Global analysis of gene expression in an rpoN mutant of Listeria monocytogenes

Safia Arous1, Carmen Buchrieser2, Patrice Folio3, Philippe Glaser2, Abdelkader Namane4, Michel Hébraud3 and Yann Héchard1

1 Equipe de Microbiologie Fondamentale et Appliquée, Université de Poitiers, 40 avenue du Recteur Pineau, 86022 Poitiers Cedex, France
2 Laboratoire de Génomique des Micro-organismes Pathogènes, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France
3 Station de Recherches sur la Viande, Institut National de la Recherche Agronomique de Theix, 63122 Saint-Genes Champanelle, France
4 Plateforme de protéomique, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France

Correspondence
Yann Héchard
yann.hechard{at}univ-poitiers.fr


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The role of the alternative {sigma}54 factor, encoded by the rpoN gene, was investigated in Listeria monocytogenes by comparing the global gene expression of the wild-type EGDe strain and an rpoN mutant. Gene expression, using whole-genome macroarrays, and protein content, using two-dimensional gel electrophoresis, were analysed. Seventy-seven genes and nine proteins, whose expression was modulated in the rpoN mutant as compared to the wild-type strain, were identified. Most of the modifications were related to carbohydrate metabolism and in particular to pyruvate metabolism. However, under the conditions studied, only the mptACD operon was shown to be directly controlled by {sigma}54. Therefore, the remaining modifications seem to be due to indirect effects. In parallel, an in silico analysis suggests that {sigma}54 may directly control the expression of four different phosphotransferase system (PTS) operons, including mptACD. PTS activity is known to have a direct effect on the pyruvate pool and on catabolite regulation. These results suggest that {sigma}54 is mainly involved in the control of carbohydrate metabolism in L. monocytogenes via direct regulation of PTS activity, alteration of the pyruvate pool and modulation of carbon catabolite regulation.


Abbreviations: CCR, carbon catabolite repression; CRE, catabolite responsive element; PTS, phosphotransferase system


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The sigma 54 ({sigma}54) factor, encoded by rpoN, is an alternative subunit of bacterial RNA polymerase. It was first identified in Escherichia coli and in other Gram-negative bacteria as a {sigma} factor required for transcription of nitrogen-regulated genes. Today, a wide variety of {sigma}54-dependent genes of Gram-negative and Gram-positive bacteria are known to be involved in various cellular functions: nitrogen and carbon utilization, as well as in flagellar synthesis and virulence (Studholme & Buck, 2000). The {sigma}54 factor enables the RNA polymerase to initiate transcription from a class of –24/–12 promoters that differ considerably from the vegetative –35/–10 promoters ({sigma}70-dependent). The consensus of the {sigma}54-dependent promoters is TGGCAC-N5-TTGCa/t in which the GG and GC pairs are highly conserved (Morett & Buck, 1989). In bacterial species where the promoter sequence has been characterized it is more conserved than that of {sigma}70. In contrast to {sigma}70, the {sigma}54-dependent RNA polymerase needs an activator to form the open promoter complex to initiate transcription. Those {sigma}54-dependent activators (also reported as enhancer-binding proteins; EBPs) bind specifically at an upstream activating sequence (UAS) generally localized between 100 and 200 bp upstream of the promoter (Shingler, 1996).

Among Gram-positive bacteria, {sigma}54-dependent genes have been thoroughly studied only in Bacillus subtilis, where the levanase operon was the first described as strictly dependent on the presence of both {sigma}54 and the LevR activator (Martin et al., 1989). This operon encodes a fructose phosphotransferase system (PTS) permease and the levanase enzyme. LevR has a unique structure among the {sigma}54-dependent activators (Débarbouillé et al., 1991). In particular, it contains two PTS regulation domains (PRD) and an EIIA domain that can be phosphorylated by the PTS components to control LevR activity (Deutscher et al., 2002; Stulke et al., 1995). Four other {sigma}54-dependent activators were described in B. subtilis: RocR (Calogero et al., 1994), AcoR (Ali et al., 2001; Huang et al., 1999), BkdR (Débarbouillé et al., 1999) and YplP (Beckering et al., 2002). The {sigma}54-dependent operons described in B. subtilis are involved in carbon metabolism, lev for levan utilization (Martin et al., 1989) and aco for acetoin utilization (Ali et al., 2001), as well as in amino acid metabolism, roc for catabolism of arginine (Calogero et al., 1994; Gardan et al., 1995) and bkd for catabolism of isoleucine and valine (Débarbouillé et al., 1999).

Listeria monocytogenes is a food-borne, facultative pathogen. {sigma}54 of L. monocytogenes has been described by our group to be involved in sensitivity to antibacterial peptides, the subclass IIa bacteriocins (Robichon et al., 1997). Only one {sigma}54-dependent operon, mptACD, has been identified so far in L. monocytogenes. Its expression is controlled by the ManR activator, which belongs to the LevR family (Dalet et al., 2001). The mptACD operon encodes the A, B, C and D subunits of a PTS permease of the mannose family, . The lack of mptACD expression, in an mpt or an rpoN mutant, leads to resistance of L. monocytogenes to subclass IIa bacteriocins. The permease was thus proposed to be the receptor for these antibacterial peptides (Dalet et al., 2001; Duché et al., 2002; Gravesen et al., 2002).

