Marker genes for the metabolic adaptation of Pseudomonas aeruginosa to the hypoxic cystic fibrosis lung environment.

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Marker genes for the metabolic adaptation of Pseudomonas aeruginosa to the hypoxic cystic fibrosis lung environment Anja Eichner, Nicole Günther, Martin Arnold, Max Schobert, Jürgen Heesemann, Michael Hogardt ∗ Johann Wolfgang Goethe-University, Frankfurt/Main, Germany

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Article history: Received 9 March 2014 Received in revised form 13 June 2014 Accepted 21 July 2014 Keywords: Pseudomonas aeruginosa Cystic fibrosis Oxygen-limitation Metabolic adaptation Artificial sputum medium Isocitrate dehydrogenase

a b s t r a c t Pseudomonas aeruginosa is the leading pathogen of chronic cystic fibrosis (CF) lung infection. Life-long persistence in the inflamed and ever fluctuating CF lungs results in the selection of a variety of changes in P. aeruginosa physiology. Accumulating evidence suggests that especially metabolic changes support the survival and growth of P. aeruginosa within the hypoxic and nutritious CF mucus. To investigate if metabolic adaptations we described for hypermutable P. aeruginosa from late CF lung disease (Hoboth et al., 2009. J. Infect. Dis., pp. 118–130) may represent specific changes in response to the selective conditions within the oxygen-restricted CF mucus, we determined the expression of a set of genes during aerobic and hypoxic growth in LB and the artificial sputum medium ASM. We further focused on the regulation of the two isocitrate dehydrogenases Icd and Idh. Interestingly, both isoenzymes may replace each other under aerobic and hypoxic conditions. The NADPH- and RpoS-dependent Icd seems to be the leading isoenzyme under prolonged oxygen limitation and stationary growth phase. LacZ reporter analysis revealed that oxygen-restriction increased the expression levels of azu, cbb3-1, cbb3-2, ccpR, icd, idh and oprF gene, whereas himD and nuoA are increasingly expressed only during hypoxic growth in ASM. Overexpression of the anaerobic regulator Anr improved the expression of azu, ccpR, cbb3-2 and icd. In summary, expression of azu, cbb3-1, cbb3-2, ccpR, icd, idh, oprF, himD, and nuoA appeared to be beneficial for the growth of P. aeruginosa under hypoxic conditions indicating these genes may represent marker genes for the metabolic adaptation to the CF lung environment. © 2014 Elsevier GmbH. All rights reserved.

Introduction P. aeruginosa is a ubiquitous and highly versatile soil bacterium and an important opportunistic human pathogen. In line with this, P. aeruginosa may conquer a variety of different niches ranging from nutrient-poor water reservoirs to the hostile but quite nutritious habitats of the chronically infected cystic fibrosis (CF) airways. During lifelong survival in the CF lung, P. aeruginosa adapts to this heterogeneous environment in response to enduring selective challenges such as the inflammatory host defense, inflammatory/oxidative stresses, recurrent antibiotic treatments, oxygen restriction, interspecies competition as well as variations in pH, electrolyte concentration and nutrient availabilities (Hogardt

∗ Corresponding author. Institut für Medizinische Mikrobiologie und Krankenhaushygiene, Universitätsklinikum Frankfurt, Johann Wolfgang Goethe-Universität, Paul-Ehrlich-Str. 40, D-60596 Frankfurt/Main, Germany. Tel.: +49 0 69 6301 5945; fax: +49 0 69 6301 5767. E-mail address: [email protected] (M. Hogardt).

and Heesemann, 2012). Mucoidity, attenuation of virulence, multidrug resistance as well as an outstanding phenotypic and genotypic diversity characterize P. aeruginosa isolates recovered from chronic stages of CF lung disease. Additionally, accumulating evidence suggests that metabolic adaptations affecting biosynthetic processes, energy metabolism, uptake and utilization of nutrients are positively selected during lung persistence (Son et al., 2007; Hoboth et al., 2009; Döring et al., 2011; Palmer et al., 2005). P. aeruginosa may utilize a wide range of compounds such as carbohydrates, diand tri-carboxylic acids, fatty acids, mono- and polyalcohols and amino acids as carbon, nitrogen or energy sources. In the CF airways the accumulation of copious amounts of mucus and infiltrated neutrophils may serve as a source of nutrition for P. aeruginosa which probably benefits from adapting its metabolism to this nutritionaly richness. For chronic CF lung disease a high prevalence (30–60%) of hypermutable (or mutator) strains is characteristic. Due to defects in DNA mismatch repair pathways mutator strains exhibit up to 1000-fold increased spontaneous mutation rates (Oliver et al., 2000). Hypermutability is linked to the accelerated acquisition of mutations and

http://dx.doi.org/10.1016/j.ijmm.2014.07.014 1438-4221/© 2014 Elsevier GmbH. All rights reserved.

Please cite this article in press as: Eichner, A., et al., Marker genes for the metabolic adaptation of Pseudomonas aeruginosa to the hypoxic cystic fibrosis lung environment. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.07.014

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Fig. 1. Schematic representation of the tricarboxylic acid (TCA) cycle, glyoxylate shunt (A) and the respiratory chain (B). The TCA cycle provides precursors for biosynthetic processes and reducing factors (NADH and FADH). Isocitrate dehydrogenase Icd (dimeric) and Idh (monomeric) are located at the branch point of TCA cycle and (anaerobic glyoxylate shunt. The respiratory chain drives the generation of ATP via oxidative phosphorylation by using O2 (aerobic respiration) or N-oxides such as NO− 3 respiration/denitrification) as terminal electron acceptor. Electrons are transferred to the different oxidases/reductases via the ubiquinone pool or cytochrome bc1 complex. Aditionally, Ndh, NADH dehydrogenase; Azu, azurin; CoxBA, cytochrome c oxidase; Cbb3-1, cytochrome oxidase cbb3-1; Cbb3-2, cytochrome oxidase cbb3-2; CIO, cyanide insensitive oxidase; and CcpR, cytochrome C551 peroxidase are depicted. Enzymes of the denitrification pathway are not shown for the sake of clarity.

obviously confers an evolutionary advantage during adaptation of P. aeruginosa to the CF airways (Mena et al., 2008; Oliver, 2010). Recently, we have shown that P. aeruginosa mutator strains with a defect mutS gene as compared to non-mutator strains showed adaptations in various gene functions that are related e.g. to antibiotic resistance, virulence and metabolism (Hoboth et al., 2009; Son et al., 2007). Metabolic genes found to be up-regulated in late lungadapted mutator strains include those of tricarboxylic acid (TCA) cycle, lipid and amino acid metabolism. The increased expression of Anr-regulated genes such as the redox-active protein azurin (azu), the cytochrome peroxidase (ccpR), the high affinity cytochrome oxidase (cbb3-2) and genes of arginine deiminase pathway, indicate that oxygen restriction is indeed a dominant selective condition within the CF lungs (Vijgenboom et al., 1997; Alvarez-Ortega and Harwood, 2007). In line with this, we showed by RT-PCR that in endstage mutator strains the expression of the anr gene was increased (Hoboth et al., 2009). Anr (anaerobic regulator of arginine deiminase and nitrate reductase) belongs to the Fnr (fumarate and nitrate reductase regulator) family of transcriptional regulators that sense the oxygen tension by its [4Fe-4S]2+ cluster (Zimmermann et al., 1991; Winteler and Haas, 1996). Anoxic conditions induce the formation of the dimeric (active) form of Anr that binds to a conserved consensus sequence (5 -TTGATNNNNATCAA-3 ) located upstream of various P. aeruginosa promoters to regulate anaerobic gene transcription (Ye et al., 1995; Chen et al., 2006). Anr is also required for the transcription of the downstream regulator DNR (dissimilatory nitrate respiration regulator), that in the presence of N-oxides promotes the expression of the denitrification genes nir, nor and nos (Arai et al., 1995). Thus, increased expression of Anr or genetically fixed alterations in the Anr regulon may subsequently result in secondary adaptations of P. aeruginosa.

