A novel cyanide-inducible gene cluster helps protect Pseudomonas aeruginosa from cyanide

June 20, 2017 | Autor: Emanuela Frangipani | Categoria: Microbiology, Ecology, Pseudomonas aeruginosa, Pseudomonas Aeruginosa
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Environmental Microbiology Reports (2013)

doi:10.1111/1758-2229.12105

A novel cyanide-inducible gene cluster helps protect Pseudomonas aeruginosa from cyanide Emanuela Frangipani,1*† Isabel Pérez-Martínez,1‡ Huw D. Williams,2 Gaëtan Cherbuin1§ and Dieter Haas1 1 Département de Microbiologie Fondamentale, Université de Lausanne, CH-1015 Lausanne, Switzerland. 2 Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, UK. Summary Pseudomonas aeruginosa produces the toxic secondary metabolite hydrogen cyanide (HCN) at high cell population densities and low aeration. Here, we investigated the impact of HCN as a signal in cell-cell communication by comparing the transcriptome of the wild-type strain PAO1 to that of an HCN-negative mutant under cyanogenic conditions. HCN repressed four genes and induced 12 genes. While the individual functions of these genes are unknown, with one exception (i.e. a ferredoxin-dependent reductase), a highly inducible six-gene cluster (PA4129-PA4134) was found to be crucial for protection of P. aeruginosa from external HCN intoxication. A double mutant deleted for PA4129-PA4134 and cioAB (encoding cyanide-insensitive oxidase) did not grow with 100 μM KCN, whereas the corresponding single mutants were essentially unaffected, suggesting a synergistic action of the PA4129-PA4134 gene products and cyanide-insensitive oxidase. Introduction Hydrogen cyanide (HCN) is a potent inhibitor of cytochrome c oxidases and a variety of other metalloenzymes (Solomons, 1981). Despite its toxicity, cyanide is produced or degraded by a small number of bacterial species. In Pseudomonas spp., HCN

Received 27 May, 2013; revised 3 August, 2013; accepted 13 September, 2013. *For correspondence. E-mail emanuela.frangipani@ uniroma3.it; Tel. (+39) 06 57336408; Fax (+39) 06 57336321. Present addresses: †Dipartimento di Scienze, Università Roma Tre, V.le G. Marconi 446, I-00146 Roma, Italy; ‡Area de Genetica, Facultad de Ciencias, Universidad de Málaga, E-29010 Málaga, Spain; §Institut für Medizinische Mikrobiologie, Universität Zürich, CH-8006 Zürich, Switzerland.

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biosynthesis occurs by enzymatic decarboxylation of glycine, catalysed by a membrane-bound protein complex termed HCN synthase (Castric, 1977; Pessi and Haas, 2004). This complex is sensitive to molecular oxygen and has been only partially purified (Wissing and Andersen, 1981). In P. aeruginosa, the HCN synthase protein complex is encoded by the hcnABC operon, and its expression is tightly regulated at transcriptional and posttranscriptional levels (Pessi and Haas, 2000; 2001; Carterson et al., 2004; Cody et al., 2009). Main factors favouring cyanogenesis are low oxygen tension and high cell population density. Glycine and iron stimulate cyanogenesis (Castric, 1975; Blumer and Haas, 2000; Pessi and Haas, 2001). The promoter of the hcnABC operon is positively regulated by the anaerobic transcriptional regulator ANR and the quorum-sensing activators LasR and RhlR (Pessi and Haas, 2000). Furthermore, positive regulation of the hcnABC genes by the GacS/ GacA two-component system acts indirectly at the transcriptional level via upregulation of the RhlRI quorumsensing system and directly at the post-transcriptional level by relieving RsmA-mediated repression (Pessi and Haas, 2001). Moreover, the AlgR transcriptional regulator activates cyanide production in mucoid clinical strains of P. aeruginosa (Carterson et al., 2004). Finally, the transcriptional regulator RsaL negatively controls hcnABC transcription by binding to the hcnA promoter (Rampioni et al., 2007). In Pseudomonads, HCN has the characteristics of a typical secondary metabolite as it is produced at the end of the exponential growth phase under oxygen limitation, has no apparent toxic effect on the producer strain and likely provides a selective advantage to the producer (Castric, 1975; Blumer and Haas, 2000). HCN appears to act as a virulence factor in severely burned patients (Goldfarb and Margraf, 1967) and contributes to killing in Caenorhabditis elegans and Drosophila melanogaster models of infection (Gallagher and Manoil, 2001; Broderick et al., 2008). Cyanide is present in the sputum of P. aeruginosa-infected cystic fibrosis patients at concentrations of up to 130 μM, and its presence is associated with a reduced lung function (Ryall et al., 2008; Sanderson et al., 2008). In P. aeruginosa cultures, HCN can reach high concentrations of up to 300 μM (Pessi and Haas, 2000). The organism avoids cyanide intoxication by at least two

