• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Apr 2005; 187(8): 2565–2572.
PMCID: PMC1070385

Genome-Wide Transcriptional Profiling of the Steady-State Response of Pseudomonas aeruginosa to Hydrogen Peroxide

Abstract

The steady-state response of Pseudomonas aeruginosa to the oxidative-stress-generating agent hydrogen peroxide was analyzed by PAO1 transcriptome profiling. In total, 694, 411, and 237 genes were upregulated and 668, 576, and 468 genes were downregulated in P. aeruginosa strains TB, 892, and PAO1, respectively. The expression profiles of the two variants of the TB clone were significantly more related to each other than the expression profile of either strain was to that of PAO1. Exposure to H2O2 activated by more than 10-fold the expression of the cyoABCD operon, which is key for aerobic respiration, and of oxidative-stress response elements such as the catalase KatB, the alkyl hydroperoxide reductase AhpF, and the thioredoxin reductase 2 operon. Genes for iron and sulfur homeostasis were upregulated. Most enzymes necessary for the conversion of amino acids into the citric acid cycle were globally downregulated at the transcriptional level. Nitrate respiration and arginine fermentation were shut off in the clone TB strains and attenuated in the PAO strain. The transcriptional profiles indicate that the two clone TB strains are more proficient in coping with H2O2-mediated oxidative stress than the reference strain PAO. According to this data, we recommend study of the transcriptome of strain PAO1 in parallel with those of at least two strains of another clone in order to differentiate common responses from clone- and strain-specific responses and to minimize overinterpretations of microarray data.

Pseudomonas aeruginosa is an increasingly prevalent opportunistic human pathogen and is the most common gram-negative bacterium found in nosocomial and life-threatening infections of immunocompromised patients (48). Patients with cystic fibrosis (CF) are especially disposed to P. aeruginosa infections, and for these persons the bacterium is responsible for high rates of morbidity and mortality (13, 26, 35). The bacterium synthesizes and secretes a number of virulence determinants that are postulated to enhance survival within the host by providing nutrients, evading the host immune response, and allowing the spread of the organism to other tissues. It has a remarkable ability to adapt to and persist in a variety of environments, including human hosts (15).

P. aeruginosa generates metabolic energy primarily through aerobic respiration. This process, involving a four-electron reduction of molecular oxygen (O2) to water, can be potentially dangerous to the cell. Specifically, aberrant electron flow from the electron transport chain or cellular redox enzymes to O2 can lead to the production of reactive oxygen intermediates (ROIs). These include superoxide (O2¯), hydrogen peroxide (H2O2), and hydroxyl radical (HO·). Furthermore, bacteria can be exposed to exogenous ROIs, especially during infection of humans, where phagocytes (e.g., neutrophils) mount a dramatic oxygen-dependent antimicrobial response (17, 30). The unchecked production or accumulation of these species can lead to cell damage, mutations, or death. P. aeruginosa possesses an impressive antioxidant armament for defense against ROIs, including two superoxide dismutases (SODs) cofactored by either iron (Fe-SOD) or manganese (Mn-SOD) (18, 20) to disproportionate O2¯ to H2O2 and O2, three catalases (KatA, KatB, and KatC) (4, 27), and four alkyl hydroperoxide reductases (AhpA, AhpB, AhpCF, and Ohr) (34).

Polymorphonuclear-leukocyte-mediated phagocytosis and killing are the most important host defense mechanisms against P. aeruginosa (9). We were interested to know whether P. aeruginosa can evade oxidative stress and, if so, how. With limited exceptions, information regarding the nature and regulation of antioxidant enzymes in P. aeruginosa has been derived from studies of laboratory strains. There have not been extensive studies of these enzymes in clinical isolates of this organism. Recent technological advances have made it possible to study global gene expression in both prokaryotic and eukaryotic organisms by using high-density oligonucleotide microarrays. Completion of the entire P. aeruginosa PAO1 genome sequence (44), as well as the availability of high-density oligonucleotide arrays (Affymetrix P. aeruginosa GeneChip arrays), has permitted global gene expression profiling of P. aeruginosa. This approach has already been successfully used in studying P. aeruginosa gene expression under a number of different growth conditions (8, 10, 11, 33, 36, 39, 40, 41, 50). To compare the array data with those from the candidate gene approach (4, 19, 32, 34) and to compare the steady-state response with the acute-stress response (36), the model compound hydrogen peroxide was selected as the oxidative-stress-generating agent because it is a natural effector of innate immunity (30). The transcriptional profile of the sequenced reference strain PAO1, a burn wound isolate from the 1950s, was compared with that of the two clonal variants TB and 892, isolated from the lungs of patients with cystic fibrosis, which differ substantially in their intracellular-survival ability in polymorphonuclear leukocytes (46). After exposure to hydrogen peroxide, a differential expression of the 1362, 987, and 705 genes was observed in strains TB, 892, and PAO1. The transcriptome data revealed a complex regulation of the cellular response to oxidative stress.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

