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Journal of Bacteriology, August 1999, p. 5024-5032, Vol. 181, No. 16
Grupo de Patogénesis Molecular
Bacteriana, Unidad de Microbiología e Inmunología,
Facultad de Veterinaria, Universidad Complutense, 28040 Madrid,
Spain,1 and Lehrstuhl für
Mikrobiologie, Theodor- Boveri-Institut für Biowissenschaften,
Universität Würzburg, 97074 Würzburg,
Germany2
Received 16 March 1999/Accepted 31 May 1999
The The gram-positive, facultative
intracellular bacterium Listeria monocytogenes causes
listeriosis, a severe, often fatal infectious disease of humans and
animals. L. monocytogenes is an opportunistic pathogen that
affects predominantly debilitated individuals with a defective
cell-mediated immune response. Its natural habitat is soil, from which
it reaches the vertebrate host via the food chain (15, 45).
As in many other bacterial pathogens that can live saprophytically in
inanimate environments, virulence gene expression is tightly regulated
in L. monocytogenes. Almost all known listerial virulence
determinants are coordinately expressed under the positive control of
the PrfA protein, a transcription factor structurally and functionally
related to cyclic AMP receptor protein (CRP) (6, 24, 25, 51,
54). A model has been proposed for the regulatory mechanism of
PrfA in which, as for CRP, the protein becomes transcriptionally active
via an allosteric conformational transition brought about by a putative
low-molecular-weight cofactor (41, 54). There is evidence
that the PrfA regulon is subject to complex environmental control in
L. monocytogenes. Virulence genes are expressed at 37°C
(i.e., the body temperature of warm-blooded animals) but not at 26°C
or below (i.e., environmental temperatures) (10, 27, 41),
suggesting that temperature sensing may enable L. monocytogenes to detect its transition from the free environment
to the animal host. However, an increase in temperature is not
sufficient for the full activation of the PrfA regulon
(40-42). L. monocytogenes produces little or no
virulence factors in rich medium (e.g., brain heart infusion [BHI])
at 37°C, but a clear induction of virulence genes takes place at this
temperature if BHI is either supplemented with the absorbent activated
charcoal (40-43) or replaced by minimal essential medium
(4). Clearly, therefore, chemical components of the
extracellular environment also play a critical role in regulating the
expression of the PrfA regulon in L. monocytogenes.
Park and Kroll reported in 1993 (35) that the presence in
the growth medium of the In this study we present evidence that reconciles the two hypotheses:
that of Park and Kroll (35) suggesting that Bacterial strains, media, and culture conditions.
Wild-type
L. monocytogenes strains were from a previously described
panel comprising recent clinical and environmental isolates and
collection or laboratory strains of various serotypes (41, 42). The strains tested met with the phenotypic criteria of the
wild-type prfA genotype (41, 42): they exhibited
weak to undetectable hemolytic and lecithinase reactions in BHI at 37°C, and the production of the corresponding activities was strongly activated in charcoal-treated medium. Escherichia coli
DH5 DNA methods.
Restriction and modification enzymes were
purchased from Pharmacia and were used according to the manufacturer's
instructions. Listeria chromosomal DNA was isolated as
previously described (20). Specific DNA was amplified with
the Expand High Fidelity PCR system (Boehringer Mannheim). PCR products
were purified with the Quiaquick Gel Extraction Kit (Qiagen) and were
cloned with the Sure Clone Kit (Pharmacia). Plasmid DNA was extracted
from E. coli with the Plasmid Purification Kit (Qiagen).
Sequences were determined from both strands of plasmid DNA with an
Applied Biosystems 377 apparatus. For Southern blot analysis, DNA
fragments were subjected to electrophoresis in 1% agarose in
Tris-borate-EDTA, denatured, and transferred to nitrocellulose membrane
(Schleicher & Schuell) according to standard protocols (1).
DNA probes consisted of PCR or restriction fragments internal to the
genes of interest. They were radiolabeled with the Boehringer Mannheim Random Primed DNA Labeling Kit and [ PlcB (lecithinase) activity determinations.
