Department of Food Science, Cornell University, 412 Stocking Hall, Ithaca, NY 14853, USA
Correspondence
Martin Wiedmann
mw16{at}cornell.edu
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ABSTRACT |
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INTRODUCTION |
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Bacteria can alter gene expression patterns at appropriate times in response to changing environments and stressful conditions. Both Gram-positive and Gram-negative bacteria have the ability to regulate patterns of gene expression at the transcriptional level (Becker et al., 2000; Fang et al., 1992
; Helmann et al., 2001
). The holoenzyme RNA polymerase (RNAP) catalyses the transcription of DNA into mRNA (Burgess et al., 1969
). A sigma factor is a protein subunit of RNAP that is required for recognition of specific promoter sequences and for initiation of transcription (Helmann & Chamberlin, 1988
). The association of different alternative sigma factors with RNAP is one mechanism that enables a bacterial cell to rapidly induce expression of specific genes within a regulon in response to specific stimuli. The general stress-responsive alternative sigma factor
S has been identified in many Gram-negative bacteria, including Escherichia coli, Salmonella spp. and Yersinia spp. (Badger & Miller, 1995
; Fang et al., 1992
; McCann et al., 1991
). In both E. coli and Salmonella Typhimurium,
S plays a crucial role in protection against conditions of starvation, hyperosmolarity, oxidative and acid stresses (Cheville et al., 1996
; Small et al., 1994
). For Gram-positive bacteria, the general stress-responsive alternative sigma factor
B was first identified and characterized in Bacillus subtilis (Boylan et al., 1993
). The
B-dependent general stress regulon of B. subtilis consists of well over 100 genes that are induced by exposure to stressful conditions such as heat, acid, ethanol or high osmolarity, or by deprivation of glucose, oxygen or phosphate (Helmann et al., 2001
; Petersohn et al., 2001
; Price et al., 2001
). Previous studies have demonstrated roles for stress-responsive sigma factors in regulating expression of virulence genes in some bacterial pathogens, including Staphylococcus aureus, Yersinia enterocolitica and Salmonella (Deora et al., 1997
; Humphreys et al., 1999
; Kullik et al., 1998
), suggesting a link between stress response and virulence in these organisms.
Mounting evidence also supports an association between the ability of L. monocytogenes to survive exposure to environmental stresses and to infect host cells. For example, B contributes to L. monocytogenes survival and growth under certain environmental stress conditions [e.g. acid stress, low-temperature stress, salt stress (Becker et al., 1998
, 2000
)], as well as to persistence within a host and to host cell infection (Nadon et al., 2002
; Wiedmann et al., 1998
). The stress-responsive compatible solute transporter opuCA has also been demonstrated to contribute to host infection in an animal model (Sleator et al., 2001
). While transcription of selected L. monocytogenes genes (e.g. opuCA, lmo1421) has been shown to be reduced in a sigB null mutant background (Ferreira et al., 2003
; Fraser et al., 2003
), the temporal nature of
B-dependent contributions to transcription induction of these genes has not yet been quantified. To test our hypothesis that
B coordinates a rapid response that aids L. monocytogenes in survival of environmental and host-imposed stress conditions, we identified and confirmed the
B dependence of L. monocytogenes genes that had previously been demonstrated to contribute to survival under these conditions and characterized their induction and expression patterns. Specifically, we confirmed the
B dependence of L. monocytogenes opuCA, lmo1421 and bsh. These genes represent general stress-response genes (opuCA and lmo1421 encode known and putative compatible solute transporter proteins, respectively) and a virulence gene [bsh encodes a conjugated bile salt hydrolase (BSH)]. We have shown that all three genes are expressed under conditions of environmental stress and that expression of opuCA and lmo1421 is induced following exposure to salt stress.
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METHODS |
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For detection of BSH activity, the L. monocytogenes wild-type, L. innocua wild-type (negative control) and sigB null mutant strains were spotted onto deMan, Rogosa and Sharpe (MRS) agar medium (BD Biosciences) containing 0·5 % (w/v) glycodeoxycholic acid (Sigma) as originally described by Dashkevicz & Feigner (Dashkevicz & Feighner, 1989
; Dussurget et al., 2002
).
