the nit1c gene cluster of pseudomonas pseudoalcaligenes cect5344 involved in assimilation of...

The nit1C gene cluster of Pseudomonaspseudoalcaligenes CECT5344 involved in assimilationof nitriles is essential for growth on cyanideemi4_337 326..334

Jessica Estepa,† Victor M. Luque-Almagro,†

Isabel Manso, M. Paz Escribano,Manuel Martínez-Luque, Francisco Castillo,Conrado Moreno-Vivián and M. Dolores Roldán*Departamento de Bioquímica y Biología Molecular,Campus de Rabanales, Edificio Severo Ochoa,1a Planta, Universidad de Córdoba, 14071, Córdoba,Spain.

Summary

A proteomic approach was used to identify severalproteins induced by cyanide in the alkaliphilic bacte-rium Pseudomonas pseudoalcaligenes CECT5344,two of them, NitB and NitG, encoded by genes thatbelong to the nit1C gene cluster. The predicted prod-ucts of the nit1C gene cluster are a Fis-like s54-dependent transcriptional activator (NitA), a nitrilase(NitC), an S-adenosylmethionine superfamily member(NitD), an N-acyltransferase superfamily member(NitE), a trifunctional polypeptide of the AIRS/GARSfamily (NitF), an NADH-dependent oxidoreductase(NitH) and two hypothetical proteins of unknownfunction (NitB and NitG). RT-PCR analysis suggestedthat nitBCDEFGH genes were co-transcribed,whereas the regulatory nitA gene was divergentlytranscribed. Real-time RT-PCR revealed that expres-sion of the nitBCDEFGH genes was induced bycyanide and repressed by ammonium. TheP. pseudoalcaligenes CECT5344 nit1C gene clusterwas found to be involved in assimilation of free andorganic cyanides (nitriles) as deduced for the inabilityto grow with cyanides showed by the NitA, NitB andNitC mutant strains. The wild-type strain CECT5344showed a nitrilase activity that allows growth oncyanide or hydroxynitriles. The NitB and NitC mutantsonly presented low basal levels of nitrilase activitythat were not enough to support growth on either freecyanide or aliphatic nitriles, suggesting that nitrilase

NitC is specific and essential for cyanide and aliphaticnitriles assimilation.

Introduction

Cyanide causes problems of pollution in some areasdue to human activities like gold-extraction mines, woodprocessing and metal and jewellery industries. Biologicaltreatments to remove cyanide are based on the use ofmicroorganisms (Raybuck, 1992; Dubey and Holmes,1995) that are able to utilize it as nitrogen source bydifferent cyanide degradation pathways (Fig. 1), includ-ing hydrolytic, oxidative and substitution/addition reac-tions (Ebbs, 2004; Gupta et al., 2010; Luque-Almagroet al., 2011a). The hydrolysis of cyanide produces formicacid and ammonium (Kunz et al., 2001). Cyanide can beoxidized to produce CO2 and NH4

+ with cyanate as aputative intermediate (Harris and Knowles, 1983; Guptaet al., 2010). Two types of substitution/addition reactionshave been described; the production of thiocyanate fromcyanide and S2O3

2- (Dubey and Holmes, 1995) and thesynthesis of 3-cyanoalanine from cyanide and eitherserine or cysteine (Ebbs, 2004). However, most of themicrobes that use cyanide show an optimum pH forgrowth near to 7.0 and significant amounts of cyanidemay evaporate as HCN since the pKa of HCN/CN-

is 9.2.Cyanide is a toxic chemical that inhibits the function of

metalloproteins and strongly binds metals like iron to formmetal–cyanide complexes (Gupta et al., 2010). Therefore,a cyanotrophic microorganism must produce sidero-phores to break down the metal–cyanide complexes foriron scavenging under conditions of low availability of thismetal (Clarke et al., 2001; Huertas et al., 2006). Cyanidealso causes modifications in the electron transport chainssince it inhibits cytochrome c oxidase blocking aerobicrespiration (Ashcroft and Haddock, 1975; Jünemann,1997).

Pseudomonas pseudoalcaligenes CECT5344 is a suit-able candidate for bioremediation of cyanide-containingindustrial effluents since uses free- and metal–cyanidecomplexes as the sole N-source (Luque-Almagro et al.,2005a,b; Huertas et al., 2006; 2010). In the presence offree cyanide, P. pseudoalcaligenes CECT5344 produces

Received 25 May, 2011; revised 24 February, 2012; accepted 1March, 2012. *For correspondence. E-mail [email protected]; Tel.(+34) 957218318; Fax (+34) 957218592. †Authors have contributedequally to this work.

