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Applied and Environmental Microbiology, March 2004, p. 1749-1757, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1749-1757.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Elucidation of the Antibacterial Mechanism of the Curvularia Haloperoxidase System by DNA Microarray Profiling

Eva H. Hansen,1,2 Mark A. Schembri,3* Per Klemm,3 Thomas Schäfer,1 Søren Molin,3 and Lone Gram2

Novozymes A/S, DK-2880 Bagsværd,1 Department of Seafood Research, Danish Institute for Fisheries Research,2 Centre for Biomedical Microbiology, BioCentrum-DTU, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark3

Received 3 October 2003/ Accepted 26 November 2003


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ABSTRACT
 
A novel antimicrobial enzyme system, the Curvularia haloperoxidase system, was examined with the aim of elucidating its mechanism of antibacterial action. Escherichia coli strain MG1655 was stressed with sublethal concentrations of the enzyme system, causing a temporary arrest of growth. The expression of genes altered upon exposure to the Curvularia haloperoxidase system was analyzed by using DNA microarrays. Only a limited number of genes were involved in the response to the Curvularia haloperoxidase system. Among the induced genes were the ibpA and ibpB genes encoding small heat shock proteins, a gene cluster of six genes (b0301-b0306) of unknown function, and finally, cpxP, a member of the Cpx pathway. Knockout mutants were constructed with deletions in b0301-b0306, cpxP, and cpxARP, respectively. Only the mutant lacking cpxARP was significantly more sensitive to the enzyme system than was the wild type. Our results demonstrate that DNA microarray technology cannot be used as the only technique to investigate the mechanisms of action of new antimicrobial compounds. However, by combining DNA microarray analysis with the subsequent creation of knockout mutants, we were able to pinpoint one of the specific responses of E. coli—namely, the Cpx pathway, which is important for managing the stress response from the Curvularia haloperoxidase system.


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INTRODUCTION
 
A vast of array of antimicrobial compounds are used in the fight against microorganisms. For instance, disinfection of surfaces in the clinical sector or in pharmaceutical and food processing settings involves the use of compounds such as chlorite, hydrogen peroxide, iodophors, or quaternary ammonium compounds. Many of these compounds have adverse side effects, such as leaving active residues, causing corrosion and degradation of surfaces and equipment, or being unsafe for the personnel exposed thereto. Hence, there has been a drive for development of milder, more environmentally friendly antimicrobial agents (14, 20). One such agent is the Curvularia haloperoxidase system, which has a lethal effect against bacteria, yeasts, and filamentous fungi and is active against both planktonic microorganisms and surface-associated microorganisms (16). In the presence of hydrogen peroxide, Curvularia haloperoxidase facilitates the oxidation of halides, such as chloride, bromide, and iodide, to antimicrobial compounds. The level of hydrogen peroxide used in the enzyme system is much lower than if hydrogen peroxide is used as the sole compound, thus reducing the corrosive action of the system. It has been hypothesized that the antimicrobial effect of the Curvularia haloperoxidase system is due to the production of highly oxidative intermediates; however, this is not known factually. Further assessment of areas of application of the Curvularia system would be greatly facilitated by an understanding of its mechanism of action. Also, understanding the mechanism would allow evaluation of potential side effects such as development of resistance to the system.

Elucidating the mechanisms of action of antimicrobial compounds can be approached in different ways, including creation of mutants that are resistant or hypersensitive to the effect, determining cellular damage (e.g., respiration effects and leakage of intracellular materials), or using stress-gene reporter fusions (2, 10, 12, 17, 26, 32, 38). Comparison of global gene transcripts as performed by using DNA microarrays offers a unique way of analyzing the effects of antimicrobial compounds on microorganisms. The expression profiles may reveal the mode of action of the compound and may also provide information on potential resistance mechanisms. Examples of areas investigated by use of microarray analysis are oxidative stress and effect of antibacterial compounds intended for both disinfection and pharmaceutical use (27, 40, 43).

