| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Microbiology, National University of Ireland, Galway, University Road, Galway, Ireland
2 Produce Quality and Safety Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Beltsville, MD 20705-2350, USA
Correspondence
Conor P. O'Byrne
conor.obyrne{at}nuigalway.ie
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Tuite et al., 2005). Growth inhibition by Hcy appears to be caused by a perturbation of branched-chain amino acid (BCAA) biosynthesis that gives rise to a partial starvation for isoleucine (Tuite et al., 2005). Supplementation of the growth medium with isoleucine fully reverses the inhibitory effects of 0.5 mM Hcy. Hcy inhibits threonine deaminase, the enzyme that catalyses the first committed step of isoleucine biosynthesis, and this inhibition is the likely cause of the isoleucine starvation and growth inhibition that occur in Hcy-treated E. coli cells (Tuite et al., 2005).
High levels of serum Hcy are associated with serious diseases in humans, including cardiovascular disease (Anderson et al., 2000; Vollset et al., 2000), birth defects and pregnancy complications (van der Put et al., 2001; Vollset et al., 2000), as well as neurodegenerative diseases such as Alzheimer's disease (Seshadri et al., 2002). More recently it has been shown that there is a causal link between elevated serum-Hcy levels and stroke (Casas et al., 2005). The underlying mechanisms linking high-serum Hcy levels and disease in humans remain to be elucidated. However, a number of different theories have been proposed and these have been reviewed recently (Jakubowski, 2004). Hcy can induce cellular damage through modification of cellular proteins. Proteins can become homocysteinylated by the formation of an amide bond between Hcy and the 
-amino group of lysine residues (Jakubowski, 1999, 2002). Proteins modified in this way are known to elicit an immune response and this is thought to contribute to the pathology of disease (Jakubowski, 2004). In addition, S-nitrosylated Hcy, a methionine analogue that is formed when Hcy reacts with nitric oxide, can be misincorporated into proteins during translation (Jakubowski, 2000). An alternative model accounting for Hcy toxicity suggests that Hcy might trigger oxidative stress in human cells (Loscalzo, 1996; Lynch & Frei, 1997; Starkebaum & Harlan, 1986; Upchurch et al., 1997). In E. coli, cysteine, an amino acid that is closely related to Hcy, is known to trigger oxidative stress by enhancing the Fenton reaction (Berglin et al., 1982; Park & Imlay, 2003). However, at present there is no evidence that Hcy induces oxidative stress in E. coli.
In E. coli Hcy is produced by two routes: the majority of the Hcy in the cell results from the cleavage of cystathionine by cystathionine 
-lyase; a smaller, undefined amount is generated by the action of LuxS on S-ribosylhomocysteine during the production of the intracellular signalling molecule autoinducer-2 (AI-2) (Greene, 1996; Winzer et al., 2002). Hcy is converted to methionine in a methylation reaction that is catalysed by either of two methionine synthases, MetE or MetH. The methyl donor in this reaction is 5-methyltetrahydrofolate (CH3-THF). The synthesis of CH3-THF requires two enzymes, serine hydroxymethyltransferase (GlyA) and methylenetetrahydrofolate reductase (MetF), whose function is to transfer a single carbon atom from serine to tetrahydrofolate to produce methylene-tetrahydrofolate (methylene-THF) and then to reduce methylene-THF to CH3-THF (Greene, 1996). The same biochemical steps are employed in human cells to convert Hcy to methionine. Indeed MetH, GlyA and MetF all share significant homology with their human counterparts. The similarities in the metabolism of Hcy between bacteria and humans have prompted us to use E. coli as a simple model system for investigating the toxic effects of this thiol-containing amino acid.
In the present study we sought to further elucidate the molecular basis for the inhibitory effects of Hcy on the growth of E. coli cells by using DNA macroarray technology to investigate the global effects of Hcy on transcription. We found that two principal classes of genes exhibited reproducible changes in expression in the presence of Hcy: those involved in translation and those involved in amino acid biosynthesis. Genes involved in the oxidative stress response were found to be repressed. We also found that cspA, the gene encoding the major cold-shock protein CspA, was induced strongly during growth in the presence of Hcy and this induction was prevented when isoleucine was present in the growth medium. Taken together with our previous work (Tuite et al., 2005), these data suggest that in E. coli growth in the presence of Hcy results in perturbation of amino acid biosynthesis and translation.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). All growth experiments were carried out at 37 °C with aeration in a minimal medium (MM) that was adjusted to pH 6.0. MM contained 20 mM citric acid, 56 mM Na2HPO4, 5 mM K2HPO4, 0.4 mM MgSO4.7H2O, 7.6 mM (NH4)2SO4, 6 µM ammonium ferrous sulphate and 3 µM thiamine. Cells were grown overnight in 25 ml MM supplemented with limiting glucose (0.04 %, w/v) in a 250 ml flask at 37 °C with vigorous shaking. The next day glucose (0.4 %, w/v) was added to this culture, and growth was continued to an OD600 of 0.8. This culture was then used to inoculate 25 ml MM (0.4 %, w/v, glucose) to an OD600 of 0.05. Cell growth was measured spectrophotometrically (Jenway 6305 spectrophotometer) by measuring the OD600 of 1 ml samples at regular intervals during growth. Hcy was added to growth media at a concentration of 0.5 mM when required. Unless specified, all chemicals used in this study were supplied by Sigma and BDH.