This study was aimed to verify if metabolic adaptations (increased expression as determined by proteome and/or transcriptome) of lung-selected CF mutator isolates are indeed related to oxygen-limitation and/or the composition of CF mucus. We therefore examined whether a set of genes with important metabolic functions, namely accB, atuA, cbb3 -oxidases PA1554 and PA1557, himD, icd, idh, lpdG, nuoA and oprF are differentially regulated under hypoxic conditions, during overexpression of Anr (to simulate the phenotype of CF mutators), and growth in the artificial sputum medium ASM (to simulate CF mucus composition). The coxBA gene encoding aerobic cytochrome oxidase was decreased in late mutator CF isolates and selected for control (Hoboth et al., 2009; Son et al., 2007). Further, for a more detailed analysis of Pseudomonas lung adaptation, we focused on the isocitrate dehydrogenase (IDH) isoenzymes Icd and Idh of P. aeruginosa for several reasons. Isocitrate dehydrogenase is a key metabolic enzyme that converts isocitrate to oxoglutarate during TCA cycle, but is also involved in energy generation (e.g. ATP), the production of intermediates for the biosynthesis of amino acids and provides glutamic acid. IDH further directs the carbon flux through TCA cycle and/or glyoxylate shunt and typically provides NADPH for biosynthetic processes, cellular replication and ATP synthesis by aerobic and anaerobic respiration/denitrification (Fig. 1A and B). Moreover, NADPH is pivotal to maintain the function of anti-oxidative enzymes such as catalase, superoxide dismutase (SOD) and glutathione peroxidase and therefore contributes to the maintenance of a reducing environment in aerobic organisms. Prokaryotic isocitrate dehydrogenases utilize either NADP [EC:1.1.1.42] or NAD [EC:1.1.1.41] as a cofactor and act as either monomers (Idh) or dimers (Icd). The much more common dimeric Icd is formed by two identical subunits of about 40–45 kDa and found in several bacterial species including


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Table 1 P. aeruginosa strains and plasmids used in this study. Strain or plasmid Strains P. aeruginosa PAO1 PA14 PAO1 SS24 PAO6261 PA14 Tn::icd PA14 Tn::idh PA14 Tn::rpoS PA14 Tn::aceK PAO1 (accB’-lacZ) PAO1 (atu’-lacZ) PAO1 (azu’-lacZ) PAO1 (cbb3-1 -lacZ) PAO1 (cbb3-2 -lacZ) PAO1 (cppR’-lacZ) PAO1 (coxBA’-lacZ) PAO1 (himD’-lacZ) PAO1 (icd’-lacZ) PAO1 (idh’-lacZ) PAO1 (lpdG’-lacZ) PAO1 (coxBA’-lacZ) PAO1 (nuoA’-lacZ) PAO1 (oprF’-lacZ) PAO1 (anr’-lacZ) Plasmids pRK2013 pBBR1MCS pBBRanr50 pMP220

Genotype or relevant characteristicsa

Reference or source

Wild-type Wild-type rpoS deletion mutant of PAO1; rpoS101::aacCI, GmR anr deletion mutant of PAO1 PA14 carrying MAR2xT7 transposon mutation in icd gene, GmR PA14 carrying MAR2xT7 transposon mutation in idh gene, GmR PA14 carrying MAR2xT7 transposon mutation in rpoS gene, GmR PA14 carrying MAR2xT7 transposon mutation in aceK gene, GmR PAO1 carrying transcriptional accB’-lacZ fusion PAO1 carrying transcriptional atu’-lacZ fusion PAO1 carrying transcriptional azu’-lacZ fusion PAO1 carrying transcriptional cbb3-1 -lacZ fusion PAO1 carrying transcriptional cbb3-2 -lacZ fusion PAO1 carrying transcriptional cppR’-lacZ fusion PAO1 carrying transcriptional coxBA’-lacZ fusion PAO1 carrying transcriptional himD’-lacZ fusion PAO1 carrying transcriptional icd’-lacZ fusion PAO1 carrying transcriptional idh’-lacZ fusion PAO1 carrying transcriptional lpdG’-lacZ fusion PAO1 carrying transcriptional coxBA’-lacZ fusion PAO1 carrying transcriptional nuoA’-lacZ fusion PAO1 carrying transcriptional oprF’-lacZ fusion PAO1 carrying transcriptional anr’-lacZ fusion

Suh et al. (1999) Liberati et al. (2006) Suh et al. (1999) Ye et al. (1995) Liberati et al. (2006) Liberati et al. (2006) Liberati et al. (2006) Liberati et al. (2006) This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study

Helper plasmid, KmR , tra+ , mob+ , CarbR , Expression vector, tra- , rep+ , mob+ , CmR (50 copies/cell) pBBR1MCS containing a 1155 bp fragment of anr, CmR Promotorless lacZ reporter plasmid, oriT+ , IncP, TcR

Figurski and Helinski (1979) Kovach et al. (1994) This study Spaink et al. (1987)

a TcR , tetracycline resistance; CarbR , carbenicillin resistance; CmR , chloramphenicol resistance; KmR , kanamycin resistance; GmR , gentamicin resistance tra+ , tranfer genes; rep+ replication genes; mob+ , conjugative mobilization genes; MCS, multiple cloning site.

Escherichia coli (Burke et al., 1974). In contrast, Corynebacterium glutamicum, Azotobacter vinelandii and Streptomyces avermitilis (Eikmanns et al., 1995; Sahara et al., 2002; Wang et al., 2011) possess only the monomeric Idh with a molecular weight between 80 and 100 kDa. Except for P. aeruginosa only very few species such as Ralstonia eutropha, Collwellia spp., Mycobacterium tuberculosis and several species from the genus Pseudomonas. are endowed with both IDH isoenzymes (Banerjee et al., 2005; Ishii et al., 1987; Maki et al., 2006; Wang et al., 2003). In P. aeruginosa PAO1 and PA14 (http//:www.pseudomonas.com) the icd gene (PA2623 and PA 30190, respectively) has a 1257 bp sequence that codes for a 418 aa subunit of about 45.5 kDa. The idh gene (PA2624 and PA 30180, respectively) which is arranged opposite to icd, encodes for a monomeric 2226 bp gene product of 81.6 kDa (Winsor et al., 2011). These differences in structure, function and distribution suggest that different types of IDH enzymes evolved different catalytic activities and mechanisms of regulation. So far, for P. aeruginosa the adaptive benefits of expressing both Icd and Idh are unknown. We therefore characterized the IDH isoenzymes of P. aeruginosa with respect to their relative enzyme activities, expression under aerobic and hypoxic conditions in routine Luria Bertani medium as well as in the artificial sputum medium (ASM). Through this approach that simulates different aspects of lung environment we expected to identify potential marker genes for the adaptation of P. aeruginosa during chronic CF lung disease. These data will probably help to better understand the habitat-specific adaptation process of P. aeruginosa in order to identify new treatment strategies and targets to prevent chronic CF lung infection.