The expression
of plcB was quantified via the lecithinase activity of its
protein product, the wide-substrate-range phospholipase C, PlcB
(53), by using a previously described turbidimetric assay
(42) and type IV phosphatidylcholine from egg yolk (Sigma) as the substrate. We use plcB as a reporter of
PrfA-dependent expression (40, 41) because it has several
advantages over the hly (hemolysin) gene used by others
(2, 3, 32). First, unlike hly, which is expressed
from PrfA-dependent and -independent promoters (12), the
expression of plcB is driven uniquely by a PrfA-dependent
promoter, PactA (53). This promoter also has a threshold for activation by PrfA that is higher than that of
hly (41), which helps eliminate any constitutive
background activity. Second, the PlcB assay gives more clearcut, linear
results than do hemolysin determinations (41). PlcB activity
was determined in the supernatant of cultures collected when the
optical density at 600 nm (OD600) reached 2.0. PrfA-dependent virulence gene expression is normally silenced in
wild-type L. monocytogenes growing in BHI at 37°C (see
above) (40-42). Therefore, to enable the repressor effect
exerted by sugars on plcB expression to be observed,
wild-type strains were cultured in charcoal-treated BHI, in which the
PrfA regulon is fully activated (40-43).
Cloning and sequencing of the bvr locus.
A
4.4-kb DNA fragment was amplified by PCR from EGDCR by
using the oligonucleotide B35031 (5'-AGGTTGTGATGTTTATGGAAA),
specific for parB, and the degenerate primer A35030
(5'-RTTNCCYTCYTCNGTRTATAAYTT; where R = purine, Y = pyrimidine, and N = any base), designed from the Listeria
seeligeri kat-encoded oligopeptide KFYTEEGN (7) (see
below and Fig. 2). Several attempts to clone the full-length fragment
in E. coli by using pUC18 failed, suggesting that this DNA
region was toxic for bacteria if present in multiple copies. However, a
plasmid was isolated (pKLB3A) that harbored a deletion at the
parB-proximal part of the 4.4-kb fragment. The 2.1-kb insert of pKBL3A was partially sequenced and two further oligonucleotides, 94C230 (5'-GTTGCTCCTGCAGCCGGTGT-3') and 94C229
(5'-TAATTCCAAGCGCATGTCCT), were designed which, when used
with B35031 and A35030, respectively, gave rise to 2.1- and 2.4-kb PCR
products. These PCR fragments were inserted into pUC18, yielding the
plasmids pKLB3B (B35031-94C230 fragment) and pKLB3C (A35030-94C229
fragment). The entire sequence of the bvr locus, encompassed
by the parB and kat genes (see Fig. 2), was
determined from the overlapping inserts of pKLB3B and pKLB3C.
Construction of the bvrAB:: RNA procedures.
Total RNA was extracted from
mid-exponential-phase cultures of L. monocytogenes by using
a hot-acid-phenol protocol as described elsewhere (43). The
operon structure of the bvr locus was investigated by using
5 µg of total RNA and the Titan One Tube reverse transcription-PCR (RT-PCR) Kit from Boehringer Mannheim. The effect of Evidence that cellobiose downregulates virulence gene expression
via a specific mechanism.
Milenbachs et al. (32) showed
that NCTC 7973 has an anomalous pattern of carbon source regulation,
with cellobiose being the only sugar, of a variety of fermentable
carbohydrates tested, causing virulence gene repression. This behavior
suggests that (i) NCTC 7973 is probably a deregulated mutant with a
defect in an aspect of carbon source regulation (2) and that
(ii) cellobiose represses virulence genes via a specific pathway that
is different from that used by other common carbohydrates.
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The bvr Locus of Listeria
monocytogenes Mediates Virulence Gene Repression by
-Glucosides
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-glucoside cellobiose has been reported to specifically
repress the PrfA-dependent virulence genes hly and
plcA in Listeria monocytogenes NCTC 7973. This
led to the hypothesis that -glucosides, sugars of plant origin, may
act as signal molecules, preventing the expression of virulence genes
if L. monocytogenes is living in its natural habitat
(soil). In three other laboratory strains (EGD, L028, and 10403S),
however, the effect of cellobiose was not unique, and all fermentable
carbohydrates repressed hly. This suggested that the
downregulation of virulence genes by -glucosides is not a specific
phenomenon but, rather, an aspect of a global regulatory mechanism of
catabolite repression (CR). We assessed the effect of carbohydrates on
virulence gene expression in a panel of wild-type isolates of L. monocytogenes by using the PrfA-dependent phospholipase C gene
plcB as a reporter. Utilization of any fermentable sugar
caused plcB repression in wild-type L. monocytogenes. However, an EGD variant was identified in which,
as in NCTC 7973, plcB was only repressed by -glucosides.