RNA preparation for gene macroarray analysis.
Cells were grown in 25 ml MM (in 250 ml flasks) to an OD600 of 0.2, before the addition of Hcy (0.5 mM) to half of the samples. The flasks were returned to the incubator for 1 h, before 20 ml cells was removed and added to a sterile centrifuge tube containing 3.3 ml RNA stabilization reagent RSR-3 [95 % ethanol, 5 % phenol (v/v)] (Bhagwat et al., 2003). Cells were then harvested by centrifugation (15 min, 4 °C, 8200 g), the supernatant decanted and the pellets stored at –20 °C until required. RNA was prepared using a modified version of the protocol described in the Panorama E. coli Gene Arrays Manual (Sigma-Genosys). The cell pellet was resuspended in 250 µl resuspension buffer [0.3 M sucrose, 10 mM sodium acetate (pH 4.2)] and 37.5 µl 0.5 M EDTA. Resuspended cells were then transferred to a fresh microcentrifuge tube before the addition of 375 µl lysis buffer [2 % SDS, 10 mM sodium acetate (pH 4.2)], mixed well by vortexing then incubated on ice for 3 min. RNA was then prepared by phenol/chloroform extraction. After the first phenol step, the RNA/DNA mix was ethanol precipitated and the nucleic acid pellet was resuspended in 50 µl TE buffer containing 5 U RNase-free DNase I (Roche), and incubated at room temperature for 30 min. This was followed by a further phenol extraction step, then a phenol/chloroform/isoamyl alcohol (25 : 24 : 1) step and finally a chloroform/isoamyl alcohol (24 : 1) step. The RNA was then concentrated by ethanol precipitation, before being resuspended in 50 µl deionized H2O and stored at –70 °C until required.
Probe synthesis and hybridization of gene arrays.
Hybridization probes were generated using E. coli gene-specific primers covering the entire genome (Sigma-Genosys) and following the protocol provided by the manufacturer, which was suitable for achieving >50 % incorporation of the [
-33P]dCTP. Panorama E. coli gene arrays were obtained from Sigma-Genosys and prehybridized in 5 ml hybridization buffer [5x SSPE (1x SSPE is 0.18 M NaCl, 10 mM NaH2PO4 and 1 mM EDTA, pH 7.7), 2 % SDS, 1x Denhardt's reagent and 100 µg denatured, sonicated salmon sperm DNA ml–1]. After incubation for 1 h at 65 °C, the prehybridization solution was replaced with the denatured radioactively labelled cDNA probe. The probes were denatured in 3 ml hybridization buffer and incubated at 95 °C for 4 min immediately before use. After hybridization for 16 h at 65 °C in a hybridization incubator (Robbins Scientific), the filters were washed briefly three times at room temperature with 50 ml wash solution (0.5x SSPE, 0.2 % SDS) and then washed three times at 65 °C in 100 ml wash solution. The washed filters were blotted on Whatman 3MM paper for 5 min and then placed in a plastic wrap. The filters were exposed to a phosphorimager screen for 24–72 h and the screen was scanned at 50 µm resolution using a Fujix BAS1800-II phosphorimager.
Data acquisition and analysis.
The Fujix BAS image files were analysed using Array-Vison software running on a Microsoft Windows 2000 workstation. The signal intensity for each spot was determined using the integrated intensity function, which calculates the volume of each spot by summing the value of each pixel within the boundaries of the spot (minus the local background). The pixel density values were exported to a tab-delimited text file. Each open reading frame (ORF) on the array is represented by duplicate spots of 10 ng PCR product. To compare the signal intensities between filters, a relative intensity for each ORF was calculated by dividing the mean intensity for a given ORF by the total signal intensity on the filter and multiplying by 100. The total signal intensity on the filter was calculated by summing the intensities of all ORFs on the array (n=4290).
Two macroarray experiments were performed for each condition, using independently isolated RNA preparations. Thus the data were derived from a total of four macroarrays (two untreated controls and two plus Hcy). Genes that showed a mean expression ratio of twofold or greater (between treated and untreated) were short-listed for further analysis. Genes were eliminated from this list if the expression ratio of one replicate was less than ±1.5-fold. A statistical analysis of this subset of genes was performed using SigmaStat 3.0 (Systat Software). This subset of genes consisted of two subgroups; one up-regulated in response to Hcy and one down-regulated. The median expression ratios for each of these subgroups were compared to the median expression ratio for the remainder of the genes on the macroarray using a Kruskal–Wallis one-way analysis of variance (ANOVA) on ranks. In both cases the median expression ratios were significantly different with P
0.001.
RT-PCR.