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mechanisms. One involves the enzyme rhodanese, which converts HCN to the less toxic compound thiocyanate in the presence of thiosulfate (Cipollone et al., 2007). Another well-characterized mechanism is mediated by cyanide insensitive oxidase (CIO), a copper-independent terminal oxidase that allows P. aeruginosa to respire oxygen in the presence of cyanide, whereas the other branches of respiration are sensitive to cyanide (Cunningham et al., 1997; Williams et al., 2007; Frangipani et al., 2008; Arai, 2011). Intriguingly, a CIOnegative (cioAB) mutant still produces cyanide similarly to the wild-type parental strain, and the mutant’s viability is not affected during prolonged incubation with HCN, suggesting the existence of additional mechanisms of resistance to cyanide (Zlosnik et al., 2006). In the present study, we have investigated the effect of endogenously produced cyanide on P. aeruginosa, by comparing the transcriptome of the wild-type to that of an hcnB-negative mutant under conditions that are optimal for HCN production. We have discovered a cluster of six genes (PA4129-PA4134) whose expression is greatly induced during cyanogenesis and provides protection against high levels of cyanide in a cioAB-negative background.

Results and discussion Production of HCN by P. aeruginosa affects only a small portion of the transcriptome In order to investigate the effects of HCN as a signal in cell-cell communication of P. aeruginosa, we performed a transcriptomic analysis of the P. aeruginosa wild-type PAO1 versus the isogenic hcnB-defective mutant PAO6344. Cells were grown to late exponential phase (OD600 ≅ 1.2, corresponding to approximately 3.5 × 109 cells per ml) in minimal medium MMC (Castric, 1975) under low aeration. Culture vessels were tightly closed to avoid outgassing of HCN. Under these growth conditions, chosen to maximize HCN production, the expression of an hcnA’-‘lacZ translational fusion (carried on plasmid pME3826) as well as the production of HCN increased along the growth curve in the wild-type strain (Fig. 1; lines with open squares and white bars, respectively). HCN concentrations were maximal (about 50 μM) at the end of the exponential growth phase. The expression of hcnA was similar in the wild-type and in the HCN-negative mutant (Fig. 1; lines with filled squares). The latter failed to produce any detectable amount of HCN (Fig. 1; black bars) in agreement with earlier findings (Zlosnik et al., 2006). Microarray analysis showed that among the 5901 genes represented on the P. aeruginosa Affymetrix chip, only four genes exhibited a ≥ 2-fold decrease in transcript abundance, whereas only 12 genes showed a ≥ 2-fold

Fig. 1. Conditions of cyanogenesis. The activity of a β-galactosidase reporter plasmid containing an hcnA’-‘lacZ translational fusion (pME3826) was measured in the wild-type PAO1 (line with open squares) and in the ΔhcnB mutant PAO6344 (line with filled squares). HCN production was determined in PAO1 (open bars) and in PAO6344 (black bars). Cultures were grown in MMC in tightly closed bottles with limiting aeration. Each value is the average of three different cultures ± standard deviation.