Salt-stress conditions.
To monitor induction of target gene expression following exposure to salt stress, bacterial strains were grown to mid-exponential phase in BHI as described above. Specifically, 20 ml aliquots of cells grown to an OD600 value of 0·4 were centrifuged as described above, then each pellet was resuspended in 20 ml of 0·154 M NaCl (representing a physiological salt concentration of 0·9 %, w/v; pH 5·9), 0·5 M NaCl (pH 5·8) or 0·5 M KCl (pH 5·8). Following exposure times of 15, 30, 60 or 120 min, 1 ml samples of the wild-type and sigB opuCAgus fusion strains (FSL S1-063 and FSL S1-059, respectively) were collected for
-glucuronidase (GUS) activity measurement, as described below.
L. monocytogenes wild-type and sigB strains (10403S and FSL A1-254, respectively) were also exposed to 0·121 M KCl (0·9 %) and 0·5 M KCl as described above. Samples were collected at 5, 10 and 15 min post-exposure for total RNA isolation and RT-PCR.
Total RNA isolation.
For the RT-PCR experiments, total RNA was purified from cells collected during exposure to salt stress and throughout growth, as described above. Bacterial cells collected at the specified time points were centrifuged and immediately resuspended in 10 ml Trizol reagent (Invitrogen) per 30 ml of culture harvested. The resuspension was immediately placed on ice and sonicated for three 20 s intervals (output: 20 W) using a Sonicator 3000 (Misonix). A 20 ml aliquot of chloroform (Shelton Scientific) was added for each 10 ml of original cell culture. After vigorous vortexing and 10 min incubation at room temperature, tubes were centrifuged (2190 g) for 60 min. Nucleic acids from the aqueous layer were precipitated with an equal volume of 2-propanol and centrifuged (17 900 g). The resulting pellets were washed twice with 100 % ethanol, resuspended in RQ1 10x DNase Buffer and treated with RQ1 DNase (Promega). Nucleic acids were subsequently purified by phenol/chloroform extraction and ethanol precipitation with 0·3 M sodium acetate (Sambrook et al., 1989). DNase treatment, phenol/chloroform extraction and ethanol precipitation steps were repeated two additional times to remove any contaminating DNA. The final RNA pellet was resuspended in 60 µl diethyl pyrocarbonate (DEPC)-treated water (Invitrogen). Total nucleic acid concentrations were estimated using absorbance readings (260 nm/280 nm) on a DU Series 600 Spectrophotometer (Beckman Coulter).
RT-PCR.
Reverse transcription was performed using the Superscript First-Strand Synthesis RT-PCR System (Invitrogen) with 50 ng of total RNA for each reaction. Primers to amplify opuCA, lmo1421, bsh and rpoB, which were designed using PRIMEREXPRESS software (Applied Biosystems), are shown in Table 2. Reverse transcription reactions were cycled once at 42 °C for 50 min and then at 70 °C for 15 min. PCR amplification of cDNA was performed using 10 µl of each reverse transcriptase reaction and the AmpliTaq Gold DNA Polymerase system (Applied Biosystems). PCR cycling conditions included an initial 9 min hold at 95 °C, followed by 30 cycles of 1 min at 94 °C, 1 min at 55 °C, 30 s at 72 °C, and a final hold of 5 min at 72 °C. Reverse transcription and PCR amplification reactions were performed in a GeneAmp 9600 (Perkin Elmer). To monitor for possible contamination by genomic DNA, an aliquot of each RT-PCR was run in the absence of Superscript II enzyme. RT-PCR products (10 µl) were subjected to gel electrophoresis using 3 % Metaphor (BioWhittaker Molecular Applications) agarose gels. The pGEM DNA ladder was used as a molecular mass marker and as a standard for PCR product quantification.
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Quantitative GUS assay.
