Department of Microbiology and Immunology,
Emory University Health Sciences Center, Atlanta, Georgia
30322,1 and
Department of
Biophysical Chemistry, Section of Microbial Ingredients, Netherlands
Institute for Dairy Research (NIZO), 6710 BA Ede, The
Netherlands2
 |
INTRODUCTION |
Many gram-positive cocci, such
as Streptococcus pyogenes (group A streptococcus),
Streptococcus agalactiae (group B streptococcus), Streptococcus pneumoniae, and some strains of
Enterococcus faecalis (previously classified as group D
streptococcus), are important pathogens that are major causes of a
variety of infectious diseases in humans. The illnesses caused by these
pathogenic streptococci range from local infections of moderate
severity, such as impetigo, pharyngitis, sinusitis, and otitis media,
to life-threatening invasive diseases, such as pneumonia, meningitis,
endocarditis, bacteremia, myositis, necrotizing fasciitis, and
streptococcal toxic shock syndrome. In addition, some types of
streptococcal infections can result in severe complications, such as
arthritis, rheumatic fever, and acute glomerulonephritis
(2). Major progress has been made in the last decade in the
genetic study of pathogenic streptococci due to the development of
molecular tools such as vectors, reporter genes, transducing phages,
and transposons that are functional in these bacteria (6,
13). However, the absence of other important tools, such as
controlled gene expression systems, significantly limits the study of
these gram-positive pathogens.
Bacterial pathogens often regulate the expression of virulence genes in
a coordinated manner in response to changes in the environment. The
ability to manipulate the expression of different genes is an important
tool for understanding the regulatory cascade mechanisms and the
importance of gene regulation in pathogenesis. In addition, to study
the importance of the relative quantities of gene products on their
interactions, it is essential to be able to control their expression
independently. For these purposes, a regulated heterologous promoter
system is essential.
In gram-negative bacteria, a variety of promoters (native or chimeric)
and vectors (ranging from low to high copy number) have been developed
to allow quantitative modulation of gene expression over a range of
levels. Unfortunately, many of these systems do not function in
gram-positive organisms, probably because the requirements for promoter
usage are more stringent in these bacteria than in
Escherichia coli (34, 35). Only a few
regulated promoters have been described for gram-positive
bacteria, and some of these, such as the Bacillus
subtilis sporulation promoters, require factors unique to the
native species that are not present in heterologous systems,
like specific sigma factors and their regulators (19). Some
sugar-regulated promoters might not be functional in all hosts because
they depend both on sugar entry and on some initial steps to convert
the sugar into the inducer that relieves repression of the promoter
involved. The appropriate enzymes to carry out these processes might
not be present in all hosts. A considerable portion of the
available information regarding gene expression in gram-positive
bacteria was obtained from the extensively studied species B. subtilis and Lactococcus lactis (10, 11).
One well-characterized lactococcal gene expression system is based on
the autoregulatory properties of the L. lactis nisin gene
cluster. Two genes in the cluster, nisA and nisF,
are induced by nisin via a two-component signal transduction
pathway consisting of a histidine protein kinase, NisK, and a response
regulator, NisR. Expression of both nisR and nisK
is driven from the constitutive promoter of nisR (9,
29). Nisin acts as an inducer on the outside of the cell and is
sensed by NisK. Recently, it has been reported that a two-plasmid
system in which the nisA promoter and the regulatory genes
nisR and nisK are used allows efficient control
of gene expression by nisin in a variety of lactic acid bacteria
(27, 30).
Another controllable expression system of L. lactis is based
on the lactose-inducible transcription of the lac
operon. Expression of this operon is repressed during
growth on glucose and is regulated at the transcriptional level by the
LacR repressor (40). LacR expression is repressed during
growth on lactose, which allows expression from the lacA
promoter. The lacA promoter and the LacR repressor have been
used to express several heterologous genes in a
lactose-glucose-dependent manner in L. lactis (40,
42).
In B. subtilis, expression of the xylose utilization
operon is inducible via a repressor-mediated mechanism
(18). The xylA promoter and the XylR repressor
have been used to control expression of heterologous genes in bacillus
species. In addition to such native promoters, a chimeric promoter,
designated Spac, consisting of the E. coli lac
operator fused to the SPO-1 phage promoter was constructed to regulate
gene expression in bacillus (45). The promoter and the
ribosome-binding site of the penicillinase gene of Bacillus
licheniformis were linked to the E. coli lacI repressor
gene to allow expression of lacI in bacillus. In this system, transcription from Spac is repressed by the LacI
repressor and can be induced by lactose and the gratuitous inducer
isopropyl-
-D-thiogalactoside (IPTG).