Cells were grown as described above for gene macroarray analysis, and then 20 ml cells was transferred to a sterile centrifuge tube containing RSR-3 (see above), pre-incubated on ice. Cells were then harvested by centrifugation (15 min, 4 °C, 8200 g). RNA was prepared using the GenElute Mammalian Total RNA Miniprep kit (Sigma), following the manufacturer's instructions. The RNA was then treated to remove DNA contamination using the DNA-free kit (Ambion) following the manufacturer's protocol. cDNA was synthesized from 20 µl diluted RNA by using Expand reverse transcriptase with random primer p(dN)6 (both supplied by Roche). Aliquots (2 µl) of the resulting cDNA were subjected to 18, 24 and 30 cycles of PCR and run on agarose gels. Primers for the gene encoding polymerase A (polA) were used as controls to detect contaminating DNA and allow normalization of cDNA template concentration. Non-reverse-transcribed RNA was used as template for PCRs to ensure complete removal of genomic DNA. Specific primers for cspA, argF, argH, cysN and gatZ (Table 1) were used in conjunction with cDNA generated from E. coli Frag1 grown in the presence or absence of 0.5 mM Hcy. RNA and cDNA were prepared twice, from independent cultures, and the RT-PCRs for each set of primers were repeated at least twice from each cDNA preparation.
|
. The E. coli strain JM83 cspA : : cat (Mitta et al., 1997) was used as the donor. The P1vir phage was a gift from Charles Dorman (Trinity College, Dublin). Disruption of the cspA gene was confirmed by PCR. The resultant strain was designated COB229.
Measurement of -galactosidase activity.
-Galactosidase activity was measured as described by Miller (1972). Briefly, cells were grown in MM (see above) to the desired sampling point and the OD600 measured. Then 75 µl cells was aliquoted to a sterile centrifuge tube containing 675 µl Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4 and 50 mM -mercaptoethanol), 50 µl chloroform and 25 µl 0.1 % (w/v) SDS stock. The tube was mixed thoroughly and incubated at 28 °C for 5 min. Then 150 µl of a 4 mg ml–1 ONPG stock was added to the tube, and incubation continued until the contents turned yellow. The reaction was then stopped by the addition of 375 µl 1 M Na2CO3 and the time noted. Cell debris was removed by centrifugation at 19 800 g for 4 min, and 1 ml of supernatant was used to measure OD420. -Galactosidase activity, in Miller units, was then calculated using the following formula: Miller units=[OD420/(tx0.075xOD600)]x(1000/1), where t is the reaction time in minutes.
Cell extract preparation and measurement of catalase activity.
Crude cell extracts were prepared according to the method of Harris (1981) with some modifications. At an OD600
0.28, 200 ml cultures were harvested by centrifugation at 12 000 g for 10 min and washed once with MM. The resulting pellets were resuspended in 4 ml buffer containing 0.05 M Tris/HCl buffer (pH 7.6), 0.05 M MgCl2, 0.025 M KCl, 1 mM 2-mercaptoethanol and 5 % (v/v) glycerol. The cell suspension was disrupted by sonication using an MSE Soniprep 150 set at 
16 µm amplitude for six 30 s pulses, with a 30 s interval between pulses. Sonication was carried out on ice. The extracts were clarified by centrifugation at 14 000 g for 20 min, and the supernatant was stored at –80 °C. The amount of protein in samples was determined using the Bio-Rad RC DC kit, which is a version of the well-documented Lowry method, using BSA as a standard. Catalase activity measurements were subsequently carried out using the method of Beers & Sizer (1952). It is based on the principle that the breakdown of substrate, H2O2, which has an absorbance peak at 240 nm, can be followed spectrophotometrically. A 4 ml quartz cuvette was soaked overnight in concentrated HCl, then rinsed thoroughly with 100 % ethanol and allowed to air dry. Then 250 µl cell extract [2 mg (ml protein)–1] and 2.25 ml phosphate buffer [144 mM K2HPO4, 56 mM KH2PO4 (pH 7.2)] were added to the cuvette and used to zero the spectrophotometer at 240 nm. A 750 µl volume of substrate solution (a 10–2 dilution of a 30 %, v/v, H2O2 stock in phosphate buffer) was added and this was taken to be zero time point. Spectrophotometric readings were taken every 10 s for the first 400 s; after this time, gas bubbles accumulated within the cuvette, leading to erroneous measurements. The relative levels of catalase activity could be determined by plotting the decrease in absorbance versus time. The relative rates of catalase activity were then determined by calculating the slope of these lines.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Tuite et al., 2005), with 50 % inhibition occurring in the presence of 0.5 mM Hcy (Tuite et al., 2005). Hcy has been shown to cause a perturbation of isoleucine biosynthesis, through inhibition of threonine deaminase, the enzyme responsible for catalysing the first dedicated step in the biosynthesis of isoleucine (Tuite et al., 2005). In the present study we sought to investigate the effect of Hcy on gene expression in E. coli using gene macroarrays.
Four cultures were grown to early exponential phase (OD6000.2) in MM. Two of these were used as untreated controls and 0.5 mM Hcy was added to the other two. RNA was prepared from all four cultures 1 h after the addition of Hcy (See Methods). Four Panorama genome macroarrays (Sigma-Genosys) were probed with labelled cDNA prepared from these RNA extracts, as described in Methods. Genes exhibiting reproducible changes in expression greater than twofold, in the presence of Hcy, are presented in Table 2 grouped by function. A total of 68 genes fulfilled these criteria, showing a reproducible alteration in expression in the presence of 0.5 mM Hcy. This group comprised 38 genes that were up-regulated in response to Hcy and 30 genes whose expression decreased in the presence of Hcy. Using the Kruskal–Wallis one-way ANOVA, the median expression ratio for the 38 up-regulated genes was found to be significantly different from the median expression ratio for the remainder of the genes (n=4169), with P0.001. Likewise, the median expression ratio for the 30 down-regulated genes was also significantly different (P0.001) from the median expression ratio for the remainder of the genes (n=4169).
|
), was also induced. In the same operon as yfjA is trmD, a gene encoding a tRNA methyltransferase (Bystrom & Bjork, 1982), and this was also found to be induced in response to Hcy. These results suggest that translation may be perturbed in E. coli cells exposed to high levels of Hcy. These results are unlikely to arise simply because of the effect of Hcy on growth rate (50 % reduction in the presence of Hcy); a reduced growth rate would usually be expected to reduce, not increase, the expression of translation-related functions in the cell. This observation also gives us confidence that many of the changes in expression observed represent specific responses to the presence of Hcy rather than a general response to reduced growth rate per se. However, it is accepted that some of the changes in expression detected may simply arise as a consequence of reduced growth rate.