Assays for GUS activity were performed essentially as described by Youngman (Harwood & Cutting, 1990). Specifically, cells from 1 ml aliquots were pelleted and washed in 1 ml Buffer AB Light (60 mM K2HPO4, 40 mM KH2PO4, 0·1 M NaCl, pH 7·0). The washed cell pellet was resuspended in 400 µl of Buffer AB Light for GUS assay and standard plate counts on BHI agar. To quantify GUS activities, 100 µl of the cell suspension were thoroughly mixed with 100 µl of Buffer AB Plus (Buffer AB Light containing 0·2 % Triton X-100) and incubated at room temperature for 60 min to lyse the cells. In a black, flat-bottomed Packard OptiPlate 96-well plate (Perkin Elmer), 50 µl of the lysed cells were mixed with 10 µl of 4-methylumbelliferyl-
-D-glucuronide (4-MUG; Sigma) in 0·4 mg ml-1 DMSO (Fisher Scientific) and held at room temperature for at least 60 min. Exact incubation times were recorded to calculate activity units as described below. Fluorescence was measured in a Packard Fusion Instrument (Perkin Elmer) using an excitation filter of 360 nm and an emission filter of 460 nm. Fluorescence units were converted to picomoles of methylumbelliferone (MU-) using a standard curve of known MU- (Sigma) concentrations. GUS activities were expressed in activity units defined as picomoles of 4-MUG hydrolysed per millilitre of cells at OD600=1·0, per minute. For salt-stress experiments, percentage changes between GUS activities of the salt-exposed cells (0·5 M NaCl and 0·5 M KCl) and non-exposed cells (0·154 M NaCl) were calculated at each collection time point. The one-sample t-test was used to identify significant differences in activity units between samples exposed to the different test conditions at each time point. Normality of observations was satisfied using the AndersonDarling test (P<0·05). All statistical analyses were performed using MINITAB version 13 (Minitab).
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RESULTS |
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BSH assay
To confirm the role of B in expression of bsh, which encodes a conjugated BSH (Dussurget et al., 2002
), L. monocytogenes wild-type 10403S, L. innocua DD608 (as a negative control) and the
sigB strain FSL A1-254 were spotted onto MRS medium agar containing 0·5 % glycodeoxycholic acid. A heavy white precipitate of free bile salt was observed only around the L. monocytogenes wild-type strain, but not around the
sigB or the L. innocua strains, indicating the presence of BSH activity only in the wild-type strain, but not in the others (Fig. 2
).
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DISCUSSION |
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Identification of B-dependent genes and promoters
Semi-quantitative RT-PCR and opuCAgus reporter fusions confirmed opuCA, lmo1421 and bsh as members of the L. monocytogenes B regulon. Previous investigations by Fraser and co-workers identified a putative
B-dependent promoter sequence upstream of the opuC operon (Fraser et al., 2000
) and lmo1421 and provided initial evidence for
B-dependent transcription of these genes (Fraser et al., 2003
). Confirmation of opuCA and lmo1421 as
B-dependent genes further illustrates the importance of
B in regulating transcription of osmotic stress genes in L. monocytogenes. In L. monocytogenes, OpuC is one of several compatible solute transporters (e.g. transporters encoded by betL, gbu) which confer increased osmotolerance to L. monocytogenes under osmotic stress environments (Fraser et al., 2000
; Ko & Smith, 1999
; Sleator et al., 1999
). For example, under chill- and salt-stress conditions (0·5 M KCl), an opuCB mutant strain of L. monocytogenes 10403S exhibited a reduced ability to accumulate carnitine as compared to the wild-type strain (Angelidis et al., 2002
). lmo1421 is predicted to encode the ATPase subunit of another ABC compatible solute (choline) transporter, based on sequence similarities to known transporter genes in L. monocytogenes and B. subtilis (Fraser & O'Byrne, 2002
). Based on the high sequence homology with the B. subtilis opuBA, which encodes a choline transporter, other groups also have referred to lmo1421 as opuBA (Sleator et al., 2003
; Wemekamp-Kamphuis et al., 2002
). OpuB has been demonstrated in B. subtilis to contribute to choline uptake (Kappes et al., 1999
) and thus is likely to contribute to osmotolerance in L. monocytogenes, although Sleator et al. (2003)
proposed a possible role for OpuB in carnitine uptake in L. monocytogenes strain LO28. The demonstrated
B dependence of opuCA and lmo1421 is consistent with the previous observation that a L. monocytogenes sigB null mutant shows a reduced ability to accumulate betaine and carnitine under osmotic- and cold-stress conditions (Becker et al., 1998
, 2000
). Interestingly, opuE, which also encodes an osmoprotectant, is regulated by
B in B. subtilis (von Blohn et al., 1997
). In combination, these observations support a broad role for
B in regulation of osmolyte uptake systems in low-G+C-content Gram-positive bacteria.
