Project description: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 PCC 7002 and other cyanobacteria at low concentrations. Inhibition was overcome by a loss of function mutation in the gene acsA. 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 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).
Project description: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 PCC 7002 and other cyanobacteria at low concentrations. Inhibition was overcome by a loss of function mutation in the gene acsA. 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 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). Cultures were grown in medium modified with 5mM acrylic acid at pH 8 and compared to cultures grown in unmodified medium. Samples were processed in duplicate.
Project description:Microbial autotroph-heterotroph interactions influence biogeochemical cycles on a global scale, but the diversity and complexity of natural systems and their intractability to in situ manipulation make it challenging to elucidate the principles governing these interactions. The study of assembling phototrophic biofilm communities provides a robust means to identify such interactions and evaluate their contributions to the recruitment and maintenance of phylogenetic and functional diversity overtime. To examine primary succession in phototrophic communities, we isolated two unicyanobacterial consortia from the microbial mat in HotLake, Washington, characterizing the membership and metabolic function of each consortium. We then analyzed the spatial structures and quantified the community compositions of their assembling biofilms. The consortia retained the same suite of heterotrophic species, identified as abundant members of the mat and assigned to Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes. Autotroph growth rates dominated early in assembly, yielding to increasing heterotroph growth rates late in succession. The two consortia exhibited similar assembly patterns, with increasing relative abundances of members from Bacteroidetes and Alphaproteobacteria concurrent with decreasing relative abundances of those from Gamma proteobacteria. Despite these similarities at higher taxonomic levels, the relative abundances of individual heterotrophic species were substantially different in the developing consortial biofilms. This suggests that, although similar niches are created by the cyanobacterial metabolisms, the resulting webs of autotroph-heterotroph and heterotroph-heterotroph interactions are specific to each primary producer. The relative simplicity and tractability of the Hot Lake unicyanobacterial consortia make them useful model systems for deciphering interspecies interactions and assembly principles relevant to natural microbial communities.