In pathogenic streptococci, no regulated foreign promoters have been
identified. Furthermore, there is a paucity of information regarding
comparative expression of specific promoters in different pathogenic
streptococcal species. We demonstrate here that the L. lactis
nisA promoter is active in all of the gram-positive species that
we used, including the pathogenic streptococci. Furthermore, expression
from the nisA promoter can be efficiently regulated experimentally in all of these organisms. When the same reporter gene
was used, the nisA promoter was found to be stronger than the lacA, xylA, or Spac promoter in
both B. subtilis and S. pyogenes and to show the
greatest degree of induction in S. pyogenes. We expect the
nisA promoter to allow manipulation of gene expression in
pathogenic streptococci and to bring the level of sophistication of
genetic studies of these gram-positive bacteria closer to that of
gram-negative pathogens.
| |
MATERIALS AND METHODS |
Bacterial strains.
Experiments were performed with the
following gram-positive strains: S. pyogenes JRS4 (group A
streptococcus) (15), S. agalactiae COH31 (group B
streptococcus) (38), S. pneumoniae 1131 (47), E. faecalis OG1RF (12), and
B. subtilis W168 (7). L. lactis FMCB1,
derived from MG1363 (5), was used as a host for plasmids pNZ9520 and pNZ9530 (Table 1), and
E. coli JM109 (44) and DH5
(20)
were used as hosts for all of the other plasmids (Table 1).
Media.
S. pyogenes, S. agalactiae, and
E. faecalis were grown in Todd-Hewitt broth supplemented
with 0.2% yeast extract (16). S. pyogenes was
also grown on L3 medium (22). S. pneumoniae cells were grown in Todd-Hewitt broth supplemented with 0.5% yeast extract. B. subtilis and E. coli cells were grown in Luria
broth (39). L. lactis cells were grown in M17
broth (Becton-Dickinson) supplemented with 0.5% glucose. The following
antibiotics were used: for S. pyogenes, S. agalactiae, and S. pneumoniae, 0.5 µg of erythromycin per ml and 2 µg of chloramphenicol per ml; for E. faecalis, B. subtilis, and L. lactis, 5 µg
of erythromycin per ml and 10 µg of chloramphenicol per ml; and for
all species, 100 µg of spectinomycin per ml.
Plasmid DNA preparation and transformation.
Plasmid DNA was
prepared from L. lactis by using glass beads for cell lysis
(17) and from E. coli by the alkaline lysis method (32). Naturally competent S. pneumoniae
and B. subtilis cells were transformed with plasmid DNA as
previously described (43, 46). Electroporation was used as
previously described to transform S. pyogenes
(36), S. agalactiae (14), and E. faecalis (33).
Plasmid construction.
The plasmids used in this study are
described in Table 1. Plasmid pEU308 was obtained by ligating the
2.1-kb StyI-EcoRI fragment of plasmid pDH88
(45), containing the Spac promoter and the laclq repressor gene, to a 3.4-kb
HindIII-EcoRI fragment of plasmid pLZ12Spec
(23) carrying the pSH71 origin of replication and the
spectinomycin resistance gene add9. The protruding ends of StyI and HindIII were filled in by using the
Klenow fragment of DNA polymerase I prior to digestion with
EcoRI. Plasmid pEU352 was generated by ligating the 1.85-kb
HindIII-BamHI fragment of plasmid pMLK99
(26) carrying a promoterless gusA gene to plasmid pEU308 cut with HindIII and BglII, creating a
transcriptional fusion of the Spac promoter to the
gusA gene.
Plasmid pEU327 was constructed by ligating the 3.4-kb
HindIII-EcoRI fragment of plasmid pLZ12Spec
containing the pSH71 origin of replication and the add9
spectinomycin resistance gene to the 1.5-kb
HindIII-EcoRI fragment of plasmid pDG1832
(1) carrying the xylR repressor and the
xylA promoter. Plasmid pEU356 was generated by ligating the
1.85-kb NotI-HindIII fragment of plasmid
pMLK100 (26) carrying a promoterless gusA gene to
plasmid pEU327 cut with HincII and HindIII,
creating a transcriptional fusion of the xylA promoter and
the gusA gene. The protruding ends of NotI were
filled in with the Klenow fragment of DNA polymerase I prior to cutting
with HindIII.