In order to assess hydrogen peroxide-mediated oxidative stress, three P. aeruginosa strains were selected: TB (TBCF10839), isolated from the sputum of a CF patient, Sehnde, Germany, in 1984; 892 (TBCF121838), isolated from the sputum of a CF patient, Hannover, Germany, in 1984 (47); and PAO1, the genomic and genetic reference strain isolated from a burn wound of a patient in Melbourne, Australia (44). P. aeruginosa TB and 892 belong to the same clone (47). All bacterial cultures were grown in 15 ml of Luria broth at 37°C at 300 rpm in 125-ml Erlenmeyer flasks to a turbidity of 5.0 (stationary phase) (optical density at 600 nm). The cultures grown to stationary phase (3 × 1010 cells) were resuspended in fresh Luria broth and kept in a dialysis tube (14-kDa cutoff; tube diameter, 25 mm) with an effective length of 6 cm for the exchange of fluids. Then the dialysis tube was resuspended in a 1-liter Erlenmeyer flask containing 600 ml of Luria broth without or with 10 mM hydrogen peroxide. The flasks were incubated at 37°C on a rotary shaker at 200 rpm for 2 h. After the incubation period, bacteria were immediately recovered from the dialysis tube and subjected to RNA isolation.

RNA isolation.

The cells from the dialysis bags were recovered and harvested by centrifugation at 3,800 × g for 2 min at 4°C. Total RNA from approximately 3 × 1010 cells was extracted with a modified hot phenol method (45). Bacteria were quickly resuspended in 0.5 ml of distilled water and lysed in 7.5 ml of preheated (65°C) phenol-lysis buffer mix (5 ml of phenol [pH 5.5], 2.5 ml of 2% sodium dodecyl sulfate, 30 mM Na-acetate, 3 mM EDTA [pH 5.5]) with vigorous shaking for 10 min. The cell lysate was centrifuged at 3,800 × g for 20 min, and the supernatant was extracted with 3 ml of phenol-chloroform-isoamyl alcohol (25:24:1, vol/vol) and then subsequently with 3 ml of chloroform-isoamyl alcohol (24:1, vol/vol). To pellet the nucleic acids, 0.1 volume of 3 M Na-acetate (pH 5.2) and 2.5 volumes of ethanol were added and the mixture was incubated at −20°C overnight and centrifuged for 30 min at 3,800 × g. The pellet was washed with 5 ml of 70% ethanol and resuspended in 175 μl of diethyl pyrocarbonate-treated water. DNA was digested by the addition of 40 U of DNase I (Roche, Mannheim, Germany) and 20 U of SUPERaseIn (Ambion, Cambridgeshire, United Kingdom) in DNase I buffer (50 mM Na-acetate, 10 mM MgCl2, 2 mM CaCl2, pH 6.5) for 30 min at 37°C in a total volume of 200 μl. Then the RNA was purified with the use of RNeasy columns (QIAGEN, Hilden, Germany) according to the manufacturer's instructions, and the yield of total cellular RNA was quantified by measuring the light absorption at 260 nm. RNA with a size below 200 bp (e.g., tRNAs and 5S rRNA) is below the cutoff of the column and therefore could not be recovered. All the steps were carried out at 4°C, or RNA was also incubated on ice intermittently during the whole RNA isolation procedure. RNA samples were separated electrophoretically in 1.2% agarose with 2% formaldehyde as the denaturing reagent, and 1× MOPS (20 mM 4-morpholinepropanesulfonic acid, 10 mM Na-acetate, 1 mM EDTA, pH 7.0) buffer was used as the running buffer. The agarose gels were stained with ethidium bromide to check the purity and integrity of RNA preparation, with the 16S and 23S ribosomal bands as references.

Gene chip experiments and analyses.

cDNA synthesis, fragmentation, and labeling and P. aeruginosa PAO1 gene chip hybridization and washing were performed according to the instructions of the manufacturer (Affymetrix, Santa Clara, Calif.) and as described by Salunkhe et al. (39). For each gene chip hybridization experiment, the culturing of bacteria, exposure to hydrogen peroxide, and subsequent RNA isolation were performed in duplicate on the same day, and the RNA preparations were then pooled for cDNA synthesis and hybridization onto a single GeneChip. A total of two GeneChips per strain and growth condition were scanned at 570 nm with a 3-μm resolution by the Affymetrix scanner. Data analysis was performed using Affymetrix Microarray Suite software 5.0 with Affymetrix default parameters. The average microarray hybridization signal intensity was scaled to 150. Two GeneChips per strain for growth in the presence and absence of hydrogen peroxide, respectively, were compared by the four-comparison survival method (2, 5). The data were imported into a Microsoft Access database capable of searching for genes, which were found in all four pairings that were (i) at least twofold regulated and (ii) defined by the Affymetrix Microarray Suite Software as having significant changes in their signal intensities. The arithmetic average and the standard deviation were calculated. Finally, a Bonferroni correction for multiple testing was applied as a rigorous criterion for significantly changed signal intensities (39). Data were combined with the annotation (dated 15 December 2004) from the website of the P. aeruginosa PAO1 sequence and the community annotation project provided at http://www.pseudomonas.com. Information about all significantly differentially expressed PAO genes in the three investigated strains is available at the authors' personal website (http://www.mh-hannover.de/kliniken/kinderheilkunde/kfg/pub.htm).

RESULTS AND DISCUSSION

Global microarray data analysis of the response of P. aeruginosa to hydrogen peroxide.