Bacteria are able to respond to stress conditions in order to circumvent stress factors such as oxidative stress, heat shock, cold shock, etc. Oxidative stress is caused by exposure to reactive oxygen intermediates and has been shown to cause damage to proteins, nucleic acids, and lipids (13). The regulatory protein SoxRS controls the defense against superoxide, whereas OxyR regulates the hydrogen peroxide response (13, 36, 43). Another defense mechanism is the synthesis of heat shock proteins that stabilize and protect intracellular proteins not only from heat but also from other stresses such as oxidative stress. Escherichia coli strains overexpressing the molecular chaperone DnaK or the small heat shock proteins IbpA and IbpB exhibit increased tolerance to hydrogen peroxide or superoxide, respectively (12, 19).

In the present study, we exposed the E. coli K-12 strain MG1655 to the Curvularia haloperoxidase system. The completion of the full genome sequence of this strain (3) has allowed the development of commercial DNA microarray chips, and the vast biological knowledge of this organism facilitates the interpretation of expression profiles from the microarray analysis. Other E. coli strains such as verotoxigenic E. coli O157:H7 are important human pathogens, and outbreaks have been caused by contaminated drinking water (15) and cross-contamination of foods probably due to improper disinfection of food process equipment (5, 22). E. coli is therefore an important organism when the need arises to evaluate a potential antimicrobial agent.

Since the Curvularia haloperoxidase system has an almost instantaneous bactericidal effect, we analyzed the changes in gene expression during a short-term sublethal exposure of E. coli strain MG1655 to this system. This analysis allowed the identification of both single genes and gene clusters affected by the system, and we subsequently generated knockout mutants to confirm their role in the response of E. coli to the enzyme system. Our data allow us to suggest a mechanism by which the enzyme system may exert its killing effect. This study presents a novel method for investigating new antimicrobial compounds with the aim of elucidating the mechanism of antibacterial action.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and growth conditions.
The E. coli K-12 reference strain MG1655 was used in this work (1). Cells were routinely grown at 37°C on solid or in liquid Luria-Bertani medium supplemented with the appropriate antibiotics. Cells for stress induction experiments were grown at 37°C in morpholinepropanesulfonic acid (MOPS) minimal medium supplemented with 0.4% glucose (25). MOPS minimal medium was supplemented with 50 µg of kanamycin per ml when used as growth medium for knockout mutants.

Monitoring growth and enumeration of bacteria.
Growth of bacterial strains was followed by measurements of optical density at 450 nm (OD450). Flasks were kept under agitation (~100 rpm) during sampling to ensure constant growth conditions. Plate counting was used to determine cell numbers after stress induction. Serial 10-fold dilutions were prepared in sterile physiological saline (0.85% NaCl) with 0.1% peptone (211677; BD). The first 10-fold dilution was prepared in sterile physiological saline with 0.1% peptone and 0.5% bovine serum albumin (A-7906; Sigma) to inhibit any residual antimicrobial effect of the Curvularia haloperoxidase enzyme (16). Appropriate dilutions were surface plated on Tryptone soya agar (CM131; Oxoid), and all plates were incubated overnight at 37°C.

Killing experiments were carried out as described previously by Hansen et al. (16). A 24-h E. coli cell culture was diluted to an OD450 of 0.05 in a total volume of 10 ml and grown to an OD450 of 0.4 under agitation (150 rpm) at 37°C. Separate solutions of Curvularia haloperoxidase, hydrogen peroxide (1.07209; Merck), and KBr (P-9881; Sigma) were made 30.3 times stronger than the final concentration used in the experiment. Stock solutions of Curvularia haloperoxidase and KBr were prepared in MOPS minimal medium, and the hydrogen peroxide solution was mixed in Milli-Q water. A volume of 900 µl of cell suspension, 33 µl of Curvularia haloperoxidase, 33 µl of hydrogen peroxide, and 100 µl of KBr were mixed in a sterile Eppendorf tube. The final concentrations varied between 0.6 and 1.0 mg of enzyme liter-1, 0.7 and 1.0 mM hydrogen peroxide, and 3 and 5 mM bromide. Suspensions of bacteria were incubated with the Curvularia haloperoxidase system for 20 min at 37°C under agitation (270 rpm), and cell numbers were determined by plate counting. Counts of bacteria were converted to a log scale, and levels before and after treatment were compared by using Student's t test. All experiments were performed in duplicate.