maintained in Luria-Bertani (LB) medium at 37 ◦ C with shaking at 200 rpm. When indicated, artificial sputum medium (ASM), applied to simulate the CF mucus, was used. For oxygen-restricted growth experiments 25 ml flasks fulfilled with medium that contained 50 mM NO− 3 were used. After 1–2 h of bacterial growth the oxygen concentration reached ≤1% representing hypoxic to anoxic conditions (determined by the oxygen sensor Oxy 4 microsensor, Presens GmbH Regensburg; Germany data not shown). Bacterial growth was measured in triplicate at an optical density of OD600 . ASM was prepared except minor modifications as previously described (Sriramulu et al., 2005). Briefly, for 1 L ASM 100 ml 10× sputum buffer (690 mM NaCl, 30 mM CaCl2 , 200 mM Hepes) was mixed with 10 ml of 80 mg/ml glucose, 1 ml of 500 mM urea and 10 ml of 25 mg/ml of each of 20 common amino acids. Then the mixture was adjusted to pH 6.9 with 5 M NaOH, filled up to 495 ml with water and was subsequently sterile filtered (0.22 ␮m). Finally, 200 ml of a sterile filtered albumin solution (125 mg/ml), 250 ml of 20 mg/ml 4× mucine solution (Sigma M2378), 40 ml of 67.5 mg/ml fish sperm DNA, 10 ml heat-inactivated FBS and 5 ml egg yolk emulsion (Sigma E7899) were added. Freshly prepared ASM was stored for a maximum of four weeks at 4 ◦ C. Antibiotics were added to growth media as required at the following concentrations: for P. aeruginosa tetracycline 150 ␮g/ml, gentamicin 150 ␮g/ml, and chloramphenicol 200 ␮g/ml. E. coli strains were transformed by heat shock for 90 s at 42 ◦ C, while plasmids were introduced into P. aeruginosa by triparental conjugation using helper E. coli (pRK2013) as described (Hogardt et al., 2006). Construction of ˇ-galactosidase transcriptional fusions

Material and methods Bacterial strains, plasmids and culture conditions P. aeruginosa strains and cloning plasmids used in this study are listed in Table 1. P. aeruginosa and E. coli strains were routinely

␤-galactosidase (LacZ) reporter plasmids used in this study are listed in Table 2. Corresponding cloning primers (Metabion, Martinsried, Germany) are shown in supplemental Table A.1. Primers with flanking XbaI and KpnI restriction sites were used to amplify fragments carrying the putative promotor region of P.


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Table 2 P. aeruginosa ␤-galactosidase (lacZ) fusions used in this study. Reporter constructa

Gene function (PA number)

LBMb

ASMc

accB’-lacZ atu’-lacZ azu’-lacZ cbb3-1 -lacZ cbb3-2 -lacZ ccpR’-lacZ coxBA’-lacZ himD’-lacZ icd’-lacZ idh’-lacZ lpdG’-lacZ nuoA’-lacZ oprF’-lacZ anr’-lacZ

Biotin carboxyl carrier protein (PA4847) Citronellol catabolism (PA2886) Redox-active protein azurin (PA4922) Cytochrome oxidase cbb3-1 (PA1554) Cytochrome oxidase cbb3-2 (PA1557) Cytochrome peroxidase c551 (PA4587) Cytochrome c oxidase (PA0105) IHF ␤-subunit (PA3161) Isocitrate dehydrogenase (PA2623) Isocitrate dehydrogenase (PA2624) ␣-Ketoglutarate dehydrogenase (PA1587) NADH dehydrogenase (PA2637) Outer membrane porin (PA1777) Transcriptional regulator Anr (PA1544)

(aerobe → hypoxic growth) – – + ++ + +++ – – + + – – + +

– – +++ ++ +++ + – ++ + ++ – +++ + n.d.

a This study is the reference for all reporter constructs. PA numbers are listed according to P. aeruginosa PAO1 genome (http//www.pseudomonas.com). Semiquantitative classification of the increase in ␤-galactosidase activities during aerobic versus hypoxic growth in LB medium (LBM) and artificial sputum medium (ASM): 2-fold (+), 4-fold (++), 6-fold (+++). b Significance values for the hypoxic upregulation in LBM are P ≤ 0.001 (for azu, cbb3-1, cbb3-2, icd, idh, oprF) and P ≤ 0.01 (for ccpR). c In ASM are P ≤ 0.001 (for cbb3-2) and P ≤ 0.01 (for azu, cbb3-1, icd, idh, oprF) and P ≤ 0.05 (for ccpR).

aeruginosa genes, selected due to their up-regulation in late lungadapted CF isolates (Hoboth et al., 2009). PCR amplifications were performed using Pfu DNA Polymerase (Fermentas, St. Leon-Rot, Germany) and genomic DNA from P. aeruginosa PAO1 as template. Resulting PCR fragments (103–667 bp in size) were digested with KpnI and XbaI and cloned into the corresponding sites of the promotorless reporter plasmid pMP220 resulting in the following fourteen transcriptional ␤-galactosidase reporter fusions: accB’-lacZ, atu’-lacZ, azu’-lacZ, cbb3-1 -lacZ, cbb3-1 -lacZ, ccpR’-lacZ, coxBA’-lacZ, himD’-lacZ, icd’-lacZ, idh’-lacZ, lpdG’-lacZ, nuoA’-lacZ, oprF’-lacZ, and anr’-lacZ. LacZ-Reporters for azu, ccpR and cbb3-2 up-regulated in late CF mutators and known to be increasingly transcribed during oxygen restriction were included. Correct cloning of each reporter fusion was confirmed by sequencing using primer f-pMP220 5 -CAGGGTTGCGCCCTGTGC-3 and primer r-pMP220 5 -TATCAACGGTGGTATATCCAG-3 . Resulting lacZ reporter fusions were conjugated into P. aeruginosa PAO1 and PA14. To investigate icd/idh expression in more detail corresponding reporters were conjugated into rpoS deletion mutants SS24 and PA14 Tn::rpoS as well as into aceK deletion mutant PA14 Tn::aceK. DNA cloning, plasmid preparation and sequencing were performed according to standard methods. ˇ-galactosidase assay P. aeruginosa strains harboring lacZ fusions were grown overnight in 25 ml LB at 37 ◦ C to late stationary phase. ␤galactosidase assays were performed according to the procedure of Miller (1972). In brief, OD600 of overnight culture was determined, then 250 ␮l cell suspension was washed once with PBS and added to 750 ␮l Z-Puffer (0.06 M Na2 HPO4 , 0.04 M NaH2 PO4 × H2 O, 0.01 M KCl, 0.001 M MgSO4 × 7 H2 O, 0.05 M ␤-mercaptoethanol, pH 7), 5 ␮l 0.1% sodium dodecyl sulfate (SDS) and 10 ␮l chloroform (≥99%). The mixture was incubated for 5 min at 28 ◦ C. ␤-galactosidase reaction was started by adding 200 ␮l ortho-nitrophenyl-␤-galactoside (ONPG) to the cell extract and stopped at appropriate colorization of the mixture by adding 500 ␮l stop solution (1 M Na2 CO3 ). Then, optical density of the 1.5 ml reaction mixture was determined photometrically (Ultrospec 3100pro, Amersham Biosciences) at 420 and 550 nm. One Miller unit was determined by the formula: 1000 × [(OD420 − 1.75 × OD550 )]/(T × V × OD600 ), while V = volume of culture in mL used in the assay, and t = time of the reaction in minutes. Assays were done in three independent experiments with at least five replicates per measurement. Control samples lacking cell