DNA microarrays were used to study the global expression profile of P. aeruginosa in response to H2O2-mediated oxidative stress. Bacterial cultures grown in Luria broth to stationary phase were placed into a dialysis bag that was then placed in a 100-fold-larger surrounding volume of broth containing a bactericidal concentration of 10 mM hydrogen peroxide. Hence, a large discontinuous concentration gradient was established at the barrier of the dialysis membrane, generating a constant flow of hydrogen peroxide into the bag. By this procedure we allowed for an initial diffusion-controlled asymptotic increase in the H2O2 concentration until the steady state of influx and decomposition of H2O2 was established. This approach enabled most of the 3 × 1010 bacteria to adapt to the H2O2 stress and to remain viable. Prior to and after the 2-h exposure to 10 mM H2O2, numbers of CFU were the same (within the experimental error) for all three strains analyzed in this study, although a statistically nonsignificant trend to lower cell numbers was noted. In previous studies, the bacteria were typically directly exposed to H2O2 (16, 34, 36, 38, 52). The absence of any lag phase is of course a stronger perturbation of cell homeostasis and, depending on the H2O2 concentration, will evoke an immediate response ranging from virtually no reaction to 100% killing within seconds that is difficult to control. The separation of the bacteria from the stressor by a diffusion barrier enabled us to select a lethal concentration of H2O2 and hence to induce the maximal stress response in the bacteria without killing them. Moreover, H2O2 is notoriously unstable in aqueous solutions at 37°C, and although we started with 10 mM, a concentration of 7 to 8 mM, which is still lethal, was measured after 2 h. Hence, our experimental setting combined an initial asymptotic increase in stressor concentration with stationary maximal stress conditions thereafter. In summary, at the time point of 2 h, we could identify the specific steady-state response of P. aeruginosa to hydrogen peroxide.

According to the GeneChip analyses of the microarrays, the Pearson correlation coefficients were 0.98, 0.95, and 0.98 for the hydrogen peroxide-treated replicates and 0.45, 0.44, and 0.61 between hydrogen peroxide-treated and untreated control samples of TB, 892, and PAO1, respectively. The very low interexperimental variance of the results from replicates is reflected by the fact that after application of the Bonferroni correction for multiple testing of 5,900 independent items, all values indicating a 1.9-fold change or more in mRNA levels between H2O2-treated and untreated preparations from the same strain were classified as a significant change. After exposure for 2 h to hydrogen peroxide, 24, 17, and 12% of the 5,900 open reading frames were significantly differentially regulated in P. aeruginosa TB, 892, and PAO1, respectively. In total, 694, 411, and 237 genes were significantly upregulated and 668, 576, and 468 genes were significantly downregulated in hydrogen peroxide-treated TB, 892, and PAO1 than in the untreated controls. Interestingly, the number of genes classified by the software as expressed differed by strain. Whereas more than 70% of genes were expressed by the TB and 892 strains in the presence or absence of hydrogen peroxide, the PAO1 strain expressed only 64% of genes in Luria broth medium. Exposure to hydrogen peroxide increased the proportion of expressed genes to 72%. Moreover, only 441 genes were consistently differentially expressed in the three strains. Seven hundred twenty-nine genes showed strain-specific regulation, and 501 genes were similarly regulated in two strains (Fig. (Fig.1).1). The expression profiles of the two TB clones were significantly more related to each other than they were to that of PAO1 (P < 0.001). The 892 strain shared 84 and 88% of up- and downregulated genes with its more virulent clonal variant TB, which exhibited a more strain-specific expression profile, with 50 and 76% of up- and downregulated genes being held in common with 892. It is noteworthy that strains TB and 892 exhibit identical informative single-nucleotide-polymorphism genotypes (31) and highly related SpeI and DraI macrorestriction fragment patterns (47). No nucleotide substitution was identified during comparative sequencing of 50 kb, and the sum of pulsed-field gel electrophoresis-separated SpeI fragment sizes yielded the same estimate of genome size (data not shown). The proteomes of the two strains are 96% identical under standard growth conditions in vitro (1). Nevertheless, the global mRNA expression profiles of the two strains were highly divergent when the strains were cultivated simultaneously under three different conditions (39; this work). In other words, genetically very closely related P. aeruginosa strains of matching genome sizes present strain-specific mRNA expression profiles that under carefully controlled identical conditions are highly reproducible but distinct from those of variants of the same clone. These individual signatures impede any generalizations about mRNA expression and gene regulation of the taxon. Hence, we chose two related isolates of a clone and the unrelated genetic reference strain PAO1 to get a clue about the relative impact of strain- and clone-specific responses versus general responses of P. aeruginosa.

FIG. 1.
Differential gene expression in response to hydrogen peroxide in the analyzed P. aeruginosa strains PAO1, 892, and TB. The number of genes showing increased or decreased expression and their levels of induction or repression are indicated.

The differentially regulated genes were classified by metabolic category (44). Genes encoding transporters and elements of amino acid metabolism were significantly overrepresented among the more strongly expressed genes in the presence of hydrogen peroxide, whereas the categories of chemotaxis, energy metabolism, and putative enzymes contained the relatively largest proportions of downregulated genes. The TB strain exhibited the largest number of upregulated genes in all but one category, whereas the patterns of downregulated genes were more similar among the three tested strains.

