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J Bacteriol, September 1998, p. 4547-4554, Vol. 180, No. 17
Department of Food Science and Technology,
University of Nebraska, Lincoln, Nebraska 68583-0919
Received 4 May 1998/Accepted 6 July 1998
Listeria monocytogenes is well known for its robust
physiology, which permits growth at low temperatures under conditions of high osmolarity and low pH. Although studies have provided insight
into the mechanisms used by L. monocytogenes to allay the
physiological consequences of these adverse environments, little is
known about how these responses are coordinated. In the studies
presented here, we have cloned the sigB gene and several rsb genes from L. monocytogenes, encoding
homologs of the alternative sigma factor Listeria monocytogenes is
a ubiquitous gram-positive bacterium that occasionally causes outbreaks
and sporadic cases of food-borne illness. It is estimated that more
than 1,700 cases occur annually in the United States, with outcomes
ranging from flu-like illness to meningitis, meningoencephalitis,
septicemia, abortion, and death, particularly in pregnant women and
immunocompromised individuals (24, 41).
As an intracellular parasite that is transmitted primarily by
contaminated food, L. monocytogenes must reach and/or
maintain a cell density in the food environment that is necessary for
infection. This population must further mitigate the physiological
consequences of passage through the gastrointestinal tract and entry
into host cell phagosomes. Although genetic analysis of virulence has
provided many of the details of how virulence genes allow L. monocytogenes to elude potentially lethal outcomes of entry into
host cells (23, 40, 43), little is known about the role of
adaptive physiological responses in promoting the growth and survival
of this bacterium in the food, intestinal, and intracellular
environments.
L. monocytogenes is well known for its robust physiological
characteristics and is one of the few pathogenic bacteria capable of
growth at refrigeration temperatures, at low pHs, and/or under conditions of high osmolarity (21, 22, 32, 36, 55). These
characteristics play an integral role in its potential as a food-borne
pathogen, since they permit the growth of L. monocytogenes under conditions that prohibit the growth of many commensal organisms. Physiological studies have previously demonstrated that specific transport systems which mediate the uptake of osmoprotectants and
cryoprotectants facilitate growth under these environmental conditions
(9, 31, 32). A further role for adaptive physiological responses in pathogenesis has also been proposed on the basis of
genetic experiments demonstrating that genes participating in acid
tolerance (35) and stress-induced proteolysis
(42) are necessary for full virulence in the mouse model.
Thus, significant evidence is beginning to mount for a central role of
adaptive physiological responses of L. monocytogenes in
pathogenesis as well as in growth and survival in the environment.
To further our understanding of the connection between physiology and
the transmission of food-borne illness, we have begun an analysis of
genetic pathways that modulate adaptive responses in L. monocytogenes. One candidate for mediating such responses in
gram-positive organisms is The activity of To examine how the Strains and plasmids.
L. monocytogenes Scott A
was obtained from R. Hutkins (University of Nebraska), and L. monocytogenes LO4035 was obtained from N. Freitag (Wayne State
University). L. monocytogenes cells were grown in brain
heart infusion (BHI) (Difco, Detroit, Mich.) at 30°C unless otherwise
specified. Where specified, kanamycin (30 µg/ml) or chloramphenicol
(6 µg/ml) was added. Escherichia coli DH5 Cloning of sigB from L. monocytogenes.
Preliminary Southern blot experiments using a 540-bp
EcoRI-PstI fragment of the B. subtilis
sigB gene (10) as a probe indicated that a single
3.8-kb EcoRI fragment of the L. monocytogenes
Scott A or LO4035 chromosome hybridized under conditions of moderate stringency (with 5× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate] at 56°C). A plasmid minilibrary was therefore constructed by cloning 3- to 4-kb EcoRI fragments of L. monocytogenes chromosomal DNA into EcoRI-digested
pUC19. The resulting plasmid library was transformed into a DH5 DNA sequence analysis.
Both strands of the 3.75-kb
EcoRI fragment of pLM413 were subjected to DNA sequence
analysis on an automated sequencer (LiCor, Inc.) by primer walking.
Sequence alignments were performed with the PILEUP and BESTFIT programs
from the Genetics Computer Group.