Until now, a global analysis of an rpoN mutant has not been reported for any bacteria. We took advantage of the availability of the complete genome sequence of L. monocytogenes EGDe (Glaser et al., 2001) to study gene expression at the RNA level using DNA macroarrays, at the protein level by two-dimensional (2D) gel electrophoresis and to perform an in silico analysis of the {sigma}54 regulon. The results show that although the lack of {sigma}54 expression has a pleiotropic effect, the major consequences are on carbohydrate metabolism of L. monocytogenes.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
Wild-type L. monocytogenes EGDe and the derivative rpoN mutant, EGK50 (Dalet et al., 2001), were grown to late exponential phase (OD600 0·8–1) in BHI medium (Difco) at 42 °C. This temperature was chosen to stabilize the plasmid integration in the rpoN mutant. Erythromycin (5 µg ml–1) was added for growth of the mutant strain.

Total protein extraction.
Bacterial cells from a 20 ml culture were harvested by centrifugation, washed twice in 2 ml buffer A (20 mM Tris/HCl, 5 mM EDTA, 5 mM MgCl2) and adjusted to pH 7·5. Pellets were resuspended in 1 ml buffer A, adjusted to pH 9, and sonicated three times for 2 min each (Vibra cell) at 50 % of the active cycle, power level 5, with chilling of the tubes on ice between each cycle. Cell lysates were treated with 500 µl buffer B (20 mM Tris, 5 mM EDTA, 5 mM MgCl2, 4 % CHAPS, 7 M urea, 4 M thiourea, pH 9) and 10 µl 0·2 M tributylphosphine (TBP), and then with 10 µl 1 % RNase A and 1 % DNase; the mix was incubated for 1 h at 4 °C. Suspensions were centrifuged (13 000 g, 20 min, 4 °C) to pellet the cell debris and the protein concentration in the supernatant was determined using the Bradford protein assay (Bio-Rad) with bovine serum albumin as standard. Proteins were precipitated from the supernatant with 3 vols cold acetone for at least 2 h at –20 °C and then pelleted by centrifugation (13 000 g, 40 min, 4 °C). Pellets were resuspended in isoelectric focusing buffer [7 M urea, 2 M thiourea, 4 % (w/v) CHAPS with traces of bromophenol blue] at a final concentration of 5 µg protein µl–1 and stored at –20 °C.

2D gel electrophoresis.
2D electrophoresis of bacterial soluble proteins was performed according to a modified O'Farrell (1975) method. The first dimension consisted of isoelectric focusing (IEF) with precast Immobiline pH Gradient (IPG) Drystrips, pH 3–6 or pH 3–10 (Bio-Rad). Samples containing 50 µg total protein were mixed with buffer C (7 M urea, 2 M thiourea, 4 % CHAPS, 2 mM TBP, 1·5 % ampholytes, pH 3–6) and used to rehydrate the Drystrips for 16 h. IEF was performed with the Multiphor II electrophoresis unit (Amersham Biosciences) for a total of 60·0 kVh according to the manufacturer's instructions. The strips were equilibrated for 15 min in buffer 1 (50 mM Tris/HCl, 6 M urea, 2 % SDS, 30 % glycerol, 5 mM TBP, pH 8·8) and 20 min in buffer 2 (modified buffer 1, 2·5 % iodoacetamide instead of TBP, and traces of bromophenol blue). The second dimension was carried out with 12·5 % acrylamide in a Multicell Protean II XL system (Bio-Rad). A constant current of 10 mA per gel was applied over 1 h for migration in the stacking gel; then 15 mA per gel were applied over 15 h for protein separation. Gels were silver-stained according to the method described by Rabilloud (1992).

Analysis of protein patterns.
For each strain, six gels from two independent bacterial cultures (three gels per extraction) were performed. 2D gels were scanned by a GS-700 imaging densitometer (Bio-Rad) and the protein patterns of the two strains were compared using Melanie 3 software (Genebio). Only spots present in at least five gels were taken into account. For protein identification, spots of interest were excised from the gel and submitted to tryptic digestion as described by Shevchenko et al. (1996). The resulting peptides were analysed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS on a Voyager DE STR (PerSeptive Biosystems) equipped with a nitrogen laser (337 nm). The instrument was operated in the delayed extraction mode and the delay time was 150 ns. Each mass spectrum was a mean of 250 laser shots. External calibration was performed. For searching the L. monocytogenes EGDe genome (http://genolist.pasteur.fr/ListiList/), monoisotopic masses were assigned using a local copy of the MS-Fit 3.2 part of the Protein Prospector package (University of California, Mass Spectrometry Facility, San Francisco). The parameters were set as follows: no restriction on the isoelectric point of proteins, the maximum mass error allowed was 50 p.p.m. and one incomplete cleavage per peptide was considered.