Stress induction experiments.
An exponentially growing culture was diluted to an OD450 of 0.05 in a total volume of 100 ml and thereafter grown to an OD450 of approximately 0.8 under agitation (150 rpm) at 37°C in flasks containing baffles to ensure proper aeration of the culture. At an OD450 of 0.8, cultures were diluted to an OD450 of 0.05 in MOPS minimal medium. The Curvularia enzyme system (or individual components thereof) was added in sublethal concentrations as the OD450 again reached 0.4. This ensured a minimum of seven generations in steady-state exponential growth prior to the addition of the enzyme system or control solutions. The total volume in the flasks was at that time 88 to 93 ml. Solutions of Curvularia haloperoxidase (Novozymes A/S), hydrogen peroxide (1.07209; Merck) and KBr (P-9881; Sigma) were therefore made 883 to 933 times stronger than the final concentration used in the experiment when stress was induced with the complete enzyme system, or alternatively, the solutions were made 881 to 931 times stronger when stress was induced with a single component. Stock solutions of Curvularia haloperoxidase and bromide were prepared in MOPS minimal medium, whereas stock solution of hydrogen peroxide was prepared in Milli-Q water. One hundred microliters of each stock solution was added, giving a final concentration of 0.4 mg of Curvularia haloperoxidase liter-1, 0.8 mM hydrogen peroxide, and 2.0 mM KBr. Samples for microarray analysis (5 ml) were taken before, during, and after the stress induction and were resuspended immediately into an equal volume of ice-cold RNAlater (7021; Ambion). Growth was monitored until the cultures reached stationary phase.

RNA isolation and cDNA labeling.
Total RNA was isolated and purified from cells resuspended in RNAlater by using an RNeasy mini kit (part no. 74104; Qiagen). After purification, RNA was treated with RNase-free DNase I to remove contaminating DNA and was then repurified by using Qiagen RNeasy columns. RNA samples were quantified spectrophotometrically at 260 nm and additionally checked by gel electrophoresis. Purified total RNA was precipitated with ethanol and stored at -80°C until further use. Conversion of RNA to cDNA and microarray analysis were performed according to the Affymetrix expression analysis technical manual. Briefly, 10 µg of RNA was mixed with 750 ng of random hexamer primers (Invitrogen), denatured at 70°C for 10 min, and then allowed to anneal. cDNA synthesis was performed by using Superscript II reverse transcriptase (Invitrogen) in 1x first-strand synthesis buffer containing dithiothreitol (10 mM), deoxynucleoside triphosphate mix (500 µM), and SUPERase inhibitor (30 U; Ambion). The following incubations were performed: 25°C for 10 min, 37°C for 60 min, 42°C for 60 min, and 70°C for 10 min. The mixture was cooled to 4°C and treated with NaOH (65°C for 30 min) to degrade the RNA strands, followed by neutralization with HCl. cDNA was further purified by using a QIAquick PCR purification kit (Qiagen). Fragmentation was performed with DNase I (0.6 U/µg of cDNA) in One-Phor-All buffer (Amersham Pharmacia Biotech). DNase I was inactivated by heating at 98°C for 10 min, and the 3' terminus of fragmented cDNA products was labeled with biotin by using the Enzo BioArray terminal labeling kit with biotin ddUTP (Affymetrix).

DNA microarray analysis.
E. coli GeneChip microarrays were purchased from Affymetrix (Santa Clara, Calif.). An antisense oligonucleotide array essentially the same as that described by Selinger et al. (34) (with the exception that probe sequences were the same as the coding region sequence) was employed. Biotin-labeled fragmented cDNA was hybridized to E. coli GeneChip microarrays overnight at 45°C in morpholineethanesulfonic acid (MES) buffer containing herring sperm DNA (100 µg/ml) and bovine serum albumin (500 µg/ml). Probe array washing and staining procedures were carried out as described by using GeneChip analysis suite software (version 4.0). Hybridized cDNA was fluorescently labeled in a three-step affinity binding procedure that involved binding of streptavidin to biotin-labeled cDNA, binding of biotin-conjugated streptavidin antibody to streptavidin, and finally, binding of phycoerythrin-conjugated streptavidin to biotin-labeled antibodies. Probe arrays were scanned twice at 570 nm at a 3-µm resolution with an Affymetrix scanner, and a quantitative analysis of hybridization patterns and intensities was performed by using the GeneChip analysis suite software's expression analysis window.