increase in mRNA levels during cyanogenesis (Table 1). Endogenous cyanide production mostly affected the expression of genes with unknown function, with one exception, i.e. a ferredoxin-dependent NADP+ reductase (fpr). We decided to focus on a cluster of genes whose expression was most strongly affected (PA4129-PA4134). These genes are predicted to encode hypothetical proteins (PA4129, PA4132 and PA4134), proteins possibly involved in electron transfer (PA4131 and PA4133), and a protein hypothetically involved in dissimilatory reduction of nitrite or sulfite (PA4130) (Table 1). The PA4129PA4134 gene cluster is predicted to be organized in three transcriptional units, with promoters located upstream of PA4130, PA4131 and PA4133, respectively (Fig. S1; http://www.pseudomonas.com). This is corroborated by a recent single-nucleotide resolution transcriptome analysis performed in P. aeruginosa PA14 (Wurtzel et al., 2012). To confirm the microarray results, we monitored the expression of a representative gene, PA4130, under the same growth conditions using a PA4130’-‘lacZ translational fusion. This choice was made as PA4130 and PA4129 are homologues of two genes that form a transcriptional unit together with cioAB in P. pseudoalcaligenes (Fig. S1) and appear likely to account for the cyanide resistance phenotype of that species. As expected, the expression of the PA4130’-‘lacZ construct was upregulated in the wild type by comparison with the hcnB mutant, whereas the expression of a constitutive housekeeping gene, measured with a transla-

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports

Cyanide resistance determinant in P. aeruginosa Table 1. P. aeruginosa genes repressed or induced in response to endogenously produced HCN. Gene (name) Repressed genes PA0433 PA0434

PA0435 PA2299 Induced genes PA2328 PA2329

Fold change −8.26 −5.86 −3.64 −2.06 2.13 2.78

PA2330 PA2331 PA3022 PA3397 (fpr) PA4129 PA4130

2.80 4.82 7.23 2.68 24.98 42.99

PA4131 PA4132

51.41 22.28

PA4133

110.26

PA4134

31.10

Protein (function) Hypothetical protein Hypothetical protein (TonB-dependent receptor, not regulated by irona) Hypothetical protein Putative transcriptional regulator Hypothetical protein (periplasmic binding protein ?) Hypothetical protein (ABC transporter protein ?) Hypothetical protein Hypothetical protein Hypothetical protein Ferredoxin-NADP+ reductase Hypothetical protein Hypothetical protein (sulfite or nitrite reductase ?) Putative iron-sulfur protein Hypothetical protein (transcriptional regulator ?) Orphan cytochrome c oxidase subunit (cbb3-type) Hypothetical protein

a. Cornelis and colleagues (2009).

tional proC’-‘lacZ fusion (pME3641), was comparable in the PAO1 wild-type and in the hcnB mutant throughout growth (Fig. 2). These results validate specific HCNmediated regulation of PA4130 under the growth condition chosen.

The PA4129-PA4134 locus does not specify an alternative branch of cyanide-resistant respiration during cyanogenesis Given that cyanide is a potent poison that inhibits several metalloenzymes, in particular Cu-containing terminal oxidases, we hypothesized that HCN production in P. aeruginosa might induce the expression of defense mechanisms capable of protecting the producer strain from the toxicity of HCN. Judging from previous data (Zlosnik et al., 2006), it seemed possible that CIO alone might not be sufficient for cyanide-resistant respiration in P. aeruginosa. We considered the possibility that the most highly upregulated genes in our microarray analysis (i.e. PA4129-PA4134) might encode a new cyanideinsensitive respiratory pathway. We also note that in a cyanide-degrading strain of P. pseudoalcaligenes genes homologous to PA4129-PA4130 (encoding a putative ferredoxin-dependent sulfite or nitrite reductase, and a protein with unknown function, respectively) are likely to be transcriptionally coupled to cioAB homologues (Quesada et al., 2007; Luque-Almagro et al., 2013), sug-