Major stress responses and changes in metabolic pathways to H2O2 treatment.

Table Table11 highlights the genes that changed consistently more than 10-fold in all three strains. Exposure to H2O2 activated the expression of the cyoABCD operon, which is key for aerobic respiration (6, 7), and of numerous determinants of the known elements of the oxidative-stress response (Table (Table2).2). The strongest induction was observed for the catalase KatB (PA4613), followed by alkyl hydroperoxide reductase AhpF (PA0140) and the thioredoxin reductase 2 operon (PA0848-PA0849). Constitutive high expression that increased by about a further 50% was observed for AhpC (PA0139), KatA (PA4236), and the superoxide dismutase SodB (PA4366). On the other hand, as already reported for the 10-min exposure to H2O2 (36), the OxyR-regulated genes ahpB, katE, katN, and soxR (34, 42, 43) were not induced by H2O2. Ohr, which directly detoxifies organic and inorganic hydroperoxides to less toxic metabolites (25), was 4- to 10-fold upregulated during continuous exposure to H2O2 (Table (Table22).

TABLE 1.
Genes exhibiting a >10-fold change in mRNA levels in all three analyzed P. aeruginosa strains after exposure to hydrogen peroxide
TABLE 2.
Expression of mRNA transcripts known to be involved in the oxidative stress response

The oxidative-stress response is intimately linked with iron homeostasis. Fe2+ is essential for bacterial growth but also produces hydroxyl radicals from H2O2 that are regarded as the most potent nonselective oxidizing agent in biological systems (30). The bacteria managed to control iron homeostasis by increased expression of genes encoding the biosynthesis enzymes and receptor for the siderophore pyochelin (PA4221-PA4231, 3- to 26-fold upregulated), the hemin receptor (PA4664, 3-fold upregulated), and the HitAB iron binding and transport system (PA4687-PA4688, 2- to 8-fold upregulated), and they controlled the downregulation of bacterioferritins (2- to 9-fold downregulated). Moreover, individual strains upregulated heme biosynthesis, iron-sulfur proteins, and iron transport systems. Another important element affected by oxidative stress is sulfur. On one hand, free thiol groups are oxidized to disulfide bonds, and on the other, sulfur is an essential part of iron-sulfur proteins that are particularly prone to oxidative damage (23). In particular, the two strains of the TB clone were proficient in activating the sulfate transport operon cysAWT, the sulfate binding proteins Sbp and CysP, sulfite reductase CysI, and the ATP sulfurylase CysND to supply sulfur to the cell. Sulfur was kept as reduced thiol by high levels of thioredoxin reductase.

Besides the direct impact of H2O2 on the activation of direct defense mechanisms, the strong stationary exposure to this detrimental agent caused responses in primary metabolism and signal transduction. Numerous transcriptional regulators became differentially expressed; in particular, two-component systems associated with chemotaxis were downregulated, e.g., PA0174-PA0180 (7- to 25-fold) and PA2571-PA2573 (4- to 28-fold). Most enzymes necessary for the conversion of amino acids into the citric acid cycle were globally downregulated at the transcriptional level. Nitrate respiration and arginine fermentation were shut off in the clone TB strains and attenuated in the PAO strain. Table Table33 lists these strikingly different levels of 2- to 500-fold downregulation of mRNA transcripts of the operons PA0509-PA0517, PA0523-PA0525, PA3391-PA3394, PA3870-PA3877, and PA5171-PA5173, which represent the major pathways of anaerobic energy metabolism in P. aeruginosa (53). Anaerobic respiration requires molybdenum as a cofactor for catalytic activity (53), and correspondingly, the elements for molybdenum transport and molybdopterin biosynthesis were significantly less expressed in the presence of H2O2.

TABLE 3.
Expression of mRNA transcripts involved in microaerophilic and anaerobic growth

Reactive oxygen intermediates cause damage to nucleic acids and proteins. Accordingly, numerous elements of DNA repair, such as RecA, RecR, LexA, MutS, and DnaX, were upregulated at the transcriptional level. Protein biosynthesis was maintained by stronger expression of mRNAs for ribosomal proteins and aminoacyl-tRNA-synthetases, and in parallel, the ribosome modulation factor that inactivates the ribosome (51) was downregulated. Several genes encoding prolyl-peptidyl isomerases were more strongly transcribed to reverse damage by misfolding. clpA protease genes were less transcribed to ensure a decreased rate of protein degradation. In summary, these data indicate that the bacteria tried to keep the mutation rate and the loss of protein to a minimum.

Exposure to H2O2 also affected secondary metabolism and major pathways of bacterial virulence. The three tested P. aeruginosa strains exhibited lower mRNA levels of genes encoding the biosynthesis of the PQS signal (37), effector proteins of type I and type II secretion (24), and extracellular polymers, such as exopolysaccharides (29) and polyhydroxyalkanoic acid (21).