extract were assayed in parallel and subtracted from data obtained from complete reaction mixtures. Resulting values are given as mean and standard deviation. Construction of Anr overexpression plasmid pBBRanr50 To verify the Anr-dependent regulation of lacZ promoter fusions with increased expression under hypoxic growth, ␤galactosidase activities of respective reporter constructs were tested during Anr overexpression. Briefly, by using primer fpBBR1anr 5 -TGATAAGGTACCTGCCTGGGAAAGCTGTACATG-3 and primer r-pBBR1anr 5 -AAGGCTGGATCCTCAGCCTTCCAGCTGGCCG3 a 1155 bp fragment containing the entire anr gene and its promotor was amplified and cloned into the broad host range vector pBBR1MCS (copy number 30–50) via KpnI and BamHI restriction sites. The resulting expression plasmid pBBRanr50 was then conjugated into P. aeruginosa PAO1 and its anr deletion mutant PA6261 by triparental mating. In addition, both strains harboured one of those lacZ reporter plasmid that showed an increased ␤galactosidase activity under hypoxic conditions, namely either azu’-lacZ, cbb3-1 -lacZ, cbb3-2 -lacZ, ccpR’-lacZ, himD’-lacZ, icd’-lacZ, idh’-lacZ, nuoA’-lacZ, or oprF’-lacZ. The reporter plasmid of the Anrdependent azu gene (azu‘-lacZ) was used as positive control to confirm the Anr production from pBBRanr50 while the plasmid pMP220 represents the negative vector control. Correct cloning of the anr gene into pBBR1MCS was verified by sequencing with primers pBBR1MCS 5 -ACAATTTCACACAGGAAAC-3 and primer rpBBR1MCS 5 -GTTGTAAAACGACGGCCAG-3 . Determination of isocitrate dehydrogenase (IDH) activity Isocitrate dehydrogenase (IDH) activity was determined in U/mg of protein as described elsewhere (Eikmanns et al., 1995). Briefly, P. aeruginosa strains were grown in 25 ml LB medium at 37 ◦ C with 180 rpm shaking to early stationary phase grown cells (OD600 of ∼2.5–3.0), harvested by centrifugation at 4 ◦ C (10 min, 3400 rpm, Sorvall Super T21, rotor ST-H750, Thermo Fisher Scientific Inc., Waltham, MA, USA), washed twice with 1× PBS, and disrupted by sonication for 1–2 min (Branson Sonifier 250, Duty Circle 50%, Output Control 3). For the determination of growthdependent IDH activities cells were grown to individual densities as indicated. The supernatant obtained after centrifugation for 15 min at 4 ◦ C was used as cell-free extract to start the enzyme reaction. The reduction of NAD(P)+ to NAD(P)H was monitored at 340 nm


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Fig. 2. ␤-galactosidase activities of P. aeruginosa PAO1 containing different lacZ reporter fusions. Bacterial cells were grown either in LB (A) or in ASM (B) under aerobic () and hypoxic ( ) conditions at 37 ◦ C until stationary growth phase. Each value represents the average of at least three different experiments with standard deviation (P values see legend of Table 2).

(Ultrospec 3100pro, Amersham Biosciences) during the first 3 min. Reaction mixture (2 ml) contained 0.1 M Tris–HCl (pH 7.5), 0.66 mM NAD(P)+ , 1 mM MnSO4 , and 1 mM DS -(+)-threo-isocitrate. Protein concentration was estimated by the method of Bradford (Bradford, 1976) with bovine serum albumin as a standard. One unit (U) of enzyme activity is defined as 1 ␮mol NAD(P)H formed per min by using a molar extinction coefficient of 6.22 mmol−1 cm−1 L. Enzyme activity was determined in triplicate from cell-free extracts from at least five replicates per measurement. Mixture without cell-free extract was used as control. Negative controls lacking NAD(P)+ , isocitrate or cell-free extract, failed to demonstrate any measurable activity (data not shown).

(azu, cbb3-1, cbb3-2, ccpR, icd, idh, oprF, himD and nuoA) were analyzed for the presence of a putative Anr-binding motif by using the algorithm of promscan (http://molbiol-tools.ca/promscan/) and a cut-off value of 80 (supplemental Table A.2). Statistical correlations of data were done by the student t test using SigmaStat software (Systat GmbH, Erkrath, Germany), while P values of ≤0.05 were considered significant. Results Expression of P. aeruginosa metabolic gene functions characteristic for late CF mutator isolates under aerobic and hypoxic growth conditions

Sequence alignments and statistical analysis Simultaneously to the overexpression of Anr, the promotor regions of genes with increased expression under hypoxic growth

Assuming that oxygen restriction and/or CF mucus composition accounts for several adaptations of P. aeruginosa CF isolates we tested increased transcripts/proteins described for end-stage




Fig. 3. ␤-galactosidase activities of lacZ reporter fusions azu’-lacZ, cbb3-2 -lacZ, icd’-lacZ and idh’-lacZ in P. aeruginosa PAO1 and the anr-mutant PA6261 with ( ) or without () overexpression of anr by expression plasmid pBBRanr50. For comparison control plasmid pMP220 is shown. Each experiment was performed at least in triplicate. Each value per experiment was calculated from at least five technical replicates (* P ≤ 0.01; ** P ≤ 0.001).

CF isolates (Hogardt et al., 2007; Hoboth et al., 2009) for their oxygen-dependent gene expression. By using transcriptional lacZ reporter fusions of respective genes we first compared aerobic versus hypoxic ␤-galactosidase activities after growth of P. aeruginosa in standard LB medium in order to test for hypoxic, possibly Anr-dependent gene expression. As expected, for reporter fusions azu’-lacZ, ccpR’-lacZ and cbb3-2 -lacZ increased hypoxic gene expression was found. Likewise, hypoxic ␤-galactosidase activities of cbb3-1 -lacZ, icd’-lacZ, idh’-lacZ and oprF’-lacZ exceed the expression levels that were measured after aerobic growth (Fig. 2A). When P. aeruginosa cells were harvested after growth in the artificial sputum medium ASM, mimicking CF sputum composition, additional reporter constructs himD’-lacZ and nuoA’-lacZ were found to be increasingly transcribed (Fig. 2B). Almost no ␤-galactosidase activity was detected for coxBA’-lacZ, lpdG’-lacZ, accB’-lacZ and atuA’-lacZ in LB medium or in ASM. Remarkably, cbb3-2 -lacZ, nuoA’-lacZ, azu’-LacZ, idh’-lacZ and himD’-lacZ reporters exhibited a higher expression signal after growth in ASM than in LB medium indicating that oxygen-limitation, sputum composition and its combination may represent relevant selective

conditions. Results from corresponding ␤-galactosidase assays in LB and ASM are summarized in Table 2. For nine out of 13 preselected genes an increased hypoxic gene expression was detected in LB and/or ASM medium. Thus, up-regulation of cbb3-1, cbb32, himD, icd, idh, nuoA, oprF, azu and ccpR may be beneficial for P. aeruginosa survival under oxygen limitation within the sticky CF mucus. Anr-dependent expression of reporter fusions with increased ˇ-galactosidase activities under hypoxic growth conditions To verify whether the hypoxic increase in gene expression of genes cbb3-1, cbb3-2, himD, icd, idh, nuoA, oprF, azu and ccpR is Anr-dependent, we applied an experimental strategy that may detect a direct or indirect Anr-dependent gene regulation, since we assumed that the phenotypes of clinical mutator strains also exhibited changes secondary to mutations in Anr-regulated gene functions. Briefly, in P. aeruginosa wild-type PA14 and anr mutant strain PA6261 we overexpressed the Anr protein by pBBRanr50 in the presence of each of the reporter fusions with increased