Synthesis and labelling of cDNA.
Bacterial cells, grown in a 10 ml culture, were harvested by centrifugation at 12 000 g for 5 min. Pellets were resuspended in 400 µl lysis buffer (5 mM Tris/EDTA, 5 mg lysozyme ml–1, 100 g glucose l–1, pH 8·0) and incubated for 1 h at 37 °C. Total RNA extraction was carried out by the addition of 1 ml RNAwiz solution (Ambion) and incubation for 5 min at 25 °C. Then 200 µl chloroform was added to the sample which was subsequently incubated for 10 min at 25 °C. RNA extracts were clarified by centrifugation at 12 000 g for 15 min at 4 °C and supernatants were transferred to new tubes. Total RNA was precipitated by adding 1 ml –20 °C-chilled 2-propanol and leaving for at least 1 h at –20 °C before centrifugation at 12 000 g for 30 min at 4 °C. Supernatants were carefully removed and pellets were washed with 75 % ethanol. RNA pellets were dried and dissolved in 70 µl diethylpyrocarbonate (DEPC)-treated water. To eliminate contamination by DNA, 4 units RNase-free DNase (Invitrogen) was added. The RNA concentration was determined using a spectrophotometer at 260 nm. RNA quality was checked on a formaldehyde agarose gel containing ethidium bromide. cDNA synthesis and labelling for macroarray hybridization was performed as follows. Total RNA (1 µg) was mixed with 3 µl 5x reverse transcriptase buffer, 1 µl each dNTP (10 mM), 2 µl L. monocytogenes ORF-specific primers (33 nM each primer) and DEPC-treated water to a final volume of 25 µl. The mixture was incubated for 2 min at 90 °C, cooled to 42 °C before addition of 3 µl [{alpha}-33P]dCTP (20 µCi) and 2 µl AMV-RT (50 U) (Roche). The mixture was incubated for 2 h at 42 °C. Salts and unincorporated nucleotides were removed using a QIAquick column (Qiagen) according to the manufacturer's instructions. Labelled cDNA was denatured by heating at 99 °C for 5 min before hybridization.

Transcriptome and data analysis.
Macroarrays were pre-hybridized for 2 h at 65 °C with 10 ml hybridization buffer (5x SSPE, 2 % SDS, 1 % Denhardt's reagent, 100 µg sonicated salmon sperm DNA ml–1) in roller bottles. Hybridizations were performed overnight at 65 °C with 5 ml fresh hybridization solution containing the labelled cDNA probe. Macroarrays were then washed twice at room temperature for 3 min and twice at 65 °C for 20 min with washing solution (0·5x SSPE, 0·2 % SDS). Macroarrays were exposed to a phosphoimager screen and scanned with a Typhoon (Pharmacia-Amersham). Membranes were stripped for 30 min at room temperature with 0·5 M NaOH and then stored at –20 °C. For both the wild-type and the rpoN mutant strains, two hybridizations for each of two independent RNA extractions were performed. Consequently, a set of four macroarrays was used. The signal intensity of each spot was quantified using ArrayVision analysis software (Imaging Research). For each spot, the hybridization intensity value was normalized by dividing by the mean of all significant intensity values on each filter. The global background for each macroarray was defined as the mean signal intensity from the gathered negative templates; the mean background was subtracted from each normalized value. SAM software (Significance Analysis of Microarrays) (Tusher et al., 2001) was used to identify genes with significantly different expression between the wild-type strain and the rpoN mutant.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Transcriptome analysis
DNA macroarrays containing 99 % (2816 ORFs) of the 2853 predicted ORFs of the L. monocytogenes EGDe genome (Milohanic et al., 2003) were used to study the transcriptional profiles of the wild-type EGDe strain and an rpoN mutant. For a first statistical analysis of the macroarray results using the SAM program (Tusher et al., 2001), we chose a minimum fold change of two as a selection criterion. This analysis identified 240 genes (23 false significant) whose expression was significantly different. The expression of all these genes should actually be considered as potentially regulated in the rpoN mutant. However, to concentrate on the more significantly affected genes, we decided to select only genes with the highest differences in expression by choosing a minimum fold change of three as a selection criterion. Seventy-eight genes (1 false significant) fulfilled this criterion when using the SAM program. Twenty of the 77 genes were repressed, whereas 57 were induced in the rpoN mutant compared to the wild-type strain. These genes belonged mainly to three different functional categories: carbohydrate metabolism-related genes (16 genes, Table 1), amino acid metabolism-related genes (10 genes, Table 2) and genes encoding other functions (51 genes, Table 3).


View this table:
[in this window]
[in a new window]
 
Table 1. Carbohydrate metabolism-related genes with at least threefold differential expression in the {sigma}54 mutant

 

View this table:
[in this window]
[in a new window]
 
Table 2. Amino acid metabolism-related genes with at least threefold differential expression in the {sigma}54 mutant

 

View this table:
[in this window]
[in a new window]
 
Table 3. Genes encoding other functions with at least threefold differential expression in the {sigma}54 mutant

 
Global analysis of protein content
In parallel to the transcriptome analysis, we compared the protein content of the wild-type L. monocytogenes EGDe strain and the rpoN mutant. Total extracts of proteins were subjected to 2D gel electrophoresis (first dimension IEF 3–6 or 3–10 and second dimension 12·5 % SDS-PAGE). As no difference in protein content between the two strains was observed above pH 6, we decided to focus on the pH 3–6 range. The comparison of the proteomic profiles of the wild-type strain and the rpoN mutant, and the analysis by MS of spots with significantly different intensities, led to the identification of nine proteins whose expression was modulated. Seven of these proteins were induced while two were repressed in the rpoN mutant compared to the wild-type (Fig. 1 and Table 4). Most of these identified proteins were related to carbohydrate metabolism, and particularly to pyruvate metabolism (Table 4).



View larger version (65K):
[in this window]
[in a new window]
 
Fig. 1. Differential protein synthesis patterns of the rpoN mutant of L. monocytogenes (a) and the wild-type EGDe strain (b). 2D gels were stained using the silver stain method. White arrows indicate protein spots differentially expressed between the two strains.