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Environmental Microbiology Reports (2012) 4(3), 326–334 doi:10.1111/j.1758-2229.2012.00337.x

© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd

a nitrile, identified as the 2-hydroxynitrile of oxaloacetate.A malate:quinone oxidoreductase activity that oxidizesL-malate to oxaloacetate is associated to the cyanide-insensitive alternative oxidase (Quesada et al., 2007;Luque-Almagro et al., 2011b). The cioA and cioB genesare clustered together and encode the subunits I and II ofthe terminal bd-oxidase. A gene encoding a sulfite/nitritereductase and two putative genes coding for uncharacter-ized proteins are also found in the cio gene cluster. Thetargeted disruption of the cioA gene eliminates bothexpression of the cyanide-stimulated respiratory activityand growth on cyanide (Quesada et al., 2007). It hasbeen described that 2-oxoacids chemically react withfree cyanide to produce 2-hydroxynitriles, which can beconverted into ammonium by either a nitrilase or anitrile hydratase/amidase system (Luque-Almagro et al.,2011b). However, up to date, the strain CECT5344 is thefirst organism in which the cyanide degradation pathwayincludes a link between free cyanide and 2-hydroxynitriles(Fig. 1).

In this work we describe that the nit1C gene cluster isinvolved in free cyanide and 2-hydroxynitriles degradation/assimilation in P. pseudoalcaligenes CECT5344. Thelink between cyanide and the nit1C gene cluster is high-lighted by the phenotype of the mutants in nitA, nitCand nitB genes, which are unable to use cyanide or2-hydroxynitriles as the sole N-source. The nitC genebelongs to the nit1C cluster and likely codes for a nitrilasethat uses several aliphatic nitriles as substrate. The nitB-CDEFGH genes are induced by cyanide and repressed byammonium, but expression of the regulatory nitA gene,

which is divergently transcribed from the others nit1Cgenes, is found in all nitrogen sources.

Results and discussion

The nit1C gene cluster of P. pseudoalcaligenesCECT5344

A differential proteomic study was carried out in the strainCECT5344 by analysing proteins induced under cyan-otrophic conditions compared with those present innitrate-containing media. Pseudomonas pseudoalcali-genes CECT5344 cells were grown in M9 minimalmedium (Sambrook and Russel, 2001) with sodiumacetate as C-source and with either 2 mM cyanide or2 mM nitrate (as control) as the sole N-source. After ~70%of the N-source was consumed, cells were harvested andbroken by cavitation. The crude extracts were centrifugedand supernatants corresponding to the subcellular solublefractions were analysed by two-dimensional polyacryla-mide gels as previously described (Luque-Almagro et al.,2007). A preliminary proteomic study in this bacteriumrevealed that cyanide induces several proteins relatedwith defence mechanisms against iron deprivation, oxida-tive damage and nitrogen stress (Luque-Almagro et al.,2007). In this work we have identified different spotsinduced by cyanide (Fig. 2A) that were excised from thegels and identified by MALDI-TOF/TOF. A preliminarydraft of the whole-genome sequence of P. pseudoalcali-genes CECT5344 (V.M. Luque-Almagro et al., unpub-lished) has been used to identify the spots isolated from

Fig. 1. Microbial cyanide degradation pathways. In most cases degradation of cyanide includes one or two steps in a specific pathway thatgenerates ammonium, which is further assimilated through glutamine synthase. 1, nitrilase (Pseudomonas pseudoalcaligenes CECT5344); 2,cyanidase (P. fluorescens NCIMB 11764 and Escherichia coli); 3, cyanide hydratase and 4, formamidase (P. stutzeri and pathogen fungi); 5,cyanide dioxygenase (Pseudomonas and Bacillus); 6, rhodanase and 7, thiocyanate hydrolase (Bacillus and Thiobacillus); 8, 3-cyanoalaninesynthase and 9, nitrilase or nitrile hydratase/amidase (Bacillus).

327 J. Estepa et al.

© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 4, 326–334

the proteomic analysis. Two of these spots correspond tothe same protein of unknown function encoded by the nitBgene of the nit1C gene cluster, which also contains aputative nitrilase-encoding nitC gene. Another spot was ahypothetical protein encoded by the nitG gene that alsobelongs to the nit1C gene cluster (Fig. 2A).

The nit1C gene cluster (Figs 2B and 3A) is a DNAfragment of about 7.8 kb (GenBank Accession No.JF748722), which includes eight genes that code for pro-teins whose putative functions are: NitA (640 amino acidresidues), a Fis-like s54-dependent transcriptional activa-

tor; NitB (160 residues), a hypothetical protein; NitC (326residues), a nitrilase that belongs to the subfamily 1 of thenitrilase branch (Podar et al., 2005); NitD (366 residues), amember of the S-adenosylmethionine (SAM) superfamilyinvolved in unreactive C–H bonds cleavage for biosynthe-sis of vitamins, coenzymes and antibiotics, and for nucleicacid repair and modification (Wang and Frey, 2007); NitE(186 residues), a member of the N-acyltransferase (GNAT)superfamily that uses acyl-CoA to acylate its cognatesubstrates (Vetting et al., 2004); NitF (340 residues), atrifunctional polypeptide that belongs to the AIRS (also

Fig. 2. A. Differential proteomic analysis of P. pseudoalcaligenes CECT5344. This bacterial strain was isolated from the Guadalquivir River,Córdoba (Spain), as previously described (Luque-Almagro et al., 2005b). Cells were cultured with either nitrate (NO3