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hypoxic gene expression as identified above. We hypothesized that the increased production of Anr would lead to an increased cellular amount of the inactive Anr monomer and thus also to the increased formation of the active Anr dimer. The increasingly available Anr dimer should then bind to its target DNA to activate gene transcription that would subsequently result in the increase in ␤galactosidase activity of reporter plasmids carrying a promotor fusion that is Anr-dependent. As expected ␤-galactosidase activity of ccpR’-lacZ (data not shown) significantly increased in PA14 (P ≤ 0.001) and PA6261 (P ≤ 0.05) in the presence of pBBRanr50 as compared to respective controls that do not contain the expression plasmid. The aerobic overexpression of Anr may thus activate the expression of Anr-regulated gene functions. Similarly, expression levels of azu’-lacZ and cbb3-2 -lacZ and icd’-lacZ (Fig. 3) but not cbb3-1 -lacZ, idh’-lacZ, himD’-lacZ, nuoA’-lacZ, and oprF’-lacZ were significantly increased in PA14 and PA6261. The control plasmid pMP220 exhibited no relevant ␤-galactosidase activity under these conditions. Finally, these data showed that azu, ccpR, cbb3-2 as well as the icd gene depend on the expression of Anr. The Anr effect may be mediated either directly, by binding to the promotor region of the respective gene or indirectly, by a secondary factor that is expressed due to the overall Anr effect. A direct Anr-dependent regulation has been shown for azu, ccpR and cbb3-2 but not for icd that carries a putative Anr-box in the promotor region (Table A.2). In contrast, from these experiments the reason for the hypoxic increase of idh, himD, oprF and cbb3-1 gene expression remains unclear. Due to the insufficient growth yield of the anr mutant PA6261 we were not able to perform lacZ reporter analysis under hypoxic conditions. Furthermore, by the co-expression of the reporter construct anr’-lacZ and pBBRanr50 we were able to test whether the anr gene is subject to an autoregulation. As depicted in Fig. 4 no increase in ␤-galactosidase activity of anr’-lacZ was found during overexpression of Anr in PAO1 or in the anr negative background of PA6261. Besides, we also determined the LacZ activity of anr’-lacZ during hypoxic growth. As the anr mutant PA6261 can only grow sufficiently aerobically this experiment was performed solely with the wild-type PAO1. Interestingly, under hypoxic conditions we found a significant increase of anr’-lacZ activity from 2569 ± 166 to 3586 ± 449 when pBBRanr50 was present (P ≤ 0.05), indicating a minimal direct or indirect autoregulation of the anr gene (Fig. 4). Cofactor specificity and specific enzyme activities of P. aeruginosa isocitrate dehydrogenase (IDH) isozymes Icd and Idh P. aeruginosa possesses two isocitrate dehydrogenase isoenzymes, Icd and Idh. So far, nothing is known about the specific function of each isoenzyme, differences in their gene expression or enzymatic function. Strikingly, both enzymes were increasingly transcribed under hypoxic conditions (Fig. 2A and B). However, expression of icd but not idh is controlled by Anr which is suggestive of their role during adaptation to varying oxygen conditions. During further analyses we investigated in more detail the function of isocitrate dehydrogenase (IDH) activity of P. aeruginosa. Initially, we determined the specific enzyme activities of Icd and Idh during aerobic and hypoxic growth. To discriminate between Icd and Idh we used single transposon mutants PA14 Tn::idh and PA14 Tn::icd that express either Icd or Idh activity, respectively. Secondly an IDH enzyme assay was established and applied to determine the cofactor specificity of Icd (PA14 Tn::idh) and Idh (PA14 Tn::icd). Interestingly, the two mutant strains PA14 Tn::idh and PA14 Tn::icd exhibited more or less identical growth curves under aerobic as well as hypoxic conditions in LB medium (supplemental Fig. A.1). This indicated that Icd may take over the function of Idh and vice versa. The dependency of P. aeruginosa isocitrate dehydrogenase Icd and Idh on NAD+ or NADP+ as cofactors, was investigated by

Fig. 4. ␤-galactosidase activities of the lacZ reporter fusion anr’-lacZ in P. aeruginosa PAO1 either with ( ) or without () overexpression of anr by expression plasmid pBBRanr50 under aerobic (A) and hypoxic (B) conditions. For comparison control plasmid pMP220 is shown. Each experiment was performed at least in triplicate. Each value per experiment was calculated from at least five technical replicates (* P ≤ 0.05).

determining the concomitant formation of NADH or NADPH during decarboxylation of isocitrate in cell free extracts from wild-type strain PA14 (expressing both Idh and Icd), its isogenic transposon mutants PA14 Tn::icd (expressing only Idh) and PA14 Tn::idh (expressing only Icd). Interestingly, enzyme assays with NADP+ as a cofactor showed a significant reduction of NADP+ to NADPH, whereas if NAD+ was added no NADH formation was detectable (supplemental Fig. A.2). Therefore, both isoenzymes Icd and Idh are strictly specific for NADP+ as cofactor and exhibited no in vitro activity in the presence NAD+ . P. aeruginosa IDH activity contributes only to the formation of NADPH and not NADH. In contrast to their capacity to support P. aeruginosa growth and cofactor specificity, the isocitrate decarboxylation assay (IDH assay) revealed that Icd (PA14




stationary growth. Furthermore, no difference in ␤-galactosidase activity of icd’-lacZ was detected for strain pairs PAO1/SS24 and PA14/PA14 Tn::rpoS during exponential growth. In contrast, during stationary growth phase a significant increase in lacZ expression levels was found for the two rpoS mutant strains as compared to wild-type (for SS24 2959 ± 391 miller units versus PAO1 1359 ± 392 miller units; P ≤ 0.01 and for PA14 Tn::rpoS 1639 ± 490 miller units versus PA14 860 ± 166 miller units; P ≤ 0.05). To further confirm the RpoS-mediated regulation of icd gene and to elucidate whether this effect is directly translated into an increase in enzyme activity, we determined the IDH activity of P. aeruginosa pairs PAO1/SS24 and PA14/PA14 Tn::rpoS during exponential (OD600 ∼ 0.7; i.e. at 4 h after inoculation) and stationary growth (OD600 ∼ 4.0; i.e. at 8 h after inoculation). As expected, IDH activity was significantly increased in both rpoS mutant strains during stationary but not during exponential growth (Fig. 6). Similar enzyme activities were observed for PAO1 and its isogenic rpoS mutant SS24 during exponential growth (IDH activity: 0.483 ± 0.109 U/mg protein for PAO1 and 0.543 ± 0.067 U/mg protein for SS24). During stationary growth IDH enzyme activity of the PAO1 strain increased slightly up to 0.692 ± 0.104 U/mg protein, whereas the activity of SS24 increased to 1.150 ± 0.069 U/mg protein (significance P ≤ 0.01). During stationary growth, IDH activity of PA14 achieved 0.842 ± 0.077 U/mg versus 1.236 ± 0.129 U/mg protein for PA14 Tn::rpoS (significance P ≤ 0.001). These data indicate that during stationary growth phase RpoS has a negative regulatory effect on the expression of IDH activity. Although a differentiation between Icd and Idh activity is not possible, it is likely that the increase in IDH activity of rpoS mutant results from the increased transcription of the icd gene shown above. Fig. 5. Growth-phase-dependent isocitrate dehydrogenase (IDH) activity of P. aeruginosa PA14 and its isogenic transposon mutants Tn::icd ( ) and Tn::idh () under both aerobic (A) and hypoxic (B) conditions. Cells were grown at 37 ◦ C in 25 ml LB medium either aerobically with rigorous shaking (200 rpm) or for hypoxic growth in a tightly sealed 25 ml flask containing LB medium supplemented with . Each experiment was performed at least in triplicate. Enzyme activities 50 mM NO− 3 are presented as average and standard deviation.