Preparation and analysis of RNA.
RNA samples were prepared
from 200 ml of mid-logarithmic-phase L. monocytogenes cells
(optical density at 600 nm [OD600], ~0.4) before and 20 min after the addition of either 4% NaCl, 1 mM EDTA, 2% ethanol,
0.15% H2O2, or glacial acetic acid to a pH of
5.3. Additional samples were prepared after an aliquot of the cells was
allowed to enter stationary phase (OD600, ~3.0) and after
mid-logarithmic-phase cells were shifted from 25 to 48°C for 20 min
or to 4°C for 24 h. After treatment, the cells were harvested by
centrifugation at 8,000 × g for 5 min at 4°C, and
the cell pellets were frozen at Primer extension analysis of sigB transcripts.
For each reaction, 10 pmol of the oligonucleotide VPROM2 (5'
CGCTGTATAAGCATCGATCAC 3') was end labeled with 50 µCi of
[ Construction of the sigB::km
strain.
A null mutation in the L. monocytogenes sigB
gene (sigB::km) was generated by
cloning an aph3'5'' gene encoding kanamycin resistance from
pDG783 (26) into the unique StyI site of pLM413, which lies within the sigB coding region. The
aph3'5'' gene was removed from pDG783 as a
HindIII fragment and was subsequently blunt ended with
Klenow fragment prior to ligation to StyI-restricted pLM413
that had also been blunt ended with Klenow fragment. The resulting
plasmid was designated pLM425 and was restriction mapped to confirm
insertion of the aph3'5'' gene into the appropriate position. The entire EcoRI fragment of pLM425, containing
the rsbU, rsbV, rsbW,
sigB::km, and rsbX genes,
was then cloned into the EcoRI site of the
temperature-sensitive integration vector pKSV7, which carries a
chloramphenicol resistance gene (44). The resulting plasmid,
designated pKSV7K8, was restriction mapped to ensure its integrity.
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of the Gene Encoding the Alternative
Sigma Factor
B from Listeria monocytogenes
and Its Role in Osmotolerance
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
B and the
RsbUVWX proteins, which govern transcription of a general stress
regulon in the related bacterium Bacillus subtilis. The L. monocytogenes and B. subtilis sigB and
rsb genes are similar in sequence and physical
organization; however, we observed that the activity of
B in L. monocytogenes was uniquely
responsive to osmotic upshifting, temperature downshifting, and the
presence of EDTA in the growth medium. The magnitude of the response
was greatest after an osmotic upshift, suggesting a role for
B in coordinating osmotic responses in L. monocytogenes. A null mutation in the sigB gene led
to substantial defects in the ability of L. monocytogenes
to use betaine and carnitine as osmoprotectants. Subsequent
measurements of betaine transport confirmed that the absence of
B reduced the ability of the cells to accumulate
betaine. Thus, B coordinates responses to a variety of
physical and chemical signals, and its function facilitates the growth
of L. monocytogenes under conditions of high osmotic
strength.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
B, a secondary subunit of RNA
polymerase that is known to govern a large stress response regulon in
the related bacterium Bacillus subtilis. The
B regulon of B. subtilis comprises at least
40 genes (52) and includes the katE gene,
encoding a catalase (19, 20), the opuE gene,
encoding transport machinery for osmoprotectants (53), the
clpC gene, which is similar to stress-induced ATPase
subunits of ClpP-type proteases (33), the gtaB
gene, encoding a UDP-glucose pyrophosphorylase believed to participate
in trehalose biosynthesis (46), and several genes whose
functions cannot be inferred from their sequences (3, 11,
47).
B is joined to several physical and
chemical signals through a postranslational mechanism that partitions B between inactive complexes with an anti-sigma factor
protein, RsbW (for regulator of sigma B), and free B,
which is capable of forming holoenzyme complexes with core RNA polymerase (6). At least two independent pathways exist
which can alter the affinity of RsbW for its antagonist, RsbV, or
B. First, it has been proposed that ATP stimulates the
formation of RsbW-B complexes and activates a serine
kinase activity in RsbW that phosphorylates its antagonist, RsbV, into
an inactive state (2, 18, 58). Physical signals such as
temperature, pH, and osmolarity can also invoke B
activity (7, 11-13, 51) by stimulating the function of the RsbV-phosphate phosphatase RsbU (29, 58). At least four
other Rsb proteins (RsbR, RsbS, RsbT, and RsbX) modulate the activity of RsbU and serve as distinct points of signal input into the system
(1, 29).