-) or cyanide (CN-) as thesole N-source at 2 mM final concentration. When about 70% of the nitrogen source was consumed, cells were harvested and subcellularfractionation was carried out as previously described (Luque-Almagro et al., 2007). Triplicate 2D-PAGE separations were generated for eachsample. Proteins were identified by peptide mass fingerprinting (PMF) and confirmed by MS/MS analysis of most abundant peptide ions.MASCOT searching engine (Matrixscience) was used for protein identification over the non-redundant NCBI database of proteins and a localdatabase with the annotated whole-genome sequence of P. pseudoalcaligenes CECT5344 (V.M. Luque-Almagro et al., unpublished). Theconfidence in the PMF matches (P < 0.05) was based on the MOWSE score (higher than 65) and CI > 99.8%, and confirmed by the accurateoverlapping of the matched peptides with the major peaks of the mass spectrum. Protein identification was carried out in the UCO-SCAIproteomic unit, a member of Carlos III Networked Proteomics Platform, ProteoRed-ISCIII. The spots marked by arrows were identified as NitBand NitG proteins.B. The nit1C gene cluster of P. pseudoalcaligenes CECT5344 and other bacteria. These genes code for the following putative products: nitA,transcriptional regulator with GAF, AAA-type ATPase and DNA-binding domains; nitB, hypothetical protein 1; nitC, nitrilase; nitD, radical SAMdomain-containing protein; nitE, GCN5-related N-acetyltransferase; nitF, AIR synthase-like protein; nitG, hypothetical protein 2; nitH,NADH-dependent flavin oxidoreductase; gntR, GntR family transcriptional regulator; and gst, glycosyltransferase.

Role of nitriles in cyanide degradation 328


called GARS) family with phosphoribosylglycinamideformyltransferase, phosphoribosylglycinamide synthetaseand phosphoribosylaminoimidazole synthetase activitiesrequired for de novo purine synthesis; NitG (105 residues),a hypothetical protein; and NitH (421 residues), an NADH-dependent flavin oxidoreductase. Curiously, another puta-tive phosphoribosylglycinamide formyltransferase, withdifferent molecular mass and pI to NitF protein, was previ-ously found to be induced by cyanide in P. pseudoalcali-genes and its role in siderophore biosynthesis waspostulated (Luque-Almagro et al., 2007).

The predicted nitrilase NitC of P. pseudoalcaligenes isclosely related to nitrilases found in non-cultivable micro-organisms and in some g-proteobacteria (Fig. S1), and itbelongs to the subfamily 1 of the nitrilase superfamily(Podar et al., 2005). Database search analysis revealsthat the nit1C gene cluster is found with the same orsimilar gene composition and arrangement in otherg-proteobacteria, like Acinetobacter sp. ATCC 27244,Photorhabdus luminecens and Xenorhabdus nemato-phila, and several a- and b-proteobacteria, such as theopportunistic pathogen Burkholderia vietnamiensis G4,the simbiont Rhizobium leguminosarum bv. viciae 3841,the cyanobacterium Prochlorococcus marinus MIT 9301and the Gram-positive bacterium Rhodococcus jostiiRHA1 (Fig. 2B). In the nit1C gene cluster of R. jostii RHA1there are two genes coding for putative nitrilases; onegene is homologous to the P. pseudoalcaligenes nitCgene and the other gene is also homologous to another

gene of P. pseudoalcaligenes CECT5344 (Ppsal_1647)that is present in a different genome locus (V.M. Luque-Almagro et al., unpublished). However, up to date none ofthese bacterial strains has been tested for cyanide deg-radation. Among pseudomonads, P. pseudoalcaligenesCECT5344 is the only strain with both, the nit1C genecluster and the capacity to degrade cyanide. Curiously,the genes flanking the P. pseudoalcaligenes nit1C genecluster are found in a correlative position in the genome ofother evolutionary closely related pseudomonads likePseudomonas mendocina and P. stutzeri (Fig. S2). Thepredicted NitC nitrilase of the strain CECT5344 has fourconserved residues (Arg129, His168, Glu182 and His185)present in other nitrilases that use aliphatic nitriles(Fig. S3), but lacks the residues His129, Asn168 andMet170 conserved in aromatic nitrilases (Sharma et al.,2009). This agrees with the substrate specificity found forthis enzyme (Table 1). The nitB gene is also found in thenit1C gene cluster of other proteobacteria (Fig. S4).

Transcriptional analysis of the nit1C gene cluster

The P. pseudoalcaligenes nit1C gene region contains aputative regulatory gene, nitA, which is transcribed diver-gently from nitBCDEFGH genes. NitA codes for a Fis-likes54-dependent transcriptional activator. Fis (factor forinversion stimulation) is a multifunctional protein involvedin different site-specific recombination processes, regula-tion of gene expression and oriC-directed initiation