Tn::idh) and Idh (PA14 Tn::icd) exhibit differences in their specific enzyme activities dependent on growth phase. Under aerobic conditions Idh activity remains consistent reaching an activity of about 0.2–0.3 U/mg protein, whereas Icd activity steadily increases with cell density reaching its maximum activity of 0.760 ± 0.109 U/mg protein in early stationary growth phase. At early stationary growth Icd activity was about three times higher than Idh activity, while only during growth at 4 h and 5 h (OD600 < 1.0) after inoculation Idh activity exceeded that of Icd (Fig. 5A). However, there were no significant differences in cell-density-dependent specific enzyme activities for Icd and Idh when determined under hypoxic or aerobic conditions (Fig. 5B). RpoS-dependent expression of P. aeruginosa icd but not idh Due to the cell-density-dependent expression of Icd we assumed that the icd gene of P. aeruginosa may be regulated by the stationary transcription factor RpoS, as shown for E. coli (Jung et al., 2006). Therefore, we measured the ␤-galactosidase activity of icd’-lacZ and idh’-lacZ in P. aeruginosa wild-type PAO1 and PA14 and their isogenic rpoS-mutant strains SS24 and PA14 Tn::rpoS, respectively, both during exponential (OD600 ∼ 0.7) and stationary growth phase (OD600 ∼ 4.0). Initially, we showed that the two rpoS mutant strains exhibited no detectable growth defect as compared to wild-type (supplemental Fig. A.1). The resulting LacZ activities of wild-type/rpoS mutant pairs are summarized in Table 3. The ␤galactosidase activity of idh’-lacZ showed no difference between wild-type and isogenic rpoS mutants during exponential or

AceK-dependent expression of P. aeruginosa isocitrate dehydrogenase activity In E. coli isocitrate dehydrogenase activity is postregulated by isocitrate dehydrogenase transcriptionally phosphatase/kinase AceK that phosphorylates or dephosphorylates IDH, resulting in inactivation or activation of IDH. In P. aeruginosa a homolog of the bifunctional phosphatase/kinase AceK exists, namely PA1376 (PAO1) or PA14 46450 (PA14). To verify if AceK may play a role in the activation/inactivation of P. aeruginosa isocitrate dehydrogenases we compared specific enzyme activities of wild-type PA14 and its isogenic aceK transposon mutant PA14 Tn::aceK, both during exponential and stationary phase. As the rpoS mutant strains PA14 Tn::aceK exhibited no detectable growth defect (supplemental Fig. A.1). While during exponential growth almost identical enzyme activities were found for PA14 and mutant Tn::aceK, the wild-type exhibited a two-fold higher activity during stationary growth phase (Fig. 7). Upon aceK inactivation the cell-density-dependent increase of IDH activity was absent. These results suggest that during the switch from exponential to stationary growth, activation of IDH by AceK occurs (at least during aerobic growth in amino acid-rich LB medium) (Sezonov et al., 2007). Although differentiation between Icd and Idh is not possible it seems likely that the loss-of-cell-density-dependent increase in Icd activity is the basis for the reduced IDH activity of PA14 Tn::aceK during stationary growth. Discussion The characterization of the habitat-specific adaptation process of P. aeruginosa during long-term survival in the CF lung has become a relevant research area that will probably help to deduce the specific selective conditions P. aeruginosa is faced with. The aim of this study was to investigate potential marker genes for the metabolic


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Table 3 RpoS dependent activity of icd and idh reporter fusions exponential growth stationary growth. Strain

Phenotype

␤-galactosidase activitya

P value

␤-galactosidase activitya

P value

PAO1 (icd‘-lacZ) SS24 (icd‘-lacZ) PAO1 (idh‘-lacZ) SS24 (idh‘-lacZ) PA14 (icd‘-lacZ) Tn::rpoS (icd‘-lacZ) PA14 (idh‘-lacZ) Tn::rpoS (idh‘-lacZ)

Wild-type rpoS Wild-type rpoS Wild-type rpoS Wild-type rpoS

325 ± 76 469 ± 45 492 ± 304 480 ± 269 380 ± 105 485 ± 59 372 ± 179 399 ± 195

n.s.

1359 ± 392 2959 ± 391 659 ± 222 767 ± 206 860 ± 166 1639 ± 490 190 ± 77 214 ± 166

P ≤ 0.01

n.s. n.s. n.s.

n.s. P ≤ 0.05 n.s.

a Data are given as average ± standard deviation from at least three independent experiments with at least five replicates each. P values were determined by SigmaStat software (Systat GmbH, Erkrath, Germany). P values of P ≤ 0.01 were considered significant.

Fig. 6. Isocitrate dehydrogenase (IDH) enzyme activities of P. aeruginosa wild-type ( ) PAO1 and PA14 as compared to their respective isogenic rpoS mutant strains ( ) SS24 and PA14 Tn::rpoS. Bacterial cells were grown aerobically at 37 ◦ C and harvested during exponential (OD600 ∼ 0.7; 4 h of growth) and stationary OD600 ∼ 4.0; 8 h of growth) growth phase. Data are given as average and standard deviation of at least three independent experiments with five replicates. During stationary growth phase IDH activity of rpoS mutant strains was significantly increased as compared to wild-type strains (* P ≤ 0.01; ** P ≤ 0.001).

adaptation of P. aeruginosa by simulating the CF lung environment through a combination of oxygen restriction and artificial sputum medium (ASM). Importantly, CF sputum is characterized by nutritional richness due to high amounts of mucin, DNA, lipids, amino acids and proteins that likely promote efficient P. aeruginosa growth and energy metabolism and probably select likewise

Fig. 7. Isocitrate dehydrogenase (IDH) enzyme activities of P. aeruginosa wild-type PA14 ( ) and its isogenic aceK mutant strain PA14 Tn::aceK ( ). Bacterial cells were grown aerobically at 37 ◦ C and harvested during exponential (OD600 ∼ 0.7; 4 h of growth) and stationary OD600 ∼ 4.0; 8 h of growth) growth phase. Data are given as average and standard deviation of at least three independent experiments with five replicates. During stationary growth phase IDH activity of aceK mutant strain was significantly decreased as compared to wild-type strain (* P ≤ 0.01).

oxygen limitation for specific metabolic adaptations (Worlitzsch et al., 2002; Hogardt and Heesemann, 2012). This assumption is corroborated by the frequent recovery of auxotrophic variants which also underlines the sufficient availability of amino acids within CF airway secretions (Barth and Pitt, 1996; Taylor et al., 1992; Thomas et al., 2000). Considerable data have also shown that peptides, amino acids and fatty acids are sufficient to sustain the growth of P. aeruginosa CF isolates (Palmer et al., 2005; Son et al., 2007; Barth and Pitt, 1996). Artificial sputum medium (ASM) containing major components of CF mucus is easy to use, induces the formation of biofilm-like microcolonies, and seems to yield in vitro results that are much more relevant to CF than those from standard media (Sriramulu et al., 2005; Fung et al., 2010; Kirchner et al., 2012). For a further analysis of CF lung adaptation we selected accB, atuA, azu, cbb3-1, cbb3-2, ccpR, coxBA, himD, icd, idh, lpdG, nuoA and oprF since these genes encode important metabolic gene functions and were increasingly expressed in late CF mutators as previously shown (Hoboth et al., 2009). Interestingly, the activities for reporter constructs of azu, cbb3-1, cbb3-2, ccpR, icd, idh, oprF, himD and nuoA reporters were significantly increased during hypoxic growth in either LB and/or ASM, supporting the hypothesis that the function of these genes (nine out of 13 selected genes) are advantageous for survival of P. aeruginosa in the oxygen-restricted CF airway secretions. Remarkably, cbb3-2, nuoA, azu, idh and himD reporters exhibit a higher reporter activity in ASM than in LB pointing to the fact that beside oxygen restriction the nutritional composition of ASM (amino acids, DNA, mucin, albumin) additively supports the expression of these genes. Since increasingly expressed under oxygen-limitation, by in silico analysis we tested these genes for the presence of a putative Anr-binding site in their promotor region and thus a possible direct Anr-dependent gene regulation (see supplemental Table A.2).