Besides these responses common to all three investigated strains, multiple clone- and strain-specific responses were observed (Fig. (Fig.2).2). Each strain activated other members of the extracytoplasmic-function σ70 family of global transcription factors (from strain PAO1, PA0472; from TB, PA1300; and from 892, PA2387 and PA3285) (28). Strain PAO1, but not the clone TB strains, induced the expression of the FtsYE cell division operon (PA0373-PA0374) and reduced the expression of the global transcriptional regulator GacA and numerous elements in the degradation and secondary metabolism of aromatic amino acids. The expression of a total of 13 ABC transporters, symporters, efflux systems, and permeases was downregulated in strain PAO1. With respect to the expression of transporters, an opposite response of the two clone TB strains was observed. The TB and 892 strains increased the mRNA levels for 23 different transport systems, including five ABC transporters, the MexEFOprN efflux pump, the Na+/H+ antiporter, and transporters or permeases for citrate, lactate, and amino acids.

FIG. 2.
PAO1 genes with significantly changed expression levels in hydrogen peroxide-treated P. aeruginosa TB, 892, and PAO1. Genes that were found to be up- or downregulated in two or three strains are indicated in the intersections.

Exposure to H2O2 had a major impact on energy metabolism at the transcriptional level. Levels of cytochrome c oxidase mRNA transcripts (PA0105-PA0108 and PA1555-PA1557) in one of the terminal energy-transducing respiratory complexes were moderately (PA1555-PA1557, 2- to 13-fold) or strongly (PA0105-PA0108, 7- to 50-fold) reduced in the presence of H2O2. Alternative routes of anaerobic energy metabolism to synthesize ATP were also downregulated. Strain PAO reduced the concentration of all mRNA species of nitrate respiration. Strains 892 and TB reacted even more strongly to H2O2 by complete elimination of nitrate respiration and acetate and arginine fermentation. As alternatives for ATP synthesis, two systems were upregulated: the cyoABCDE operon (PA1317-PA1321), encoding cytochrome o ubiquinol oxidase, the main terminal oxidase of the electron transport chain under highly aerobic conditions (14), and the nqrFEDCBA operon (PA2994-PA2999), encoding the Na+-translocating NADH-quinone oxidoreductase (3, 12), which can probably function as complex I of the respiratory chain, by which it pumps sodium ions instead of protons (12).

Comparison with published transcriptome studies of the response of P. aeruginosa to oxidative stress.

Exposure to hydrogen peroxide influenced the expression of hundreds of P. aeruginosa genes belonging to numerous metabolic categories. The damaging hydroxyl radicals are produced not only by decomposition of hydrogen peroxide but also by superoxide. The superoxide anion is an unavoidable by-product of aerobic metabolism (22). It leaches free iron from iron-sulfur clusters and promotes hydroxyl-radical formation and consequent DNA damage. We previously measured the genome-wide transcription profile against the superoxide-generating agent paraquat in the same P. aeruginosa strains employed in this study, PAO1, TB, and 892 (39). One operon of conserved hypotheticals (PA0939-PA0942) was consistently 20- to 200-fold upregulated in the three strains, but otherwise only 24 to 29 subtle strain-specific changes in the oxidative-stress response (ahpF), energy metabolism (cox operon), iron-responsive elements, and transport were noted. Of these strain-specific responses, all of the 17 paraquat-repressed genes and 13 of the 38 paraquat-induced genes were also down- and upregulated by the exposure to hydrogen peroxide. Hence, paraquat modulates a small subset of the genes that are differentially regulated by hydrogen peroxide. However, with the exception of a single operon, no changes in the mRNA expression pattern common to all three strains occurred when the bacteria were exposed to 0.5 mM paraquat for 2 h under the same experimental conditions as applied here. We would like to conclude that in our experimental setup, hydrogen peroxide with its massive production of hydroxyl radicals and oxygen more strongly affects metabolism and signaling in P. aeruginosa than the superoxide anion.

We report here on the steady-state response to hydrogen peroxide, whereas Palma and colleagues (36) have analyzed the global transcriptional profile of the early adaptation phase. P. aeruginosa PAO1 bacteria were directly exposed to a sublethal concentration of 1 mM hydrogen peroxide for 10 min. Only 60 of 2,525 genes detected as significantly differentially regulated in one or the other study were consistently up- or downregulated in both transcriptome analyses (Fig. (Fig.3).3). Two hundred eleven genes were discordantly regulated in the two studies. All genes but the cyo operon that were found to be consistently upregulated in our study by more than 10-fold in all three strains (Table (Table1)1) were also reported by Palma et al. (36) to be strongly activated. The key players of an inducible oxidative-stress response, such as alkylhydroperoxide reductase, catalase, and thioredoxin reductase (Table (Table2),2), were highly expressed in the PAO1 strain under both experimental conditions. In contrast, the overlap of the subset of the 55 consistently >10-fold-downregulated genes in our study with the complete set of 1,080 downregulated PAO1 genes in Palma's study is nil. The low similarity of the transcriptome profiles suggests that P. aeruginosa copes differently with hydrogen peroxide-induced stress during the initial adaptation and the later stationary phase. However, as already outlined in an article in this journal (49) on the quorum-sensing transcriptome of PAO1 (41, 50), differential handling and growth conditions of PAO1 cells and differences in the genotypes and phenotypes of the PAO1 strain (44) may also account for divergent mRNA transcription data. Hence, we analyzed in parallel three strains in order to differentiate between common responses and clone- and strain-specific responses and thereby to identify the signature of the species-typical response to the stressor (Fig. (Fig.2).2). To date when independent investigators have studied the PAO1 transcriptome with the same or similar objectives, discordant profiles have been obtained, and we recommend for future transcriptome studies that PAO1 and at least two strains of another clone always be incorporated in order to estimate more reliably inherent and experimental variances.