 

View this table:
[in this window]
[in a new window]
 
Table 4. Proteins identified by 2D electrophoresis

 
Carbohydrate metabolism-related genes
Many regulated genes or proteins, including those displaying the highest modification in their expression, were involved in carbohydrate metabolism. In this category, we identified five genes as being down-regulated and 10 genes as being up-regulated (Table 1) in the rpoN mutant as compared to the wild-type strain.

The most repressed gene was mptD (ratio 0·06). mptD is part of a large locus (lmo0096 to lmo0104), all genes of which were down-regulated in the rpoN mutant. mptD is organized in an operon with mptC, whose expression was also significantly decreased (ratio 0·26), and mptA. mptA was not selected by the SAM analysis, although its expression ratio was 0·17, due to a too high standard deviation. This operon was previously described to be {sigma}54-dependent (Dalet et al., 2001). In addition, the mptACD operon is preceded by a –24/–12 consensus promoter. At the protein level, MptA completely disappeared in the rpoN mutant, as shown by 2D electrophoresis (Fig. 1). MptC and MptD were probably not present in the gel, due to their localization in the cytoplasmic membrane and extraction of integral membrane proteins is known to be poorly achieved with classical procedures. Therefore, a change in expression was not detected by this method.

The lmo0099 and lmo0102 genes, located downstream from mptACD, were also repressed up to threefold. In addition, lmo0100, lmo0101, lmo0103 and lmo0104 all displayed an expression ratio of about 0·5. Another strongly repressed gene was lmo0520 (ratio 0·09, Table 1). It encodes a protein similar to transcriptional regulators of the NagC/XylR families as it shows 19 % identity with the xylose repressor, XylR, of B. subtilis (Kreuzer et al., 1989) and 23 % identity with the N-acetylglucosamine repressor of E. coli, NagC, which has been shown to be also an activator (Plumbridge, 2001). lmo0519, localized just upstream and divergent from lmo0520, was also highly repressed (ratio 0·22). The lmo0519 product is similar to multidrug resistance proteins and sugar transporters. Additional evidence for the important impact of {sigma}54 on carbon metabolism was the observation of the up-regulation of many genes or proteins related to this part of the metabolism (Table 1 and Table 4). lmo0027 was the most induced gene (ratio 16·3, Table 1). It encodes a putative {beta}-glucoside PTS permease, EIIbgl, as it shows high identity with a {beta}-glucoside permease of related bacterial species. lmo0319 (ratio 4·4) is likely to encode a phospho-{beta}-glucosidase. Interestingly, the transcription of these two genes (lmo0027 and lmo0319) was previously shown to be increased in spontaneous mutants resistant to class IIa bacteriocins as well as in mutants of the above-described mptACD operon (Gravesen et al., 2000, 2002). lmo0517 and lmo1871 encode proteins similar to phosphoglycerate mutase and phosphoglucomutase, respectively. The former is involved in glycolysis and the latter catalyses the isomerization reaction between glucose 1-phosphate and glucose 6-phosphate.

The pflA and pflB products are similar to pyruvate formate lyases, which convert pyruvate to formate and acetyl coenzyme A under fermentative conditions, and PflC is similar to pyruvate formate lyase-activating enzymes. The three genes are organized in two putative transcriptional units, the pflA and pflBC operons. glpD, glpF and lmo1538, which were induced in the rpoN mutant, belong to three different operons encoding proteins involved in glycerol metabolism. GlpD is similar (56 % identity) to the glycerol-3-phosphate dehydrogenase of B. subtilis, GlpF shows 58 % identity with glycerol uptake facilitators and Lmo1538 shows 72 % identity with glycerol kinases. Three proteins, identified from 2D gel electrophoresis, are similar to pyruvate dehydrogenase subunits (Table 4); one corresponds to the B subunit and the two others correspond to isoforms of the D subunit, probably related to different states of phosphorylation, which could explain the variation in their pI. The PdhABCD complex is involved in the transformation of pyruvate to acetyl-coenzyme A, a key component providing an energy source and a metabolite precursor. Similar to what is found in B. subtilis, the enzymic complex of pyruvate dehydrogenase is organized in a single transcriptional unit of four genes (pdhABCD). The expression level of these four genes was also two- to threefold-induced, as revealed by macroarrays. Therefore, the increase in the amount of the PdhB and PdhD proteins might be partly due to enhanced transcription of the operon. Finally, the L-lactate dehydrogenase, Ldh and the Lmo1579 protein, which is similar to alanine dehydrogenases, were also induced in the rpoN mutant, as shown by 2D electrophoresis (Table 4). However, differences in their expression were not observed at the RNA level (ratios 1·06 and 1·51, respectively).

Amino acid metabolism-related genes
The second largest group of genes affected by the lack of {sigma}54 expression was related to amino acid metabolism, mainly encoding transporters.

Five repressed genes in the rpoN mutant were similar to oligopeptide or amino acid transporters (lmo0136, lmo0137, lmo0152, lmo0645 and arpJ) (Table 2). lmo0136 and lmo0137 are predicted to belong to the same operon. The corresponding proteins are similar to the B and C membrane subunits of oligopeptide ABC permeases. Lmo0152 is highly similar to OppA, the cytoplasmic subunit A. lmo0645 forms a putative operon with another down-regulated gene, lmo0646. Lmo0646 displays about 33 % identity with proteins of the glyoxalase family and is predicted to be a ring cleavage extradiol dioxygenase. arpJ encodes an amino acid ABC transporter of the arginine family, previously described to be preferentially expressed by L. monocytogenes within mammalian cells (Klarsfeld et al., 1994).