-Glucuronidase activity.
Nisin was obtained from Sigma as
a 2.5% nisin solution in sodium chloride containing denatured milk
solids. The nisin concentrations referred to below are in micrograms of
total solids per milliliter. Stock solutions of nisin were made by
suspending 100 mg of nisin per ml in 0.05% acetic acid and then
diluting the preparations 10-fold with dimethyl sulfoxide and were
stored at 
20°C. Further dilutions were made with water and were
used immediately.
Expression of the gusA gene was determined by assaying the
rate of hydrolysis of the substrate
p-nitrophenyl--D-glucuronide. Bacterial
cultures were inoculated at concentrations of approximately 106 CFU/ml into selective media along with various
concentrations of nisin. Most cultures were grown overnight at 37°C
statically; the only exception was B. subtilis cultures,
which were grown with aeration. A 1.0-ml aliquot of each culture was
pelleted, resuspended in 0.4 ml of lysis buffer [60 mM
K2HPO4, 33 mM KH2PO4, 7.4 mM (NH4)2SO4, 1.7 mM sodium
citrate; pH 7.0], and lysed with a FastPrep cell disrupter (model
FP120; Bio 101, Inc.) in tubes containing a glass bead matrix (Bio 101, Inc.) at a speed of 5 m/s for 30 s. A 0.1-ml aliquot of cell
extract was added to 0.9 ml of reaction buffer (60 mM
Na2HPO4, 40 mM NaH2PO4,
10 mM MgSO4 [pH 7.0], 20 mM dithiothreitol), and 0.2 ml
of a 4-mg/ml solution of
p-nitrophenyl--D-glucuronide in lysis buffer
was then added. Samples were incubated at 30°C until yellow color
development reached an optical density at 420 nm of approximately 0.2 to 0.6. Reactions were stopped by adding 0.5 ml of 1 M
Na2CO3. Enzyme activity (units [U]) is given
below as 1,000 times the increase in absorbance at 420 nm per minute
per unit of optical density at 600 nm of the culture.
| |
RESULTS AND DISCUSSION |
Nisin gene expression system.
To characterize regulated
expression driven from the L. lactis nisA promoter, we used
a plasmid-based system with E. coli gusA as a reporter gene.
This system consists of a reporter plasmid and a regulatory plasmid,
each derived from a broad-host-range replicon. The reporter plasmid,
pNZ8008, is a pSH71 derivative that carries a transcriptional fusion of
the nisA promoter to gusA (9) (Table
1). The nisA regulatory genes, nisR and
nisK, were carried on either of two alternative plasmids,
both of which are pAM1 replicons. One of the regulatory
plasmids, pNZ9530, carries the native pAM1 origin of
replication, and the other, pNZ9520, is a derivative that has a
deletion in the replication repressor gene, repF, which
results in increased plasmid copy number in L. lactis
(27) (Table 1). Consistent with their copy numbers, plasmids
pNZ9530 and pNZ9520 are expected to provide low and high gene dosages,
respectively, of the regulatory genes nisR and
nisK.
Nisin sensitivity among the species tested.
Nisin has
bactericidal activity against many gram-positive bacteria, including
staphylococci, streptococci, bacilli, clostridia, and mycobacteria
(24). However, nisin sensitivity has been reported to vary
significantly, even between members of the same species (3, 4,
8). We found that there is considerable variation in nisin
sensitivity among S. pyogenes, S. agalactiae,
S. pneumoniae, B. subtilis, and E. faecalis (Fig. 1), which is
consistent with observations made for other gram-positive bacteria
(4, 8, 24, 41).
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FIG. 1.
Nisin sensitivity. Bacteria harboring both the reporter
vector and one of the regulatory plasmids were inoculated into media
containing different nisin concentrations. Cell growth is expressed as
the culture optical density at 600 nm (O.D.600) following overnight
incubation at 37°C. (A) S. pyogenes cells carrying pNZ9520
and pNZ8008. (B) Strains of the following species carrying pNZ8008 and
pNZ9530: S. agalactiae ( ), S. pneumoniae
( ), B. subtilis ( ), and E. faecalis
( ).
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The nisA system is regulated by nisin in all strains
tested.