Amino acid metabolism is disrupted by Hcy
Several genes encoding proteins involved in amino acid metabolism were expressed at significantly altered levels in Hcy-treated cells. The most striking finding was the up-regulation of a large number of genes involved in arginine biosynthesis. The up-regulation of the argF and argH genes, which are located in separate operons, was confirmed using RT-PCR (Fig. 1). In addition, several genes encoding components of the artPIMQJ arginine transporter also showed some degree of up-regulation (>1.5-fold) (data not shown). Of the genes showing down-regulation in the presence of Hcy, the largest effects were observed in genes encoding enzymes involved in the biosynthesis of cysteine, isoleucine and methionine. In the case of cysteine biosynthesis, the largest changes were seen for the cysD, cysK, cysM and cysN genes (Table 2
). The down-regulation of cysN was confirmed by RT-PCR (Fig. 1). We also observed a degree of down-regulation (>1.5-fold) of many of the other genes required for the conversion of sulphate to cysteine, including down-regulation of the sulphate ABC transport system encoding genes (data not shown). Two isoleucine biosynthetic genes showed reduced expression (Table 2): ilvI, which encodes a subunit of the AHAS III enzyme responsible for the conversion of -ketobutyrate to acetohydrobutyrate (Umbarger, 1996), and ilvC, which encodes the enzyme required for the subsequent conversion of acetohydrobutyrate to dihydromethylvalerate (Umbarger, 1996). The livH gene, which encodes the permease subunit of the BCAA high-affinity transport system (Adams et al., 1990), also showed reduced expression. Two genes involved in methionine metabolism were also down-regulated: the first was the metE gene, which encodes the vitamin-B12-independent homocysteine transmethylase, responsible for the transfer of a methyl group from CH3-THF to Hcy to generate methionine (Greene, 1996); the second was the yaeC gene, encoding the binding protein for the MetD DL-methionine transporter (Merlin et al., 2002).
|
Repression of genes involved in carbon utilization
There was a down-regulation of many genes required for central metabolism, including six genes encoding enzymes involved in glycolysis and the TCA cycle (gltA, mdh, pgk, sucA, sucD and tpiA) (Table 2). In addition, a further five genes encoding enzymes for these pathways, gpmA, eno, icd, sucB and sucC, showed a degree of down-regulation (>1.7-fold; data not shown). Genes required for utilization of alternative carbon sources also exhibited down-regulation, in particular the galacticol utilization genes gatB, gatZ and gatC, which showed the largest degree of down-regulation of all the genes tested (Table 2). The repression of gatZ by 0.5 mM Hcy was confirmed using RT-PCR (Fig. 1).
The major cold-shock protein CspA is induced strongly by Hcy
The gene showing the largest degree of up-regulation (5.5-fold) in response to Hcy was cspA, which encodes the major cold-shock protein CspA. This result was surprising since increased levels of cspA mRNA are mainly thought to occur in E. coli when cells have been subjected to a cold shock (Goldstein et al., 1990). The function of CspA remains uncertain but it is thought to act as an RNA chaperone and may act to facilitate translation at low temperatures by preventing the formation (or destabilization) of secondary structures in mRNA (Phadtare et al., 2000). RT-PCR was used to confirm that cspA mRNA is present at elevated levels in Hcy-treated cells (Fig. 1). Although the RT-PCR data were qualitatively similar to the macroarray data, there appeared to be a difference in the relative induction levels of cspA and argH using these two experimental approaches. The argH gene appeared to give the largest induction using RT-PCR, whereas cspA gave the largest induction in the macroarray experiments. The reason for this difference is not clear but it may be due to handling differences in the preparation of cDNA for the two approaches.
Next we used a cspA : : lacZ translational fusion, carried on plasmid pMM016 (Bae et al., 1997), to measure the expression of cspA in response to Hcy. -Galactosidase activity was measured in control and Hcy-treated (0.5 mM) cultures of Frag1(pMM016) at suitable time intervals before and for 3 h after the addition of Hcy. The growth rate of the Hcy-treated culture was reduced by 31 % compared to the untreated control (Fig. 2a). -Galactosidase activity was found to drop during growth in the control cultures (Fig. 2a), consistent with a previous study showing a rapid drop in cspA mRNA as cell density increases (Brandi et al., 1999). In contrast, there was a marked increase in cspA expression in the Hcy-treated cultures; after 3 h expression was approximately fivefold higher in Hcy-treated cultures than in the control cultures (Fig. 2a). This increase in cspA expression was not simply due to a decrease in the growth rate, since cultures whose growth rates were reduced by the addition of 0.25 M NaCl (growth rate approximately 35 % slower than untreated controls) showed no increase in cspA expression (Fig. 2b).