Several amino acid metabolism-related genes were induced in the rpoN mutant (Table 2). lmo2569 encodes a protein predicted to be a dipeptide-binding lipoprotein. lmo2114 and lmo2115 likely encode subunits of an amino acid ABC transporter, the ATP-binding subunit and the permease subunit, respectively.

Genes encoding other functions
The remaining genes or proteins, which were differentially expressed in the rpoN mutant, are involved in various pathways and functions (Table 3).

lmo2829 was highly repressed in the rpoN mutant as shown at both the RNA and the protein level (Table 3 and Table 4). The lmo2829 product is similar (54 %) to the Frm2 protein of Saccharomyces cerevisiae. Frm2 was predicted to be an oxidoreductase related to the nitroreductase family and has been described as being involved in the fatty acid signalling pathway (McHale et al., 1996).

Among the genes which are induced, an entire locus of 14 genes was found to be highly expressed in the rpoN mutant (ratios 2·6–15·3) (Table 3). The first four genes, lmaD, lmaC, lmaB and lmaA, belong to the same putative operon. lmaDCBA encodes antigens unique to pathogenic Listeria and the lack of one of these genes caused virulence attenuation in an animal model (Schaferkordt & Chakraborty, 1997). The cluster of ten genes (lmo0119 to lmo0129, except lmo0121), localized just downstream of the lmaDCBA operon, was also clearly induced. These genes encode unknown or bacteriophage-related proteins. The dlt operon, comprising a putative transcriptional unit of four genes (dltABCD), was up-regulated in the rpoN mutant as compared to the wild-type. dltA and dltC were more than threefold-induced (Table 3) while dltB and dltD were twofold-induced in the rpoN mutant. These proteins are involved in the incorporation of D-alanine into the teichoic acids of Gram-positive bacteria. A relationship between the expression of the dlt operon and sugars transported by the PTS was previously described in Streptococcus mutans (Spatafora et al., 1999). The phosphopentomutase (Drm) and the purine nucleoside phosphorylase (Pnp) proteins were induced in the rpoN mutant, as shown by 2D gel electrophoresis (Fig. 1 and Table 4). In addition, macroarray analysis showed that the corresponding genes, which belong to the same operon, were 2·16- and 2·5-fold more expressed in the rpoN mutant, respectively. They are involved in salvage of purine nucleosides from external environments or arising from the intracellular breakdown of nucleotides. The Pnp enzyme catalyses the cleavage of purine nucleosides to the free base plus deoxyribose 1-phosphate. The latter is isomerized to 5-phosphate by the Drm enzyme.

In silico analysis of the {sigma}54 regulon
To identify putative {sigma}54-dependent genes, the intergenic regions of the complete genome sequence of L. monocytogenes EGDe were searched for the presence of the consensus –24/–12 consensus promoter sequence defined previously (TGGCA<6-6>TTGCW) (Barrios et al., 1999). We identified six putative promoters with perfect match to this –24/–12 consensus sequence (Table 5). Four of these six putative {sigma}54-regulated genes belong to the PTS family: lmo0096, lmo1720, lmo2683 and lmo2708. lmo0096 (mptA) has been described as the first gene of the mpt operon which is controlled by the ManR activator (Dalet et al., 2001). lmo1720 (lpoB) has been described as belonging to the lpo operon and to be controlled by the LacR activator in another strain, L. monocytogenes L028 (Dalet et al., 2003). The two other putatively {sigma}54-regulated genes (lmo2172 and lmo0105) encode proteins similar to propionate CoA transferase and chitinase B, respectively.


View this table:
[in this window]
[in a new window]
 
Table 5. Genes with totally conserved {sigma}54 promoters

 
It is known that the central domain is highly conserved in all {sigma}54-dependent activators. Thus it was used to search the L. monocytogenes genome sequence in order to identify putative activators. We identified three: Lmo2173 and two that were described previously, ManR (Lmo0785) (Dalet et al., 2001) and LacR (Lmo1721) (Dalet et al., 2003). Lmo2173 is a 455 aa protein whose central and C-terminal parts (position 150–455) are highly similar to the NifA/NtrC families of {sigma}54-dependent activators. lmo2172 is located just downstream of the gene encoding the Lmo2173 activator, suggesting that it is actually controlled by this activator. The two other activators, ManR and LacR, are similar to LevR of B. subtilis (31 and 38 % identity, respectively) that control the expression of a PTS permease.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Although {sigma}54 is a global regulator that has been studied in depth in various bacteria, until now an analysis of gene or protein expression of an rpoN mutant at the genome level has not been reported. To determine the impact of rpoN on gene and protein expression, we undertook a transcriptome and proteome analysis of the wild-type L. monocytogenes EGDe strain and its rpoN mutant derivative. The number of proteins identified as modulated is smaller than the number of genes shown to have altered expression, though we observed an excellent agreement between the two approaches.

An in silico analysis of the complete genome sequence of L. monocytogenes EGDe (Glaser et al., 2001) was undertaken, with the aim to identify putative {sigma}54 promoters and possible {sigma}54 activators. A conserved {sigma}54 promoter was identified upstream of six genes (Table 5). Among them, only expression of mptA was shown to be affected in the rpoN mutant, establishing that, under our experimental conditions, only mpt is directly controlled by {sigma}54. Interestingly, the five remaining genes, showed very low expression in the EGDe strain (data not shown), suggesting that repression of these genes may not have been perceptible in the rpoN mutant. Probably, these five genes were not or were poorly expressed because their cognate activator was not active. Indeed, it is known that {sigma}54-dependent activators become functional under certain conditions only, e.g. LevR is activated in the presence of fructose in B. subtilis (Martin et al., 1989). These conditions are unknown so far in L. monocytogenes and might be different for each activator. In our experiment, only the ManR activator might be active, allowing expression of mptACD. Therefore, the rpoN mutation seems to lead mainly to indirect modifications.