Thus, the regulation of L. monocytogenes virulence genes by
sugars appears to be mediated by two separate mechanisms, one
presumably involving a CR pathway and another specifically responding
to -glucosides. We have identified in L. monocytogenes a
4-kb operon, bvrABC, encoding an antiterminator of the BglG
family (bvrA), a -glucoside-specific enzyme II permease component of the phosphoenolpyruvate-sugar phosphotransferase system
(bvrB), and a putative ADP-ribosylglycohydrolase
(bvrC). Low-stringency Southern blots showed that this
locus is absent from other Listeria spp. Transcription of
bvrB was induced by cellobiose and salicin but not by
arbutin. Disruption of the bvr operon by replacing part of
bvrAB with an interposon abolished the repression by
cellobiose and salicin but not that by arbutin. Our data indicate that
the bvr locus encodes a -glucoside-specific sensor that
mediates virulence gene repression upon detection of cellobiose and
salicin. Bvr is the first sensory system found in L. monocytogenes that is involved in environmental regulation of
virulence genes.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-glucoside cellobiose, but not of other common fermentable carbohydrates, strongly repressed the expression of
two genes of the PrfA virulence regulon (hly, encoding the hemolysin, and plcA, encoding a
phosphatidylinositol-specific phospholipase C) in L. monocytogenes NCTC 7973. Another -glucoside, arbutin, was shown
to have a similar effect in the same strain (34). In soil,
L. monocytogenes is thought to utilize decaying vegetation
as its primary growth substrate. This is perhaps most clearly
illustrated by silage, a fermented vegetable fodder which, if
incorrectly made, supports the growth of high numbers of L. monocytogenes bacteria and causes listeriosis outbreaks in
domestic ruminants (52). Both cellobiose and arbutin are
unique to the plant kingdom and are presumably abundant in decaying
vegetation, so Park and Kroll reasoned that these plant-derived
-glucosides may act as specific "signature molecules," allowing
L. monocytogenes bacteria to sense that they are present in
a soil environment and, consequently, to repress their virulence genes
(35). This notion was recently refuted by Milenbachs et al.
(32), who found that in L. monocytogenes strains
other than NCTC 7973 not only cellobiose, but several other readily
metabolized sugars, such as glucose or fructose, caused virulence gene
downregulation. They observed that sugar-mediated inhibition of
virulence gene expression only occurred in the presence of amounts of
carbohydrates sufficient to significantly stimulate growth, indicating
that the repression of virulence genes by sugar metabolism in L. monocytogenes might result from a general catabolite repression
(CR) response (32). This view has been challenged by a
recent report from the same group (3), showing that an
L. monocytogenes homolog of the transcription factor CcpA,
an important mediator of CR in low G+C content gram-positive bacteria
(21, 46), appears not to be involved in the carbon source
regulation of virulence genes. Nevertheless, CR in gram-positive
bacteria is still poorly understood, and there are other possible CR
mediators and pathways that may account for the repression of virulence
genes in L. monocytogenes.
-glucosides act as specific "environmental signature" repressor molecules and
that of Milenbachs et al. (32) that the regulation of
virulence genes by sugars is part of a global CR mechanism.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
was used as cloning host. Bacteria were cultured at 37°C in
BHI (Difco) or Luria-Bertani (LB) media, with antibiotics if required.
Liquid cultures were shaken at 170 rpm. Fresh stock solutions of
carbohydrates were filter sterilized and added to the culture medium to
a final concentration of 10 to 25 mM. Charcoal-treated BHI was prepared as previously described (42).
-32P]dATP
(Amersham). Prehybridization (2 h) and hybridization (16 h) were
performed in 6× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium
citrate) at 56°C. Filters were then washed twice for 30 min at 45°C
each in 5× SSC-0.1% sodium dodecyl sulfate and autoradiographed.