Modulation of the nisA promoter activity by
nisin was characterized in S. pyogenes, S. agalactiae, S. pneumoniae, B. subtilis, and
E. faecalis. Cells harboring both the reporter
vector pNZ8008 and one of the regulatory plasmids were incubated in
media containing different amounts of nisin. The -glucuronidase
activity (expressed from the nisA promoter-gus
transcriptional fusion) was determined following overnight incubation.
In all strains tested, cells harboring only the gusA
reporter plasmid exhibited very low levels of -glucuronidase activity. This indicates that nisA promoter function is
dependent on its regulatory components, NisR and NisK, even in
heterologous hosts. However, the plasmid supplying the regulatory
proteins NisR and NisK for optimal induction differed in the different hosts (Table 2).
It was expected that pNZ9520 would have a higher copy number than
pNZ9530 because of a deletion in the replication repressor gene
(27). Although this was the case in L. lactis
(27), we found that it was not true in all of the strains
which we used. We compared the amount of linearized plasmids pNZ9520
and pNZ9530 with the amount of the coresident plasmid pNZ8008 by using
ethidium bromide-stained agarose gel electrophoresis and/or Southern
blot analysis (data not shown). In B. subtilis, pNZ9520 had
a copy number similar to that of pNZ8008, while pNZ9530 had a lower
copy number. However, in S. pyogenes and in S. pneumoniae, there was no detectable difference between the amounts
of pNZ9520 and pNZ9530, and their copy numbers appeared to be much
lower than the copy number of pNZ8008. Therefore, the copy numbers of
these broad-host-range plasmids, which replicate by a rolling-circle
mechanism, cannot be predicted in different species, and the amounts of
NisR and NisK expressed from each plasmid cannot be predicted either.
In S. pyogenes cells harboring both the expression plasmid
pNZ8008 and the regulatory plasmid pNZ9520, very low constitutive activity was observed in the absence of nisin, and nisin caused about
59-fold induction (Table 2 and Fig. 2A).
In S. pyogenes, the regulatory plasmid pNZ9530 was
less useful than pNZ9520 because the constitutive activity was high,
and little induction was observed (Table 2). In contrast, in the
E. faecalis strain carrying pNZ9520, although the
constitutive -glucuronidase activity was low, addition of nisin to
the medium resulted in no significant induction (Table 2). In the other
strains investigated, the S. agalactiae, S. pneumoniae, and B. subtilis strains, cells harboring
pNZ9520 exhibited such high levels of constitutive
-glucuronidase activity that little induction above these levels
was detected in the presence of nisin (Table 2).
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FIG. 2.
Dose response of the nisA promoter. Cells
harboring both pNZ8008 and pNZ9520 (A) or pNZ9530 (B through E) were
inoculated into media containing different nisin concentrations, and
-glucuronidase (GUS) activity was determined in cell extracts
following overnight growth at 37°C. The enzyme activity is given as
1,000 times the increase in absorbance at 420 nm per minute per optical
density unit of the culture. (A) S. pyogenes. (B) E. faecalis. (C) S. agalactiae. (D) S. pneumoniae. (E) B. subtilis.
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In all of the strains tested in which pNZ9520 caused constitutive
nisA promoter activity, the regulatory plasmid pNZ9530
resulted in controlled expression from the nisA promoter
(Table 2 and Fig. 2). In response to nisin, 20-fold induction was
observed in E. faecalis (Fig. 2B) and 10- to 11-fold
induction of -glucuronidase activity was observed in S. agalactiae, S. pneumoniae, and B. subtilis
(Fig. 2C through E).
The nisin concentration required for maximal induction of the
nisA promoter was different for each of the strains. In
general, the greatest induction was obtained with a concentration just below the inhibitory level (Fig. 1 and 2). A comparison of the growth
curve obtained in the presence of this sublethal concentration of nisin
to the growth curve obtained in the absence of nisin showed that the
high nisin concentration had no effect on the growth rate (data not
shown). The optimal nisin concentrations used for induction were 1 µg/ml in S. pyogenes, 5 µg/ml in S. agalactiae, 1 to 5 µg/ml in S. pneumoniae, and 20 µg/ml in B. subtilis and E. faecalis (Fig. 2).
These concentrations are higher than those used to control the
system in L. lactis, in which induction was observed with a
nisin concentration far below the inhibitory concentration
(27).
Characterization of nisA promoter strength.