Fig. 3. Transcriptional analysis of the nit1C gene cluster of P. pseudoalcaligenes CECT5344 by RT-PCR and real-time RT-PCR. Cells werepreviously cultured with 2 mM ammonium and when this compound was depleted (about 24 h), different N-sources were added as follows:2 mM cyanide, 3 mM ammonium or 3 mM ammonium plus 2 mM cyanide. A control without additional N-source (-N) was also carried out.When about 50% of the N-source was consumed, cells were harvested and washed in TEG buffer containing 25 mM Tris-HCl (pH 8.0) with1% glucose and 10 mM EDTA. RNA isolations were performed following the Qiagen RNA extraction kit (RNeasy midi kit). DNase incubationwas carried out in the column with RNase-free DNase set (Qiagen) and an additional post-column treatment was required with DNase I(Ambion). The concentration and purity of the RNA samples were measured by using a ND1000 spectrophotometer (Nanodrop Technologies).Synthesis of total cDNA was achieved in 20 ml final volume containing 500 ng RNA, 0.7 mM dNTPs, 200 U SuperScript II ReverseTranscriptase (Invitrogen), and 3.75 mM random hexamers (Applied Biosystems). Samples were initially heated at 65°C for 5 min and thenincubated at 42°C for 50 min, followed by incubation at 70°C for 15 min.A. The P. pseudoalcaligenes CECT5344 nit1C gene cluster and specific oligonucleotides used for PCR reactions (Table S1).B. RT-PCR reactions with RNA isolated from wild-type (WT) cells grown without nitrogen or with cyanide (upper panel) and with RNA from theNitA, NitB and NitC mutants grown with cyanide (lower panel). To carry out PCR reactions, 2 ml of each cDNA was initially heated at 94°C for5 min, followed by 30 cycles of amplification: 94°C, 30 s; 65°C, 30 s and 69°C, 1 min. Polymerase extension reactions were completed by anadditional incubation at 72°C for 10 min. Both panels: M, molecular marker X (Roche); lane 1, negative control without template, lane 2,positive control with genomic DNA as template. Upper panel: lanes 3 and 4, RT-PCR reactions with RNA isolated from cells cultured undernitrogen depletion (–N), without or with reverse transcriptase respectively; lanes 5 and 6, RT-PCR reactions with RNA isolated from cellscultures with cyanide, without or with reverse transcriptase respectively. Lower panel: (-), (+), RT-PCR reactions without or with reversetranscriptase respectively.C. Real-time RT-PCR analysis of the expression of nitA (grey) and nitC (black) genes either under different N-sources in the wild type-strain(left) or under cyanotrophic conditions in the wild-type and NitB and NitC mutant strains (right). RNA isolation and cDNA synthesis werecarried out as indicated above. For real-time assays, the cDNA was purified using Favorprep Gel/PCR purification kit (Favorgen) and theconcentration was measured using a ND1000 spectrophotometer. The iQ5 Multicolour Real-Time PCR Detection System (Bio-Rad) was usedin a 25 ml reaction (final volume), containing 2 ml of diluted cDNA (12.5, 2.5 and 0.5 ng) and 0.2 mM of each primer: NitA-1 and NitA-2 (nitAgene), NitC-1 and NitC-2 (nitC gene), RpoB-3 and RpoB-4 (rpoB gene), and 12.5 ml of iQ SYBR Green Supermix (Bio-Rad). Target cDNAsand reference samples were amplified three times in separate PCR reactions. Samples were initially denatured by heating at 95°C for 3 min,followed by 40 cycles of amplification (95°C, 30 s; test annealing temperature, 60°C, 30 s; elongation and signal acquisition, 72°C, 30 s).For relative quantification of the fluorescence values, a calibration curve was made using dilution series from 80 to 0.008 ng ofP. pseudoalcaligenes CECT5344 genomic DNA sample. Represented data were normalized by using the rpoB gene as housekeeping(Table S1). Error bars represent standard deviation calculated from the results of three independent experiments.



of chromosomal replication (Finkel and Johnson, 1992).The nitC and nitD genes overlap 14 bp, and nitE and nitFgenes also overlap 8 bp. RT-PCR analysis suggeststhat nitBCDEFGH genes are probably co-transcribed(Fig. 3B). Several mutant strains were constructed in dif-ferent nit genes. Thus, NitB and NitC mutant strainsof P. pseudoalcaligenes CECT5344 were generated byinsertion of a gentamicin resistance cassette (Beckeret al., 1995) in the nitB and nitC genes respectively. Polareffect over the downstream genes can be rule out in themutant strains NitB and NitC, as deduced for the pres-ence of a positive band when primers NitE-2 and AIRS5′were used in the RT-PCR reactions (Table S1 andFig. 3B). Also, this result was corroborated by real-timePCR when analysing nitC gene expression level oncyanide that was similar in the wild-type and NitB and NitCmutant strains. In addition, a NitA mutant strain was gen-erated by insertion of a kanamycin-resistant cassette inthe regulatory nitA gene. The absence of expression ofthe nitE and nitF genes in the NitA mutant demonstratesthat the regulator NitA is an activator essential for theexpression of the nit1C genes in the presence of cyanide(Fig. 3B). Real-time RT-PCR analysis also revealed thatcyanide induces the expression of the nitBCDEFGHgenes, whereas ammonium acts as repressor. Expres-sion of the nitC gene in cyanide was ~ 11-fold higher thanin the absence of nitrogen (Fig. 3C). This type of regula-tion is usual for systems involved in inorganic nitrogenassimilation. On the contrary, expression of the regulatorynitA gene, which is transcribed in the opposite direction,was found in all N-sources tested, although at low levels.

