The cyanotrophic bacterium <i>Pseudomonas pseudoalcaligenes</i> CECT5344 responds to cyanide by defence mechanisms against iron deprivation, oxidative damage and nitrogen stress

Luque-Almagro, Víctor M.; Huertas, María José; Roldán, María Dolores; Moreno‐Vivián, Conrado; Martı́nez-Luque, Manuel; Blasco, Rafael; Castillo, Francisco

doi:10.1111/j.1462-2920.2007.01274.x

Cited by 21 publications

(23 citation statements)

References 48 publications

(62 reference statements)

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“…Nevertheless, the reason for the induction of cyanase activity in cells growing in cyanide is still an open question ( Table 2). Nitrogen limitation conditions generated by cyanide, recently described for P. pseudoalcaligenes (25), are not sufficient to explain the high level of cyanase activity detected in cells growing in cyanide, which was similar to that observed in cells growing in cyanate (Table 2, Fig. 2).…”

Section: Fig 5 Tolerance Of the Wild Type (White Bars) And Cynssupporting

confidence: 64%

Characterization of the Pseudomonas pseudoalcaligenes CECT5344 Cyanase, an Enzyme That Is Not Essential for Cyanide Assimilation

Luque-Almagro

Huertas

Sáez

et al. 2008

Appl Environ Microbiol

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Cyanase catalyzes the decomposition of cyanate into CO 2 and ammonium, with carbamate as an unstable intermediate. The cyanase of Pseudomonas pseudoalcaligenes CECT5344 was negatively regulated by ammonium and positively regulated by cyanate, cyanide, and some cyanometallic complexes. Cyanase activity was not detected in cell extracts from cells grown with ammonium, even in the presence of cyanate. Nevertheless, a low level of cyanase activity was detected in nitrogen-starved cells. The cyn gene cluster of P. pseudoalcaligenes CECT5344 was cloned and analyzed. The cynA, cynB, and cynD genes encode an ABC-type transporter, the cynS gene codes for the cyanase, and the cynF gene encodes a novel 54 -dependent transcriptional regulator which is not present in other bacterial cyn gene clusters. The CynS protein was expressed in Escherichia coli and purified by following a simple and rapid protocol. The P. pseudoalcaligenes cyanase showed an optimal pH of 8.5°C and a temperature of 65°C. An insertion mutation was generated in the cynS gene. The resulting mutant was unable to use cyanate as the sole nitrogen source but showed the same resistance to cyanate as the wild-type strain. These results, in conjunction with the induction pattern of the enzymatic activity, suggest that the enzyme has an assimilatory function. Although the induction of cyanase activity in cyanide-degrading cells suggests that some cyanate may be generated from cyanide, the cynS mutant was not affected in its ability to degrade cyanide, which unambiguously indicates that cyanate is not a central metabolite in cyanide assimilation.

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Section: Fig 5 Tolerance Of the Wild Type (White Bars) And Cynssupporting

confidence: 64%

Characterization of the Pseudomonas pseudoalcaligenes CECT5344 Cyanase, an Enzyme That Is Not Essential for Cyanide Assimilation

Luque-Almagro

Huertas

Sáez

et al. 2008

Appl Environ Microbiol

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“…After ∼70% of the N‐source was consumed, cells were harvested and broken by cavitation. The crude extracts were centrifuged and supernatants corresponding to the subcellular soluble fractions were analysed by two‐dimensional polyacrylamide gels as previously described (Luque‐Almagro et al ., 2007). A preliminary proteomic study in this bacterium revealed that cyanide induces several proteins related with defence mechanisms against iron deprivation, oxidative damage and nitrogen stress (Luque‐Almagro et al ., 2007).…”

Section: Resultsmentioning

confidence: 99%

The nit1C gene cluster of Pseudomonas pseudoalcaligenes CECT5344 involved in assimilation of nitriles is essential for growth on cyanide

