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1 Wageningen Centre for Food Sciences (WCFS), Diedenweg 20, 6703 GW Wageningen, The Netherlands
2 Division of Health and Safety, NIZO Food Research, Kernhemseweg 2, 6718 ZB Ede, The Netherlands
3 Laboratory of Food Microbiology, Wageningen University and Research Centre, Bomenweg 2, 6703 HD Wageningen, The Netherlands
4 Institute of Medical Microbiology, Justus-Liebig University, Frankfurter Strasse 107, 35392 Giessen, Germany
Correspondence
Stijn van der Veen
Stijn.vanderVeen{at}wur.nl
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ABSTRACT |
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These authors contributed equally to this work.
Supplementary tables of the sequences of the Q-PCR primers and the genes that show differential expression at different time points after exposure to a temperature of 48 °C are available with the online version of this paper.
The microarray platform and microarray data discussed in this publication have been deposited in ArrayExpress (http://www.ebi.ac.uk/arrayexpress) under accession numbers A-MEXP-752 and E-MEXP-1118, respectively.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Health Protection Agency, 2005; Koch & Stark, 2006). This bacterium shows relatively high resistance to environmental insults compared with many other non-spore-forming food-borne pathogens. It is able to grow at a wide pH range (from pH 5 to pH 9), at high salt concentrations (up to 12 %), and at a wide range of temperatures (–0.4 to 44 °C) (Kallipolitis & Ingmer, 2001; Karatzas et al., 2005). The ability of L. monocytogenes to proliferate under adverse conditions and survive environmental insults is mediated by various mechanisms that allow for rapid responses and adaptations to changing environments. Due to consumers' demands for less heavily preserved foods and more convenience foods, processing conditions in the food industry are becoming milder. L. monocytogenes is able to adapt to such milder conditions, making it of major concern to the food industry.
Variation in temperature is a stress that is commonly encountered in nature and during the processing of foods. DNA microarrays provide an excellent tool to study the expression profiles of a complete genome during exposure to heat stress. Previous studies involving transcriptional analysis of the heat-shock response in various bacteria have shown induction of several protection mechanisms, including general protection mechanisms and specific heat-shock responses. The heat-shock response is a common phenomenon among bacteria that enables them to survive a wide variety of stresses, in particular heat stress. Most heat-stress-induced genes encode molecular chaperones or proteases that can either protect other proteins/enzymes against misfolding and damage or mediate degradation when this fails. Maintenance of protein quality is important for normal growth of cells and is essential under stress conditions. In L. monocytogenes, two specific heat-shock-response mechanisms and a general stress-response mechanism can be distinguished, namely, the class I and class III heat-shock response, and the class II stress response (Benson & Haldenwang, 1993; Kruger & Hecker, 1998; Schulz & Schumann, 1996). Class I heat-shock genes are controlled by the HrcA repressor, which binds to the CIRCE operator sequence (TTAGCACTC-N9-GAGTGCTAA) preceding this class of genes. Class I heat-shock genes include dnaK, dnaJ, groES and groEL, encoding chaperones. Class III heat-shock genes encode chaperones and ATP-dependent Clp proteases, which degrade damaged or misfolded proteins. This class is regulated by the CtsR repressor, which binds specifically to a heptanucleotide repeat in the promoter region (A/GGTCAAANANA/GGTCAAA). The class II stress genes encode general stress proteins, of which the expression is regulated by the alternative sigma factor SigB. This sigma factor recognizes alternative –35 and –10 sequences (GTTT-N13–17-GGGWAT) in the promoter region of the class II stress genes (Kazmierczak et al., 2003).
The complete heat-shock regulon of L. monocytogenes that acts in response to a temperature increase has, apparently, not been investigated before, even though heating is an important preservation strategy for the food industry during minimal processing. The aim of this study was to determine the global transcriptional response of L. monocytogenes to heat stress. Our data show that exposure to elevated temperatures triggers the classical heat-shock genes, and, in addition, a transient effect on expression of genes involved in the cell replication machinery was observed. Another novel finding is that heat shock triggers the SOS response in L. monocytogenes.
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METHODS |
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) was grown in brain heart infusion (BHI) broth (Difco) with shaking (200 r.p.m., New Brunswick C24KC) using 10 ml culture medium in 100 ml conical flasks. An exponentially growing culture was used to inoculate 100 ml fresh pre-warmed BHI broth in a 500 ml flask. This culture was incubated at 37 °C with agitation at 200 r.p.m. until OD600 1.0 was reached. At this point (designated time zero) a 5 ml aliquot was removed for RNA extraction and 10 ml aliquots were transferred to pre-warmed 100 ml flasks at 48 °C. The cultures were incubated in a shaking water bath at 48 °C (GFL type 1083, 60 % shaking speed), and samples for RNA extraction and microscopic analysis were taken after 3, 10, 20 and 40 min.