FIG. 3.
Global comparison of the genes identified to be differentially regulated upon exposure to hydrogen peroxide by Palma et al. (36) and in this study.

Acknowledgments

Provision of reference RNA transcripts by Steve Lory, Boston, Mass., is gratefully acknowledged.

Financial support by the European Union (contract QL K2-CT-2001-01339 to B.T.) is gratefully acknowledged. Subsidized PAO1 genome arrays were made available by the Cystic Fibrosis Foundation and Mukoviszidose e.V. P.S. has been a recipient of a predoctoral stipend provided by the DFG-sponsored European Graduate School program “Pseudomonas: Pathogenicity and Biotechnology.”

REFERENCES

1. Arevalo-Ferro, C., J. Buschmann, G. Reil, A. Gorg, L. Wiehlmann, B. Tummler, L. Eberl, and K. Riedel. 2004. Proteome analysis of intraclonal diversity of two Pseudomonas aeruginosa TB clone isolates. Proteomics 4:1241-1246. [PubMed]
2. Bakay, M., Y. W. Chen, R. Borup, P. Zhao, K. Nagaraju, and E. P. Hoffman. 2002. Sources of variability and effect of experimental approach on expression profiling data interpretation. BMC Bioinform. 3:4. [PMC free article] [PubMed]
3. Barquera, B., W. Zhou, J. E. Morgan, and R. B. Gennis. 2002. Riboflavin is a component of the Na+-pumping NADH-quinone oxidoreductase from Vibrio cholerae. Proc. Natl. Acad. Sci. USA 99:10322-10324. [PMC free article] [PubMed]
4. Brown, S. M., M. L. Howell, M. L. Vasil, A. J. Anderson, and D. J. Hassett. 1995. Cloning and characterization of the katB gene of Pseudomonas aeruginosa encoding a hydrogen peroxide-inducible catalase: purification of KatB, cellular localization, and demonstration that it is essential for optimal resistance to hydrogen peroxide. J. Bacteriol. 177:6536-6544. [PMC free article] [PubMed]
5. Chen, Y. W., P. Zhao, R. Borup, and E. P. Hoffman. 2000. Expression profiling in the muscular dystrophies: identification of novel aspects of molecular pathophysiology. J. Cell Biol. 151:1321-1336. [PMC free article] [PubMed]
6. Cotter, P. A., V. Chepuri, R. B. Gennis, and R. P. Gunsalus. 1990. Cytochrome o (cyoABCDE) and d (cydAB) oxidase gene expression in Escherichia coli is regulated by oxygen, pH, and the fnr gene product. J. Bacteriol. 172:6333-6338. [PMC free article] [PubMed]
7. Cotter, P. A., S. B. Melville, J. A. Albrecht, and R. P. Gunsalus. 1997. Aerobic regulation of cytochrome d oxidase (cydAB) operon expression in Escherichia coli: roles of Fnr and ArcA in repression and activation. Mol. Microbiol. 25:605-615. [PubMed]
8. Dasgupta, N., M. C. Wolfgang, A. L. Goodman, S. K. Arora, J. Jyot, S. Lory, and R. Ramphal. 2003. A four-tiered transcriptional regulatory circuit controls flagellar biogenesis in Pseudomonas aeruginosa. Mol. Microbiol. 50:809-824. [PubMed]
9. Döring, G., R. Knight, and G. Bellon. 1995. Immunology of cystic fibrosis, p. 99-129. In M. E. Hodson and D. Geddes (ed.), Cystic fibrosis. Chapmen and Hall, London, United Kingdom.
10. Firoved, A. M., and V. Deretic. 2003. Microarray analysis of global gene expression in mucoid Pseudomonas aeruginosa. J. Bacteriol. 185:1071-1081. [PMC free article] [PubMed]
11. Frisk, A., J. R. Schurr, G. Wang, D. C. Bertucci, L. Marrero, S. H. Hwang, D. J. Hassett, and M. J. Schurr. 2004. Transcriptome analysis of Pseudomonas aeruginosa after interaction with human airway epithelial cells. Infect. Immun. 72:5433-5438. [PMC free article] [PubMed]
12. Gemperli, A. C., P. Dimroth, and J. Steuber. 2003. Sodium ion cycling mediates energy coupling between complex I and ATP synthase. Proc. Natl. Acad. Sci. USA 100:839-844. [PMC free article] [PubMed]
13. Gilligan, P. H. 1991. Microbiology of airway disease in patients with cystic fibrosis. Clin. Microbiol. Rev. 4:35-51. [PMC free article] [PubMed]
14. Gohlke, U., A. Warne, and M. Saraste. 1997. Projection structure of the cytochrome bo ubiquinol oxidase from Escherichia coli at 6 Å resolution. EMBO J. 16:1181-1188. [PMC free article] [PubMed]
15. Govan, J. R., and V. Deretic. 1996. Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol. Rev. 60:539-574. [PMC free article] [PubMed]
16. Hassett, D. J., E. Alsabbagh, K. Parvatiyar, M. L. Howell, R. W. Wilmott, and U. A. Ochsner. 2000. A protease-resistant catalase, KatA, released upon cell lysis during stationary phase is essential for aerobic survival of a Pseudomonas aeruginosa oxyR mutant at low cell densities. J. Bacteriol. 182:4557-4563. [PMC free article] [PubMed]