In addition to opuCA and lmo1421, we also experimentally identified and confirmed bsh as a B-dependent gene using a semi-quantitative RT-PCR approach. bsh, which encodes a conjugated BSH, was first identified in L. monocytogenes by Dussurget et al. (2002)
. Using the assay described by Dussurget et al. (2002)
, we demonstrated that only the L. monocytogenes wild-type strain hydrolysed bile salt, while no visually apparent bile salt hydrolysis was observed in the
sigB strain (Fig. 2
). Thus, we phenotypically confirmed the importance of an intact sigB on expression of BSH activity in L. monocytogenes. Since a functional bsh was shown to be required for full virulence in guinea pig infections (Dussurget et al., 2002
), our results provide evidence for a role of
B in virulence gene expression and virulence in L. monocytogenes. The role of bsh as a virulence gene is further supported by the observation that its expression is regulated by PrfA, a general activator of virulence gene expression in L. monocytogenes (Dussurget et al., 2002
).
In combination with other studies, our results provide further evidence for a broad role of L. monocytogenes B during hostpathogen interactions (Milohanic et al., 2003
). Specifically, previous work has shown that
B directly contributes to the regulation of prfA transcription (Nadon et al., 2002
), and that both opuCA and bsh contribute to intra-host survival by L. monocytogenes (Dussurget et al., 2002
; Sleator et al., 2001
; Wemekamp-Kamphuis et al., 2002
). An intact opuC was required for wild-type colonization of the mouse upper small intestine following peroral inoculation by L. monocytogenes LO28 (Sleator et al., 2001
; Wemekamp-Kamphuis et al., 2002
). Dussurget et al. (2002)
described a reduction in the recovery of a L. monocytogenes EGD bsh null mutant as compared to its wild-type parent strain in guinea pig stools 48 h after intragastric inoculation (Dussurget et al., 2002
). While bsh appears to be specific to L. monocytogenes and is absent from the non-pathogenic L. innocua and B. subtilis, similarity searches for opuCA and lmo1421 show that homologues for both these genes exist in both B. subtilis strain 168 and L. innocua CLIP11262 (Glaser et al., 2001
). Thus,
B appears to be involved in the transcriptional regulation of both classical virulence genes (e.g. bsh, prfA) and of general stress-response genes, which are important for bacterial survival during hostpathogen interaction as well as for survival in non-host environments. During gastrointestinal passage, pathogens such as L. monocytogenes experience dramatic changes in environmental conditions, such as exposure to low pH in the stomach and to high osmolarity in the small intestine (Davenport, 1982
). As
B fulfils functional roles in both virulence and general stress response and as the gastrointestinal environment encountered by L. monocytogenes during passage through a host imposes a set of physiological stresses on bacteria, we hypothesize that
B contributes to ensuring appropriate gene expression enabling bacterial survival under these conditions.