The most striking result of the transcriptome and proteome analyses is the important modification in the expression of carbohydrate metabolism genes. The regulation of carbohydrate metabolism is well described in B. subtilis (Deutscher et al., 2002) and is likely to be similar in other Gram-positive bacteria such as L. monocytogenes (Behari & Youngman, 1998). Briefly, it occurs from the control of catabolic operons by transcriptional activators, repressors, antiterminators and carbon catabolite repression (CCR). The latter is a mechanism whereby the expression of many genes is repressed in the presence of a readily metabolizable carbon source such as glucose. CCR is mediated by the CcpA transcriptional regulator and its co-repressor, HPr(ser-P), a phosphorylated intermediate of the HPr PTS component. This protein complex targets specific regulating sequences known as catabolite responsive elements (CREs) (Bruckner & Titgemeyer, 2002; Hueck & Hillen, 1995; Moreno et al., 2001; Titgemeyer & Hillen, 2002).

In our experiments, the highest induced gene, lmo0027, a {beta}-glucoside PTS enzyme, was previously described to be induced in spontaneous mutants of L. monocytogenes that lack mptACD expression (Dalet et al., 2001; Gravesen et al., 2002). Thus, the induction of lmo0027 is likely to be a secondary effect of the rpoN mutation and might be mediated via a relief of CCR since a CRE was identified upstream of lmo0027. Three genes involved in glycerol metabolism, glpD, glpF and lmo1538 (a glpK homologue), were up-regulated. Their paralogues in B. subtilis are controlled by both CCR and the GlpP antiterminator (Darbon et al., 2002). Among the carbohydrate metabolism-related genes whose expression is induced in the rpoN mutant, putative CRE boxes were found upstream of pflA, pdhABCD, lmo0517 and lmo0027. In addition, we noticed that the transcription of ccpA in L. monocytogenes was induced in the rpoN mutant (ratio 2·3). Interestingly, the ccpA gene has been already described in L. monocytogenes to mediate CCR and a conserved CRE is located upstream of this gene (Behari & Youngman, 1998), indicating that CcpA might control its own expression. Based on our results and on the mechanisms described in B. subtilis (Deutscher et al., 2002), we propose that the drop in mptACD expression might result in an increase in P-His-HPr, leading to both a phosphorylation of different enzymes, such as GlpK or PTS components, and a decrease in P-Ser-HPr formation responsible for relief of CCR. The regulation of the expression of other PTS systems by a mannose PTS has also previously been described as part of CCR in Streptococcus salivarius and Lactobacillus pentosus (Bourassa & Vadeboncoeur, 1992; Chaillou et al., 2001).

Remarkably, many enzymes that use pyruvate as a substrate, i.e. pyruvate dehydrogenase, alanine dehydrogenase, lactate dehydrogenase and pyruvate formate lyase, were induced at either the RNA and/or the protein level. In the first step of the PTS phosphorylation cascade, phosphoenolpyruvate is transformed into pyruvate. Thus, we suggest that lack of {sigma}54 directly modifies the PTS pathway, which in turn affects the pyruvate to PEP ratio. Conversely, it has been shown that this ratio is a major determinant of the phosphorylation state of PTS proteins in E. coli (Hogema et al., 1998). This modification may finally influence the expression of pyruvate metabolism-related enzymes. For example, pyruvate is a putative coeffector for the expression of the pdh operon in E. coli (Quail et al., 1994). In addition, 2D electrophoresis identified two isoforms of the PdhD subunit of pyruvate dehydrogenase with a slight difference in their pI. This might be due to a different level of phosphorylation of the protein, suggesting that the activity of this enzyme may be modulated post-translationally. It is conceivable that PTS components are involved in the modulation of phosphorylation.

As L. monocytogenes is a pathogen, we wanted to know if the rpoN mutation also had an impact on virulence. Therefore, the virulence of the rpoN mutant was assessed in a mouse model. However, we did not observe any significant differences in the LD50, after intravenous injection, between the wild-type EGDe strain and the rpoN mutant (data not shown).

In conclusion, this first global analysis of the effect of a disruption of the rpoN gene will be a valuable basis for a better understanding of the physiological role of {sigma}54, which has to be tested in different conditions. It will now pave the way for advanced biochemical and physiological experiments to gain comprehensive knowledge of its regulatory functions.