Data analysis.
Gene expression data was analyzed by using Microarray Suite 5.0 software (Affymetrix). The software calculates change calls, change in P values, and signal-log ratios. Change calls indicate an increase or decrease between a baseline array and an experimental array, and change in P value indicates the statistical significance for the change calls. The signal-log ratio is the relative change between the baseline and the experimental array expressed as the loge ratio. In these studies, the signal-log ratios were calculated as the difference between expression levels after stress induction and expression values before stress induction. All experiments were performed in duplicate or triplicate unless indicated otherwise. The fluorescence of each array was normalized by scaling total chip fluorescence intensities to a common value of 5,000. Changes in expression levels that had a change call of decrease or increase together with a P value of <0.001 and a signal-log ratio of <2 or >2 were considered significant. Expression ratios of all genes for each experiment along with all the results are available at http://www.dfu.min.dk/micro.

Construction of knockout mutants.
Mutant strains were constructed by using the {lambda} Red recombinase gene replacement system (9). Briefly, the kanamycin gene from plasmid pKD4 was amplified by primers containing 50-nucleotide homology extensions for the ykgB-D region (P1, 5'-AGCGATGCTGGCCAGCGATTCGACGGTCCAGGCGTGTCCTGGCATTTCCAGTGTAGGCTGGAGCTGCTTC-3', and P2, 5'-TCAATACCAGGCGACCAGCACCGGATAAATAAGGGAAACCATGATGAGCGCATATGAATATCCTCCTTAG-3'), the cpxP (b3913 and b3914) region (P3, 5'-TAAGTTTAACCGAACATCAGCGTCAGCAGATGCGAGATCTTATGCAACAGGTGTAGGCTGGAGCTGCTTC-3', and P4, 5'-CGTTAACAGGCGATACATTTGGTTGCGGACTTTTGCCATCTCAACCTGACCATATGAATATCCTCCTTAG-3'), and the cpxP (b3913 and b3914)-cpxAR region (P5, 5'-CAGTTCCTTGCTTTCACCGCTACGACGGCGCAGTAACGCCGTACCCAGTTGTGTAGGCTGGAGCTGCTTC-3', and P6, 5'-CGTTAACAGGCGATACATTTGGTTGCGGACTTTTGCCATCTCAACCTGACCATATGAATATCCTCCTTAG-3'). The amplified products were digested with DpnI and transformed into MG1655 (pKD46), and kanamycin-resistant colonies were selected. Correct double-crossover and recombination events were checked by PCR. The {lambda} Red helper plasmid pKD46 was cured by growth at 37°C, and the subsequent strains were designated MS669 (MG1655 {Delta}ykgBICD), MS710 (MG1655 {Delta}cpxP), and MS719 (MG1655 {Delta}cpxARP).


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RESULTS
 
Killing of E. coli MG1655 with Curvularia haloperoxidase system.
In order to assess the killing kinetics of the Curvularia haloperoxidase system, cells of E. coli MG1655 were exposed to a range of different enzyme concentrations (Fig. 1). The initial cell number was 8.4 ± 0.1 log CFU ml-1, corresponding to an OD450 of 0.4. Low concentrations of the enzyme system (0.6 mg of enzyme liter-1, 0.7 mM hydrogen peroxide, 3 mM bromide) had no effect on cell numbers following a 20-min exposure time. However, increasing the concentration of all three components (1 mg of enzyme liter-1, 1 mM hydrogen peroxide, 5 mM bromide) resulted in a log reduction of 3.2 ± 0.5.



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  		FIG. 1. Killing of Escherichia coli strain MG1655 with different concentrations of the Curvularia haloperoxidase system. The initial cell number was 8.4 ± 0.1 log CFU ml-1, corresponding to an OD450 of 0.4. The following concentrations were used: treatment 1, 1 mg of Curvularia haloperoxidase liter-1, 1 mM hydrogen peroxide, 5 mM bromide; treatment 2, 0.8 mg of Curvularia haloperoxidase liter-1, 0.9 mM hydrogen peroxide, 4 mM bromide; and treatment 3, 0.6 mg of Curvularia haloperoxidase liter-1, 0.7 mM hydrogen peroxide, 3 mM bromide. Data are means, and error bars indicate standard deviations.

