Variations in the Rhythms of Respiration and Nitrogen Fixation in Members of the Unicellular Diazotrophic Cyanobacterial Genus Cyanothece

In order to accommodate the physiologically incompatible processes of photosynthesis and nitrogen fixation within the same cell, unicellular nitrogen-fixing cyanobacteria have to maintain a dynamic metabolic profile in the light as well as the dark phase of a diel cycle. The transition from the photosynthetic to the nitrogen-fixing phase is marked by the onset of various biochemical and regulatory responses, which prime the intracellular environment for nitrogenase activity. Cellular respiration plays an important role during this transition, quenching the oxygen generated by photosynthesis and by providing energy necessary for the process. Although the underlying principles of nitrogen fixation predict unicellular nitrogen-fixing cyanobacteria to function in a certain way, significant variations are observed in the diazotrophic behavior of these microbes. In an effort to elucidate the underlying differences and similarities that govern the nitrogen-fixing ability of unicellular diazotrophic cyanobacteria, we analyzed six members of the genus Cyanothece. Cyanothece sp. ATCC 51142, a member of this genus, has been shown to perform efficient aerobic nitrogen fixation and hydrogen production. Our study revealed significant differences in the patterns of respiration and nitrogen fixation among the Cyanothece spp. strains that were grown under identical culture conditions, suggesting that these processes are not solely controlled by cues from the diurnal cycle but that strain-specific intracellular metabolic signals play a major role. Despite these inherent differences, the ability to perform high rates of aerobic nitrogen fixation and hydrogen production appears to be a characteristic of this genus.Nitrogen fixation is an important global phenomenon by which molecular nitrogen, one of the most abundant components of the earth’s atmosphere, is converted into a more reduced form suitable for incorporation into living systems. The majority of this nitrogen fixation is achieved by biological means through the activity of microorganisms (Burris and Roberts, 1993; Raymond et al., 2004; Rubio and Ludden, 2008). This process is energy intensive, and nitrogenase, the enzyme complex involved in the biological nitrogen fixation reaction, is generally known to be extremely sensitive to oxygen (Robson and Postgate, 1980; Hill et al., 1981; Berman-Frank et al., 2005). Thus, most microbes participating in this process fix nitrogen only when suitable anaerobic or microaerobic conditions are established in an otherwise oxygen-rich environment. However, some nitrogen-fixing (diazotrophic) microbes have the advantage of being able to fix nitrogen in aerobic environments. Outstanding among these are the photosynthetic prokaryotes called cyanobacteria, an extremely successful group of microbes with plant-like traits. These microbes are considered to be the progenitors of plant chloroplasts. Cyanobacteria perform both oxygen-evolving photosynthesis and oxygen-sensitive nitrogen fixation, thereby providing a platform to power the most metabolically expensive biological process (Simpson and Burris, 1984) with solar energy.Among the nitrogen-fixing cyanobacteria, filamentous strains have been extensively studied for their contribution to the nitrogen cycle in marine and terrestrial ecosystems (Mulligan and Haselkorn, 1989; Kaneko et al., 2001; Meeks et al., 2001; Sañudo-Wilhelmy et al., 2001; Wong and Meeks, 2001; Gomez et al., 2005). Some of these filamentous strains develop specialized cells called heterocysts that allow the spatial segregation of photosynthesis and nitrogen fixation. These heterocysts also have higher rates of respiratory oxygen consumption, which results in a virtually anoxic environment conducive for the nitrogenase enzyme (Bergman et al., 1997). All heterocystous strains are known to fix nitrogen aerobically. In contrast, nonheterocystous cyanobacteria lack any specialized oxygen-free compartments and often require incubation under microoxic or anaerobic conditions for nitrogen fixation (Rippka and Waterbury, 1977; Rippka et al., 1979; Brass et al., 1992). However, some nonheterocystous cyanobacterial strains can fix nitrogen under aerobic conditions. These include some filamentous genera like Trichodesmium spp., Lyngbya spp., and Oscillatoria spp. (Jones, 1990; Janson et al., 1994; Finzi-Hart et al., 2009) as well as unicellular genera like Gloeothece spp. and Cyanothece spp. (Wyatt and Silvey, 1969; Rippka and Waterbury, 1977; Huang and Chow, 1988; Van Ni et al., 1988; Schütz et al., 2004).In comparison with filamentous cyanobacteria, which