RNA isolation, labelling, hybridization, imaging and microarray analysis.
Samples (0.5 ml) were rapidly removed and diluted in 1.0 ml RNAprotect (Qiagen). After incubation for 5 min at room temperature and centrifugation at 5000 g for 5 min pellets were stored at –80 °C. Microarray experiments (including microarray generation, total RNA extraction, labelling, hybridization, imaging and microarray analysis) were performed as described in detail previously (Chatterjee et al., 2006). The Significance Analysis of Microarrays (SAM) program was used to analyse the data. The cut-off for significantly differentially expressed genes was set with a q value (false discovery rate) of 
1 % and a fold-change of 
2. In three independent experiments, the whole-genome expression profiles of cells that had undergone heat shock for 3, 10, 20 and 40 min were compared with those of cells at time zero in a dye-swap hybridization experiment.
Microscopy and image analysis.
Samples (1 ml) were removed from cultures (see above) and centrifuged at 5000 g for 2 min. Cells were dissolved in nigrosin solution (Sigma-Aldrich) and dried on glass slides. Images of the cells were taken at x100 magnification with a Dialux 20 microscope (Leica). The ImageJ program (http://rsb.info.nih.gov/ij/download.html) was used to analyse the images. The images were loaded in eight-bit type and the threshold was adjusted to black and white. The number of pixels per cell was counted and distribution graphs were constructed in Excel (Microsoft) by analyses of seven images from two cell preparations for each time point.
Quantitative PCR (Q-PCR).
Superscript III reverse transcriptase (Invitrogen) was used to synthesize first-strand cDNA using 1 µg DNase-treated total RNA. RNA samples were controlled for DNA contamination by omitting this cDNA synthesis step. Q-PCR reactions were performed using 10 µl 2xSybr Green PCR Master Mix (Applied Biosystems), 200 nM primers and 1 µl cDNA sample in a 20 µl final volume. For each primer set a standard curve was generated using both genomic DNA and cDNA, and negative control samples using pure water were included. Reactions were run on the 7500 Real-Time PCR system (Applied Biosystems) with an initial step of 10 min at 95 °C, and 40 cycles of 15 s at 95 °C and 1 min at 60 °C. To verify single-product formation, a dissociation cycle was added. Forward and reverse primers (Supplementary Table S1) were designed with an amplicon length of about 100 bp and a Netprimer rating (http://www.premierbiosoft.com/netprimer) of above 80.
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RESULTS |
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1 % (significant values are given in Supplementary Table S2). Of these 714 genes, 427 showed increased expression and 287 showed decreased expression upon heat shock, constituting 15 and 10 % of the total of 2857 genes, respectively. The maximum level of gene induction observed was 50.3-fold (lmo1883), while the maximum level of gene repression was 31.6-fold (lmo0048). Most of the differentially expressed genes showed a transient pattern. The highest number of differentially expressed genes, which account for 24 % of all genes, was observed 3 min after the temperature upshift. After 40 min, only 2 % of all genes were differentially expressed. The differentially expressed genes were grouped into functional classes (Fig. 1). The classes containing the highest numbers of differentially expressed genes were carbohydrate transport and metabolism, amino-acid transport and metabolism, transcription and translation, with 84, 63, 59 and 52 genes, respectively. Most genes involved in carbohydrate transport and metabolism (82 %) and transcription (81 %) showed upregulation, and most genes involved in translation (92 %) showed down-regulation. Numerous genes belonging to the amino-acid transport and metabolism class showed differential expression, both up- and down-regulation (36 and 27 genes, respectively).
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. A detailed list of the differentially expressed genes belonging to these different classes is presented in Table 2 and described further below.
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also detected fivefold induction of two htrA paralogues in Bacillus subtilis, and designated this general class of heat-shock genes class U.
