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Alginate synthesis in Azotobacter vinelandii is increased by reducing the intracellular production of ubiquinone

  • Applied Genetics and Molecular Biotechnology
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Abstract

Azotobacter vinelandii, a soil nitrogen fixing bacterium, produces alginate a polysaccharide with industrial and medical relevant applications. In this work, we characterized a miniTn5 mutant, named GG101, that showed a 14-fold increase in the specific production of alginate when grown diazotrophically on solid minimal medium comparing to the parental E strain (also named AEIV). Quantitative real-time reverse transcription PCR analysis indicated that this increased alginate production was due to higher expression levels of several biosynthetic alg genes such as algD. Sequencing of the locus interrupted in GG101 indicated that the miniTn5 was inserted in the positive strand, and 10 bp upstream the start codon of the gene ubiA, encoding the enzyme for the second step in the biosynthesis of ubiquinone (Q8). Both the transcription of ubiA and the content of Q8 are decreased in the mutant GG101 when compared to the wild-type strain E. Genetic complementation of mutant GG101 with a wild-type copy of the ubiCA genes restored the content of Q8 and reduced the production of alginate to levels similar to those of the parental E strain. Furthermore, respirometric analysis showed a reproducible decrease of about 8 % in the respiratory capacity of mutant GG101, at exponential phase of growth in liquid minimal medium. Collectively, our data show that a decreased content in Q8 results in higher levels of alginate in A. vinelandii.

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Acknowledgment

We thank S. Ainsworth, A. Ocádiz, and J. M. Hurtado for their technical support. This research was supported by projects PAPIIT UNAM (IN207911) and by CONACyT (101643). AVB is indebted to RFBR for financial support (grant 10-04-00352).

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Authors and Affiliations

  1. Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa, Cuernavaca, Morelos, 62210, México

    Cinthia Núñez, Alberto Hernández-Eligio, Soledad Moreno, Josefina Guzmán & Guadalupe Espín

  2. Departamento de Ingeniería celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa, Cuernavaca, Morelos, 62210, México

    Carlos Peña

  3. AVT. Biochemical Engineering, RWTH Aachen University, Aachen, 52074, Germany

    Wolf Kloeckner & Jochen Büchs

  4. Department of Molecular Energetics of Microorganisms, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119992, Russia

    Alexander V. Bogachev

  5. Departamento de Microbiología Molecular, Instituto de Biotecnología, UNAM, Apdo Postal 510-3, Cuernavaca, Morelos, 62251, México

    Cinthia Núñez

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  1. Cinthia Núñez
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  2. Carlos Peña
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  3. Wolf Kloeckner
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  4. Alberto Hernández-Eligio
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  5. Alexander V. Bogachev
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  6. Soledad Moreno
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  7. Josefina Guzmán
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  8. Jochen Büchs
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  9. Guadalupe Espín
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Correspondence to Cinthia Núñez.

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Núñez, C., Peña, C., Kloeckner, W. et al. Alginate synthesis in Azotobacter vinelandii is increased by reducing the intracellular production of ubiquinone. Appl Microbiol Biotechnol 97, 2503–2512 (2013). https://doi.org/10.1007/s00253-012-4329-0

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  • DOI: https://doi.org/10.1007/s00253-012-4329-0

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