|
). Since Hcy can be used by the cell to synthesize methionine, we tested whether the addition of this amino acid was sufficient to account for the observed induction of cspA expression. Methionine did give rise to a small increase in cspA expression (0.5-fold induction 3 h after addition) but this was not sufficient to account for the large induction observed in the presence of Hcy (Fig. 2c). High levels of cspA expression in the presence of Hcy suggested the possibility that growth under these conditions may require a functional CspA protein. To investigate this, an E. coli Frag1 strain harbouring a cspA : : cat disruption was generated by P1 transduction and designated COB229. When grown in the presence of a range of Hcy concentrations, COB229 exhibited the same pattern of growth inhibition as the wild-type parent (data not shown), suggesting that CspA is not essential for survival in the presence of Hcy.
Induction of cspA by Hcy does not require a functional CspA protein
In a mutant strain carrying a truncated allele of cspA the cspA : : lacZ translational fusion present on pMM016 is expressed at higher levels than in a wild-type background in response to cold shock (Bae et al., 1997). This finding suggests that cspA is subject to negative autoregulation. Other reports also indicate that CspA acts to negatively regulate cspA expression (Brandi et al., 1999; Graumann et al., 1997). It was possible therefore that Hcy-induced expression of cspA could result if Hcy interfered with the activity of the CspA protein. We investigated this possibility by examining the expression of a cspA : : lacZ translational fusion in a background deficient for cspA. Plasmid pMM016 was transformed into COB229 (cspA : : cat), generating strain COB230. Hcy was found to stimulate cspA expression in this genetic background by approximately 3.5-fold (Fig. 3). Furthermore, levels of cspA expression were found to be higher in the cspA : : cat background than in the wild-type (Fig. 3). These data suggest that Hcy-induced expression of cspA does not require the CspA protein and indicate that, consistent with reports in the literature, cspA expression is subject to negative autoregulation.
|
). This finding is explained by the fact that threonine deaminase, the first enzyme on the isoleucine biosynthetic pathway, is inhibited by Hcy, thereby depleting the cell of isoleucine (Tuite et al., 2005). We investigated whether the induction of cspA by Hcy was related to the partial auxotrophy for isoleucine caused by Hcy treatment. -Galactosidase assays were performed on Frag1 (pMM016) growing in the presence and absence 0.5 mM Hcy, both with and without added isoleucine (0.15 mM). As expected, isoleucine supplementation alone had no significant effect on cspA expression. However, the addition of isoleucine to Hcy-treated cultures almost fully prevented the induction of cspA (Fig. 4). This result indicates that cspA induction is likely to be caused, directly or indirectly, by the depletion of isoleucine that arises in Hcy-treated cells.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Genes involved in the biosynthesis of cysteine are repressed. In minimal medium E. coli synthesizes cysteine by combining sulphide with O-acetyl-L-serine in a reaction catalysed by O-acetylserine lyase, which is encoded by the cysK and cysM genes. Both cysK and cysM are repressed in the presence of Hcy. Sulphide is produced from sulphate in a series of four biochemical reactions. Several of the genes involved in these reactions (cysD, N, H, and J) are also repressed by Hcy. All of these genes belong to the CysB regulon. CysB is a LysR-like transcriptional activator that up-regulates the transcription of the cysteine biosynthetic genes in the presence of the inducer N-acetyl-L-serine, which is produced spontaneously from O-acetyl-L-serine (Kredich, 1996). Reduced expression of the cysteine biosynthetic genes occurs when cysteine is present in the medium. Cysteine causes feedback inhibition of serine acetyltransferase, the enzyme required for O-acetyl-L-serine synthesis, and this leads to a depletion of the CysB inducer (Kredich, 1996). One plausible explanation for the decreased expression of the cysteine biosynthetic genes in the presence of Hcy is that serine acetyltransferase may be inhibited by Hcy, which is chemically very similar to cysteine, giving rise to inappropriately low levels of the inducer, N-acetyl-L-serine. Apart from the cys genes indicated here, none of the other genes detected in the macroarray analyses were found to have CysB-binding sites (data not shown), making it unlikely that those changes were influenced by CysB.
The metE gene, which encodes methionine synthase, is also repressed in the presence of Hcy (Table 2). This is probably accounted for by an increased availability of methionine within the cell, which arises because of an increase in the availability of the MetE substrate, Hcy. Increased intracellular methionine would then trigger a repressive effect through the MetJ repressor together with its corepressor S-adenosylmethionine (SAM). Interestingly, increased Hcy levels have been reported to stimulate metE transcription through the activation of MetR, a positive transcriptional regulator of metE. However, these studies were conducted using metJ mutant strains of E. coli (Byerly et al., 1990; Urbanowski & Stauffer, 1989; Wu et al., 1995), making it difficult to predict what impact the increase in methionine levels might have on metE transcription. It appears that under the growth conditions tested in the present study the repressive effect of MetJ-SAM on metE transcription outweighs the inducing effect of MetR-Hcy. Indeed, an in vitro study of metE transcription has shown that MetR-Hcy fails to induce metE in the presence of MetJ-SAM (Cai et al., 1989).