17. Hassett, D. J., and M. S. Cohen. 1989. Bacterial adaptation to oxidative stress: implications for pathogenesis and interaction with phagocytic cells. FASEB J. 3:2574-2582. [PubMed]
18. Hassett, D. J., H. P. Schweizer, and D. E. Ohman. 1995. Pseudomonas aeruginosa sodA and sodB mutants defective in manganese- and iron-cofactored superoxide dismutase activity demonstrate the importance of the iron-cofactored form in aerobic metabolism. J. Bacteriol. 177:6330-6337. [PMC free article] [PubMed]
19. Hassett, D. J., P. A. Sokol, M. L. Howell, J. F. Ma, H. T. Schweizer, U. Ochsner, and M. L. Vasil. 1996. Ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa demonstrate defective siderophore-mediated iron uptake, altered aerobic growth, and decreased superoxide dismutase and catalase activities. J. Bacteriol. 178:3996-4003. [PMC free article] [PubMed]
20. Hassett, D. J., W. A. Woodruff, D. J. Wozniak, M. L. Vasil, M. S. Cohen, and D. E. Ohman. 1993. Cloning and characterization of the Pseudomonas aeruginosa sodA and sodB genes encoding manganese- and iron-cofactored superoxide dismutase: demonstration of increased manganese superoxide dismutase activity in alginate-producing bacteria. J. Bacteriol. 175:7658-7665. [PMC free article] [PubMed]
21. Hoffmann, N., A. Steinbuchel, and B. H. Rehm. 2000. The Pseudomonas aeruginosa phaG gene product is involved in the synthesis of polyhydroxyalkanoic acid consisting of medium-chain-length constituents from non-related carbon sources. FEMS Microbiol. Lett. 184:253-259. [PubMed]
22. Keyer, K., and J. A. Imlay. 1996. Superoxide accelerates DNA damage by elevating free-iron levels. Proc. Natl. Acad. Sci. USA 93:13635-13640. [PMC free article] [PubMed]
23. Keyer, K., and J. A. Imlay. 1997. Inactivation of dehydratase [4Fe-4S] clusters and disruption of iron homeostasis upon cell exposure to peroxynitrite. J. Biol. Chem. 272:27652-27659. [PubMed]
24. Lazdunski, A. M., I. Ventre, and J. N. Sturgis. 2004. Regulatory circuits and communication in Gram-negative bacteria. Nat. Rev. Microbiol. 2:581-592. [PubMed]
25. Lesniak, J., W. A. Barton, and D. B. Nikolov. 2002. Structural and functional characterization of the Pseudomonas hydroperoxide resistance protein Ohr. EMBO J. 21:6649-6659. [PMC free article] [PubMed]
26. Lyczak, J. B., C. L. Cannon, and G. B. Pier. 2002. Lung infections associated with cystic fibrosis. Clin. Microbiol. Rev. 15:194-222. [PMC free article] [PubMed]
27. Ma, J. F., U. A. Ochsner, M. G. Klotz, V. K. Nanayakkara, M. L. Howell, Z. Johnson, J. E. Posey, M. L. Vasil, J. J. Monaco, and D. J. Hassett. 1999. Bacterioferritin A modulates catalase A (KatA) activity and resistance to hydrogen peroxide in Pseudomonas aeruginosa. J. Bacteriol. 181:3730-3742. [PMC free article] [PubMed]
28. Martinez-Bueno, M. A., R. Tobes, M. Rey, and J. L. Ramos. 2002. Detection of multiple extracytoplasmic function (ECF) sigma factors in the genome of Pseudomonas putida KT2440 and their counterparts in Pseudomonas aeruginosa PA01. Environ. Microbiol. 4:842-855. [PubMed]
29. May, T. B., and A. M. Chakrabarty. 1994. Pseudomonas aeruginosa: genes and enzymes of alginate synthesis. Trends Microbiol. 2:151-157. [PubMed]
30. Miller, R. A., and B. E. Britigan. 1997. Role of oxidants in microbial pathophysiology. Clin. Microbiol. Rev. 1:1-18. [PMC free article] [PubMed]
31. Morales, G., L. Wiehlmann, P. Gudowius, C. van Delden, B. Tummler, J. L. Martinez, and F. Rojo. 2004. Structure of Pseudomonas aeruginosa populations analyzed by single-nucleotide polymorphism and pulsed-field gel electrophoresis genotyping. J. Bacteriol. 186:4228-4237. [PMC free article] [PubMed]
32. Ochsner, U. A., D. J. Hassett, and M. L. Vasil. 2001. Genetic and physiological characterization of ohr, encoding a protein involved in organic hydroperoxide resistance in Pseudomonas aeruginosa. J. Bacteriol. 183:773-778. [PMC free article] [PubMed]
33. Ochsner, U. A., P. J. Wilderman, A. I. Vasil, and M. L. Vasil. 2002. GeneChip expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genes. Mol. Microbiol. 45:1277-1287. [PubMed]