Stress induction of E. coli MG1655 with Curvularia haloperoxidase system.
E. coli strain MG1655 was subsequently exposed to sublethal levels of the Curvularia haloperoxidase system (0.4 mg of Curvularia haloperoxidase liter-1, 0.8 mM hydrogen peroxide, and 2.0 mM KBr). The Curvularia haloperoxidase system caused a temporary arrest of growth, after which the normal growth rate was resumed (Fig. 2A) (Table 1). The doubling times of E. coli MG1655 before and after stress induction were 63.3 and 69.3 min, respectively. During the 17 min of growth arrest, the doubling time increased to 630.1 min. Each of the individual components of the Curvularia haloperoxidase system was also tested for growth interference. Hydrogen peroxide affected growth, although not to the same extent as did the Curvularia haloperoxidase system (Fig. 2B) (Table 1). Exposure to hydrogen peroxide caused an increase in the doubling time (from 64.2 to 161.2 min, over an arrest time of 19 min) before returning to 71.5 min. Induction with either Curvularia haloperoxidase or bromide alone did not significantly affect the growth rate (Fig. 2C and D) (Table 1).



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  		FIG. 2. Stress induction of MG1655 with Curvularia haloperoxidase system (A), hydrogen peroxide (B), Curvularia haloperoxidase (C), and bromide (D). Arrows indicate times of stress induction. Data are means, and error bars indicate standard deviations of duplicate determinations.


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  		TABLE 1. Doubling times of E. coli MG1655 and mutant strains MS669, MS710, and MS719 before, during, and after stress induction with the Curvularia haloperoxidase system or individual components thereof

DNA microarray analysis of E. coli MG1655 stress induced with Curvularia haloperoxidase system.
The Curvularia haloperoxidase enzyme system consists of three individual components, and gene induction or repression can be a consequence of individual components of the system or of the combined effect when Curvularia haloperoxidase is mixed with bromide and hydrogen peroxide. DNA microarray analysis revealed that several genes were affected either by the complete enzyme system or by the individual components, especially hydrogen peroxide. In order to evaluate genes affected by the Curvularia haloperoxidase system, it was therefore essential to differentiate between genes which were also altered in expression after induction with each of the individual components administered alone.

Identification of genes altered in expression in response to stress induction with hydrogen peroxide.
Upon exposure to hydrogen peroxide, the regulatory protein OxyR induces the expression of several genes belonging to the OxyR regulon. Therefore, we specifically evaluated the expression of genes belonging to the OxyR regulon after exposure to 0.8 mM hydrogen peroxide (Table 2). The expression of 73% of the genes in the OxyR regulon was induced with a loge factor of >2 in the present study. Similarly, Zheng et al. (43) found a fourfold or greater induction of 73% of the genes in the OxyR regulon after stress induction with 1 mM hydrogen peroxide. Most of the previously described OxyR-regulated genes that were not induced in the present study (e.g., fur, gor, dsbG, flu, and fhuF) were also not induced in the study by Zheng et al. (43). The strong correlation between the two data sets serves as an additional internal control to indicate the reliability of our microarray data.


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  		TABLE 2. Induction of genes in the OxyR regulon of Escherichia coli MG1655 after exposure to H2O2

Identification of genes altered in expression in response to stress induction with Curvularia haloperoxidase or bromide.
Several genes were affected by stress induction with pure Curvularia haloperoxidase, but only yieC, which is a putative membrane protein, was down-regulated in both experiments. No genes were up-regulated in both experiments. In the response to bromide, 50 genes were either up-regulated or down-regulated. The down-regulated genes were primarily ribosomal genes. Other genes had a low baseline expression level, and a relatively small increase in expression level resulted in a log ratio of >2.

Identification of genes altered in expression in response to stress induction with the Curvularia haloperoxidase system.
When identifying genes that were up-regulated by the Curvularia haloperoxidase system, we specifically addressed genes that were affected by the complete enzyme system and not by any of the components individually. This resulted in only a limited number of genes being involved in the response to the Curvularia haloperoxidase system (Table 3).


