An organic acid based counter selection system for cyanobacteria

PLoS One. 2013 Oct 1;8(10):e76594. doi: 10.1371/journal.pone.0076594. eCollection 2013.

Abstract

Cyanobacteria are valuable organisms for studying the physiology of photosynthesis and carbon fixation, as well as metabolic engineering for the production of fuels and chemicals. This work describes a novel counter selection method for the cyanobacterium Synechococcus sp. PCC 7002 based on organic acid toxicity. The organic acids acrylate, 3-hydroxypropionate, and propionate were shown to be inhibitory towards Synechococcus sp. PCC 7002 and other cyanobacteria at low concentrations. Inhibition was overcome by a loss of function mutation in the gene acsA, which is annotated as an acetyl-CoA ligase. Loss of AcsA function was used as a basis for an acrylate counter selection method. DNA fragments of interest were inserted into the acsA locus and strains harboring the insertion were isolated on selective medium containing acrylate. This methodology was also used to introduce DNA fragments into a pseudogene, glpK. Application of this method will allow for more advanced genetics and engineering studies in Synechococcus sp. PCC 7002 including the construction of markerless gene deletions and insertions. The acrylate counter-selection could be applied to other cyanobacterial species where AcsA activity confers acrylate sensitivity (e.g. Synechocystis sp. PCC 6803).

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acrylates / pharmacology
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Coenzyme A Ligases / genetics*
  • Coenzyme A Ligases / metabolism
  • Drug Resistance, Bacterial / drug effects
  • Drug Resistance, Bacterial / genetics*
  • Lactic Acid / analogs & derivatives
  • Lactic Acid / pharmacology
  • Metabolic Engineering / methods
  • Microbial Sensitivity Tests
  • Mutagenesis, Insertional
  • Mutation
  • Propionates / pharmacology
  • Pseudogenes
  • Selection, Genetic*
  • Synechococcus / drug effects
  • Synechococcus / genetics*
  • Synechococcus / growth & development

Substances

  • Acrylates
  • Bacterial Proteins
  • Propionates
  • Lactic Acid
  • hydracrylic acid
  • Coenzyme A Ligases
  • acrylic acid
  • propionic acid

Grants and funding

This work was funded by the National Science Foundation (EFRI-1240268, CBET-1149678, SEES 12-15871), the US Air Force Office of Scientific Research (FA9550-11-1-0038), the US Department of Energy Genomics: GTL and SciDAC Programs (DE-FG02-04ER25627) and the University of Wisconsin-Madison graduate school. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.