Applied Microbiology and Biotechnology

, Volume 97, Issue 6, pp 2503–2512 | Cite as

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

  • Cinthia Núñez
  • Carlos Peña
  • Wolf Kloeckner
  • Alberto Hernández-Eligio
  • Alexander V. Bogachev
  • Soledad Moreno
  • Josefina Guzmán
  • Jochen Büchs
  • Guadalupe Espín
Applied Genetics and Molecular Biotechnology

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.

Keywords

Azotobacter vinelandii Alginate Ubiquinone algD expression Gene regulation Respirometric analysis 

Notes

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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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Cinthia Núñez
    • 1
    • 5
  • Carlos Peña
    • 2
  • Wolf Kloeckner
    • 3
  • Alberto Hernández-Eligio
    • 1
  • Alexander V. Bogachev
    • 4
  • Soledad Moreno
    • 1
  • Josefina Guzmán
    • 1
  • Jochen Büchs
    • 3
  • Guadalupe Espín
    • 1
  1. 1.Departamento de Microbiología Molecular, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMéxico
  2. 2.Departamento de Ingeniería celular y Biocatálisis, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMéxico
  3. 3.AVT. Biochemical EngineeringRWTH Aachen UniversityAachenGermany
  4. 4.Department of Molecular Energetics of Microorganisms, A.N. Belozersky Institute of Physico-Chemical BiologyMoscow State UniversityMoscowRussia
  5. 5.Departamento de Microbiología Molecular, Instituto de BiotecnologíaUNAMCuernavacaMéxico

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