   ACKNOWLEDGEMENTS
 
We are grateful to Anne Lise Gravesen for critical reading of the manuscript and Olivier Dussurget for the virulence tests in the animal model. This work was supported by an ‘Action Concertée Incitative’ of the French Ministry of research, ‘Génomique fonctionelle de Listeria monocytogenes: interactions avec l'hôte et survie dans l'environnement’, ‘Réseaux des génopoles’, GIP Aventis and European Commission contract QLG2-CT-1999-00932 (REALIZ).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ali, N. O., Bignon, J., Rapoport, G. & Débarbouillé, M. (2001). Regulation of the acetoin catabolic pathway is controlled by sigma L in Bacillus subtilis. J Bacteriol 183, 2497–2504.[Abstract/Free Full Text]

Barrios, H., Valderrama, B. & Morett, E. (1999). Compilation and analysis of sigma(54)-dependent promoter sequences. Nucleic Acids Res 27, 4305–4313.[Abstract/Free Full Text]

Beckering, C. L., Steil, L., Weber, M. H., Volker, U. & Marahiel, M. A. (2002). Genomewide transcriptional analysis of the cold shock response in Bacillus subtilis. J Bacteriol 184, 6395–6402.[Abstract/Free Full Text]

Behari, J. & Youngman, P. (1998). A homolog of CcpA mediates catabolite control in Listeria monocytogenes but not carbon source regulation of virulence genes. J Bacteriol 180, 6316–6324.[Abstract/Free Full Text]

Bourassa, S. & Vadeboncoeur, C. (1992). Expression of an inducible enzyme II fructose and activation of a cryptic enzyme II glucose in glucose-grown cells of spontaneous mutants of Streptococcus salivarius lacking the low-molecular-mass form of IIIman, a component of the phosphoenolpyruvate : mannose phosphotransferase system. J Gen Microbiol 138, 769–777.[Medline]

Bruckner, R. & Titgemeyer, F. (2002). Carbon catabolite repression in bacteria: choice of the carbon source and autoregulatory limitation of sugar utilization. FEMS Microbiol Lett 209, 141–148.[CrossRef][Medline]

Calogero, S., Gardan, R., Glaser, P., Schweizer, J., Rapoport, G. & Débarbouillé, M. (1994). RocR, a novel regulatory protein controlling arginine utilization in Bacillus subtilis, belongs to the NtrC/NifA family of transcriptional activators. J Bacteriol 176, 1234–1241.[Abstract]

Chaillou, S., Postma, P. W. & Pouwels, P. H. (2001). Contribution of the phosphoenolpyruvate : mannose phosphotransferase system to carbon catabolite repression in Lactobacillus pentosus. Microbiology 147, 671–679.[Abstract/Free Full Text]

Dalet, K., Cenatiempo, Y., Cossart, P. & Héchard, Y. (2001). A sigma(54)-dependent PTS permease of the mannose family is responsible for sensitivity of Listeria monocytogenes to mesentericin Y105. Microbiology 147, 3263–3269.[Abstract/Free Full Text]

Dalet, K., Arous, S., Cenatiempo, Y. & Héchard, Y. (2003). Characterization of a unique {sigma}54-dependent PTS operon of the lactose family in Listeria monocytogenes. Biochimie 85, 633–638.[CrossRef][Medline]

Darbon, E., Servant, P., Poncet, S. & Deutscher, J. (2002). Antitermination by GlpP, catabolite repression via CcpA and inducer exclusion triggered by P~GlpK dephosphorylation control Bacillus subtilis glpFK expression. Mol Microbiol 43, 1039–1052.[CrossRef][Medline]

Débarbouillé, M., Martin-Verstraete, I., Klier, A. & Rapoport, G. (1991). The transcriptional regulator LevR of Bacillus subtilis has domains homologous to both sigma 54- and phosphotransferase system-dependent regulators. Proc Natl Acad Sci U S A 88, 2212–2216.[Abstract]

Débarbouillé, M., Gardan, R., Arnaud, M. & Rapoport, G. (1999). Role of BkdR, a transcriptional activator of the sigL-dependent isoleucine and valine degradation pathway in Bacillus subtilis. J Bacteriol 181, 2059–2066.[Abstract/Free Full Text]

Deutscher, J., Galinier, A. & Martin-Verstraete, I. (2002). Carbohydrate uptake and metabolism. In Bacillus subtilis and its Closest Relatives: from Genes to Cells. Edited by A. L. Sonenshein, J. A. Hoch & R. Losick. Washington, DC: American Society for Microbiology.

Duché, O., Tremoulet, F., Glaser, P. & Labadie, J. (2002). Salt stress proteins induced in Listeria monocytogenes. Appl Environ Microbiol 68, 1491–1498.[Abstract/Free Full Text]

Gardan, R., Rapoport, G. & Débarbouillé, M. (1995). Expression of the rocDEF operon involved in arginine catabolism in Bacillus subtilis. J Mol Biol 249, 843–856.[CrossRef][Medline]

Glaser, P., Frangeul, L., Buchrieser, C. & 52 other authors (2001). Comparative genomics of Listeria species. Science 294, 849–852.[Abstract/Free Full Text]

Gravesen, A., Warthoe, P., Knochel, S. & Thirstrup, K. (2000). Restriction fragment differential display of pediocin-resistant Listeria monocytogenes 412 mutants shows consistent overexpression of a putative beta-glucoside-specific PTS system. Microbiology 146, 1381–1389.[Abstract/Free Full Text]

Gravesen, A., Ramnath, M., Rechinger, K. B., Andersen, N., Jansch, L., Héchard, Y., Hastings, J. W. & Knochel, S. (2002). High-level resistance to class IIa bacteriocins is associated with one general mechanism in Listeria monocytogenes. Microbiology 148, 2361–2369.[Abstract/Free Full Text]

Hogema, B. M., Arents, J. C., Bader, R., Eijkemans, K., Yoshida, H., Takahashi, H., Aiba, H. & Postma, P. W. (1998). Inducer exclusion in Escherichia coli by non-PTS substrates: the role of the PEP to pyruvate ratio in determining the phosphorylation state of enzyme IIAGlc. Mol Microbiol 30, 487–498.[CrossRef][Medline]