Under cyanotrophic conditions, nitA gene expression was~ 41-fold lower than the expression of the nitC gene(Fig. 3C).

NitA, NitB and NitC mutant strains ofP. pseudoalcaligenes CECT5344 are unable to growwith inorganic cyanide and several aliphatic nitrilesas N-source

The strain CECT5344 can grow with cyanide, several2-hydroxynitriles and other aliphatic nitriles, like glu-taronitrile and the 2-hydroxynitriles of oxaloacetate,2-oxoglutarate, formaldehyde (glycolonitrile) or acetalde-hyde (lactonitrile) as the sole N-source (Table 1; Luque-Almagro et al., 2011b). Under cyanotrophic conditions thisbacterium induces an alternative oxidase for insensitive-cyanide respiration coupled to a malate:quinone oxi-doreductase that produces oxaloacetate from L-malate.Curiously, malate dehydrogenase activity was undetect-able in the strain CECT5344 (Luque-Almagro et al.,2011b). Oxaloacetate, among other 2-oxoacids, reactswith cyanide to produce a 2-hydroxynitrile that isfurther metabolized through ammonium formation(Luque-Almagro et al., 2011b). The NitC mutant strainwas unable to grow with cyanide as the sole N-source(Fig. 4). In the presence of cyanide, both wild-typeand NitC mutant strains produce oxaloacetate by thecyanide-induced malate:quinone oxidoreductase activity.Oxaloacetate reacts chemically with cyanide to produceits 2-hydroxynitrile, which was determined by HPLC using

Table 1. Growth of P. pseudoalcaligenes CECT5344 wild-type and NitC mutant strains with different nitriles as the sole nitrogen source.

Nitrile

Growth (%)*

Linear formulaWT NitC

Aliphatic2OG-CNa 100 � 12 17 � 0.5 HOOCCH2CH2C(OH)CNCOOHOAA-CNb 83 � 8 28 � 1 HOOCCH2C(OH)CNCOOH2-Hydroxyisobutyronitrilec 22 � 0.6 5 � 0.05 (CH3)2C(OH)CNGlycolonitriled 75 � 8 14 � 0.9 CH2(OH)CNLactonitrilee 47 � 5 19 � 1 CH3CH(OH)CNAcetonitrile 0 0 CH3CNAcrylonitrile 0 0 CH2CHCNGlutaronitrile 89 � 7 8 � 0.04 NC(CH2)3CNUndecyl cyanide 0 0 CH3(CH2)10CN

AromaticBenzonitrile 0 0 C6H5CNMandelonitrilef 0 0 C6H5CH(OH)CNo-Tolunitrile 0 0 CH3C6H4CN4-Cyanobenzoic acid 0 0 CNC6H4COOHPhthalonitrile 0 0 C6H4(CN)2

2-Ethoxybenzonitrile 0 0 CNC6H4(O)CH2CH3

2-Methoxybenzonitrile 0 0 CNC6H4(O)CH3

*Maximal growth expressed as percentage (%). One hundred per cent corresponds to an absorbance at 600 nm of 0.360 that was achieved withthe 2-hydroxynitrile of 2-oxoglutarate as nitrogen source. In all cases the initial absorbances at 600 mn were about 0.010 � 0.005. Among others,2-hydroxynitriles of 2-oxoglutaratea, oxaloacetateb, acetonec, formaldehyded, acetaldehydee and benzaldehydef were used.



internal standards as previously described (Luque-Almagro et al., 2011b). In the wild-type strain, this nitrile istransiently released to the media and assimilated viaammonium by the glutamine synthase. In contrast, theNitC mutant was unable to assimilate this nitrile, whichwas not further degraded and it was consequently accu-mulated in the media (Fig. 4). This is the first time that a2-hydroxynitrile is described as intermediate in a cyanidebiodegradation process. Several strains of Pseudomonashave been described to degrade cyanide (Fig. 1), includ-ing Pseudomonas fluorescens NCIMB 11764, whichdegrade cyanide through formic acid and ammoniumproduction (Kunz et al., 2001), and P. stutzeri, which pro-duces formamide from cyanide (Watanabe et al., 1998).The wild-type strain of P. pseudoalcaligenes CECT5344was also able to grow with aliphatic nitriles, like glu-taronitrile and the 2-hydroxynitriles of oxaloacetate,2-oxoglutarate, formaldehyde or acetaldehyde (Table 1).In contrast, growth of the NitC mutant strain with these

nitriles was very low or almost undetectable (Table 1).Aliphatic nitriles are substrates for the putative nitrilaseNitC of P. pseudoalcaligenes as suggested by themaximal growth achieved by the wild-type strain com-pared with that shown by the NitC mutant strain (Table 1).The NitB and NitA mutant strains showed a similar behav-iour with cyanide and nitriles to that shown by theNitC mutant (data not shown). None of the strains, wildtype, NitA, NitB and NitC mutants, was able to use thearomatic nitriles tested, including aromatic hydroxynitriles(Table 1).