Genes involved in isoleucine biosynthesis (ilvC, I) or transport (livH) were also found to be repressed in Hcy-treated cells (Table 2). We have shown elsewhere (Tuite et al., 2005) that Hcy-treated cells become depleted for isoleucine. Hcy inhibits threonine deaminase, the first enzyme on the isoleucine biosynthetic pathway, leading to a partial auxotrophy for this amino acid (Tuite et al., 2005). Here ilvC and ilvI are shown to be repressed in the presence of Hcy, although it is not clear at present why this is the case. The transcription of ilvC is positively regulated by IlvY in the presence of substrate for the IlvC enzyme (acetohydroxy acid isomeroreductase), either acetolactate or acetohydroxybutyrate (Umbarger, 1996). Acetohydroxybutyrate levels are likely to be reduced in Hcy-treated cells since the activity of threonine deaminase is impaired by Hcy, thereby reducing the metabolite flux through the isoleucine branch of the pathway. Reduced levels of acetohydroxybutyrate could account for the reduced expression of ilvC observed in Hcy-treated cells.
A number of studies of Hcy toxicity in eukaryotic systems suggest that exogenous Hcy treatment triggers oxidative stress through the increased production of hydrogen peroxide (Loscalzo, 1996; Stamler et al., 1993; Starkebaum & Harlan, 1986). In bacteria there is evidence of a link between cysteine and oxidative stress (Berglin et al., 1982; Park & Imlay, 2003). In the present study we show that catalase activity is actually reduced in Hcy-treated cells and a number of genes (sodA, fldB, katG) involved in protecting E. coli cells against oxidative stress are repressed (Table 2). These results suggest that under these growth conditions Hcy does not exert its toxic effect via oxidative stress. Furthermore, the reduced expression of these genes may indicate that the cells experience a reductive stress when exposed to high levels of extracellular Hcy. Interestingly, Hcy triggers a decrease in glutathionine peroxidase in human endothelial cells derived from umbilical veins and also acts to block the H2O2-mediated activation of HSP70 expression (Outinen et al., 1998). In Salmonella typhimurium, Hcy acts to counteract the inhibitory effects of reactive nitrogen species (De Groote et al., 1996). The thiol-containing compound DTT can prevent induction of a heat-shock response in HeLa cells and this is attributed to it reducing activity (Huang et al., 1994). It seems likely that in E. coli high levels of intracellular Hcy act to reduce the levels of reactive oxygen species that act, via OxyR and SoxRS, to stimulate the expression of the catalases and superoxide dismutase.
The finding that Hcy triggers the induction of the major cold-shock protein CspA in E. coli was surprising since the expression of this protein is mainly thought to be induced in response to cold shock (Goldstein et al., 1990) or nutritional up-shift (Brandi et al., 1999; Yamanaka & Inouye, 2001). As discussed above, Hcy treatment leads to a partial auxotrophy for isoleucine; growth inhibition by Hcy is fully reversed by supplementing the medium with isoleucine (Tuite et al., 2005). We show that the induction of cspA triggered by Hcy is essentially blocked by the inclusion of isoleucine in the growth medium (Fig. 4), suggesting that cspA induction arises in Hcy-treated cells as a result of isoleucine starvation. Cells that are starved for isoleucine will also have reduced levels of the corresponding charged tRNA, in this case isoleucyl-tRNAIle. This will have consequences for translation in the cell; ribosomes will stall on the mRNA at Ile codons. It is possible that this ribosomal stalling acts as the signal leading to induction of cspA, since it is known that inhibitors of translational elongation, such as chloramphenicol and tetracycline, stimulate the expression of CspA (Bianchi & Baneyx, 1999; Jiang et al., 1993; VanBogelen & Neidhardt, 1990). Furthermore, these antibiotics, as well as cold shock itself, lead to an increased expression of ribosomal proteins (VanBogelen & Neidhardt, 1990). It seems possible therefore that the increased expression of ribosomal proteins observed in the presence of Hcy may also be accounted for by ribosomal stalling that occurs because of reduced charged isoleucyl-tRNAIle availability. Further work will be required to clarify this point.
This study has provided us with a greater insight into the effects of the thiol-containing amino acid Hcy on the physiology of E. coli cells. It is clear that Hcy interferes with amino acid metabolism and perturbs the process of translation. Together these effects account for the toxicity of exogenous Hcy to E. coli. In contrast to the situation in some human cells, Hcy does not induce oxidative stress in E. coli. Future studies should address the possibility that eukaryotic translation might be perturbed by Hcy, perhaps contributing to the toxicity of this amino acid in humans.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Anderson, J. L., Muhlestein, J. B., Horne, B. D., Carlquist, J. F., Bair, T. L., Madsen, T. E. & Pearson, R. R. (2000). Plasma homocysteine predicts mortality independently of traditional risk factors and C-reactive protein in patients with angiographically defined coronary artery disease. Circulation 102, 1227–1232.
Bae, W., Jones, P. G. & Inouye, M. (1997). CspA, the major cold shock protein of Escherichia coli, negatively regulates its own gene expression. J Bacteriol 179, 7081–7088.
Beers, R. F., Jr & Sizer, I. W. (1952). A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 195, 133–140.
Berglin, E. H., Edlund, M. B., Nyberg, G. K. & Carlsson, J. (1982). Potentiation by L-cysteine of the bactericidal effect of hydrogen peroxide in Escherichia coli. J Bacteriol 152, 81–88.
