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

Authors

  • Jessica Estepa,

    1. Departamento de Bioquímica y Biología Molecular, Campus de Rabanales, Edificio Severo Ochoa, 1a Planta, Universidad de Córdoba, 14071, Córdoba, Spain.
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    • Authors have contributed equally to this work.

  • Victor M. Luque-Almagro,

    1. Departamento de Bioquímica y Biología Molecular, Campus de Rabanales, Edificio Severo Ochoa, 1a Planta, Universidad de Córdoba, 14071, Córdoba, Spain.
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    • Authors have contributed equally to this work.

  • Isabel Manso,

    1. Departamento de Bioquímica y Biología Molecular, Campus de Rabanales, Edificio Severo Ochoa, 1a Planta, Universidad de Córdoba, 14071, Córdoba, Spain.
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  • M. Paz Escribano,

    1. Departamento de Bioquímica y Biología Molecular, Campus de Rabanales, Edificio Severo Ochoa, 1a Planta, Universidad de Córdoba, 14071, Córdoba, Spain.
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  • Manuel Martínez-Luque,

    1. Departamento de Bioquímica y Biología Molecular, Campus de Rabanales, Edificio Severo Ochoa, 1a Planta, Universidad de Córdoba, 14071, Córdoba, Spain.
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  • Francisco Castillo,

    1. Departamento de Bioquímica y Biología Molecular, Campus de Rabanales, Edificio Severo Ochoa, 1a Planta, Universidad de Córdoba, 14071, Córdoba, Spain.
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  • Conrado Moreno-Vivián,

    1. Departamento de Bioquímica y Biología Molecular, Campus de Rabanales, Edificio Severo Ochoa, 1a Planta, Universidad de Córdoba, 14071, Córdoba, Spain.
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  • M. Dolores Roldán

    Corresponding author
    1. Departamento de Bioquímica y Biología Molecular, Campus de Rabanales, Edificio Severo Ochoa, 1a Planta, Universidad de Córdoba, 14071, Córdoba, Spain.
      E-mail bb2rorum@uco.es; Tel. (+34) 957218318; Fax (+34) 957218592.
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E-mail bb2rorum@uco.es; Tel. (+34) 957218318; Fax (+34) 957218592.

Summary

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 σ54-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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