The
level of -glucuronidase measured with the induced nisA
promoter varied considerably among strains, as has been observed previously with Lactococcus, Leuconostoc, and
Lactobacillus species (27). The highest enzyme
activity which we found, about 230 U, was observed following nisin
induction in the strains of S. pyogenes (Fig. 2A) and
S. agalactiae (Fig. 2C). In the inducible S. pneumoniae system, which required nisR and
nisK on pNZ9530, the maximal -glucuronidase activity
induced was 55 U (Fig. 2D), which is fivefold lower than the
constitutive level observed in the presence of pNZ9520 in this organism
(data not shown). In the strains of E. faecalis and
B. subtilis tested, the highest induced levels of
-glucuronidase activity were 72 and 18 U, respectively (Fig. 2B and
E). These variations may reflect differences in the efficiencies of the
transcription and translation machinery of each of the strains tested
for recognizing the nisA promoter. Consistent with this
idea, identical promoter sequences were found to have significantly
different activities in L. lactis and S. pneumoniae or B. subtilis (28, 31). It has
also been suggested that differences between the amino acid sequences
of the RNA polymerase holoenzymes in different hosts may lead to
different affinities for the same promoter sequence (25). In
addition, differences in codon usage probably affect the amount of
-glucuronidase produced in the strains studied here.
Comparison of the strength of the nisA promoter to the
strengths of the other regulated promoters in B. subtilis.
To evaluate nisA promoter strength, we compared the activity
of this promoter to the activities of other regulated promoters known
to function in gram-positive bacteria. This comparative study was
performed first with B. subtilis, because it is the gram-positive organism in which gene expression has been most extensively investigated. In this comparison, we used
transcriptional fusions of different promoters to the E. coli
gusA gene. To avoid potential differences due to gene
dosage, all fusions were carried on the same type of replicon
containing the pSH71 origin. The following promoters were studied: the
L. lactis nisA promoter, which is regulated by
nisin through NisR and NisK; the L. lactis lacA promoter,
which is regulated by lactose through the LacR repressor; the
B. subtilis xylA promoter, which is regulated by xylose through the XylR repressor; and the chimeric
Spac promoter, which is regulated by IPTG through the Lacl
repressor. The following plasmids were used (Table 1): pNZ8008 and
pNZ9530 (nisin system [see above]); pNZ276, carrying the
xylA promoter-gusA transcriptional fusion and the
repressor lacR; pEU356, carrying the xylA
promoter-gusA transcriptional fusion and the repressor
xylR; and pEU352, carrying the Spac
promoter-gusA transcriptional fusion and the lacl
repressor. To prevent catabolite repression of the promoters that are
regulated by sugars (lacA and xylA), all assays
were performed in Luria broth containing no sugar source other
than that needed for induction. B. subtilis cells harboring
the appropriate plasmids were grown under both inducing
and noninducing conditions, and the -glucuronidase activity
was determined following overnight growth (Fig.
3 and Table
3).
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FIG. 3.
nisA promoter strength in B. subtilis.
B. subtilis cells harboring the following plasmids were used:
pNZ8008 and pNZ9530 (nisA promoter), pEU352 (Spac
promoter), pEU356 (xylA promoter), and pNZ276
(lacA promoter). Cells were grown in Luria broth or in Luria
broth supplemented with 20 µg of nisin per ml, 20 mM IPTG, 2%
xylose, or 2% lactose. The -glucuronidase (GUS) activity was
determined following overnight incubation at 37°C.
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Sequence analysis of 236 promoters recognized by the
B. subtilis sigma A subunit of RNA polymerase
revealed an extended promoter structure (21). The most
highly conserved bases include the 35 and 10 hexanucleotide core
elements and a TG dinucleotide at positions 15, 14. In
addition, several weakly conserved A and T residues are
present upstream of the 35 region. All of these elements are found
with different degrees of conservation in the four promoters which we
tested (Table 4).
In B. subtilis, the nisA, Spac, and
xylA promoters were all regulated to similar extents (about
10-fold induction), while expression from the L. lactis lacA
promoter was too low to be detected even under inducing conditions
(Fig. 3 and Table 3). The maximum induced expression was highest for
the nisA promoter.
There are several possible explanations for the differences in the
strengths of these promoters. It has been shown that the sequence
between the 10 and 35 elements affects promoter strength several
hundred-fold (25), probably by altering the context in which
these elements are presented to the RNA polymerase holoenzyme. The sequences of the promoters which we compared differ in this spacer
region, and this may also have some effect on the strengths of the
promoters.