Enzymatic determination of the nitrilase (NitC) activity

The nitrilase activity responsible for cyanide assimilationin P. pseudoalcaligenes CECT5344 was determined withglutaronitrile as substrate in the wild-type and the NitCand NitB mutant strains. Several cultures were set up in200 ml conical flasks with 2 mM ammonium as N-source.When this N-source was consumed (after ~ 24 h) differentN-sources were added to cultures, including ammonium,nitrate, cyanide, ammonium plus cyanide, and the in vitrosynthesized 2-hydroxynitriles of 2-oxoglutarate and oxal-acetate, and leaved for a further 6 h. Then, cells wereharvested by centrifugation, resuspended in phosphatebuffer (with OD600 ~ 0.7) and used as source for nitrilaseactivity determinations with glutaronitrile as substrate.This nitrilase activity was undetectable when assayedin cell-free extracts. In the wild-type strain, the nitrilaseactivity was higher in cells grown with cyanide or hydrox-ynitriles than in ammonium-grown cells (Fig. 5). Thus,maximal activity was obtained in the wild-type strain whencyanide or the 2-hydroxynitriles of 2-oxoglutarate oroxaloacetate were present, with values of about13–15 nmol ml-1 min-1. Similar results were obtained withglycolonitrile as substrate for the nitrilase NitC, but themaximal activity was 9 � 0.8 nmol ml-1 min-1. Withcyanide plus ammonium or in the absence of the inducers(cyanide or hydroxynitriles), a basal level of activity wasdetected (3.5 � 0.5 nmol ml-1 min-1), probably due toother nitrilases present in the strain CECT5344 that mightshow activity with glutaronitrile as substrate (Fig. 5).On the contrary, in the NitB and NitC mutant strains thenitrilase activity was not induced by cyanide or nitrilesand similar basal levels of activity (about 3.5 �

0.5 nmol ml-1 min-1) were found in all N-sources tested(Fig. 5), suggesting that the putative nitrilase encoded bythe nitC gene is specific and essential for assimilation ofaliphatic nitriles formed during cyanide metabolism, andthat the NitB protein is required for the nitrilase NitC activ-ity, probably acting as a chaperon as described previouslyfor another nitrilase (Layh et al., 1998). Also, the inabilityof the NitB and NitC mutant strains of P. pseudoalcali-genes CECT5344 to assimilate cyanide, 2-hydroxynitriles

Fig. 4. Growth with cyanide of the wild-type and NitC mutantstrains of P. pseudoalcaligenes CECT5344. Both strains werecultured with 2 mM ammonium chloride in order to reduce the lagphase, and when this N-source was depleted, 2 mM sodiumcyanide was added as the sole N-source (time 0 in the figure). Atthe indicated times aliquots were taken and growth was determinedby following absorbance at 600 nm for the wild-type strain (opencircles and solid line) and the NitC mutant (filled circles and dashedline). Free cyanide concentration in the media was determinedcolorimetrically (Asmus and Garschagen, 1953) for the wild-typestrain (open triangles and solid line) and the NitC mutant (filledtriangles and dashed line). Nitrile production was also determinedby HPLC analysis for both wild-type (open bars) and NitC mutant(filled bars) strains. Data correspond to the average of threeindependent experiments that gave similar results.



and other nitriles (Fig. 4 and Table 1) indicates that thenit1C gene cluster is specific and essential for growth withcyanide, and also that the proteins NitB and NitC areessential for the nitrilase activity that converts the2-hydroxynitrile of oxaloacetate and other aliphatic nitrilesinto ammonium. Oxaloacetate is produced in the wild-typestrain and the NitB and NitC mutants under cyanotrophicconditions by the association of a malate:quinone oxi-doreductase that produces oxaloacetate from L-malateand a cyanide-insensitive terminal oxidase (Luque-Almagro et al., 2011b). Other three genes that code forputative nitrilases have been identified in the genome ofP. pseudoalcaligenes CECT5344 (V.M. Luque-Almagroet al., unpublished), but none of them seems to beresponsible for degradation/assimilation of cyanide in thisbacterium as deduced for the phenotype of the NitCmutant of P. pseudoalcaligenes. These other three nit-rilases found in the strain CECT5344 belong to the sub-family 1 of the nitrilase superfamily like the nitrilase NitC,although information about their predicted function isunknown.

To summarize, scarce genetic data are available oncyanide degradation/assimilation. In this work we providerelevant data demonstrating that the P. pseudoalcali-genes CECT5344 nit1C gene cluster is essential forassimilation of free and organic cyanides, mainly2-hydroxynitriles.

Acknowledgements

This work was funded by Ministerio de Ciencia e Innovación(Grants BIO2008-04542-C02-01, BIO2011-30026-C02-02and PET2008_0048, also supported by FEDER 2007-2013)and by Junta de Andalucía (Grant CVI-7560). J.E. andV.M.L.-A. are recipient of predoctoral and postdoctoral (Juande la Cierva) fellowships from Ministerio de Ciencia e Inno-vación (Spain) respectively. We also thank GEMASUR,SAVECO and AVENIR for their fruitful collaborations.