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gesting a possible involvement in CIO-dependent respiration (Fig. S1). However, in our growth media, neither nitrite nor sulfite was present, practically ruling out these compounds as terminal electron acceptors under our experimental conditions. To test the hypothesis of a new cyanide-insensitive respiration branch, we newly constructed a ΔPA4129PA4134 mutant (PAO6738) and a ΔcioAB-ΔPA4129PA4134 double mutant (PAO6739), and we assayed respiratory activity by measuring NADH-dependent oxygen uptake in whole P. aeruginosa cells, grown as for the microarray experiment, in the presence or absence of exogenously added KCN (Table 2). In PAO1 wild-type cells, roughly 40% of the total oxidase activity persisted after the addition of 1 mM KCN, whereas such residual activity was almost nil in a cioAB mutant (PAO6437), as expected. Similarly to the wild-type PAO1, strain PAO6738 lacking the PA4129-PA4134 genes showed about 40% cyanide-resistant NADH-dependent oxygen uptake, while strain PAO6739 (ΔcioABΔPA4129-PA4134) behaved like the ΔcioAB mutant. As a control, we used a strain deleted for all known cyanide-sensitive oxidase genes (PAO6650; Frangipani et al., 2008), whose aerobic respiration was not affected significantly by the addition of KCN (Table 2). These data suggest that the PA4129PA4134 genes are unlikely to encode components of a new cyanide-resistant respiratory branch and that cyanide-resistant respiration essentially depends on CIO in P. aeruginosa PAO1. We next tested strains PAO6738 (ΔPA4129-PA4134) and PAO6739 (ΔcioABΔPA4129-PA4134) for HCN production in liquid MMC under low aeration. The wild-type strain PAO1, the ΔcioAB mutant PAO6347 and the ΔhcnB

Fig. 2. Validation of the microarray data for PA4130 expression. The activities of β-galactosidase reporter plasmids containing either a PA4130’-‘lacZ translational fusion (pME9317; triangles) or a proC’-‘lacZ translational fusion (pME3641; squares) were measured in the wild-type PAO1 (filled symbols) and in the ΔhcnB mutant (PAO6344; open symbols). Cultures were grown in MMC in tightly closed bottles with limiting aeration. Each value is the average of three different cultures ± standard deviation.

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Table 2. Cyanide-insensitive respiration in P. aeruginosa mutants. NADH-dependent oxygen uptakeb [nmol O2 min−1 (mg protein)−1] Straina

Genotype

PAO1 PAO6437 PAO6738 PAO6739 PAO6650

Wild-type cioAB PA4129-34 cioAB PA4129-34 cyo cco1 cco2 cox

−KCN

+KCN

54.7 ± 10.4 51.7 ± 18.8 73.0 ± 24.5 37.0 ± 24.0 77.3 ± 9.9

21.2 ± 5.0 2.0 ± 0.9 36.7 ± 16.8 1.4 ± 0.5 71.0 ± 16.8

% Residual activity with 1 mM KCN 38.5 ± 2.1 3.9 ± 1.2 49.4 ± 8.5 5.4 ± 4.4 90.9 ± 11.6

a. Strains were grown in triplicate in MMC to an OD600 of ≅ 1.0. b. Strains were harvested and washed in phosphate-buffered saline and kept on ice until the assay, which was no later than 90 min after harvesting. NADH-dependent oxygen uptake was measured as described previously (Cunningham and Williams, 1995).

mutant PAO6344 were used as controls. All strains tested, except the ΔhcnB mutant, produced similar amounts of HCN (Fig. 3), indicating that deletion of the PA4129PA4134 gene cluster does not influence HCN production, in a wild-type as well as in a cioAB-defective background. We also investigated the potential contribution of the PA4129-PA4134 genes to protection from endogenously produced cyanide by monitoring the growth of strains PAO1, PAO6738, PAO6437, PAO6739 and PAO6344 in MMC under low aeration for 8, 24 and 72 h. Growth as measured by turbidimetry (OD600) and viable cell counts was similar for all strains tested (Table S1). Moreover, as in the experiment shown in Fig. 3, all strains except PA6344 produced similar amounts of cyanide, and cyanide was not degraded measurably during prolonged incubation (Table S1). Overall, these data suggest that the PA4129-PA4134 genes do not appear to have a role in cyanide biosynthesis or degradation and per se may not be essential for protection from self-intoxication during cyanogenesis.

PA4129-PA4134 double mutant was strongly inhibited at 100 μM KCN and clearly handicapped at 30 or 50 μM KCN (Fig. 4) by comparison with the other strains. At the end of growth with 100 μM KCN, cells of the double mutant were viable, suggesting that cyanide had a bacteriostatic effect rather than a bactericidal activity (data not shown). These data show that the PA4129-PA4134 locus and the cioAB genes together provide synergistic protection from exposure to high levels of cyanide, in particular at low cell population densities.