Anr-boxes have been predicted earlier for azu, cbb3-2, ccpR, icd and idh (Trunk et al., 2010; Vijgenboom et al., 1997; Comolli and Donohue, 2004). Taking into account, that the majority of Anr binding sites are located between 60–100 nt upstream of the ATG and the ideal distance of the centre of Anr-binding sites to the transcriptional start site (TSS) is −41.5, the predicted Anr boxes for cbb3-1, nuoA, and oprF are obviously false positive (Wurtzel et al., 2012). In line with this, during overexpression of Anr (to simulate the increased Anr expression of late CF mutators), the reporter activity of azu’-lacZ; ccpR’-lacZ, cbb3-2 -lacZ and icd’-lacZ increased (two to 10-fold), while those for cbb3-1 -lacZ, idh’-lacZ, himD’-lacZ, nuoA’-lacZ, and oprF’-lacZ remained unaffected. Our results from reporter fusions and Anr overexpression experiments suggest that azu, cbb3-2, ccpR and icd gene are part of the Anr regulon. So far, a direct Anr-dependent regulation has been shown for azu, ccpR and cbb3-2 but not for icd (Trunk et al., 2010; Vijgenboom et al., 1997; Comolli and Donohue, 2004). Finally, transcriptional activation of icd via Anr seems likely (distance of Anr binding site to TSS is −42.5). In contrast, the promotors of cbb3-1, idh, himD, nuoA and oprF seem to be Anr-independent although we could not exclude that they may contain low-affinity Anr-binding sites that are not activated by the limited amount of dimeric Anr generated during aerobic overexpression from mid copy plasmid pBBRanr50. Altogether, the aerobic overexpression of Anr in vitro seems to be a very helpful tool to simulate Anr activation and to test for Anr-dependent effects. It has been shown that Anr activation is not absolutely restricted to anoxic conditions and may be aerobically induced e.g. by choline (Jackson et al., 2013). Finally, the expression of azu, ccpR, the high-affinity terminal oxidase cbb3-2, cbb3-1, oprF, icd and idh seem to be advantageous under low oxygen tension, while expression of nuoA (NADH dehydrogenase) and himD (integration host factor ␤-subunit) increased only during anaerobic growth in the nutrient-rich ASM. Integration host factor IHF represents a global regulatory protein that is synergistically involved in the regulation of several Anr-dependent genes (Eschbach et al., 2004; Krieger et al., 2002). The increased expression of nuoA encoding NADH dehydrogenase I chain A may be indicative for an increased demand of the redox cofactor NADH. Consistently, the NADH dehydrogenase complex (nuo) genes were required for anaerobic growth in the presence of NO3 − (Platt et al., 2008). By overexpression of ANR in the presence of the reporter anrlacZ’ we showed for the first time that under hypoxic growth and in contrast to FNR (negative autoregulation), Anr exhibit a slight positive autoregulatory effect on the expression of anr gene (Mettert and Kiley, 2007). However, whether this effect is biologically relevant in the context of ANR regulon activation at physiological concentrations is unclear. Furthermore, both lacZ-reporter assays and the determination of the IDH enzyme activities clearly point to differences in the expression of the P. aeruginosa isoenzymes Icd (monomeric) and Idh (dimeric) suggesting that each enzyme exhibits specific regulatory and/or functional properties. Isoenzymes of IDH are ubiquitous in nature as they catalyze the oxidative decarboxylation of isocitrate to 2-oxoglutarate. NAD-dependent IDHs are rare and occur in various archaea and a few bacteria that typically have an incomplete TCA cycle (Rodriguez-Arnedo et al., 2005). In contrast, most prokaryotes possess a NADP-dependent homodimeric IDH, some possess a monomeric NADP-dependent enzyme but only very few species express both isoenzymes. For example, dimeric Idh of the psychrophilic bacterium C. maris exhibits maximal activities at 45 ◦ C, while its monomeric Idh is cold-adapted with optimal activity at 20 ◦ C. Here we have shown that as in other bacteria both IDHs of P. aeruginosa i.e. Icd and Idh use NADP+ as cofactor. NADPdependent IDHs are an essential component of oxidative defense mechanisms due to their ability to generate both NADPH and

2-oxoglutarate, two strong antioxidants. The availability of NADPH ensures a reductive environment that enables the bacterial cell to combat oxidative stress generated by the oxidative phosphorylation that is driven by an active TCA cycle or by inflammatory processes. Moreover, TCA cycle and its dehydrogenases fuel the cellular NADPH redox pool that has been implicated in the susceptibility of bacteria to bactericidal antibiotics such as ciprofloxacin and tobramycin (Kohanski et al., 2007). Due to its localization at the branch point between glyoxylate bypass and TCA cycle, IDH also represents an attractive target for regulating (aerobic) metabolism (Eisenreich et al., 2010). The glyoxylate shunt is needed to replenish dicarboxylic acids during growth on acetate or fatty acids that are known to be abundant in the CF mucus. Inactivation of IDH may short-circuit TCA cycle by enabling glyoxylate bypass. In E. coli IDH is phosphorylated or dephosphorylated by AceK in response to environmental changes, leading to the inactivation or activation of IDH. So far, no data are available about the differential function and regulation of P. aeruginosa Icd and Idh. Our analyses showed that under aerobic growth conditions P. aeruginosa Idh activity remains consistent, whereas activity of Icd steadily increases with cell density reaching a maximum activity of about three-fold that of Idh at the onset of the stationary growth phase. Furthermore, relative specific enzyme activities of Icd and Idh determined under oxygen-limitation exhibited no obvious difference to those measured during aerobic growth. Icd activity in particular seems to be important under oxygen limitation. After inactivation of the sigma factor RpoS, the activity of IDH and icd’-lacZ upon entry into stationary growth phase was significantly increased. This indicates that the global transcriptional regulator of stationary growth phase RpoS should act as a negative regulator of icd gene expression. This may be plausible, since major genes functions e.g. heat shock proteins, secreted factors and genes of the central intermediary metabolism may be repressed by the action of RpoS (Hogardt et al., 2004; Jung et al., 2006). However, gene repression by RpoS may be mediated by a secondary negative regulator, the expression of which is activated by RpoS (Hogardt et al., 2004; Sonnleitner et al., 2003; Suh et al., 1999). Thus far, the icd gene was not represented in the data set of the RpoS regulated P. aeruginosa genes (Schuster et al., 2004). Secondly, in the absence of IDH kinase/phosphatase AceK (aceK deletion mutant) IDH activity was found to be reduced during stationary growth phase. As is the case in E. coli, this at first underlines that AceK is involved in the regulation of IDH activity in P. aeruginosa although the exact mechanism has to be defined in further studies. In general, it is difficult to predict the overall effect of the inactivation of AceK due to its bifunctional activity (IDH activation and inactivation). Our data argue that during stationary growth in LB the activation and likely dephosphorylation of P. aeruginosa IDH (most likely of dimeric Icd that is homolog to E. coli Icd) by AceK dominates. Interestingly, NADP-dependent IDH from Mycobacterium tuberculosis is shown to be phosphorylated during the persistent stage of this pathogen (Vinekar et al., 2012). In summary, we showed that during hypoxic growth of P. aeruginosa in either LB or in ASM the expression of azu, cbb3-1, cbb3-2, ccpR, icd, idh, oprF, himD and nuoA (all positively selected in expression variants of chronic CF isolates) is increased, which indicates that these genes may be involved in the microaerobic adaptation of P. aeruginosa during CF lung survival (Fig. 1). We further demonstrated that P. aeruginosa Anr controls at least the expression of azu, ccpR, cbb3-2 (PA1557) and of icd gene. Interestingly, the IDH isoenzymes of P. aeruginosa (Icd and Idh) may replace each other in sustaining TCA cycle and efficient cell growth in LB medium. The NADPH- and RpoS-dependent Icd seems to be the dominant isoenzyme under ongoing oxygen restriction and increasing cell density as found during stationary growth phase. Whether an increase in the activity of IDH and thus an efficient