Bhagwat, A. A., Phadke, R. P., Wheeler, D., Kalantre, S., Gudipati, M. & Bhagwat, M. (2003). Computational methods and evaluation of RNA stabilization reagents for genome-wide expression studies. J Microbiol Methods 55, 399–409.[CrossRef][Medline]
Bianchi, A. A. & Baneyx, F. (1999). Stress responses as a tool to detect and characterize the mode of action of antibacterial agents. Appl Environ Microbiol 65, 5023–5027.
Brandi, A., Spurio, R., Gualerzi, C. O. & Pon, C. L. (1999). Massive presence of the Escherichia coli ‘major cold-shock protein’ CspA under non-stress conditions. EMBO J 18, 1653–1659.[CrossRef][Medline]
Byerly, K. A., Urbanowski, M. L. & Stauffer, G. V. (1990). Escherichia coli metR mutants that produce a MetR activator protein with an altered homocysteine response. J Bacteriol 172, 2839–2843.
Bystrom, A. S. & Bjork, G. R. (1982). Chromosomal location and cloning of the gene (trmD) responsible for the synthesis of tRNA (m1G) methyltransferase in Escherichia coli K-12. Mol Gen Genet 188, 440–446.[CrossRef][Medline]
Cai, X. Y., Maxon, M., Redfield, B., Glass, R. E., Brot, N. & Weissbach, H. (1989). Methionine synthesis in Escherichia coli: effect of the MetR protein on metE and metH expression. Proc Natl Acad Sci U S A 86, 4407–4411.
Casas, J. P., Bautista, L. E., Smeeth, L., Sharma, P. & Hingorani, A. D. (2005). Homocysteine and stroke: evidence on a causal link from mendelian randomisation. Lancet 365, 224–232.[Medline]
De Groote, M. A., Testerman, T., Xu, Y., Stauffer, G. & Fang, F. C. (1996). Homocysteine antagonism of nitric oxide-related cytostasis in Salmonella typhimurium. Science 272, 414–417.[Abstract]
Goldstein, J., Lehnhardt, S. & Inouye, M. (1990). Major cold shock protein of Escherichia coli. Proc Natl Acad Sci U S A 87, 283–287.
Graumann, P., Wendrich, T. M., Weber, M. H., Schroder, K. & Marahiel, M. A. (1997). A family of cold shock proteins in Bacillus subtilis is essential for cellular growth and for efficient protein synthesis at optimal and low temperatures. Mol Microbiol 25, 741–756.[CrossRef][Medline]
Greene, R. C. (1996). Biosynthesis of methioinine. In Escherichia coli and Salmonella: Cellular and Molecular Biology, pp. 514–527. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Harris, C. L. (1981). Cysteine and growth inhibition of Escherichia coli: threonine deaminase as the target enzyme. J Bacteriol 145, 1031–1035.
Huang, L. E., Zhang, H., Bae, S. W. & Liu, A. Y. (1994). Thiol reducing reagents inhibit the heat shock response. Involvement of a redox mechanism in the heat shock signal transduction pathway. J Biol Chem 269, 30718–30725.
Jakubowski, H. (1999). Protein homocysteinylation: possible mechanism underlying pathological consequences of elevated homocysteine levels. FASEB J 13, 2277–2283.
Jakubowski, H. (2000). Translational incorporation of S-nitrosohomocysteine into protein. J Biol Chem 275, 21813–21816.
Jakubowski, H. (2002). The determination of homocysteine-thiolactone in biological samples. Anal Biochem 308, 112–119.[CrossRef][Medline]
Jakubowski, H. (2004). Molecular basis of homocysteine toxicity in humans. Cell Mol Life Sci 61, 470–487.[CrossRef][Medline]
Jiang, W., Jones, P. & Inouye, M. (1993). Chloramphenicol induces the transcription of the major cold shock gene of Escherichia coli, cspA. J Bacteriol 175, 5824–5828.
Kredich, N. M. (1996). Biosynthesis of cysteine. In Escherichia coli and Salmonella: Cellular and Molecular Biology, pp. 514–527. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Loscalzo, J. (1996). The oxidant stress of hyperhomocyst(e)inemia. J Clin Invest 98, 5–7.[Medline]
Lynch, S. M. & Frei, B. (1997). Physiological thiol compounds exert pro- and anti-oxidant effects, respectively, on iron- and copper-dependent oxidation of human low-density lipoprotein. Biochim Biophys Acta 1345, 215–221.[Medline]
Maki, Y., Yoshida, H. & Wada, A. (2000). Two proteins, YfiA and YhbH, associated with resting ribosomes in stationary phase Escherichia coli. Genes Cells 5, 965–974.[Abstract]
Merlin, C., Gardiner, G., Durand, S. & Masters, M. (2002). The Escherichia coli metD locus encodes an ABC transporter which includes Abc (MetN), YaeE (MetI), and YaeC (MetQ). J Bacteriol 184, 5513–5517.