Concluding remarks HCN production in P. aeruginosa is rigorously controlled at transcriptional and post-transcriptional levels (Pessi and Haas, 2000; 2001; Rampioni et al., 2007; Cody et al., 2009). In addition, P. aeruginosa cells need to be protected from HCN. We have shown here that HCN can act as a signal capable of inducing a novel cyanide resistance determinant, which acts in concert with CIO.

The PA4129-PA4134 locus, together with CIO, ensures cyanide resistance to exogenous cyanide To test the effect of exogenous cyanide, we monitored the growth of strains PAO1 (wild-type), PAO6437 (ΔcioAB), PAO6738 (ΔPA4129-PA4134) and PAO6739 (ΔcioABΔPA4129-PA4134) in the presence of added KCN at 30, 50 or 100 μM. Cultures were grown in Luria Broth (LB) in tightly closed tubes to prevent HCN outgassing. This medium was chosen because in it endogenous HCN production by P. aeruginosa was practically negligible, and the initial cyanide concentration remained constant during growth (data not shown). All strains had the same growth rate in unamended LB (Fig. 4). In the presence of various concentrations of KCN, the wild-type and the cioAB mutant were not affected (Fig. 4). The PA4129PA4134 mutant was slightly sensitive to KCN in a concentration-dependent manner. By contrast, the cioAB

Fig. 3. Deletion of the PA4129-PA4134 cluster does not affect HCN production. Cyanide was measured in PAO1 (wild-type), PAO6344 (ΔhcnB), PAO6437 (ΔcioAB), PAO6738 (ΔPA4129-PA4134) and PAO6739 (ΔcioABΔPA4129-PA4134). Strains were grown in MMC until they reached an OD600 ≅ 1. Each value is the average of two different cultures ± standard deviation.

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Fig. 4. Effect of mutations in PA4129-PA4134 and cioAB on growth in the presence of cyanide. Incubation was carried out in LB in closed test tubes containing the wild-type PAO1 (filled squares), the ΔPA4129-PA4134 mutant PAO6738 (filled diamonds), the ΔcioAB mutant PAO6437 (filled triangles) or the ΔPA4129-PA4134 ΔcioAB double mutant PAO6739 (filled circles). Cells were grown in the presence or absence of different KCN concentrations, as indicated. Each value is the average of three different cultures ± standard deviation.

Experimental procedures Bacterial strains and culture conditions Strains and plasmids used in this study are listed in Table S2. Bacteria were routinely grown in nutrient agar, in nutrient yeast broth (Stanisich and Holloway, 1972) or in LB (Sambrook et al., 1989) at 37°C. When required, antibiotics were added to these media at the following concentrations: 100 μg ml−1 ampicillin, 12.5 μg ml−1 tetracycline, 25 μg ml−1 kanamycin for Escherichia coli, and 300 μg ml−1 carbenicillin and 100 μg ml−1 tetracycline for P. aeruginosa. For determination of HCN production, β-galactosidase experiments using an hcnA’-‘lacZ fusion and RNA extraction, P. aeruginosa strains were grown in a synthetic glycine minimal medium (MMC) described by Castric (1975) under oxygen limitation achieved by growing the cells in tightly closed 125 ml bottles containing 30 ml of medium with gentle shaking (130 r.p.m.). At the end of growth, oxygen concentrations are estimated to be < 1 μM under such conditions (Alvarez-Ortega and Harwood, 2007). Growth experiments in the presence of exogenously added KCN were performed in LB (pH ≅ 7.5); cells were grown in tightly closed 20 ml glass tubes, each containing 3 ml of medium,

with gentle shaking (130 r.p.m.). A freshly prepared KCN solution was made for each experiment in 10 mM K phosphate buffer (pH ≅ 9).