The class II stress response represents a general stress-response mechanism that is regulated by the alternative sigma factor SigB. The gene encoding SigB (lmo0895) did not show more than twofold differential expression on the microarrays during heat shock. Verification of sigB expression using Q-PCR showed 1.5-fold upregulation after 3 and 10 min (Fig. 2). In total, 51 genes previously identified as being SigB-regulated (Kazmierczak et al., 2003) showed increased expression at one or more of the selected time points. Among these upregulated genes were ctc (lmo0211), lmo1601 and ydaG (lmo2748). These genes encode proteins that show sequence similarity with general stress proteins. Increased expression was also observed for the SigB-regulated opuC operon (lmo1425–lmo1428). This operon encodes a betaine/carnitine/choline ABC transporter and has previously been shown to be responsible for the accumulation of osmolytes in response to salt, acid and cold stress (Sleator et al., 2001; Wemekamp-Kamphuis et al., 2004). Notably, these osmolytes have also been shown to provide protection during heat exposure of B. subtilis (Holtmann & Bremer, 2004) and Escherichia coli (Caldas et al., 1999), though no increase in their intracellular concentration was observed.
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) or in the introduction of adaptive point mutations (Michel, 2005; Miller et al., 2004). Numerous genes that are part of the SOS response and the DNA repair machinery showed increased expression after heat exposure. The recA (lmo1398) gene, encoding the major activator of the SOS response (RecA), showed a gradual increase in expression over time, from 1.4-fold to 5.2-fold induction between 3 and 40 min. Differential expression of lexA (lmo1302), the repressor of the SOS response, was not observed using microarrays, but Q-PCR showed a change from 1.9-fold repression at 3 min to 2.0-fold induction at 40 min (Fig. 2
). Transcription of dinB (lmo1975) and umuDC (lmo2675 and lmo2676), coding for DNA polymerases IV and V, respectively, was also upregulated. These gene products constitute important alternative polymerases in the SOS response. The genes encoding the DNA repair proteins radA (lmo0233), recN (lmo1368), uvrA (lmo2488) and uvrB (lmo2489) showed increased expression as well. Two other genes belonging to the SOS response were induced, namely ynzC (lmo0496) and yneA (lmo1303). The latter is transcribed divergently from lexA and the product has been shown to be responsible for cell elongation and suppression of cell division (Kawai et al., 2003). Thus, the expression of yneA is consistent with the observation that exposure of L. monocytogenes to elevated temperatures results in elongated cells (Fig. 3). After exposure of cells growing at 37 °C to a temperature of 48 °C, the OD600 continued to increase (Fig. 3a), whereas the concentration of cells (c.f.u. ml–1) was found to remain constant (Fig. 3b). Cell size measurements of microscopic images showed increased cell size with time after heat exposure compared with cells that were grown continuously at 37 °C (Fig. 3c–e).
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Cell-wall-associated genes
The bacterial cell wall is a complex structure and plays an important role as the first protection against environmental stresses. Cell wall hydrolases (including autolysins) play important roles in numerous cellular processes, including cell division and cell wall turnover. In line with the results described above, all genes encoding cell wall hydrolases, namely aut (lmo1076), lmo1216, lmo1855, spl (lmo2505), ami (lmo2558), murA (lmo2691) and lmo2754, showed a transiently repressed expression pattern, except for iap (lmo0582), which showed an almost constant threefold repression. The intermediate glucosamine-6-phosphate plays a central role in the regulation of cell wall biosynthesis vs glycolysis (Komatsuzawa et al., 2004). Genes controlling the regulation between these two pathways showed induction after heat exposure. These genes are lmo0877 and lmo0957, for which the encoded products show sequence similarity to glucosamine-6-phosphate isomerases, lmo0956 and lmo2108, which encode putative N-acetylglucosamine-6-phosphate deacetylases, and lmo1998 and lmo1999, which encode products that show low sequence similarity to glucosamine-fructose-6-phosphate aminotransferases. The essential gene in the biosynthetic pathway, murB (lmo1420), was down-regulated after 3 min heat shock. Another operon that plays a role in cell wall synthesis and showed decreased expression upon heat shock was the dlt operon (lmo0971–lmo0974), which encodes proteins important in catalysing the incorporation of D-alanine residues into lipoteichoic acids and wall teichoic acids. Furthermore, the lmo1075 gene, which encodes a protein with 64 % sequence similarity to B. subtilis teichoic acid translocation ATP-binding protein TagH, was also down-regulated after 3 min of heat shock. Nine genes encoding putative cell-wall-associated proteins were differentially expressed during heat exposure. Five of these genes encode products containing an LPXTG peptidoglycan-binding motif, namely lmo0610, lmo0880, lmo1413, lmo1666 and lmo2085.