34. Ochsner, U. A., M. L. Vasil, E. Alsabbagh, K. Parvatiyar, and D. J. Hassett. 2000. Role of the Pseudomonas aeruginosa oxyR-recG operon in oxidative stress defense and DNA repair: OxyR-dependent regulation of katB-ankB, ahpB, and ahpC-ahpF. J. Bacteriol. 182:4533-4544. [PMC free article] [PubMed]
35. Ojeniyi, B., B. Frederiksen, and N. Hoiby. 2000. Pseudomonas aeruginosa cross-infection among patients with cystic fibrosis during a winter camp. Pediatr. Pulmonol. 3:177-181. [PubMed]
36. Palma, M., D. DeLuca, S. Worgall, and L. E. N. Quadri. 2004. Transcriptome analysis of the response of Pseudomonas aeruginosa to hydrogen peroxide. J. Bacteriol. 186:248-252. [PMC free article] [PubMed]
37. Pesci, E. C., J. B. Milbank, J. P. Pearson, S. McKnight, A. S. Kende, E. P. Greenberg, and B. H. Iglewski. 1999. Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 96:11229-11234. [PMC free article] [PubMed]
38. Prieto-Alamo, M. J., J. Jurado, R. Gallardo-Madueno, F. Monje-Casas, A. Holmgren, and C. Pueyo. 2000. Transcriptional regulation of glutaredoxin and thioredoxin pathways and related enzymes in response to oxidative stress. J. Biol. Chem. 275:13398-13405. [PubMed]
39. Salunkhe, P., F. Götz, L. Wiehlmann, J. Lauber, J. Buer, and B. Tümmler. 2002. GeneChip expression analysis of the response of Pseudomonas aeruginosa to paraquat-induced superoxide stress. Genome Lett. 1:165-174.
40. Sauer, K., M. C. Cullen, A. H. Rickard, L. A. Zeef, D. G. Davies, and P. Gilbert. 2004. Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm. J. Bacteriol. 186:7312-7326. [PMC free article] [PubMed]
41. Schuster, M., C. P. Lostroh, T. Ogi, and E. P. Greenberg. 2003. Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J. Bacteriol. 185:2066-2079. [PMC free article] [PubMed]
42. Seaver, L. C., and J. A. Imlay. 2001. Alkyl hydroperoxide reductase is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli. J. Bacteriol. 183:7173-7181. [PMC free article] [PubMed]
43. Storz, G., L. A. Tartaglia, and B. N. Ames. 1990. Transcriptional regulator of oxidative stress-inducible genes: direct activation by oxidation. Science 248:189-194. [PubMed]
44. Stover, C. K., X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey, F. S. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, T. L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L. Brody, S. N. Coulter, K. R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D. Spencer, G. K. Wong, Z. Wu, I. T. Paulsen, J. Reizer, M. H. Saier, R. E. Hancock, S. Lory, and M. V. Olson. 2000. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406:959-964. [PubMed]
45. Tao, H., C. Bausch, C. Richmond, F. R. Blattner, and T. Conway. 1999. Functional genomics: expression analysis of Escherichia coli growing on minimal and rich media. J. Bacteriol. 181:6425-6440. [PMC free article] [PubMed]
46. Tümmler, B. 1987. Unusual mechanism of pathogenicity of Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Infection 15:311-312. [PubMed]
47. Tümmler, B., U. Koopmann, D. Grothues, H. Weissbrodt, G. Steinkamp, and H. von der Hardt. 1991. Nosocomial acquisition of Pseudomonas aeruginosa by cystic fibrosis patients. J. Clin. Microbiol. 29:1265-1267. [PMC free article] [PubMed]
48. Van Delden, C., and B. H. Iglewski. 1998. Cell-to-cell signaling and Pseudomonas aeruginosa infections. Emerg. Infect. Dis. 4:551-560. [PMC free article] [PubMed]
49. Vasil, M. L. 2003. DNA microarrays in analysis of quorum sensing: strengths and limitations. J. Bacteriol. 185:2061-2065. [PMC free article] [PubMed]
50. Wagner, V. E., D. Bushnell, L. Passador, A. I. Brooks, and B. H. Iglewski. 2003. Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment. J. Bacteriol. 185:2080-2095. [PMC free article] [PubMed]
51. Yoshida, H., H., Yamamoto, T. Uchiumi, and A. Wada. 2004. RMF inactivates ribosomes by covering the peptidyl transferase centre and entrance of peptide exit tunnel. Genes Cells 9:271-278. [PubMed]
52. Zheng, M., X. Wang, L. J. Templeton, D. R. Smulski, R. A. LaRossa, and G. Storz. 2001. DNA microarray-mediated transcriptional profiling of the Escherichia coli response to hydrogen peroxide. J. Bacteriol. 183:4562-4570. [PMC free article] [PubMed]
53. Zumft, W. G. 1997. Cell biology and molecular basis of denitrification. Microbiol. Mol. Biol. Rev. 61:533-616. [PMC free article] [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...