Estepa¹,

Luque-Almagro²,

Manso³

et al. 2012

Environ Microbiol Rep

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A proteomic approach was used to identify several proteins induced by cyanide in the alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344, two of them, NitB and NitG, encoded by genes that belong to the nit1C gene cluster. The predicted products of the nit1C gene cluster are a Fis-like σ(54) -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/GARS family (NitF), an NADH-dependent oxidoreductase (NitH) and two hypothetical proteins of unknown function (NitB and NitG). RT-PCR analysis suggested that nitBCDEFGH genes were co-transcribed, whereas the regulatory nitA gene was divergently transcribed. Real-time RT-PCR revealed that expression of the nitBCDEFGH genes was induced by cyanide and repressed by ammonium. The P. pseudoalcaligenes CECT5344 nit1C gene cluster was found to be involved in assimilation of free and organic cyanides (nitriles) as deduced for the inability to grow with cyanides showed by the NitA, NitB and NitC mutant strains. The wild-type strain CECT5344 showed a nitrilase activity that allows growth on cyanide or hydroxynitriles. The NitB and NitC mutants only presented low basal levels of nitrilase activity that were not enough to support growth on either free cyanide or aliphatic nitriles, suggesting that nitrilase NitC is specific and essential for cyanide and aliphatic nitriles assimilation.

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“…Subcellular fractionation was carried as described above, and isolated cytoplasmic fractions containing 250 μg of protein were used to rehydrate 11 cm strips (Immobiline DryStrips with the appropriate pH range; Amersham Biosciences) for 12 h. Isoelectric focusing was carried out in an IPGphor (Pharmacia) until 20 000 volt-h were reached. After isoelectric focusing, strips were equilibrated, as previously described [40], and applied to 12% (v/v) polyacrylamide gels. The second dimension (SDS-PAGE) was performed using the Hoefer SE600 system (Amersham Biosciences), and gels were stained using the Brilliant Blue G-colloidal concentrate (Sigma) and scanned with a Molecular Image FX (Bio-Rad).…”

Section: Methodsmentioning

confidence: 99%

Transcriptional and translational adaptation to aerobic nitrate anabolism in the denitrifier Paracoccus denitrificans

Luque-Almagro

Manso

Sullivan

et al. 2017

Biochemical Journal

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Transcriptional adaptation to nitrate-dependent anabolism by Paracoccus denitrificans PD1222 was studied. A total of 74 genes were induced in cells grown with nitrate as N-source compared with ammonium, including nasTSABGHC and ntrBC genes. The nasT and nasS genes were cotranscribed, although nasT was more strongly induced by nitrate than nasS. The nasABGHC genes constituted a transcriptional unit, which is preceded by a non-coding region containing hairpin structures involved in transcription termination. The nasTS and nasABGHC transcripts were detected at similar levels with nitrate or glutamate as N-source, but nasABGHC transcript was undetectable in ammonium-grown cells. The nitrite reductase NasG subunit was detected by two-dimensional polyacrylamide gel electrophoresis in cytoplasmic fractions from nitrate-grown cells, but it was not observed when either ammonium or glutamate was used as the N-source. The nasT mutant lacked both nasABGHC transcript and nicotinamide adenine dinucleotide (NADH)-dependent nitrate reductase activity. On the contrary, the nasS mutant showed similar levels of the nasABGHC transcript to the wild-type strain and displayed NasG protein and NADH–nitrate reductase activity with all N-sources tested, except with ammonium. Ammonium repression of nasABGHC was dependent on the Ntr system. The ntrBC and ntrYX genes were expressed at low levels regardless of the nitrogen source supporting growth. Mutational analysis of the ntrBCYX genes indicated that while ntrBC genes are required for nitrate assimilation, ntrYX genes can only partially restore growth on nitrate in the absence of ntrBC genes. The existence of a regulation mechanism for nitrate assimilation in P. denitrificans, by which nitrate induction operates at both transcriptional and translational levels, is proposed.

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The cyanotrophic bacterium Pseudomonas pseudoalcaligenes CECT5344 responds to cyanide by defence mechanisms against iron deprivation, oxidative damage and nitrogen stress

Cited by 21 publications

References 48 publications

Characterization of the Pseudomonas pseudoalcaligenes CECT5344 Cyanase, an Enzyme That Is Not Essential for Cyanide Assimilation

Characterization of the Pseudomonas pseudoalcaligenes CECT5344 Cyanase, an Enzyme That Is Not Essential for Cyanide Assimilation

The nit1C gene cluster of Pseudomonas pseudoalcaligenes CECT5344 involved in assimilation of nitriles is essential for growth on cyanide

Transcriptional and translational adaptation to aerobic nitrate anabolism in the denitrifier Paracoccus denitrificans

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