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Miller, J. H. (1992). A Short Course in Bacterial Genetics: a Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Mitta, M., Fang, L. & Inouye, M. (1997). Deletion analysis of cspA of Escherichia coli: requirement of the AT-rich UP element for cspA transcription and the downstream box in the coding region for its cold shock induction. Mol Microbiol 26, 321–335.[CrossRef][Medline]
Outinen, P. A., Sood, S. K., Liaw, P. C. & 7 other authors (1998). Characterization of the stress-inducing effects of homocysteine. Biochem J 332, 213–221.[Medline]
Park, S. & Imlay, J. A. (2003). High levels of intracellular cysteine promote oxidative DNA damage by driving the Fenton reaction. J Bacteriol 185, 1942–1950.
Phadtare, S., Yamanaka, K. & Inouye, M. (2000). The cold shock response. In Bacterial Stress Responses, pp. 33–45. Edited by G. Storz & R. Hengge-Aronis. Washington, DC: American Society for Microbiology.
Roe, A. J., O'Byrne, C., McLaggan, D. & Booth, I. R. (2002). Inhibition of Escherichia coli growth by acetic acid: a problem with methionine biosynthesis and homocysteine toxicity. Microbiology 148, 2215–2222.
Seshadri, S., Beiser, A., Selhub, J., Jacques, P. F., Rosenberg, I. H., D'Agostino, R. B., Wilson, P. W. & Wolf, P. A. (2002). Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. N Engl J Med 346, 476–483.
Stamler, J. S., Osborne, J. A., Jaraki, O., Rabbani, L. E., Mullins, M., Singel, D. & Loscalzo, J. (1993). Adverse vascular effects of homocysteine are modulated by endothelium-derived relaxing factor and related oxides of nitrogen. J Clin Invest 91, 308–318.[Medline]
Starkebaum, G. & Harlan, J. M. (1986). Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine. J Clin Invest 77, 1370–1376.[Medline]
Tuite, N. L., Fraser, K. R. & O'Byrne, C. P. (2005). Homocysteine toxicity in Escherichia coli is caused by a perturbation of branched-chain amino acid biosynthesis. J Bacteriol 187, 4362–4371.
Umbarger, H. E. (1996). Biosynthesis of the branched-chain amino acids. In Escherichi coli and Salmonella: Cellular and Molecular Biology, pp. 442–457. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Upchurch, G. R., Jr, Welch, G. N., Fabian, A. J., Freedman, J. E., Johnson, J. L., Keaney, J. F., Jr & Loscalzo, J. (1997). Homocyst(e)ine decreases bioavailable nitric oxide by a mechanism involving glutathione peroxidase. J Biol Chem 272, 17012–17017.
Urbanowski, M. L. & Stauffer, G. V. (1989). Role of homocysteine in metR-mediated activation of the metE and metH genes in Salmonella typhimurium and Escherichia coli. J Bacteriol 171, 3277–3281.
VanBogelen, R. A. & Neidhardt, F. C. (1990). Ribosomes as sensors of heat and cold shock in Escherichia coli. Proc Natl Acad Sci U S A 87, 5589–5593.
van der Put, N. M., van Straaten, H. W., Trijbels, F. J. & Blom, H. J. (2001). Folate, homocysteine and neural tube defects: an overview. Exp Biol Med (Maywood) 226, 243–270.
Vollset, S. E., Refsum, H., Irgens, L. M., Emblem, B. M., Tverdal, A., Gjessing, H. K., Monsen, A. L. & Ueland, P. M. (2000). Plasma total homocysteine, pregnancy complications, and adverse pregnancy outcomes: the Hordaland Homocysteine study. Am J Clin Nutr 71, 962–968.
Winzer, K., Hardie, K. R., Burgess, N. & 8 other authors (2002). LuxS: its role in central metabolism and the in vitro synthesis of 4-hydroxy-5-methyl-3(2H)-furanone. Microbiology 148, 909–922.
Wu, W. F., Urbanowski, M. L. & Stauffer, G. V. (1995). Characterization of a second MetR-binding site in the metE metR regulatory region of Salmonella typhimurium. J Bacteriol 177, 1834–1839.
Yamanaka, K. & Inouye, M. (2001). Induction of CspA, an E. coli major cold-shock protein, upon nutritional upshift at 37 °C. Genes Cells 6, 279–290.[Abstract]
Received 22 December 2005;
revised 24 March 2006;
accepted 6 April 2006.
This article has been cited by other articles:
|
|
 |
|
  N. Abed, M. Bickle, B. Mari, M. Schapira, R. Sanjuan-Espana, K. Robbe Sermesant, O. Moncorge, S. Mouradian-Garcia, P. Barbry, B. B. Rudkin, et al. A Comparative Analysis of Perturbations Caused by a Gene Knock-out, a Dominant Negative Allele, and a Set of Peptide Aptamers Mol. Cell. Proteomics, December 1, 2007; 6(12): 2110 - 2121. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Coleman, E. R. Fischer, D. C. Cockrell, D. E. Voth, D. Howe, D. J. Mead, J. E. Samuel, and R. A. Heinzen Proteome and Antigen Profiling of Coxiella burnetii Developmental Forms Infect. Immun., January 1, 2007; 75(1): 290 - 298. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||
2.0-fold change in expression in the presence of 0.5 mM Hcy
, 
) and in the presence of additions (
, 
). Additions were made at the time point indicated by the arrow. Mean values are shown ±SD (n=4).
, 
). Growth (solid symbols) and 
) and in the presence of both 0.5 mM Hcy and 0.15 mM isoleucine (
). Additions were made at the time point indicated by the arrow. Mean values are shown ±SD (n=4).