B regulon contributes to adaptive
responses of L. monocytogenes, we have cloned the genes
encoding homologs of B and several of the Rsb proteins.
In this report, we demonstrate that the L. monocytogenes
rsbVW-sigB-rsbX transcription unit is structurally analogous to
its counterpart in B. subtilis and that its activity in
L. monocytogenes is responsive to several physical and
environmental signals. Genetic and physiological studies indicate that
B facilitates growth under conditions of high osmolarity
when betaine and carnitine are supplied as the primary osmoprotectants.
Thus, the B regulon in L. monocytogenes
participates in responses to several physiological and chemical
insults, and it may serve as a primary osmosensor in this organism.
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
and E. coli MC1061 were used as cloning hosts and were grown in Luria
broth with 150 µg of ampicillin/ml or 40 µg of kanamycin/ml where
appropriate.
strain, and approximately 200 colonies containing plasmids with inserts
were patched onto replica plates and transferred to nylon membranes.
The colonies were screened by colony hybridization with the 540-bp
EcoRI-PstI B. subtilis sigB probe
under the same hybridization conditions as those used for the Southern
blots. Two positive clones with identical restriction patterns were
obtained. One clone was designated pLM413 and was used for further
studies.
70°C overnight. The cells were
subsequently resuspended in 5 ml of buffer D (4 M guanidinium thiocyanate, 25 mM sodium citrate, 0.5% Sarkosyl, 0.1 M
2-mercaptoethanol) and passed through a French pressure cell at 1,500 lb/in2. RNA was then extracted as described by Chomczynski
and Sacchi (15). The final RNA pellet was dissolved in
diethylpyrocarbonate-treated water, quantified spectrophotometerically,
and stored frozen at 70°C. The integrity and relative
concentrations of the RNA samples were also checked by agarose gel
electrophoresis and ethidium bromide staining.
-32P]ATP. The labeled primer was then mixed with 50 µg of RNA, heated to 70°C for 10 min, chilled on ice for 30 s,
and incubated at 42°C with 3 U of SUPERSCRIPT II RNase H-reverse
transcriptase (Life Technologies). After 50 min of extension, the
products were ethanol precipitated, washed in 70% ethanol, and dried.
The primer extension products were then dissolved in 90% formamide-10
mM EDTA and loaded onto a 6% denaturing polyacrylamide gel alongside a
sequencing ladder prepared by using the VPROM2 primer and the pLM413
template DNA.
B activity, and
which acquire suppressor mutations that give rise to cells with normal
growth rates (5, 11, 50). The fact that insertion of the
kanamycin resistance gene into the sigB gene of L. monocytogenes gives rise to normal-sized colonies indicates that
any polar effects on rsbX are epistatic to inactivation of sigB and are therefore negligible with regard to the
phenotype of the sigB mutant.
Growth in DM. To assess the ability of the wild-type and sigB::km strains to use betaine and carnitine as osmoprotectants, the cells were grown in a defined medium (DM) derived from formulations described by Pine et al. (39) and Beumer et al. (9). DM contains, per liter, 15 g of KH2PO4, 1 g of (NH4)2SO4, 0.2 g of MgSO4 · 7H2O, 0.02 g of CaCl2, 10 g of glucose, 0.088 g of ferric ammonium citrate, 0.1 g each of L-leucine, L-isoleucine, L-valine, L-methionine, and L-cysteine, 0.6 g of L-glutamine, 0.5 mg each of riboflavin and biotin, 1.0 mg of thiamine, and 0.005 mg of thioctic acid. After stock solutions of the components were combined, the pH was adjusted to 6.7 with potassium hydroxide and was filter sterilized. Cultures were grown in DM for 18 h at 30°C and were subsequently inoculated at 2% (vol/vol) in 250-ml baffled culture flasks containing DM with or without 3% NaCl, or DM with 3% NaCl that was supplemented either with 1 mM glycine betaine or with 1 mM carnitine. The cultures were incubated with shaking at 30°C, and growth was monitored by spectrophotometric measurements of OD600 over the course of several days.