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  		TABLE 3. Loge ratiosa of E. coli MG1655 genes significantly up-regulated only by treatment with the Curvularia haloperoxidase system in sublethal concentrations and not by the single components of the enzyme system

Based on known or proposed functions, a number of the genes induced by the Curvularia haloperoxidase system were of immediate specific interest. The ibpA and ibpB genes encode small heat shock proteins in E. coli. In one of the three experiments, ibpA was induced with a loge ratio of only 1.5, but the other two experiments showed loge inductions of 3.4 and 4.0, respectively. IbpA and IbpB are part of the cellular response to denatured proteins (35, 39). In E. coli, the Cpx pathway also senses and responds to misfolded proteins (6, 8, 28). The overlapping open reading frames designated b3913 and b3914 have recently been shown to comprise the cpxP gene (7). For simplification purposes, we have therefore referred to these two open reading frames as the cpxP gene throughout the remainder of this paper. The cpxP gene lies immediately adjacent to the cpxAR sensor response regulatory genes and encodes a periplasmic protein involved in combating extracytoplasmic protein-mediated toxicity. The transcription of cpxP was increased by a loge ratio of between 2.0 and 3.8 after the stress induction compared to the expression level before stress induction. Expression levels of cpxAR were not affected by the stress induction. Additionally, we identified a cluster of six adjacently linked genes (b0301 to b0306) to be induced at significantly high levels. The function of these genes is unknown.

A number of other single genes were also up-regulated after exposure to the Curvularia haloperoxidase system. Some of these genes encode known or putative membrane- or periplasmic-located proteins that may be directly involved in the stress-induced response. For example, nlpA encodes an inner membrane lipoprotein that may reduce cell permeability (29, 42), while the tauA gene encodes a periplasmic protein whose expression is regulated by sulfate starvation (37). Other significantly enhanced genes included the gloA gene (which encodes a protein associated with enhanced tolerance to methylglyoxal) (23), nemA (which encodes an N-ethylmaleimide [NEM] reductase) (24), and yqhD and ytfG (both of which encode putative oxidoreductases).

The growth curve of E. coli after stress induction with the Curvularia haloperoxidase system indicated that the effect was only transient, as normal exponential growth resumed hereafter. In line with this observation, we assumed that the induced genes observed from our DNA microarray profiling would also return to levels observed in the uninduced state. Indeed, 1.5 h after stress induction, the expression level of all the up-regulated genes (except for b1970) returned to levels observed before stress induction (Table 3).

No clear tendency could be observed in the down-regulation of genes in the response to the Curvularia haloperoxidase system.

Mutants lacking cpx or ykg gene clusters.
To evaluate whether the up-regulated cpx and ykg gene clusters were involved in a specific defense mechanism against the Curvularia haloperoxidase system or were part of an unspecific response, knockout mutants MS669, MS710, and MS719 were constructed in which ykgB-D, cpxP, and cpxARP, respectively, were inactivated. MS669 and MS710 were stress induced with the Curvularia haloperoxidase system and compared to the parent MG1655 strain (Fig. 3A and B) (Table 1). As observed for MG1655, the growth rate of both MS669 and MS710 decreased for a period of approximately 15 to 16 min, after which it returned to normal. The similar reaction pattern of MS669, MS710, and MG1655 to stress induction with the Curvularia haloperoxidase system suggests that the ykgB-D gene cluster and the cpxP gene are dispensable with regard to the specific defense response of E. coli to the Curvularia haloperoxidase system.



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  		FIG. 3. Stress induction of MS669 (MG1655 {Delta}ykgBICD) ({triangleup}) and MG1655 ({circ}) with Curvularia haloperoxidase system (A), MS710 (MG1655 {Delta}cpxP) ({blacktriangleup}) and MG1655 ({circ}) with Curvularia haloperoxidase system (B), MS719 (MG1655 {Delta}cpxARP) ({square}) and MG1655 ({circ}) with Curvularia haloperoxidase system (C), and MS719 ({square}) with hydrogen peroxide (D). Arrows indicate times of stress induction. Data are means, and error bars indicate standard deviations of duplicate determinations.

