Furthermore, the uptake hydrogenase is oxygen sensitive and does not function in the oxygenic SGI-1776 datasheet environment of the vegetative cell (Tamagnini et al., 2007). In addition, studies on the related cyanobacterium Nostoc PCC 7120 have shown the uptake hydrogenase to be located only in the heterocyst, even though some activity has been detected in vegetative cells under microaerobic conditions when grown in the presence of hydrogen (Houchins & Burris, 1981). Transcriptional studies

of the hupSL operon in N. punctiforme show that the structural genes are only transcribed in the heterocysts (Holmqvist et al., 2009). It is possible that the uptake hydrogenase is transported from heterocysts to vegetative cells by an unknown mechanism. However, no localization signal, such as the signal peptide of the hydrogenase 1 small subunit HyaA in E. coli (Mori & Cline, 1998), is known for either HupS or HupL. Even though diffusion of the small calcein fluorophor has been observed between heterocysts and vegetative cells of Nostoc PCC 7120 (Mullineaux et al., 2008), there was no detectable diffusion of the cytoplasmic GFP protein between heterocysts and vegetative cells of Nostoc PCC 7120 (Mariscal et al., 2007), or in N. punctiforme

(this study), making the passive diffusion of HupS or HupL from heterocysts to vegetative cells of N. punctiforme unlikely. To investigate the subcellular localization of the uptake hydrogenase of N. punctiforme, the intracellular fluorescence from the HupS–GFP Nitroxoline fusion protein in the SHG culture was observed during heterocyst differentiation selleck kinase inhibitor caused by nitrogen depletion. After 24 h of combined nitrogen starvation, a homogeneous and weak GFP fluorescence could be observed from proheterocysts. This is in agreement with the observed transcription of hupS in differentiating heterocysts of the same strain of N. punctiforme (Holmqvist,

2010). After approximately 30–34 h of nitrogen starvation, and up to 7 days, several smaller or fewer larger clusters of GFP fluorescence could be observed within the heterocysts. The appearance of fluorescence coincides with the known timing of nitrogenase activity in developing heterocysts (Elhai & Wolk, 1990; Rees & Howard, 2000), indicating that the regulation of hupS expression from the pSHG construct is similar to WT. The large clusters were frequently, however not exclusively, observed close to the polar regions of the heterocysts. The observed clusters of GFP fluorescence can be compared with the studies of the GFP signal from the septum localized-protein SepJ-GFP (Flores et al., 2007) and a periplasmic-localized GFP (Mariscal et al., 2007). As shown here in the GFP control culture, highly expressed free cytoplasmic GFP does not form fluorescent clusters and the fluorescence does not overlap with thylakoid membranes.