Km
mutant.
pKLB3B was digested with HincII and blunt-end
ligated to the 2.1-kb SmaI fragment of pBR322WKm. This
fragment contains the -Km2 interposon, which bears the
aphA-3 kanamycin resistance gene from Tn1545 and,
at both ends, transcription and translation termination signals in each
reading frame (36). In the resulting plasmid, pKLBM1, the
850-bp fragment between the two HincII sites encompassing
the 3' end of bvrA and the 5'-proximal region of bvrB was replaced by the -Km2 element (see Fig. 2). The
interposon-disrupted bvr locus was PCR amplified from pKLB1
by using the primers B35031 and 94C230 (see above). This PCR fragment
was then blunt-end ligated into the SmaI site of the shuttle
vector pLSV1 (55), giving rise to pKLBM2. pLSV1 has an
erythromycin resistance gene and a thermosensitive origin of
replication for gram-positive bacteria. pKLBM2 was introduced into
L. monocytogenes EGDCR as previously described
(41). Transformants were grown for 6 h at 30°C in BHI
broth in the presence of 25 µg of kanamycin and 5 µg of
erythromycin per ml, after which serial dilutions of the culture were
plated onto BHI agar plates containing the same antibiotics. The plates
were incubated for 48 h at 42°C to select for single crossover
events between the chromosome and pKLBM2. One of the resulting colonies
was grown overnight at 37°C in BHI broth containing 25 µg of
kanamycin per ml, and appropriate dilutions of this culture were plated
onto kanamycin BHI agar. Bacteria in which a second crossover event
took place, leading to bvr allelic exchange, were selected
by checking colonies for the loss of erythromycin resistance. The
bvrAB::Km mutation was confirmed by Southern
blot and PCR with appropriate primers.
-glucosides on
bvrB and plcB transcription was assessed by
semiquantitative RT-PCR as previously described (14).
Samples of total RNA (0.5 µg/µl) were serially diluted in
diethyl-pyrocarbonate-treated water, and cDNA was synthesized from 2 µl of each dilution by using the First-Strand Synthesis Kit from
Stratagene. Then, 2-µl volumes of each primer extension reaction were
subject to 15, 20, 25, and 30 cycles of PCR, and amplified DNA
fragments were detected after electrophoresis in 1% agarose by
ethidium bromide staining. Appropriate oligonucleotides internal to the
genes of interest were used for primer extension and PCR amplification of cDNA. As controls, primers specific for the 16S rRNA and the sod gene encoding superoxide dismutase (5) of
L. monocytogenes were used.
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-glucoside cellobiose were tested for their ability to repress
virulence gene expression. Saccharose, which is not utilized by
L. monocytogenes and consequently does not repress virulence
genes (32, 40), was used as a control. The product of the
plcB gene, the wide-substrate-range phospholipase C, PlcB
(or lecithinase), was used to monitor PrfA-dependent virulence gene
expression (see Materials and Methods).
Consistent with previous observations with hly as a reporter
(32), a strong downregulation of plcB occurred in
all strains with all of the fermentable carbohydrates tested (not
shown). There was, however, an exception. The group of wild-type
strains tested included two subcultures of L. monocytogenes
EGD from different laboratories: one was that kept by one of us (J.K.),
while the other came from T. Chakraborty (University of Giessen,
Giessen, Germany). The latter, termed EGD-e, is the strain whose DNA is being sequenced by the European Listeria genome consortium.
Interestingly, EGD-e exhibited the normal repression pattern of
wild-type strains, but our EGD subculture resembled NCTC 7973 in that
plcB expression was only downregulated by cellobiose (see
Fig. 1). Since our EGD variant (which was otherwise of wild-type
prfA background) was no longer repressed by glucose and
other common fermentable sugars, we presumed it was affected in a
central CR pathway and thus called it EGDCR.
In contrast to cellobiose and arbutin, the phenolic -glucoside
salicin has been reported to have no downregulating effect on
hly expression in NCTC 7973 (34). We thus
analyzed in more detail whether NCTC 7973 and EGDCR had
similar defects by assessing plcB expression in these two strains grown in the presence of each three -glucosides. In NCTC 7973, as expected, plcB expression was repressed in the
presence of cellobiose and arbutin but was not repressed by salicin
(Fig. 1). EGDCR, however,
differed from NCTC 7973 in that virulence gene expression was
identically downregulated by the three natural -glucosides (Fig. 1).