Betaine uptake. To measure the ability of LO4035 and LMA2B to accumulate betaine, log-phase cells grown in BHI at 30°C were harvested by centrifugation, washed twice, and resuspended in 50 mM potassium phosphate buffer (pH 6.8) to an OD600 of ca. 1.0. The cells were energized by the addition of glucose (final concentration, 5 mM), and where indicated, 3% NaCl (514 mM) was also added. After 20 min of incubation at room temperature, the assays were initiated by the addition of [14C]betaine (final concentration, 1 mM; 65 µCi/mmol; American Radiolabeled Chemicals, Inc., St. Louis, Mo.). The mixtures were incubated at room temperature, and 1-ml samples were removed and centrifuged through silicon oil as described previously (16). Radioactivity was measured by scintillation counting. The results are reported as the averages of duplicate samples. Independent experiments were conducted on successive days to confirm these results. The protein concentrations of cell suspensions were derived from a standard curve relating OD to protein concentration (54).
Nucleotide sequence accession number. The sequence of the 3.75-kb EcoRI fragment of pLM413 has been deposited in GenBank under accession no. AFO74855.
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RESULTS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cloning and sequence analysis of the sigB operon from L. monocytogenes. DNA sequence analysis of the cloned fragment revealed that five putative coding regions were present, including one with homology to the 3' end of rsbU, as well as the entire coding regions of the rsbV, rsbW, sigB, and rsbX homologs (Fig. 1). The contiguous arrangement of these genes is similar to that observed in B. subtilis and Staphylococcus aureus (28, 34, 56, 57).
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B-dependent promoter in B. subtilis
that serves as an autocatalytic device for amplifying mRNA encoding
B and the RsbVWX regulatory components in response to
stress (7, 13, 28). Secondly, there appears to be
translational coupling between rsbV and rsbW and
between rsbW and sigB, as these coding regions
overlap by at least 1 codon. The overlap is most striking between
rsbW and sigB (13 codons) and appears to be an
important feature of these genes, because it is conserved in B. subtilis (13 codons) and S. aureus (8 codons) (28,
34, 57). Translational coupling has been proposed for these genes
(28), and experimental evidence supports this hypothesis in
the case of rsbW and sigB (8). The
conservation of the rsbW-sigB overlap further supports the
importance of coupling as a device for ensuring equimolar synthesis of
B and its primary regulator; however, the mechanism
through which the coupling is mediated through this extensive overlap
remains to be determined.
Homology of the Rsb and B proteins.
Alignment
of the predicted amino acid sequences of RsbUVWX and B
with their homologs from B. subtilis and S. aureus indicates that the L. monocytogenes homologs are
more closely related to their counterparts in B. subtilis
(Fig. 1 and 2). Overall, the putative
RsbU, RsbV, RsbW, and B proteins display 60 to 75%
similarity (40 to 50% identity) with their B. subtilis and
S. aureus homologs. In contrast, however, the RsbX protein
displays only limited homology (29% identity) to its B. subtilis counterpart.
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B proteins with their counterparts from
B. subtilis and S. aureus revealed highly
conserved subregions within each (Fig. 2). Alignment of the RsbU
homologs predicts that our cloned fragment contains only about half of
the L. monocytogenes rsbU gene (Fig. 2A). Within RsbV, we
identified three subregions, designated I through III, that appear to
be highly conserved (Fig. 2B). Region II includes the conserved serine
residue that can be aligned with the serine residue in the homolog
SpoIIAA, which is the site of phosphorylation by the SpoIIAB homolog
RsbW (17, 37, 38). How domains I and III contribute to RsbV
structure, function, or both remains to be determined. As with RsbV, we
also observed three highly conserved subregions, designated regions I,
IIa, and IIb, within the L. monocytogenes RsbW homolog (Fig.