References

Ashcroft, J.R., and Haddock, B.A. (1975) Synthesis of alter-native membrane-bound redox carriers during aerobicgrowth of Escherichia coli in the presence of potassiumcyanide. Biochem J 148: 349–352.

Asmus, E., and Garschagen, H. (1953) The use of barbituricacid for the photometric determination of cyanide and thio-cyanate. Z Anal Chem 138: 414–422.

Becker, A., Schmidt, M., Jäger, W., and Pühler, A. (1995)New gentamicin-resistance and lacZ promoter-probe cas-settes suitable for insertion mutagenesis and generation oftranscriptional fusions. Gene 162: 37–39.

Clarke, T.E., Tari, L.W., and Vogel, H.J. (2001) Structuralbiology of bacterial iron uptake systems. Curr Top MedChem 1: 7–30.

Dubey, S.K., and Holmes, D.S. (1995) Biological cyanidedestruction mediated by microorganisms. World J Micro-biol Biotechnol 11: 257–265.

Ebbs, S. (2004) Biological degradation of cyanide com-pounds. Curr Opin Biotechnol 15: 231–236.

Finkel, S.E., and Johnson, R.C. (1992) The Fis protein: it’snot just for inversion anymore. Mol Microbiol 6: 3257–3265.

Gupta, N., Balomajumder, C., and Agarwal, V.K. (2010)Enzymatic mechanism and biochemistry for cyanide deg-radation: a review. J Hazard Mater 176: 1–13.

Harris, R., and Knowles, C.J. (1983) Isolation and growth ofa Pseudomonas species that utilizes cyanide as a sourceof nitrogen. J Gen Microbiol 129: 1005–1011.

Huertas, M.J., Luque-Almagro, V.M., Martínez-Luque, M.,Blasco, R., Moreno-Vivián, C., Castillo, F., and Roldán,M.D. (2006) Cyanide metabolism of Pseudomonaspseudoalcaligenes CECT5344: role of siderophores.Biochem Soc Trans 34: 152–155.

Huertas, M.J., Sáez, L.P., Roldán, M.D., Luque-Almagro,V.M., Martínez-Luque, M., Blasco, R., et al. (2010) Alkalinecyanide degradation by Pseudomonas pseudoalcaligenesCECT5344 in a batch reactor. Influence of pH. J HazardMater 179: 72–78.

Jünemann, S. (1997) Cytochrome bd terminal oxidase.Biochim Biophys Acta 1321: 107–127.

Kunz, D.A., Fernandez, R.F., and Parab, P. (2001) Evidencethat bacterial cyanide oxygenase is a pterin-dependenthydroxylase. Biochem Biophys Res Commun 287: 514–518.

Layh, N., Parratt, J., and Willetts, A. (1998) Characterizationand partial purification of an enantioselective arylacetonit-rilase from Pseudomonas fluorescens DSM7155. J MolCatal B Enzym 5: 467–474.

Fig. 5. Cyanide-induced nitrilase NitC activity ofP. pseudoalcaligenes CECT5344. The wild-type strain (white bars)and the mutants NitB (black bars) and NitC (grey bars) werepre-cultured with 2 mM ammonium chloride. When this compoundwas depleted, different N-sources were added at 2 mM finalconcentration. The nitrilase activity was assayed with 100 mMglutaronitrile as substrate. Ammonium concentration wasdetermined as previously described (Solorzano, 1969).Abbreviations: -N, without nitrogen; OGCN, 2-hydroxynitrile of2-oxoglutarate; OAACN, 2-hydroxynitrile of oxaloacetate. Thesehydroxynitriles were synthesized in vitro in a previous optimizedchemical process (Luque-Almagro et al., 2011b) since they are notcommercially available. Data correspond to the average of fourindependent experiments that gave similar results.



Luque-Almagro, V.M., Blasco, R., Huertas, M.J., Martínez-Luque, M., Moreno-Vivián, C., Castillo, F., and Roldán,M.D. (2005a) Alkaline cyanide biodegradation byPseudomonas pseudoalcaligenes CECT5344. BiochemSoc Trans 33: 168–169.

Luque-Almagro, V.M., Huertas, M.J., Martínez-Luque, M.,Moreno-Vivián, C., Roldán, M.D., García-Gil, L.J., et al.(2005b) Bacterial cyanide degradation and its metal com-plexes under alkaline conditions. Appl Environ Microbiol71: 940–947.

Luque-Almagro, V.M., Huertas, M.J., Roldán, M.D., Moreno-Vivián, C., Martínez-Luque, M., Blasco, R., and Castillo, F.(2007) The cyanotrophic bacterium Pseudomonaspseudoalcaligenes CECT5344 responds to cyanide bydefence mechanism against iron deprivation, oxidativedamage and nitrogen stress. Environ Microbiol 9: 1541–1549.

Luque-Almagro, V.M., Blasco, R., Martínez-Luque, M.,Moreno-Vivián, C., Castillo, F., and Roldán, M.D. (2011a)Bacterial cyanide degradation is under review: Pseudomo-nas pseudoalcaligenes CECT5344, a case of an alka-liphilic cyanotroph. Biochem Soc Trans 39: 269–274.