Growth-phase-dependent activation of B
Through both a reporter fusion strategy (Ferreira et al., 2003) and semi-quantitative RT-PCR, opuCA was demonstrated to be expressed in a growth-phase-dependent manner. Maximal opuCA-directed GUS activity was observed at entry into the stationary phase of growth (Ferreira et al., 2003
), which generally corresponds with cellular exposure to nutrient-limiting and other stress conditions (Kolter et al., 1993
). Our results are consistent with those of Becker et al. (1998)
, who used primer extension analysis of the
B-dependent rsbV promoter to show induction of
B activity following entry into stationary phase. Studies in B. subtilis have also shown induction of
B activity upon entry into stationary phase (Boylan et al., 1993
; Varon et al., 1996
). Growth-phase-dependent expression of opuCA thus follows the typical
B-dependent expression profiles previously established for the
B-dependent rsbV in L. monocytogenes and for
B-dependent genes in other Gram-positive bacteria.
Induction of B-dependent genes during salt stress
To further examine induction of B-dependent genes under osmotic-stress conditions, we used opuCAgus transcriptional gene fusions as well as semi-quantitative RT-PCR to monitor transcription of opuCA, bsh and lmo1421. Salt-stress conditions were selected for this study, as L. monocytogenes is likely to experience stress of this nature under food-processing conditions (e.g. in brines) and in food products associated with L. monocytogenes contamination (e.g. smoked fish and brined meat products) as well as during intra-host survival (Davenport, 1982
). RT-PCR assays showed a rapid induction of opuCA and lmo1421 under salt-stress conditions (i.e. within 5 min of salt-stress exposure). opuCA induction was also further confirmed by GUS reporter fusion assays. These data are consistent with preliminary primer extension experiments by Becker et al. (1998)
, who showed increased transcription of the
B-dependent rsbV promoter following salt stress in L. monocytogenes. Osmotic stress has also been shown to induce
B activity and transcription of the
B regulon in B. subtilis (Petersohn et al., 2001
). Interestingly, even though bsh clearly showed
B-dependent transcription, we were not able to observe consistent induction of bsh transcription following exposure of exponential-phase cells to salt-stress conditions. These results may indicate that bsh requires additional co-factors (other than the
BRNA polymerase holoenzyme complex) for transcription induction, which would be consistent with the fact that BSH may only be required under specific environmental conditions (i.e. the presence of bile salts). The existence of
B-dependent genes that require additional
B-independent stress induction mechanisms has recently been demonstrated in B. subtilis. In fact, at least 24 of 125 genes in the B. subtilis regulon may require additional
B-independent stress-induction mechanisms (Petersohn et al., 2001
). Further studies on the regulation of opuCA, lmo1421 and bsh will provide important insight into the complex mechanism(s) that likely govern expression of these stress-response genes and the L. monocytogenes
B regulon under different stress conditions, including those associated with intra-host environments.
Conclusions
L. monocytogenes has the unique ability to survive and grow under adverse environmental conditions including low pH, high osmolarity and low temperatures, and also to cause food-borne infections in mammalian hosts through survival and multiplication in the intracellular environment of host cells (Cole et al., 1990; Farber & Peterkin, 1991
; Lou & Yousef, 1999
). Our data support an emerging model that proposes a link between environmental survival and virulence in the food-borne pathogen L. monocytogenes. We have identified and confirmed three
B-dependent genes, including two osmotolerance genes (opuCA and lmo1421) and a virulence gene (bsh). Together with the previous description of a
B-dependent promoter contributing to the transcription of prfA (Nadon et al., 2002
), which encodes a key positive regulator of virulence gene expression in L. monocytogenes, and the description of a set of PrfA-regulated genes preceded by putative
B-dependent promoters (Milohanic et al., 2003
), our data suggest that
B may broadly respond to host-associated bacterial stress conditions by directing appropriate gene expression patterns. Further use and refinement of the reporter fusion and RT-PCR tools for monitoring
B activity in L. monocytogenes described here will allow us to enhance our understanding of the contributions of
B to the pathogenesis of food-borne listeriosis.
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ACKNOWLEDGEMENTS |
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Received 28 May 2003;
revised 18 July 2003;
accepted 21 July 2003.
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