Construction of plasmids and gene replacement mutants DNA cloning and plasmid preparations were performed according to standards methods (Sambrook et al., 1989). Large-scale preparations of plasmid DNA were performed using JETstar 2.0 (Genomed, Löhne, Germany). To construct strain PAO6738 (ΔPA4129-PA4134), an upstream 900 bp fragment and a downstream 600 bp fragment were amplified by PCR using the primer pairs 4129newUPHind/4129newDW and Forward4134/Reverse4134, respectively. These products were digested with HindIII plus BamHI and BamHI plus EcoRI, respectively, and cloned into the corresponding sites of the suicide vector pME3087, yielding plasmid pME9922. This construct, carried by E. coli DH5α, was introduced into P. aeruginosa strain PAO1 by triparental mating using the helper strain E. coli HB101 (pRK2013). Merodiploids were resolved as described previously (Ye et al., 1995). In the resulting P. aeruginosa strain PAO6738, the PA4129-PA4134

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deletion was confirmed by PCR, and the PCR fragment obtained was checked by sequencing. P. aeruginosa strain PAO6739 (ΔPA4129-PA4134, ΔcioAB) was constructed analogously in a PAO6437 (ΔcioAB) background. A translational PA4130’-‘lacZ fusion was constructed by inserting a 593 bp BamHI-PstI fragment carrying the proximal part of PA4130 into the BamHI-PstI sites of pME6013. This fragment was generated by PCR amplification using PAO1 genomic DNA and primers P4130FW and P4130RV.

Acknowledgements We thank Gabriella Pessi for supplying strain PAO6344 and Otto Hagenbüchle, Alexandra Paillusson and Sylvain Pradervand for performing microarray analysis. IPM acknowledges a fellowship from the VELUX Foundation. This work was supported by the Swiss National Foundation for Scientific Research.

References Assays of HCN production, respiratory activity and β-galactosidase HCN was quantified in P. aeruginosa culture supernatants as previously described (Gewitz et al., 1976). NADH-dependent oxygen uptake was assayed in whole cells using a Clark-type oxygen electrode as described previously (Cunningham and Williams, 1995). β-galactosidase specific activities were determined by the Miller method (Miller, 1972).

RNA isolation, generation of cDNA probes and transcriptome analysis Pseudomonas aeruginosa PAO1 and PAO6344 were inoculated at an OD600 of 0.01 into 30 ml of MMC in hermetically closed 125 ml bottles. The cultures where grown at 37°C with gentle shaking (130 r.p.m.) until they reached an OD600 of approximately 1.0; cells were harvested and RNA protect bacteria (Qiagen, Milan, Italy) was added. Total RNA was isolated by the hot phenol method as described elsewhere (Leoni et al., 1996), followed by DNaseI treatment (Roche, Milan, Italy). The quality of total RNA was investigated by agarose gel electrophoresis and an RNA 6000 Nano LabChip in an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Next, cDNA synthesis was obtained with random primers and Superscript II reverse transcriptase (Invitrogen Corp., Carlsbad, CA, USA) using 10 μg of total RNA. cDNA fragmentation, labelling, hybridization, staining and washing steps were performed according to the manufacturer’s protocol for the Affymetrix P. aeruginosa GeneChip arrays (Affymetrix, Inc., Santa Clara, CA, USA). The arrays were scanned with the Affymetrix GeneChip Scanner 3000. Processing of the P. aeruginosa GeneChip (Affimetrix) was performed at the University of Lausanne, Center for Integrative Genomics. For each condition tested, cultures were grown in triplicate, and RNA from these cultures was pooled before proceeding to cDNA synthesis. In addition, biological replicates for each condition were performed on a separate day and run on a different microarray chip. Data were analysed as previously described (Gaillard et al., 2008). Induced and repressed genes (Table 1) meet the following criteria: (i) the P-value obtained for each transcript analysed was < 0.05 and (ii) the absolute change in the transcript level was equal or greater than twofold. The data discussed in this publication have been deposited in National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE48587 (http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE48587).

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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Fig. S1. Genes of interest to this study in (a) P. aeruginosa PAO1 and (b) P. pseudoalcaligenes CECT 5344. Note that the sir/nir and the orf1 genes are homologous to PA4130 and PA4129, showing 54% and 37% amino acid identities respectively. The putative translation start site of the PA4131 homologue in P. aeruginosa PA14 has been relocated by a recent RNAseq analysis and is indicated by a dotted line (Wurtzel et al., 2012). Arrows indicate promoters based on the 5′ UTRs obtained in the same study. HP, hypothetical protein. Table S1. Growth and cyanide production of P. aeruginosa mutants. Table S2. Strains, plasmids and primers used in this study.

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