Comparison of the strength of the nisA promoter to the
strengths of other regulated promoters in S. pyogenes.
Each
of the promoter systems mentioned above was also tested in
S. pyogenes to learn whether it could be used to
control gene expression in this organism. All assays were carried out
in L3 medium with and without inducer. To study the effect of catabolic repression on the promoters that are regulated by sugars, expression from the Spac, xylA, and lacA
promoters was also determined in L3 medium containing glucose (Fig.
4 and Table 3).
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FIG. 4.
nisA promoter strength in S. pyogenes.
S. pyogenes cells harboring the following plasmids were used:
pNZ8008 and pNZ9520 (nisA promoter), pEU352 (Spac
promoter), pEU356 (xylA promoter), and pNZ276
(lacA promoter). Cells were grown in L3 medium or in L3
medium supplemented with 1 µg of nisin per ml, 2% glucose, 2%
lactose, 20 mM IPTG, or 2% xylose. The -glucuronidase (GUS)
activity was determined following overnight incubation at 37°C.
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As in B. subtilis, in S. pyogenes the expression
driven from the induced nisA promoter was higher than the
induced activities of all of the other promoters (specific activity,
about 67 enzyme units). The Spac and lacA
promoters had lower activities (specific activities, about 35 and 24 enzyme units, respectively), and no activity from the xylA
promoter was detectable. nisA and Spac were the
only promoters that showed increased activity in response to the
inducers. nisA activity was induced 11-fold by nisin, while only 4-fold induction of the Spac promoter was obtained with
IPTG. Induction of Spac by lactose was slightly lower than
induction by IPTG (Fig. 4).
The levels of expression driven from the lacA promoter in L3
medium and in L3 medium containing lactose were similar; however, these
levels of expression were about fivefold higher than the lacA activity in L3 medium supplemented with glucose (Fig.
4). This indicates that the differences in lacA promoter
activity resulted from glucose repression rather than induction by
lactose. Since lactose did not induce the L. lactis
lacA promoter in either B. subtilis or
S. pyogenes, it is possible that in these hosts lactose is
not processed to the inducer form.
Because the constitutive expression of the lacA promoter was
high in S. pyogenes, we investigated the effect of glucose
on this system. There was a fivefold reduction in expression from the
lacA promoter when S. pyogenes was grown in
glucose compared to when it was grown either in the presence of lactose
or with no added sugar (Fig. 4). Since the lacA promoter
contains a catabolite repression element that is functional in L. lactis (11), it is possible that glucose causes
catabolite repression of this system in S. pyogenes as well.
Expression from the uninduced xylA promoter was undetectable
in both S. pyogenes and B. subtilis. However,
although this promoter was induced in the latter, it was not in the
former. We believe that it is likely that xylose does not enter
S. pyogenes, since we also found that xylose was not able to
serve as a carbon source in this organism. An overnight culture in
which glucose was added to L3 medium reached a higher optical density
than a culture with no added sugar, while addition of xylose had no
effect (data not shown).
Conclusions.
In this study we demonstrated that the
lactococcal nisA promoter is recognized in many different
gram-positive species, including pathogenic streptococci. In B. subtilis and S. pyogenes, it was the most efficient of
the regulatable promoters studied.
For efficient induction, the regulatory protein must be able to
interact with the RNA polymerase, whose sequence differs in different
bacteria. We have extended the list of species in which NisR is able to
activate transcription to include B. subtilis and some of
the pathogenic streptococci. We concluded that nisin interacts
effectively with NisK in all of these strains since the nisA
promoter was regulated by nisin in all species of AT-rich gram-positive
bacteria which we studied.
The nisA gene expression system can be moved easily between
strains of different species because it is contained on two
broad-host-range replicons, and it should provide an alternative to the
commonly used Spac promoter, which may not be active in all
gram-positive organisms. Furthermore, in strains in which
Spac is active, nisA should be compatible with
the Spac system to allow independent regulation of different
cloned genes. For all of these reasons and because it is the first
heterologous regulatable promoter described for pathogenic
streptococci, we expect the nisA gene expression system to
be extremely useful in the future, especially for genetic studies of
important gram-positive human pathogens.
This work was performed during the tenure of a research fellowship to
Z.E. from the American Heart Association, Georgia Affiliate, and was
supported by Public Health Service grant R37-AI20723.
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Abstract
Introduction
