B. Cyanothece Genomics and Genetics
Research: Tina Summerfield, with Jorg Toepel and Sowmya Nagarajan
Cyanobacteria are important model organisms for the study of many biological processes, including photosynthesis, N2-fixation and gene regulation of metabolism. This has led to the development of genetic systems in key organisms, such as Synechococcus elongatus, Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120. We wish to develop a genetic system for a strain within the genus Cyanothece, large (3-8 μm) unicellular, diazotrophic cyanobacteria. When cells are grown in the absence of combined nitrogen under 12 h light-dark (LD) conditions, Cyanothece sp. ATCC 51142 has an extreme temporal regulation that coordinates photosynthesis during the daytime and N2 fixation at night. This is a very valuable system for the study of photosynthesis, N2 fixation, cellular morphology and gene regulation. Because of these properties, six Cyanothece genomes have been sequenced, starting with Cyanothece sp. Strain ATCC 51142 Welsh et al (2008). Subsequently, five other Cyanothece strains have been sequenced through the DOE Joint Genome Initiative (http://www.jgi.doe.gov/), including Cyanothece sp. PCC 7822 (Bandyopadhyay et al, in press 2011).
Cyanothece constitute a genus of morphologically diverse, unicellular nitrogen fixing cyanobacteria that are known to inhabit a variety of ecological niches. Cyanothece 51142, a prototypic member of this genus was isolated from the Texas gulf coast and is one of the most potent diazotrophic strains yet characterized. Systems level studies with Cyanothece 51142 revealed many novel metabolic traits of this unicellular cyanobacterium which led to the determination of its genome sequence at the Washington University sequencing center. The studies revealed robust diurnal and circadian cycling of central metabolic processes in this strain as well as a strong coordination of correlated processes at the transcriptional level. Interestingly, genome analysis of Cyanothece 51142 uncovered the presence of a 430 kb functional linear chromosomal element, the first of its kind in a photosynthetic bacterium. The arrangement of genes on this chromosome suggested a specific role for it in energy metabolism and it was hypothesized that such linear elements with regulatory functions might be unique to the genus Cyanothece. Also interesting from the genomic perspective is the finding that some atypical nitrogen fixing strains, such as the endosymbiont spheroid body of the eukaryotic diatom Rhopalodia gibba and the unicellular marine cyanobacterium UCYN-A that lacks photosystem II, have genomes closely related to Cyanothece spp. In particular, the nitrogenase gene clusters in both of these organisms is highly similar to Cyanothece 51142. These organisms may have evolved as a result of drastic and targeted gene loss from a Cyanothece 51142-like ancestor, thus demonstrating a highly plastic nature of Cyanothece genomes as well as the robustness of their nitrogen fixing machinery.
The most striking of the unique metabolic capabilities of Cyanothece 51142 is that cells can exhibit high rates of nitrogenase-mediated H2 production under aerobic conditions, an unusual metabolic trait in oxygenic phototrophs. Furthermore, the metabolic versatility of this strain was demonstrated by its ability to switch between a photoautotrophic and a photoheterotrophic mode of metabolism depending on the availability of external carbon sources, and the presence of an atypical alternative citramalate pathway for isoleucine biosynthesis.
In an effort to unravel the genomic basis of such unusual metabolic traits of unicellular diazotrophic cyanobacteria, the genomes of five additional members of the genus Cyanothece (Cyanothece PCC 7424, PCC 7425, PCC 7822, PCC 8801 and PCC 8802) were sequenced at the Joint Genome Institute, US-Department of Energy. The strains were collected from different geographical locations and exhibit considerable diversity with respect to cell size and pigment composition. A comparison of the genomes of the different Cyanothece strains revealed that members of this genus are metabolically versatile, each member having acquired unique metabolic capabilities. The key to the success of this group of organism appears to lie in their ability to retain useful archaic metabolic traits while simultaneously adapting and accommodating advanced cellular features of contemporary photosynthetic organisms. Although we made many attempts, we could not develop a stable genetic exchange system using either transformation or conjugation in Cyanothece 51142. Thus, we began working with Cyanothece 7822 and have successfully constructed a stable knockout mutant in the nifK gene. Subsequently, we have also constructed a mutant in hupL, one the genes encoding the uptake hydrogenase. The details of this work are highlighted in Min and Sherman (2010b).