, 1981; Valladares et al., 2007). The potential localization of the oxygen-sensitive uptake hydrogenase to the honeycomb membrane structures could be of relevance for the function of

this enzyme, as one of the targets for the electrons released during H2 oxidation is the respiratory electron transport chain (Houchins & Burris, 1981), and because the respiration could lead to locally decreased levels of O2. However, because the fluorescence foci are also observed outside the polar regions, alternative explanations merit consideration. One such consideration is that the HupS–GFP is targeted for localized NVP-LDE225 degradation by proteases. An interesting reflection is that protease complexes that show similarities to the eukaryotic proteasome have been identified in some bacteria, i.e. the cyanobacterium Synechoccoccus and Bacillus subtilis (Kirstein et al., 2008; Simmons et al., 2008; Andersson et al., 2009). Furthermore, the protease clusters in B. subtilis are localized to the polar regions of the cells Cyclopamine and have been shown to be colocalized with protein aggregates (inclusion bodies) (Kirstein et al., 2008). Protein inclusion bodies are most often formed

during high-level protein expression in biotechnological applications (Wang, 2009), but because of the apparent low solubility of HupS–GFP, it could not be excluded that it forms a protein aggregate. The observation of fluorescent clusters in SHG over time during increased expression of HupS–GFP could indicate some form of inclusions. Even though it has been observed that a GFP fusion protein was not fluorescent upon formation of inclusion bodies (Drew et al., 2001), another study has shown that inclusion bodies of GFP fusion proteins indeed can be fluorescent (Garcia-Fruitos et al., 2005). To investigate the possibility

of HupS–GFP CHIR-99021 in vitro inclusions, TEM was used to compare heterocysts isolated from N2-fixing cultures of WT and SHG and to search for structural differences indicating inclusion bodies (Fig. S2). No such differences could be observed, although this is difficult to determine due to the many structures within the heterocyst, i.e. membranes and cyanophycin granules. Because HupS–GFP required strong denaturing conditions to be extracted, whereas most degradation products could be extracted without detergents, indicating a more soluble form, it is likely that full-length HupS–GFP forms the clusters. In the present study, the in vivo localization of the uptake hydrogenase is determined for the first time, using a HupS–GFP fusion protein reporter, as solely localized to the heterocysts of N. punctiforme. The subcellular fluorescence in fully mature heterocysts is either homogeneously distributed or localized in clusters, which may be of relevance for the function of the uptake hydrogenase.