2C). These regions have previously been proposed to play a role in ATP
binding of the RsbW homolog SpoIIAB by virtue of their similarities to
domains I and II of histidine protein kinases (37). The
B proteins, which demonstrated the highest identity
scores, showed the highest degree of identity in regions 2.2, 2.4, and
4.2, which are believed to participate in core RNA polymerase binding
and 10 and 35 recognition, respectively (27).
Stress-dependent activation of B in L. monocytogenes.
The intergenic region between rsbU and
rsbV is the location of a known B-dependent
promoter in B. subtilis (28) that serves as an
autocatalytic device for increasing the levels of mRNA encoding the
RsbV, RsbW, RsbX, and B proteins under conditions of
B activation (7, 13). We therefore examined
this region by primer extension to determine if a
B-dependent promoter is similarly positioned in L. monocytogenes and if its activity is stress dependent. A primer
(VPROM2) that is complementary to positions +63 to +83 relative to the
rsbV initiation codon was end labeled and used in primer
extension analyses on mRNA extracted from logarithmically growing cells before and after environmental stress (4% NaCl or pH 5.3) or entrance into stationary phase. As shown in Fig.
3, no transcript from this region was
detected in logarithmically growing cells; however, increasing the
osmolarity of the medium, decreasing its pH, or permitting the cells to
enter stationary phase led to the appearance of an extension product
mapping to position 646 of the nucleotide sequence. The initiation site
of this transcript lies immediately downstream of sequences that can be
aligned with known B-dependent promoters (Fig.
4), indicating that this promoter may be
utilized by B-RNA polymerase holoenzyme under stress
conditions.
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B
dependent, we performed primer extension assays on RNA extracted from
both the wild type, LO4035, and its isogenic derivative, LMA2B, which carries an allele of sigB
(sigB::km) that has been insertionally inactivated by the introduction of a kanamycin resistance gene (see
Materials and Methods). As predicted, only RNA samples from the
wild-type strain yielded primer extension products after osmotic upshifting, temperature upshifting, acidification, or entry into stationary phase (Fig. 3B). Thus, the stress-induced transcript relies
on B and appears to be a conserved autocatalytic
mechanism for increasing the abundance of the
rsbVW-sigB-rsbX transcript upon the perception of stress.
Because of the autocatalytic nature of B, the level of
the B-dependent transcript that originates upstream of
rsbV is a convenient measure of B activity in
the cell. We therefore used our primer extension assay to measure the
relative effects of different environmental conditions on
B activity in L. monocytogenes (Fig. 3C). As
observed in the previous experiment, no transcript was detected in
logarithmically growing cells; however, significant levels of the
transcript were observed after cells had been exposed to 4% NaCl, 2%
ethanol, acidification to a pH of 5.3, 1 mM EDTA, 0.15%
H2O2, or a temperature upshift (from 25 to
48°C) or downshift (from 25 to 4°C). Each of these treatments led
to the appearance of the B-dependent transcript,
indicating that B activity was stimulated.
Although many of the conditions examined above are known to activate
B in B. subtilis, we noted unique aspects of
the L. monocytogenes responses. First, based on the
intensity of the labeled bands, osmotic upshifting gave rise to the
highest degree of stimulation relative to the other treatments. The
magnitude of this response relative to the other conditions is striking
and appears to be greater than what has been observed in B. subtilis after a similar upshift (13). We note,
however, that the dose-response curve of B activity is
complex and that the "doses" of each stress used in our experiments
may not be optimal. Next, we observed two conditions, temperature
downshifting and the addition of EDTA, which activated B
only in L. monocytogenes. Experiments with B. subtilis cells grown under similar conditions failed to yield
significant activation of B activity based on
measurements of transcription from the B-dependent
transcriptional fusion (ctc::lacZ) and
on measurements of B by Western blotting (4).
Effects of the sigB::km mutation
on osmotolerance in L. monocytogenes.