An identical pattern was observed in EGD-e (Fig. 1), indicating that
the EGDCR variant retained a wild-type response to
-glucoside sugars.
|
-glucosides cause
virulence gene repression via a specific regulatory pathway, independent from the CR mechanism presumably used by glucose and other
common utilizable sugars; and (ii) that there may also be various
sugar-sensing mechanisms, with different substrate specificities, involved in the regulation of virulence genes by -glucosides.
Cloning and sequence analysis of the bvr locus of
L. monocytogenes.
We have previously reported the
cloning and characterization of the kat gene encoding the
catalase of the nonpathogenic species L. seeligeri
(20). Upstream from kat, separated by a 370-bp intergenic region, we identified in L. seeligeri the
parB locus (7). Like kat,
parB was present in a highly conserved form in all
Listeria species (7). While attempting to isolate
the L. monocytogenes homolog of kat, we found
that, in this species, there was an insertion of about 4 kb between
this gene and parB. As such genomic differences between
pathogenic and nonpathogenic species of the same genus may correspond
to virulence-associated chromosomal islands (18, 19), we
were interested in characterizing the DNA region between
parB and kat in L. monocytogenes. A
4.4-kb fragment comprising the entire parB-kat intergenic
region of L. monocytogenes was amplified by PCR, subcloned,
and sequenced (see Materials and Methods for details). It comprised
three open reading frames (ORF) of 810, 1,919, and 980 bp arranged in
the same orientation (Fig. 2A). The locus
was called bvrABC (for -glucoside-mediated virulence gene
repression [see below]).
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10 and -glucosides. BvrB also had the multidomain structure IIBCA, which is characteristic of enzymes
II of the subfamily of -glucoside PTS permeases (8, 28).
These permeases are regulated by transcriptional antitermination, brought about by a family of structurally related antiterminator proteins (AT), the prototype of which is BglG from E. coli
(44, 47). In the -glucoside PTS operons of the enteric
bacteria E. coli and E. chrysanthemi, the AT
genes (bglG and arbG, respectively) are
immediately upstream from the genes encoding the -glucoside-specific enzymes II. In B. subtilis, in contrast, the two genetic
determinants are not located in the same chromosomal region
(44). Although L. monocytogenes is
phylogenetically very closely related to B. subtilis, the
genetic structure of the bvrAB locus was virtually identical
to that of the enterobacterial loci bglGF and
arbGF: the predicted bvrA product, BvrA (270 amino acids), was very similar to BglG (50) and related ATs
from the E. chrysanthemi arb operon (ArbG) (13)
and the B. subtilis bgl and sac regulons (LicT, SacY, and SacT) (9, 11, 48) (Fig. 3A). ATs bind specifically to conserved target sequences called "RAT" (for ribonucleic
antiterminator), which overlap the 5' part of stem-loop transcriptional
terminator structures present in the leader sequences of the controlled
genes or operons, thereby preventing the formation of the terminator and allowing readthrough transcription to the downstream (PTS permease)
genes (44). In the 110-bp region between bvrA and bvrB, we identified a putative RAT sequence for BvrA
overlapping a potential rho-independent transcriptional
terminator (Fig. 2B).
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-glucoside-specific PTS operons and regulons, the enzyme II gene is
immediately followed by the structural gene of
phospho-(P)--glycosidase (bglC, arbC, and
bglH, respectively), which is involved in the further
metabolization of the incorporated sugar (13, 26, 50). In
the L. monocytogenes bvr locus, however, no such
P--glucosidase gene was identified downstream from bvrB.
FASTA searches detected a significant degree of similarity (21.3%
identity, 51.5% similarity) between the bvrC product, BvrC,
and the ADP-ribosylglycohydrolase DraG from Rhodospirillum rubrum (16). BvrC (327 amino acids) was also very
similar to the proteins encoded by two ORFs present in the genomes of
E. coli (hypothetical protein b2099: 33.3% identity, 65.2%
similarity) and Methanococcus jannaschii (DraG-homolog:
25.8% identity, 59.3% similarity). The predicted primary structure of
these two polypeptides also showed significant similarity to that of
ADP-ribosylglycohydrolases (Fig. 3C).
bvrB expression is induced by -glucosides.