In contrast, growth of MS719 ({Delta}cpxARP) (Fig. 3C) (Table 1) was significantly affected after stress induction with the Curvularia haloperoxidase system, and the growth did not resume until approximately 2 h after stress induction (albeit very slowly). Plate counting of MS719 revealed that cell numbers before and 19 min after stress induction were the same, indicating that growth was arrested after stress induction rather than that the cells were killed because of the treatment. E. coli strains containing mutations in the cpxAR gene locus are known to be more sensitive to several compounds, including hydrogen peroxide (P. De Wulf, unpublished data). To confirm that the altered stress response of MS719 was caused by the complete enzyme system and not by hydrogen peroxide alone, MS719 was stress induced only with hydrogen peroxide with the same concentration as that used in the enzyme system (Fig. 3D) (Table 1). Growth of MS719 was arrested for 26 min, after which the doubling time returned to the level observed before stress induction. The increased sensitivity to hydrogen peroxide does not resemble the severe growth arrest observed after treatment of MS719 with the Curvularia haloperoxidase system, which seems to be a response unique to this system.


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DISCUSSION
 
That the Curvularia haloperoxidase system has a lethal effect against bacteria, yeasts, and filamentous fungi and is active against both planktonic and surface-associated microorganisms has been previously demonstrated (16). When evaluating a novel antimicrobial compound, it is crucial to understand its mechanism of action. This information allows for assessment of potential side effects such as the development of resistance and is also required in the approval process of the antimicrobial compound. The Curvularia haloperoxidase system is potentially useful as a disinfectant in many areas, such as the disinfection of contact lenses, equipment used in ocular science, or medical devices, including pacemakers and urinary tract catheters.

Bacteria possess a wide range of defense mechanisms against antibacterial compounds and are therefore able to neutralize certain harmful components, such as hydrogen peroxide, to repair damaged cell components (e.g., denatured proteins) or to mutate and develop specific resistance mechanisms. By studying induced defense mechanisms, it may be possible to elucidate how an antimicrobial compound affects the bacterial cell and thereby determine its mechanism of action. Most previous studies (10, 17, 18) of mechanisms of antimicrobial compounds have focused on changes in microbial physiology or morphology, e.g., leakage of cell components, changes in respiration, changes in intracellular pH, alterations in membrane structure, etc. DNA microarrays have previously been used to evaluate the stress response in E. coli when exposed to hydrogen peroxide (4, 43). In the present study, we demonstrate how DNA microarrays can be used to investigate gene expression in response to stress induction with sublethal doses of the Curvularia haloperoxidase system. Subsequently, mutants were created in which some of the induced genes were deleted so as to identify whether the reaction mechanisms were specific for the enzyme system.

To analyze the expression of genes in E. coli that are altered upon exposure to the Curvularia haloperoxidase system, we used an Affymetrix DNA chip based on the E. coli reference strain MG1655. The chip used here was a custom-designed antisense oligonucleotide array that requires the conversion of mRNA to cDNA as part of the process for the generation of the labeled target sequence. We observed an induction of the ibpA, ibpB, and cpxP genes (among others) upon exposure to the Curvularia haloperoxidase system. Significantly, these three genes encode proteins that are known to play a role in the bacterial response to denatured proteins. ibpA and ibpB encode small heat shock proteins, and in vitro studies have shown that the IbpB protein can bind to heat-denatured proteins and hold them in a nonaggregating state and furthermore deliver them to the DnaK/DnaJ/GrpE chaperone system for subsequent refolding (35, 39). This function has been confirmed by in vivo studies in E. coli, which showed that overproduction of IbpA and IbpB stabilizes aggregates of heat-denatured proteins (21). IbpA- and IbpB-overproducing strains of E. coli have also been shown to acquire higher levels of resistance to heat and superoxide stress (19). Furthermore, the expression of the ibpA and ibpB genes has also been observed to increase during stationary-phase growth (33). Increased expression of these two genes is therefore consistent with the pause in exponential growth induced by the Curvularia haloperoxidase system. It should be noted that of all of the genes induced by the Curvularia haloperoxidase system, only ibpA and ibpB were also induced during stationary-phase growth. Taken together, these findings indicate that the genes identified by our microarray studies are specifically activated by the Curvularia haloperoxidase system.