Because we observed
the highest level of B activity after an osmotic
upshift, we next examined whether the absence of sigB would
have any effect on the ability of L. monocytogenes to grow under conditions of high osmotic strength. We initially evaluated the
growth of LO4035 and the isogenic LMA2B derivative
(sigB::km) in BHI after logarithmically
growing cells were exposed to an osmotic upshift by the addition of 6%
NaCl. As illustrated in Fig. 5A, the
addition of NaCl to the cultures led to an abrupt decrease in the
growth rate. Relative to the parental strain, however, the
sigB::km mutant showed a slightly
reduced, but reproducible, rate after upshifting of the culture during
the latter stages of logarithmic growth, suggesting that the absence of
B only slightly impaired adaptation to the osmotic
upshift in this complex growth medium.
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, LO4035
without NaCl; 
, LO4035 with 6% NaCl; 
, LMA2B without NaCl; 
,
LMA2B with 6% NaCl. (B) Equivalent volumes of 18-h cultures of LO4035
and LMA2B in DM were inoculated into DM without NaCl (
, LO4035; 
,
LO4035; ×, LMA2B), and DM with 3% NaCl supplemented with 1 mM betaine
(
, LO4035; B impairs the
ability of L. monocytogenes to use betaine and carnitine as
osmoprotectants, most likely due to defects in the transport of the
osmoprotectants.
Betaine accumulation is defective in the
sigB::km strain.
To confirm that
the absence of B impairs the accumulation of betaine, we
measured the ability of the isogenic strains to accumulate [14C]betaine under conditions of high and low osmolarity.
Cells from both LO4035 and LMA2B were grown to mid-logarithmic phase in
BHI (OD600, ~0.4) and then were harvested and washed at
room temperature to prevent temperature-dependent induction of
B activity. As shown in Fig.
6, the two strains accumulated similar amounts of labeled betaine when incubated in phosphate buffer alone,
confirming the presence of a constitutive betaine transport system that
has been described previously (49). When 3% NaCl was added
to the cells, betaine accumulation in the wild-type strain LO4035 was
stimulated considerably. In contrast, inactivation of the
sigB gene in LMA2B prohibited osmotic stimulation of betaine accumulation. These results are consistent with a model in which B mediates a sodium-inducible or osmotically inducible
component of betaine transport. Thus, B appears to play
an integral role in coordinating the physiological adaptation of
L. monocytogenes to osmotic upshifting.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The ability of L. monocytogenes to grow under
conditions of suboptimal temperature, osmolarity, and pH may favor its
growth in contaminated food products and promote survival during
transit through the gastrointestinal tract and entry into host cell
phagosomes. In this report, we have used genetic analyses and
physiological studies to examine how L. monocytogenes
coordinates such physiological responses through the activity of an
alternative sigma subunit of RNA polymerase, B.
Analysis of the physical organization of the L. monocytogenes
rsbU, rsbV, rsbW, sigB, and
rsbX genes demonstrated that the genes are organized in much
the same manner as they are in the related gram-positive organisms
B. subtilis and S. aureus, with the exception of
rsbX, which is absent downstream in S. aureus. These conserved features include an extensive overlap of the
rsbW and sigB coding regions, further supporting
an important role for translational coupling of B to its
primary regulator, RsbW. Multiple sequence alignments of the putative
B and Rsb proteins further revealed highly conserved
subregions within the anti-anti-sigma factors RsbV and SpoIIAA, which
may facilitate further genetic and biochemical analysis of the
structure-function relationships among these proteins.
Given the high degree of conservation among the RsbUVW and
B proteins, we are intrigued by the low degree of
identity between the L. monocytogenes and B. subtilis RsbX proteins. In B. subtilis, RsbX is the
farthest-upstream member of the Rsb signal transduction cascade
(29, 50, 58). This divergence may therefore reflect adaptation of rsbX to sensing functions that are attuned to
unique aspects of the physiology and ecology of L. monocytogenes. We are currently identifying the other
rsb genes in L. monocytogenes to determine how
differences in structure might correspond to differences in function.
In addition to the divergence in the primary sequence of RsbX, we also
observed unique characteristics of the physical and chemical conditions
that elicit B activity in L. monocytogenes,
including responses to temperature downshifting and to the presence of
EDTA, and the magnitude of the response to osmotic upshifting.