ATs
act as positive regulators of bacterial catabolic operons via a
regulatory process induced by the substrate. For example, in the
absence of -glucosides, the BglF PTS permease of E. coli inactivates BglG by phosphorylating it. Thus, constitutive readthrough transcription of the operon is prevented by terminators preceding the
bglG and bglF genes. However, in the presence of
inducer -glucosides, the phosphate groups are transferred from BglF
to the sugar during uptake, creating a backward flow that
dephosphorylates BglG. Active BglG prevents termination by binding to
RAT sequences and induces the expression of the operon (38, 44,
47). The structure of the L. monocytogenes bvrAB locus
is very similar to that of the E. coli bglGF locus (see
above), suggesting functional similarity. If a similar antitermination
mechanism of positive regulation operates in bvrAB, then the
expression of the putative permease gene bvrB should be
induced if L. monocytogenes is grown in the presence of
-glucosides.
-glucosides cellobiose,
salicin, and arbutin (Fig. 4). In the 5'
region immediately upstream from bvrA, overlapping a
putative promoter, we identified a 14-bp sequence that matched the
reported consensus sequence of catabolite responsive elements (CRE)
(Fig. 2C), the cis-acting binding sites for the repressor
transcription factors (presumably, the CcpA/HPr[Ser-46-P] complex)
involved in CR in low-G+C-content, gram-positive bacteria (21,
46). This suggests that the bvr locus may be subject to CR if glucose or other readily metabolizable carbon sources are
present in the culture medium. Therefore, to avoid the effects of any
possible expressional crosstalk between the bvr locus and the putative global CR mechanism involved in carbon source regulation, we performed the experiments with EGDCR. bvrB
transcription was semiquantitatively determined by RT-PCR. The level of
bvrB expression was significantly higher in the presence of
cellobiose and salicin (Fig. 4A). These results are consistent with the
structural data, which predict that BvrAB is a -glucoside-specific, substrate-inducible PTS permease system functionally similar to BglEF
from E. coli. Arbutin, however, did not activate the
transcription of bvrB (Fig. 4A).
|
The bvr locus is L. monocytogenes specific. We were interested in determining whether bvr-homologous sequences were present elsewhere on the L. seeligeri chromosome or if the locus was present in other Listeria species. Southern blot analyses were performed with chromosomal DNA from all known Listeria species, hybridizing at low stringency with a bvr probe. Positive hybridization signals were only obtained with L. monocytogenes (Fig. 5). This is consistent with the fact that we have only observed bvr-positive PCR reactions with DNA from L. monocytogenes isolates.
|
The bvr locus is involved in -glucoside-mediated
virulence gene repression.
Our structural and functional data
suggest that BvrB is a -glucoside-specific PTS permease. It was
therefore possible that the bvr locus was somehow involved
in -glucoside-mediated virulence gene repression in L. monocytogenes. To test this, we constructed a bvr
mutant in EGDCR by replacing a HincII fragment
spanning the 3' terminal part of bvrA, the bvrAB
intergenic region, and the 5' part of bvrB with the -Km2
interposon (Fig. 2A) (see Materials and Methods). RT-PCR analyses
indicated that bvrB expression was abolished in the
bvrAB::Km mutant (not shown). No transcripts
were detected for bvrC, demonstrating that the
bvr locus constitutes an operon.
Km mutation
on virulence gene repression by -glucosides by using plcB
as a reporter. The repression exerted by cellobiose and salicin was
totally abolished in bvrAB::Km. Arbutin,
however, still downregulated plcB expression to a level
similar to that of the parent strain (Fig. 1). These results were
entirely consistent with the fact that bvrB expression is
induced by cellobiose and salicin but not by arbutin (Fig. 4) and
demonstrate that the BvrB PTS permease is involved in virulence gene
repression by -glucosides.