Virulence-associated genes
A constant induction was observed for prfA (lmo0200), which encodes the major regulator of the virulence genes. This gene is both SigB- and SigA-regulated, and its product induces genes containing a PrfA box in their promoter sequence. Accordingly, numerous PrfA-regulated virulence genes (Milohanic et al., 2003) showed upregulation after heat exposure, including plcA (lmo0201), encoding a phospholipase, inlA and inlB (lmo0433 and lmo0434), encoding Listeria-specific internalins, lmo2067, encoding a conjugated bile acid hydrolase, and lmo0596, encoding an unknown protein. However, Rauch et al. (2005) have shown that PrfA has no direct influence on the transcription of lmo2067 and lmo0596. Significant down-regulation was found for the agr locus (lmo0048– lmo0051). This locus encodes a two-component system that has been shown to play a role in bacterial virulence (Autret et al., 2003).
Validation of microarray gene expression
The microarray data were validated by Q-PCR analysis using a set of 13 genes which cover a range of expression values in the microarray data. The relative gene expression levels obtained by Q-PCR were normalized to that of three genes (tpi, rpoB and the 16S rRNA gene) that did not show fluctuating expression during temperature changes. Relative quantitative values were obtained using the comparative threshold cycle method (
CT) in which the CT value corresponds to the cycle at which the fluorescent signal crosses the threshold line. The relative expression of the genes was determined in quadruplicate for three independent temperature upshift experiments. The resulting ratios were log2 transformed and plotted against the log2 values from the microarray analysis. Fig. 2 shows a strong correlation, with r >0.8 for all time points, which is considered the threshold for strong correlation. Generally, the microarray analysis underestimated the differential expression values by an average of fivefold. Similar observations have been reported previously when using ORF arrays in microbial transcriptome analysis (Gao et al., 2004; Helmann et al., 2001; Stintzi, 2003).
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DISCUSSION |
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; Helmann et al., 2001; Stintzi, 2003). These studies also showed transient expression patterns, albeit that peak numbers of differentially expressed genes were observed at later times after heat exposure. The ability of L. monocytogenes to rapidly alter transcript levels allows for a quick response to sudden environmental changes.
In L. monocytogenes, genes belonging to the class I and class III heat-shock regulons showed increased expression during heat exposure. Interestingly, the class I heat-shock genes showed a constant induction in expression levels over the 40 min period, whereas the class III heat-shock genes showed a transient expression pattern. This is likely related to the different roles that the two classes fulfil during heat shock. The Clp-ases are specifically required at the early stages of heat shock to remove the proteins initially damaged and misfolded. Following adaptation to heat, Clp-ase levels are reduced, while higher levels of chaperones are present to prevent accumulation of damaged and misfolded proteins. Q-PCR showed 1.5-fold induction of the gene encoding SigB after 3 and 10 min. Considering the role of SigB as a regulator, this slight increase in expression appears to be enough to induce genes that contain a SigB promoter, as evidenced by the transient nature of the class II stress response. When present in B. subtilis, C. jejuni and S. oneidensis (Gao et al., 2004; Helmann et al., 2001; Stintzi, 2003), genes belonging to the class I and class III heat-shock response and SigB-regulated class II stress response were induced upon heat shock. Given the genetic resemblance and similar heat-shock conditions applied, a direct comparison of upregulated genes of L. monocytogenes and B. subtilis (Helmann et al., 2001) was possible. A total of 25 orthologues of the 130 induced class II stress genes in B. subtilis showed induction in L. monocytogenes as well. Of these 25 genes, eight had been previously identified in L. monocytogenes to be SigB-regulated (Kazmierczak et al., 2003). In B. subtilis, 76 other genes, designated class U, show increased expression upon heat shock. In L. monocytogenes, nine orthologues of these genes showed induction, including htrA. Other research has shown that HtrA is important for survival during exposure to environmental and cellular stresses (Stack et al., 2005; Wilson et al., 2006).