Luque-Almagro, V.M., Merchán, F., Blasco, R., Igeño, M.I.,Martínez-Luque, M., Moreno-Vivián, C., et al. (2011b)Cyanide degradation by Pseudomonas pseudoalcaligenesCECT5344 involves a malate:quinone oxidoreductase andan associated cyanide-insensitive electron transfer chain.Microbiology 157: 739–746.

Podar, M., Eads, J.R., and Richardson, T.H. (2005) Evolutionof a microbial nitrilase gene family: a comparative andenvironmental genomics study. BMC Evol Biol 5: 42–54.

Quesada, A., Guijo, M.I., Merchán, F., Blázquez, B., Igeño,M.I., and Blasco, R. (2007) Essential role of cytochromebd-related oxidase in cyanide resistance of Pseudomonaspseudoalcaligenes CECT5344. Appl Environ Microbiol 73:5118–5124.

Raybuck, S.A. (1992) Microbes and microbial enzymes forcyanide degradation. Biodegradation 3: 3–18.

Sambrook, J., and Russel, D.W. (2001) Molecular Cloning: ALaboratory Manual, 3rd edn. Cold Spring Harbor, NY, USA:Cold Spring Harbor Laboratory Press.

Sharma, N., Kushwaha, R., Sodhi, J.S., and Bhalla, T.C.(2009) In silico analysis of amino acid sequences in rela-tion to specificity and physiochemical properties of somemicrobial nitrilases. J Proteomics Bioinform 2: 185–192.

Solorzano, L. (1969) Determination of ammonia in naturalwaters by the phenol hypochlorite method. Limnol Ocean-ogr 14: 799–801.

Vetting, M.W., de Carvalho, L.P., Yu, M., Hegde, S.S.,Magnet, S., Roderick, S.L., and Blanchard, J.S. (2004)Structure and functions of the GNAT superfamily of acetyl-transferases. Arch Biochem Biophys 433: 212–226.

Wang, S.C., and Frey, P.A. (2007) S-adenosylmethionine asan oxidant: the radical SAM superfamily. Trends BiochemSci 32: 101–110.

Watanabe, A., Yano, K., Ikebukuro, K., and Karube, I. (1998)Cyanide hydrolysis in a cyanide-degrading bacterium,

Pseudomonas stutzeri AK61, by cyanidase. Microbiology144: 1677–1682.

Supporting information

Additional Supporting Information may be found in the onlineversion of this article:

Table S1. Oligonucleotide primers used in this study.Fig. S1. Phylogenetic tree of P. pseudoalcaligenesCECT5344 NitC homologues in bacteria. Filled circles,a-proteobacteria; open circles, b-proteobacteria; opensquares, g-proteobacteria; filled squares, d-proteobacteria;and open triangles, verrucomicrobia. NitC orthologues shownare encoded by genes arranged in nit1C-like clusters, withthe exception of Burkholderia graminis and Mycobacteriumintracellulare where information is not available. TheP. pseudoalcaligenes CECT5344 NitC is marked by an arrow.Fig. S2. Gene arrangement in other pseudomonads of theregions adjacent to P. pseudoalcaligenes nit1C gene cluster.Abbreviations: cysK, cysteine synthase; aceK, isocitratedehydrogenase kinase/phosphatase. Numbers: 1.Ppsal_2959, Pmen_1788 and PST_2477 code for an AAAfamily ATPase; 2. Ppsal_2958, Pmen_1789 and PST_2475encode a protein of unknown function; 3. Ppsal_2955,Pmen_1791 and PST_2474 code for a carboxylate/aminoacid/amine transporter; 4. Ppsal_2945, Pmen_1793 andPST_2473 encode a b-lactamase-like protein.Fig. S3. Multiple sequence alignment of aliphatic nitrilases.Abbreviations and sequence Accession No.: Ppsal,P. pseudoalcaligenes CECT5344 (JF748722); Plumin, Pho-torhabdus luminescens subsp. laumondii TTO1(NP_928542.1); Psyrin, Pseudomonas syringae pv. syringaeB728a (AAY35081.1); Pstutz, Pseudomonas stutzeri AK61(BAA11653.1); Rrhod, Rhodococcus rhodochrous J1(BAA01994.1); Afaeca, Alcaligenes faecalis JM3(BAA02684.1). The conserved residues in both aromatic andaliphatic nitrilases at their active sites (Glu48, Lys131,Cys165) are highlighted with asterisks. The residues con-served in aliphatic nitrilases (Arg129, His168, Glu182,His185) are denoted by filled triangles. Alignment was per-formed by using CLUSTALW. Identical residues are shaded ingrey.Fig. S4. Phylogenetic tree of P. pseudoalcaligenesCECT5344 NitB homologues in bacteria. Filled circles,a-proteobacteria; open circles, b-proteobacteria; opensquares, g-proteobacteria; filled squares, d-proteobacteria;and open triangles, verrucomicrobia. Microorganisms con-taining two nitrilases are highlighted (asterisks). NitB ortho-logues shown are encoded by genes arranged in nit1C-likeclusters. The P. pseudoalcaligenes CECT5344 NitB ismarked by an arrow.

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