PTS permeases mediate the transport into the bacterial cell of specific
carbohydrate substrates, thus initiating their catabolism (28, 38,
46). We therefore investigated whether the bvr locus
was important for cellobiose and salicin utilization by L. monocytogenes. The growth characteristics of
bvrAB::Km and its parent strain were determined
in LB broth supplemented with glucose (control), cellobiose, salicin,
or arbutin as the only carbon source. LB is a poor growth medium, in
which L. monocytogenes can hardly grow in the absence of an
additional carbon source (maximum OD578 of 0.5 versus 2.0 in LB plus 10 mM fermentable carbohydrate) (40).
Virtually identical growth curves and final bacterial yields were
observed for both strains in LB medium with glucose or any one of the
three -glucosides (not shown). No differences were observed when the
sugar utilization profiles of the parent and mutant strains were
investigated with the API 50 CH system.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The regulatory effect of fermentable sugars on virulence genes of L. monocytogenes has recently attracted considerable interest (2, 3, 32, 34, 35). This issue has been investigated in only four laboratory strains, at least two of which (L028 and NCTC 7973) have anomalous patterns of virulence gene expression and even regulatory mutations in prfA (41, 42). To unambiguously determine the normal pattern of this type of carbon source regulation in L. monocytogenes, we assessed the effect of a selection of carbohydrates on the expression of the PrfA-dependent gene plcB by using a panel of well-characterized wild-type strains. Our results confirmed that the repressibility of PrfA-dependent virulence genes is a general characteristic of wild-type L. monocytogenes upon growth on any fermentable sugar.
Among the strains tested, we identified a phenotypic variant of strain
EGD in which virulence gene expression was still repressed by the three
natural -glucosides (cellobiose, salicin, and arbutin) but not by
other common utilizable sugars such as glucose. We concluded that the
repression of virulence genes by sugars is mediated in L. monocytogenes by at least two different regulatory mechanisms: one
presumably involving a general CR repression pathway, as suggested by
Milenbachs et al. (32), which can be eliminated by a
spontaneous mutation, and another responding specifically to
-glucosides. In support of this conclusion, we report here the
identification of the bvr locus of L. monocytogenes, which specifically mediates virulence gene
repression by -glucoside sugars.
The bvr locus comprises three genes, bvrABC,
which are expressed as an operon. bvrAB code for a putative
-glucoside-specific, substrate-inducible PTS permease system similar
to that of the bgl and arb operons of E. coli and E. chrysamthemi, respectively, and the
bgl regulon of B. subtilis. Transcription of the
enzyme II gene, bvrB, was induced by cellobiose and salicin,
but not by arbutin, suggesting that the BvrB permease was specific for the two first -glucosides. Moreover, a knockout mutation of the bvr operon totally abolished the repression exerted by
cellobiose and salicin, whereas arbutin downregulated virulence genes
to the same extent as in the parent strain. These results demonstrate the involvement of the bvr locus in virulence gene
regulation by -glucosides, suggesting at the same time that various
regulatory mechanisms, with different substrate specificities, may be
involved in -glucoside-mediated virulence gene regulation in
L. monocytogenes. Arbutin is a major substrate for other
bacterial -glucoside PTS permeases (BglF, ArbF, and BglP from
E. coli, E. chrysanthemi, and B. subtilis, respectively) (13, 26, 50) to which BvrB is
homologous. Primary structure differences in certain conserved domains
may account for the altered substrate specificity of BvrB (Fig. 3B).
It has been previously shown that cellobiose reduces virulence gene
expression without affecting the levels of the PrfA protein (32,
39). The only PrfA-dependent virulence genes used to date to
assess the effect of cellobiose are hly and plcA
(2, 3, 23, 32, 34, 35). These genes are physically linked, divergent transcriptional units with overlapping promoter regions (31), so the possibility existed that the repressor effect
exerted over them by cellobiose was completely unrelated to PrfA and
involved a specific interaction with the common promoter region. Here
we show that cellobiose and other -glucosides also repress
plcB, another gene of the virulence regulon, the expression
of which is strictly dependent on PrfA (41). As shown in
Fig. 4B, plcB is downregulated at the transcriptional level,
as are hly and plcA (23, 32, 35).
Thus, the regulatory mechanism triggered by -glucosides appears to
somehow interfere with PrfA.