supply of the antioxidant NADPH is important to protect P. aeruginosa from oxidative stresses, e.g. due to bactericidal antibiotics and/or the exhausting intrapulmonal inflammation, is left to be determined in future studies. Deeper understanding of mechanisms involved in niche specialization of P. aeruginosa during CF lung disease will hopefully help to identify new therapeutic options to overcome the onset of chronic CF lung infection or to limit the progress of lung disease. Acknowledgments The excellent technical assistance of Monika Götzfried is gratefully acknowledged. No conflict of interest is declared by the authors. We thank Dr. Fiona F. O’Rourke for carefully proofreading of the manuscript. This study was financially supported by the DFG priority program SPP1316 ‘Host adapted metabolism’ (HO 3970/11) and in part (regarding establishment of the artificial sputum medium) by a grant of the German CF Foundation (S07/05). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.ijmm.2014.07.014. References Alvarez-Ortega, C., Harwood, C.S., 2007. Responses of Pseudomonas aeruginosa to low oxygen indicate that growth in the cystic fibrosis lung is by aerobic respiration. Mol. Microbiol. 65, 153–165. Arai, H., Igarashi, Y., Kodama, T., 1995. Expression of the nir and nor genes for denitrification of Pseudomonas aeruginosa requires a novel CRP/FNR-related transcriptional regulator, DNR, in addition to ANR. FEBS Lett. 371, 73–76. Banerjee, S., Nandyala, A., Podili, R., Katoch, V.M., Hasnain, S.E., 2005. Comparison of Mycobacterium tuberculosis isocitrate dehydrogenases (ICD-1 and ICD-2) reveals differences in coenzyme affinity, oligomeric state, pH tolerance and phylogenetic affiliation. BMC. Biochem. 6, 20. Barth, A.L., Pitt, T.L., 1996. The high amino-acid content of sputum from cystic fibrosis patients promotes growth of auxotrophic Pseudomonas aeruginosa. J. Med Microbiol. 45, 110–119. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Burke, W.F., Johanson, R.A., Reeves, H.C., 1974. NADP+ -specific isocitrate dehydrogenase of Escherichia coli. II. Subunit structure. Biochim. Biophys. Acta 351, 333–340. Chen, F., Xia, Q., Ju, L.K., 2006. Competition between oxygen and nitrate respirations in continuous culture of Pseudomonas aeruginosa performing aerobic denitrification. Biotechnol. Bioeng. 93, 1069–1078. Comolli, J.C., Donohue, T.J., 2004. Differences in two Pseudomonas aeruginosa cbb3 cytochrome oxidases. Mol. Microbiol. 51, 1193–1203. Döring, G., Parameswaran, I.G., Murphy, T.F., 2011. Differential adaptation of microbial pathogens to airways of patients with cystic fibrosis and chronic obstructive pulmonary disease. FEMS Microbiol. Rev. 35, 124–146. Eikmanns, B.J., Rittmann, D., Sahm, H., 1995. Cloning, sequence analysis, expression, and inactivation of the Corynebacterium glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme. J. Bacteriol. 177, 774–782. Eisenreich, W., Dandekar, T., Heesemann, J., Goebel, W., 2010. Carbon metabolism of intracellular bacterial pathogens and possible links to virulence. Nat. Rev. Microbiol. 8, 401–412. Eschbach, M., Schreiber, K., Trunk, K., Buer, J., Jahn, D., Schobert, M., 2004. Long-term anaerobic survival of the opportunistic pathogen Pseudomonas aeruginosa via pyruvate fermentation. J. Bacteriol. 186, 4596–4604. Figurski, D.H., Helinski, D.R., 1979. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. U.S.A. 76, 1648–1652. Fung, C., Naughton, S., Turnbull, L., Tingpej, P., Rose, B., Arthur, J., Hu, H., Harmer, C., Harbour, C., Hassett, D.J., Whitchurch, C.B., Manos, J., 2010. Gene expression of Pseudomonas aeruginosa in a mucin-containing synthetic growth medium mimicking cystic fibrosis lung sputum. J. Med. Microbiol. 59, 1089–1100. Hoboth, C., Hoffmann, R., Eichner, A., Henke, C., Schmoldt, S., Imhof, A., Heesemann, J., Hogardt, M., 2009. Dynamics of adaptive microevolution of hypermutable Pseudomonas aeruginosa during chronic pulmonary infection in patients with cystic fibrosis. J. Infect. Dis. 200, 118–130. Hogardt, M., Heesemann, J., 2012. Microevolution of Pseudomonas aeruginosa to a chronic pathogen of the cystic fibrosis lung. Curr. Top. Microbiol. Immunol. 358, 91–118.

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Adaptation of iron homeostasis pathways by a Pseudomonas aeruginosa pyoverdine mutant in the cystic fibrosis lung.

Diversity of metabolic profiles of cystic fibrosis Pseudomonas aeruginosa during the early stages of lung infection.

The deletion of TonB-dependent receptor genes is part of the genome reduction process that occurs during adaptation of Pseudomonas aeruginosa to the cystic fibrosis lung.

Cystic fibrosis, Pseudomonas aeruginosa, and selective decontamination.

Exploring the expression of Pseudomonas aeruginosa genes directly from sputa of cystic fibrosis patients.

Adherence of Pseudomonas aeruginosa to cystic fibrosis buccal epithelial cells.

Iron-Mediated Control of Pseudomonas aeruginosa-Staphylococcus aureus Interactions in the Cystic Fibrosis Lung.

Small colony variants of Pseudomonas aeruginosa in chronic bacterial infection of the lung in cystic fibrosis.

Effect of Shear Stress on Pseudomonas aeruginosa Isolated from the Cystic Fibrosis Lung.

TOBI Podhaler for cystic fibrosis patients with Pseudomonas aeruginosa.

Molecular surveillance for carbapenemase genes in carbapenem-resistant Pseudomonas aeruginosa in Australian patients with cystic fibrosis.

Selective Sweeps and Parallel Pathoadaptation Drive Pseudomonas aeruginosa Evolution in the Cystic Fibrosis Lung.

Mucoid Pseudomonas aeruginosa is a marker of poor survival in cystic fibrosis.

Electrochemistry provides a point-of-care approach for the marker indicative of Pseudomonas aeruginosa infection of cystic fibrosis patients.

Eradication of Pseudomonas aeruginosa in adults with cystic fibrosis.

Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum.

Molecular identification of Pseudomonas aeruginosa recovered from cystic fibrosis patients.

Phenotypic conversion of Pseudomonas aeruginosa in cystic fibrosis.

Nosocomial acquisition of Pseudomonas aeruginosa by cystic fibrosis patients.

Extrapulmonary sites of Pseudomonas aeruginosa in adults with cystic fibrosis.

Use of a Multiplex Transcript Method for Analysis of Pseudomonas aeruginosa Gene Expression Profiles in the Cystic Fibrosis Lung.

Lytic activity by temperate phages of Pseudomonas aeruginosa in long-term cystic fibrosis chronic lung infections.

Cystic fibrosis. 1. Pseudomonas aeruginosa infection in cystic fibrosis and its management.

Interactions between Neutrophils and Pseudomonas aeruginosa in Cystic Fibrosis.