Induction of B activity subsequent to a temperature
downshift suggests that B may contribute to the unique
psychrotrophic characteristics of this organism. Since betaine has been
demonstrated to act as a cryoprotectant as well as an osmoprotectant in
L. monocytogenes (31), the participation of
B in betaine accumulation suggests that low-temperature
induction of B activity may also stimulate betaine
accumulation after a temperature downshift. We are currently testing
this hypothesis.
Induction of B activity in response to EDTA is also
notable. The response may be due to decreased availability of divalent cations within the cytoplasm. Alternatively, it could be an indirect response to destabilization of teichoic acids or effects on membrane proteins that require divalent ions for structure or function. Given
that the Rsb cascade responds to many types of physical signals (pH,
temperature, and osmolarity), it may be that compromising the integrity
of the envelope is sufficient to elicit B activity. In
support of this idea is the fact that the S. aureus sigB
gene was originally discovered as a transposon insertion that led to
sensitivity of the cells to methicillin (57), suggesting a
role for B in cell wall-associated functions in this
organism. We are currently determining which cation limitation invokes
B activity and whether it is due to its declining
concentration inside or outside the cell.
Role of B in osmotolerance in L. monocytogenes.
The magnitude of B induction that
was observed after an osmotic upshift (Fig. 3) initially suggested that
B function might be important in mediating
osmotolerance. Experiments with the isogenic
sigB+ and sigB mutant strains
subsequently demonstrated that the absence of B impaired
the ability of L. monocytogenes to use betaine and carnitine as osmoprotectants (Fig. 5B and C). This defect was further shown to
result from the inability of the sigB mutant strain to
stimulate betaine accumulation after an osmotic upshift (Fig. 6).
B mediating a sodium-inducible or osmotically inducible
component of betaine transport. This function could be a consequence of B directing the transcription of one or more genes
encoding a betaine transporter or, alternatively, a regulatory protein
that modulates the synthesis or activity of a betaine transport system.
Since betaine is the osmoprotectant of choice, when available
(48), the B-dependent component of betaine
transport in L. monocytogenes may therefore be the primary
system for osmotic stimulation. Consistent with this hypothesis, we
observed significant induction of B activity in response
to osmotic upshifting.
The use of B as a primary means for coordinating osmotic
responses in L. monocytogenes is apparently unique. B. subtilis sigB mutants do not show obvious defects in osmotolerance
(53), likely due to the existence of both multiple
regulatory systems and redundant transporters in this organism. Indeed,
three independent transporters can be used for proline and betaine
uptake in B. subtilis (30, 53). Moreover, in the
case of the opuE gene, which encodes a proline transporter,
its B-dependent promoter is one of two osmotically
inducible promoters (53). The adoption of redundant genes
encoding transport systems in this organism may have led to
dissemination of the role of coordinating their expression among other
regulatory systems in order to bring expression into harmony with the
availability of different substrates. Given that several sophisticated
adaptive responses such as sporulation and competence are also
available to B. subtilis, the role of the B
regulon may have been further diminished to accommodate these pathways.
Thus, in organisms, such as L. monocytogenes, that do not
possess such sophisticated adaptive responses, B may
serve a primary role in coordinating responses to physical changes in
the environment. Further comparative studies of the B
regulon from related gram-positive organisms would therefore provide
important information about the role of adaptive responses in the
physiology and ecology of these organisms.
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ACKNOWLEDGMENTS |
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This work was supported by grants from Li-Cor, Inc., and from the USDA Midwest Advanced Food Manufacturing Alliance to A.K.B. L.A.B. is a recipient of the Widaman Trust Distinguished Graduate Assistant Fellowship.
We thank Abraham Oommen and Margaret Esser for performing the DNA sequence analysis and Mark Morrison and Jeff Cirillo for critical reading of the manuscript.
L. A. Becker and M. S. Çetin contributed equally to this work.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Food Science and Technology, 358 Food Industry Complex, University of Nebraska, Lincoln, NE 68583-0919. Phone: (402) 472-5637. Fax: (402) 472-1693. E-mail: abenson{at}foodsci.unl.edu.
Journal Series paper 12219 of the Nebraska Agricultural
Experimental Station.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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J Bacteriol, September 1998, p. 4547-4554, Vol. 180, No. 17
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