The cpxP gene lies immediately adjacent to cpxRA (7). The CpxRA system senses and responds to envelope protein distress in E. coli by regulating the synthesis of several enzymes involved in the folding and degradation of periplasmic proteins (6, 8, 28). The exact mechanism of action of CpxP is unknown. CpxP is a repressor of the Cpx system, but during stress conditions CpxP is strongly induced (11). It has been proposed that CpxP functions as a negative regulator during nonstress conditions but is inactivated during stress, perhaps by binding to misfolded proteins, allowing for activation of the pathway (11, 30). In addition, CpxP may have a chaperone function under stress conditions (11). The Curvularia haloperoxidase system induced the transcription of cpxP, but it apparently did not affect the expression of the cpxAR genes, which was not induced by the stress induction.

The up-regulation of ibpA, ibpB, and cpxP indicates that the Curvularia haloperoxidase system damages proteins. This observation is in agreement with the fact that the addition of protein to the enzyme reaction mixture interferes with the antimicrobial effect by increasing the survival of microorganisms exposed to the Curvularia haloperoxidase system (16). The Curvularia haloperoxidase system probably damages proteins through oxidation. Other peroxidase systems such as the lactoperoxidase system are well described in previous studies (31, 41). In the presence of hydrogen peroxide, lactoperoxidase oxidizes thiocyanate (SCN-) to thiocyanogen [(SCN)2], which hydrolyzes rapidly to hypothiocyanous acid (HOSCN) or hypothiocyanate (OSCN-). (SCN)2 and HOSCN are able to oxidize protein sulfhydryl groups to sulfenyl thiocyanate derivatives. We believe that Curvularia haloperoxidase (like lactoperoxidase) oxidizes halides (for example, bromide to hypobromite), thereby causing oxidative stress in microorganisms, although this supposition has not been experimentally verified.

In addition to protein oxidation, the gene expression profile of E. coli exposed to the Curvularia haloperoxidase system may indicate that the enzyme system also caused lipid peroxidation. nemA (encoding an NEM reductase) was induced, and it has previously been postulated that lipid peroxidation is involved in the induction of NEM reductase (24). Miura et al. (24) found that NEM reductase activity in E. coli was induced by linoleic acid but not by oleic acid, which is less susceptible to lipid peroxidation than is linoleic acid. Furthermore, menadione, which generates superoxides, also induced NEM reductase activity. Phadtare et al. (27) exposed E. coli to 4,5-dihydroxy-2-cyclopenten-1-one and observed an induction of nemA in conjunction with other genes known to respond to oxidative stress.

The results from our DNA microarray experiments prompted us to construct deletion mutants within the ykg gene cluster and the cpxP gene. These mutants did not have an altered sensitivity to the enzyme system compared to the wild type. An explanation of these results may be that they are part of a general stress response that is not specific to the Curvularia haloperoxidase system or are rather part of a broad defense specifically against the enzyme system. Alternatively, these proteins may possess a redundant phenotype that can be compensated for by as yet undefined proteins from similar or parallel functional pathways. Consequently, a mutant with a more extensive deletion of the Cpx pathway was constructed. In contrast to the cpxP mutant, the cpxARP mutant was notably more sensitive to the Curvularia haloperoxidase system than was the wild type, indicating a direct role for this pathway in coping with the stress induced by the enzyme system. These results demonstrate that the DNA microarray technology cannot be used as the sole technique when investigating mechanisms of action of new antimicrobial compounds. However, the array technique provides a very powerful and unique tool for insight into the system. By combining DNA microarray analysis and subsequent creation of knockout mutants, we were able to pinpoint one of the specific responses of E. coli—namely, the Cpx pathway, which is important for managing the stress from the Curvularia haloperoxidase system. The study shows that this novel method is suitable for investigating novel antimicrobial compounds with an aim to elucidate their mechanisms of action.


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ACKNOWLEDGMENTS
 
This study was carried out as part of an industrial Ph.D. study by Eva Holm Hansen, which was financed by Novozymes A/S and the Danish Academy of Technical Sciences. The work was additionally supported by grants from the Danish Natural Sciences Research Council (grant number 21-01-0296) and the Novo Nordisk Foundation.


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FOOTNOTES
 
* Corresponding author. Mailing address: Microbial Adhesion Group, Centre for Biomedical Microbiology, BioCentrum-DTU, Bldg. 301, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark. Phone: (45) 45252519. Fax: (45) 45932809. E-mail: msc{at}biocentrum.dtu.dk. Back


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Applied and Environmental Microbiology, March 2004, p. 1749-1757, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1749-1757.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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