How does the bvr locus brings about -glucoside-mediated
virulence gene regulation? It is well known that sugar transport by PTS
permeases initiates a regulatory cascade of CR via PEP-dependent, enzyme I-catalyzed phosphorylation of the His-15 residue of the general
PTS protein HPr both in gram-negative and gram-positive bacteria
(21, 38, 46). However, our observations with
EGDCR would rule out the possibility that a general CR
pathway is involved in virulence gene repression by -glucosides.
There is extensive functional crosstalk between the various ATs of the
bgl-sac family, of which there are three well-characterized
members in B. subtilis (see above) (44).
Therefore, one possibility is that BvrB-mediated dephosphorylation and
activation of BvrA upon the transport of cellobiose and salicin
positively regulate, by transcriptional antitermination, a putative
effector system responsible for the inhibition of the PrfA regulon.
Another possibility involves the bvrC-encoded putative
ADP-ribosylglycohydrolase (Fig. 3C). In prokaryotes, these enzymes
participate in posttranslational regulatory networks involving
reversible ADP-ribosylation of target proteins (29) (see the
legend to Fig. 3C). Thus, BvrC may well provide the link between the
BvrAB -glucoside-specific PTS permease system and the virulence
regulon by posttranslationally controlling, directly or indirectly,
PrfA function.
Mutation of the bvr locus totally eliminated the repressor
effect exerted by cellobiose and salicin without affecting the utilization of these sugars. Clearly, therefore, other sugar transport systems mediating -glucoside utilization should be present in L. monocytogenes. These systems are likely to be conserved
in Listeria because all species in this genus are able to
ferment -glucosides (reference 33 and unpublished
observations). Thus, any role of the bvr locus in the uptake
of -glucosides by L. monocytogenes would be purely
accessory or redundant. bvr is present exclusively in
L. monocytogenes, as a 4-kb chromosomal island inserted
between two housekeeping genes, parB and kat,
which are contiguous in the nonpathogenic species L. seeligeri (6). This may indicate that the function
associated with this locus is relevant only to the biology of the
pathogenic species L. monocytogenes. Virulence factors are
primarily required during infection, and their synthesis outside the
host would be a waste of energy, reducing the fitness of the bacterium
to successfully compete in the free environment and thereby limiting
its potential for transmission. Our results suggest that the
bvr locus is responsible for a regulatory mechanism that,
like thermoregulation, would be aimed at ensuring that virulence genes
are shut off outside the vertebrate host. The PTS acts as a signal
transduction system in positive chemotaxis in response to the presence
of PTS-transported carbohydrates in the extracellular medium (17,
38). It is therefore possible that the Bvr system functions as a
sensor mechanism triggering virulence gene silencing upon detection of
-glucosides, sugars specific to the plant kingdom which, as
previously suggested by Park and Kroll (35), might be used
by L. monocytogenes as signal molecules of the soil habitat.
It must be noted that the virulence gene regulatory function of the
bvr locus would have remained cryptic if L. monocytogenes were not cultured in PrfA-activating conditions
(i.e., culture at 37°C in charcoal-treated BHI). This shows that
bvr genes are responsible for a repressor mechanism that is
superimposed on the positive control pathway that activates the PrfA
virulence regulon. The environmental context sensed by L. monocytogenes in soil may sometimes become ambiguous, for example, if the ambient temperature rises above 30°C. We believe that in such
situations the Bvr system may play a key role as a fail-safe mechanism
to avoid any deleterious leaky expression of virulence genes.
| |
ACKNOWLEDGMENTS |
|---|
We thank T. Chakraborty for the gift of the EGD strain kept in his laboratory. N. Montero is acknowledged for excellent technical assistance.
This work was supported by grants from the European Commission (HRCX-CT94-451 and BMH4-CT96-659), the Fondo de Investigación Sanitaria (FIS 94/0043-02), the Dirección General de Investigación de la Comunidad de Madrid (29/97), the Deutsche Forschungsgemeinschaft (SFB 165), and the Fonds der Chemischen Industrie.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Unidad de Microbiología e Inmunología, Departamento de Patología Animal I, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain. Phone: 34-91-394.37.04. Fax: 34-91-394.39.08. E-mail: vazquez{at}eucmax.sim.ucm.es.
Present address: Institut für Hygiene un Mikrobiologie, 97080 Würzburg, Germany.
| |
REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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