481 from Lactococcus lactis
(18). Based on what is known about LanT, it is reasonable to
propose that MutT has the dual function of cleaving the leader peptide and excreting mature mutacin. Insertion inactivation of mutT
with a terminatorless antibiotic cassette completely abolished mutacin production (data not shown).
The products of the open reading frames after MutT were MutFEG. MutF
showed its strongest sequence identities with LacF (44% identity and
65% positives) (19) and several other immunity proteins and
ATP-binding membrane proteins. Thus, it is likely that MutF is involved
in mutacin immunity. MutF is an ATP-binding protein without a
transmembrane domain, and MutE and MutG are membrane proteins (data not
shown) with homology to Cdd4, Cdd3, and Cdd2 from Clostridium
difficile (2). As cdd4, cdd3, and cdd2 do in C. difficile, mutF,
mutE, and mutG code for an ABC transporter
system, which is likely to be involved in mutacin immunity. Downstream
of mutG was a 0.7-kb intergenic region followed by a
fructose biphosphate adolase (fba) gene.
Genes for lantibiotic biosynthesis (8, 11, 14) can be found
on plasmids (epidermin, Pep5, cytolysin, lacticin 481, lactocin S, and
streptococcin A-FF22), on a conjugative transposon (nisin), or on the
chromosome (subtilin, epilancin K7, salivaricin A, and variacin).
Buchman et al. (3) suggested that many, if not all,
lantibiotics probably evolved from a common ancestor, and the ability
to make lantibiotics has become dispersed among gram-positive bacteria
by transfer of mobile genetic elements such as plasmids or transposons
that encode lantibiotics. Although mutacin II biosynthesis genes are
located on the chromosome, the presence of a silent transposase gene
(tra) in the upstream region of the operon suggested that
the ability to produce mutacin II in S. mutans T8 might be
obtained from an ancestral transposon. It may be that, after entering a
new host (for example, S. mutans T8), this ancestral
transposon began to evolve to suit the needs of the new host by the
usual means of spontaneous mutation and natural selection. Once stably
situated within the host chromosome, the tra function, we
hypothesize, may have become nonfunctional.
One way to identify the genes required for lantibiotic biosynthesis is
to transfer the capacity for lantibiotic production from a producer
strain to a nonproducer strain. Conversion of a
non-bacteriocin-producing strain into a bacteriocin producer by
transformation was reported for several lantibiotics, including subtilin (13), epidermin (1), nisin
(10), and lacticin 481 (18). For subtilin,
conversion was achieved by competence transformation from a
natural producer (Bacillus subtilis ATCC 6633) into a
nonproducing strain (B. subtilis 168), and a 40-kb region of
chromosomal DNA was associated with conversion (13). For
epidermin production, the transfer of an 8-kb DNA fragment containing
six complete open reading frames into heterologous strains
(Staphylococcus carnosus and Staphylococcus
xylosus) was sufficient for antibacterial activity (1).
The genes for nisin production are within a 68- to 70-kb transposon
that can be transferred among strains of L. lactis by
conjugative transposition (10). In the case of lacticin 481, two genes, lcnA and lcnM, cloned on a plasmid
vector were sufficient to convert a nonproducing strain into a
lacticin-producing strain (18). Although mutA and
mutM have high homology with lcnA and
lcnM, this is not the case in the conversion of S. mutans UA159. Transforming only mutA and
mutM is not sufficient to convert the wild-type strain UA159 into a mutacin-producing strain (unpublished results). The conversion of strain UA159 into a mutacin producer by integration of a fragment of
T8 DNA does not conclusively prove that all of the genes required for
mutacin production reside on the integrated DNA. The integrated DNA may
merely provide specific genes required for mutacin II production.
Another required regulator gene(s), such as the diacylglycerol kinase
gene dgk (6), is already present in strain UA159.
In summary, we have identified the specific mutacin II biosynthetic
gene cluster and successfully converted S. mutans UA159 into
a mutacin II producer by competence transformation. In addition, we
have successfully transformed a strain (UA140) producing another mutacin into a mutant strain with the ability to produce the resident mutacin I as well as the introduced mutacin II.
Nucleotide sequence accession numbers.
The nucleotide
sequences of mutR, mutT, and
mutFEG have been deposited in the GenBank database under
accession no. AF007761, AF026468, and AF082183, respectively.
This work was supported by NIH grant DE09082. The mass spectrometer
was purchased by funds from an NIH Instrumentation Grant (SI0RR06487)
and from the University of Alabama at Birmingham (UAB). Operation of
the Mass Spectrometer Shared Facility was supported in part by an NCI
Core Research Support Grant to the UAB Comprehensive Cancer Center (P30 CA13148).
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