Huang, M., Oppermann-Sanio, F. B. & Steinbuchel, A. (1999). Biochemical and molecular characterization of the Bacillus subtilis acetoin catabolic pathway. J Bacteriol 181, 3837–3841.[Abstract/Free Full Text]

Hueck, C. J. & Hillen, W. (1995). Catabolite repression in Bacillus subtilis: a global regulatory mechanism for the gram-positive bacteria? Mol Microbiol 15, 395–401.[Medline]

Klarsfeld, A. D., Goossens, P. L. & Cossart, P. (1994). Five Listeria monocytogenes genes preferentially expressed in infected mammalian cells: plcA, purH, purD, pyrE and an arginine ABC transporter gene, arpJ. Mol Microbiol 13, 585–597.[Medline]

Kreuzer, P., Gartner, D., Allmansberger, R. & Hillen, W. (1989). Identification and sequence analysis of the Bacillus subtilis W23 xylR gene and xyl operator. J Bacteriol 171, 3840–3845.[Medline]

Martin, I., Débarbouillé, M., Klier, A. & Rapoport, G. (1989). Induction and metabolite regulation of levanase synthesis in Bacillus subtilis. J Bacteriol 171, 1885–1892.[Medline]

McHale, M. W., Kroening, K. D. & Bernlohr, D. A. (1996). Identification of a class of Saccharomyces cerevisiae mutants defective in fatty acid repression of gene transcription and analysis of the frm2 gene. Yeast 12, 319–331.[CrossRef][Medline]

Milohanic, E., Glaser, P., Coppee, J. Y., Frangeul, L., Vega, Y., Vazquez-Boland, J. A., Kunst, F., Cossart, P. & Buchrieser, C. (2003). Transcriptome analysis of Listeria monocytogenes identifies three groups of genes differently regulated by PrfA. Mol Microbiol 47, 1613–1625.[CrossRef][Medline]

Moreno, M. S., Schneider, B. L., Maile, R. R., Weyler, W. & Saier, M. H. (2001). Catabolite repression mediated by the CcpA protein in Bacillus subtilis: novel modes of regulation revealed by whole-genome analyses. Mol Microbiol 39, 1366–1381.[CrossRef][Medline]

Morett, E. & Buck, M. (1989). In vivo studies on the interaction of RNA polymerase-sigma 54 with the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters. The role of NifA in the formation of an open promoter complex. J Mol Biol 210, 65–77.[Medline]

O'Farrell, (1975). High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250, 4007–4021.[Abstract]

Plumbridge, J. (2001). Regulation of PTS gene expression by the homologous transcriptional regulators, Mlc and NagC, in Escherichia coli (or how two similar repressors can behave differently). J Mol Microbiol Biotechnol 3, 371–380.[Medline]

Quail, M. A., Haydon, D. J. & Guest, J. R. (1994). The pdhR-aceEF-lpd operon of Escherichia coli expresses the pyruvate dehydrogenase complex. Mol Microbiol 12, 95–104.[Medline]

Rabilloud, T. (1992). A comparison between low background silver diammine and silver nitrate protein stains. Electrophoresis 13, 429–439.[Medline]

Robichon, D., Gouin, E., Débarbouillé, M., Cossart, P., Cenatiempo, Y. & Héchard, Y. (1997). The rpoN (sigma54) gene from Listeria monocytogenes is involved in resistance to mesentericin Y105, an antibacterial peptide from Leuconostoc mesenteroides. J Bacteriol 179, 7591–7594.[Abstract]

Schaferkordt, S. & Chakraborty, T. (1997). Identification, cloning, and characterization of the ima operon, whose gene products are unique to Listeria monocytogenes. J Bacteriol 179, 2707–2716.[Abstract]

Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. (1996). Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68, 850–858.[CrossRef][Medline]

Shingler, V. (1996). Signal sensing by sigma 54-dependent regulators: derepression as a control mechanism. Mol Microbiol 19, 409–416.[Medline]

Spatafora, G. A., Sheets, M., June, R., Luyimbazi, D., Howard, K., Hulbert, R., Barnard, D., El Janne, M. & Hudson, M. C. (1999). Regulated expression of the Streptococcus mutans dlt genes correlates with intracellular polysaccharide accumulation. J Bacteriol 181, 2363–2372.[Abstract/Free Full Text]

Studholme, D. J. & Buck, M. (2000). The biology of enhancer-dependent transcriptional regulation in bacteria: insights from genome sequences. FEMS Microbiol Lett 186, 1–9.[CrossRef][Medline]

Stulke, J., Martin-Verstraete, I., Charrier, V., Klier, A., Deutscher, J. & Rapoport, G. (1995). The HPr protein of the phosphotransferase system links induction and catabolite repression of the Bacillus subtilis levanase operon. J Bacteriol 177, 6928–6936.[Abstract]

Titgemeyer, F. & Hillen, W. (2002). Global control of sugar metabolism: a gram-positive solution. Antonie van Leeuwenhoek 82, 59–71.[CrossRef][Medline]

Tusher, V. G., Tibshirani, R. & Chu, G. (2001). Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98, 5116–5121.[Abstract/Free Full Text]

Received 24 October 2003; revised 22 January 2004; accepted 23 January 2004.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Arous, S.
Articles by Héchard, Y.
Articles citing this Article
PubMed
PubMed Citation
Articles by Arous, S.
Articles by Héchard, Y.
Agricola
Articles by Arous, S.
Articles by Héchard, Y.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2004 Society for General Microbiology.