title: Alginate synthesis in Azotobacter vinelandii is increased by reducing the intracellular production of ubiquinone.
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.
authors.affiliation: Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa, Cuernavaca, Morelos 62210, México. cinthia@ibt.unam.mx
authors.forename: Cinthia
authors.initials: C
authors.lastname: Núñez
authors.forename: Carlos
authors.initials: C
authors.lastname: Peña
authors.forename: Wolf
authors.initials: W
authors.lastname: Kloeckner
authors.forename: Alberto
authors.initials: A
authors.lastname: Hernández-Eligio
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Bogachev
authors.forename: Soledad
authors.initials: S
authors.lastname: Moreno
authors.forename: Josefina
authors.initials: J
authors.lastname: Guzmán
authors.forename: Jochen
authors.initials: J
authors.lastname: Büchs
authors.forename: Guadalupe
authors.initials: G
authors.lastname: Espín
mesh_terms: D000464:Alginates; D016948:Azotobacter vinelandii / Q000235:genetics / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics; D003470:Culture Media / Q000737:chemistry; D004251:DNA Transposable Elements; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D020723:Glucuronic Acid / Q000096:biosynthesis; D006603:Hexuronic Acids; D016254:Mutagenesis, Insertional; D009586:Nitrogen Fixation; D060888:Real-Time Polymerase Chain Reaction; D014451:Ubiquinone / Q000378:metabolism*
chemical_list: D000464:Alginates; D003470:Culture Media; D004251:DNA Transposable Elements; D006603:Hexuronic Acids; D014451:Ubiquinone; D020723:Glucuronic Acid; C030778:ubiquinone 8