In L. monocytogenes, several genes that play a role in the SOS response and DNA repair machinery showed increased expression after heat exposure, including recA, which is the major activator of the SOS response. RecA is involved in various processing steps of DNA replication proteins and repair proteins, and plays a role in the restart of stalled replication forks (Cox et al., 2000), homologous recombination, repair of double-stranded DNA breaks (Lusetti & Cox, 2002) and introduction of adaptive point mutations (McKenzie et al., 2001). Furthermore, it stimulates cleavage of the LexA repressor of the SOS response, activating expression of the SOS response genes (Fig. 4). The LexA repressor is autoregulatory, and binds to the operator sequence in the promoter region of the SOS response genes (CGAACATATGTTCG in B. subtilis; Au et al., 2005). Several LexA-regulated genes showed increased expression in L. monocytogenes, including genes encoding the two alternative DNA polymerases umuDC and dinB. DNA polymerases Pol IV (DinB) and Pol V (UmuD2'C) have previously been shown to respond to inhibition of replication fork progression in a damage-independent manner (Godoy et al., 2006; Hare et al., 2006). Elevated temperatures may cause the L. monocytogenes DNA replication fork to stall, leading to exposure and possible degradation of the arrested replication fork without rescue by Pol IV and Pol V. This theory is consistent with the observation that heat shock resulted in cell elongation and prevention of cell division. This effect is called ‘nucleoid occlusion’ and is generally interpreted as prevention of Z-ring formation in the vicinity of the nucleoid, thereby preventing cell division without complete replication and segregation of the nucleoid (Rothfield et al., 2005). The expression profiles showed two mechanisms that might lead to the prevention of Z-ring formation after heat shock (Fig. 5). The first mechanism would be the result of increased expression of yneA (as part of the SOS response), which is responsible for suppression of cell division in B. subtilis by reducing the level of FtsZ proteins at the cell division site, thereby preventing FtsZ ring formation (Kawai et al., 2003). The second mechanism is the result of a decreased expression of minDE and divIVA, which are involved in spatially correct septum placement. The MinCD complex negatively regulates formation of the cytokinetic Z-ring to midcell by preventing its formation near the poles (Zhou & Lutkenhaus, 2005). Another strategy of L. monocytogenes to suppress cell division might be to decrease expression of fteX and ftsE. These encode proteins with sequence similarity to ABC transporters and are localized at the division site. In E. coli they are directly involved in cell division and are important for septal ring formation (Schmidt et al., 2004). Mutants for ftsXE constitutively showed induction of the SOS response in E. coli (O'Reilly & Kreuzer, 2004).
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). Research in B. subtilis has shown that cells depleted of RodA are impaired in cell division (Henriques et al., 1998). Reduced expression levels were also observed for the genes encoding autolysins, for murB, which is an essential gene in the biosynthetic pathway of peptidoglycan, and for the dlt operon, which encodes products that catalyse the incorporation of D-alanine residues into teichoic acids. While most bacteria show slower growth rates after mutations in the dlt operon, Streptococcus gordonii shows abnormal septation and defective cell separation (Neuhaus & Baddiley, 2003).
PrfA and various other virulence genes showed induction upon heat exposure, which has apparently not been reported before. PrfA is the major virulence regulator in L. monocytogenes (Chakraborty et al., 1992; Leimeister-Wachter et al., 1990). Previous studies have shown maximal induction of the virulence genes at 37 °C (Johansson et al., 2002; Leimeister-Wachter et al., 1992). However, expression levels at temperatures above 37 °C were not measured in these studies. Destabilizing mutations in the secondary structure of the untranslated region of prfA mRNA resulted in increased levels of PrfA at 37 °C (Johansson et al., 2002), indicating that this structure is not completely unfolded at this temperature. Increasing the temperature above 37 °C probably results in a more relaxed secondary structure, allowing more PrfA translation.
A comparative analysis of the transcription profiles of L. monocytogenes during heat shock and in the intracellular environment (Chatterjee et al., 2006) showed a high number of genes that were differentially expressed during both exposures. Of the 714 genes differentially expressed during heat shock, 236 showed differential expression in the intracellular environment as well. Among genes that were commonly regulated during both exposures, 172 showed increased expression and 64 showed decreased expression. Exposure of L. monocytogenes to the intracellular environment triggered numerous (heat) stress-response genes that were shown to be differentially expressed during heat shock in this study. All genes belonging to the class I and class III heat-shock-response classes and 24 of the SigB-regulated class II stress-response genes showed induction in the intracellular environment. Remarkably, similar expression profiles for SOS response genes, cell division genes and cell-wall-related genes were observed. This comparative analysis showed that numerous responses induced during heat exposure contribute to the pathogenicity of L. monocytogenes by allowing survival under the stressful conditions encountered intracellularly and during passage through the stomach and the intestinal tract.
This study shows the importance of the SOS response as a common protection mechanism during heat shock, and its possible role in suppression of cell division to prevent transection of the genome as a result of incomplete chromosomal segregation. Whether replication fork stalling is the actual cause that leads to both the SOS response and suppression of cell division remains to be elucidated. This opens up interesting possibilities for future research. Our transcriptome analysis has revealed several aspects of the heat-shock response of L. monocytogenes that may include targets for inactivation. The role of specific factors, such as the SOS response in L. monocytogenes stress survival and virulence, will be assessed in future studies.
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ACKNOWLEDGEMENTS |
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Edited by: J. Lindsay
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Received 5 February 2007;
revised 5 June 2007;
accepted 15 June 2007.
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) were measured. At time zero (point C) the culture was transferred to 48 °C (
) or grown on at 37 °C (