have long been recognized for their nitrogen-fixing ability, the importance of unicellular cyanobacteria as key components of the environmental nitrogen cycle has only been recently uncovered. Studies over the last decade have established unicellular strains like Crocosphaera spp., Cyanothece spp., and UCYN-A as important players in the marine nitrogen cycle (Zehr et al., 2001; Montoya et al., 2004; Zehr, 2011). Since unicellular diazotrophic cyanobacteria utilize the same cellular platform for photosynthesis and nitrogen fixation, they are required to adjust their cellular metabolism to accommodate these two antagonistic processes. Systems-level studies in the unicellular genus Cyanothece have revealed a temporal separation of the two processes, photosynthesis occurring during the day and nitrogen fixation occurring at night (Stöckel et al., 2008; Toepel et al., 2008; Welsh et al., 2008). Cellular respiration plays a critical role during the transition from one phase to the next, rapidly freeing the intracellular environment of the photosynthetically generated oxygen and rendering it conducive for the induction of nitrogenase activity. In addition, respiration also sustains the process of nitrogen fixation, not only by maintaining a low-oxygen environment required for the functioning of the nitrogenase enzyme but also by mobilizing the stored solar energy to fuel this energy-intensive process.Unicellular diazotrophs exhibit great diversity in the efficiency of nitrogen fixation as well as in the physiological regulation of the process. For instance, members of the genus Gloeothece fix nitrogen aerobically during the day, but at 0% dissolved oxygen concentration, nitrogen fixation is shifted entirely to the dark period (Ortega-Calvo and Stal, 1991; Taniuchi et al., 2008). In contrast, some Synechococcus spp. strains can fix nitrogen only when incubated under anoxic conditions (Steunou et al., 2006). Members of the genus Cyanothece have been reported to engage in both aerobic and anaerobic nitrogen fixation, with nitrogenase activity peaking during the night (Reddy et al., 1993; Bergman et al., 1997; Turner et al., 2001). This suggests that, in addition to the regulations imposed by the diurnal cycle, strain-specific intracellular cues govern the process of nitrogen fixation in unicellular cyanobacteria, which may vary according to the genotype or the ecotype of the strains.Members of the unicellular cyanobacterial genus Cyanothece are diazotrophs that thrive in marine as well as terrestrial environments. This genus was originally grouped together with Synechococcus spp. but was later separated on the basis of distinct morphological and biochemical differences between the two genera (Komárek, 1976; Rippka and Cohen-Bazire, 1983). Some of the features that define the largely heterogeneous genus Cyanothece are oval to cylindrical cells, larger than 3 µm in size (they can be as large as 24 µm in diameter), radially arranged thylakoids, and a mucilaginous layer surrounding the cells (Komárek and Cepák, 1998; Porta et al., 2000; Liberton et al., 2011).It was recently demonstrated that Cyanothece sp. ATCC 51142, a member of the genus Cyanothece, has the unique ability to produce molecular hydrogen at exceptionally high rates under aerobic conditions (Bandyopadhyay et al., 2010). This striking observation was attributed to the nitrogenase enzyme system of Cyanothece sp. ATCC 51142. Our study also indicated that high rates of respiration in this strain might contribute to its nitrogenase-mediated aerobic hydrogen production. Glycerol was found to be an efficient source of reductants and energy for this process. In an effort to investigate if this atypical cyanobacterial trait was a characteristic of the genus Cyanothece, five additional Cyanothece spp. strains from different ecological habitats were sequenced to completion. The six strains display more than 90% identity in their 16S ribosomal RNA sequence but exhibit striking variability with respect to their genome sizes (with the largest genome being 7.8 Mb and the smallest being 4.4 Mb), the number of plasmids, and the percentage of pseudogenes (Bandyopadhyay et al., 2011). In addition, two of the strains possess linear chromosomal elements, features not known to occur in any other photosynthetic bacteria sequenced to date, which may impart niche-specific advantages to these strains. Analysis of the genome sequence of the Cyanothece spp. strains showed the presence of a nitrogenase gene cluster in all five strains, and preliminary analysis showed that four of the five strains were capable of aerobic nitrogen fixation and hydrogen production (Bandyopadhyay et al., 2011). In this study, we have focused on the patterns of nitrogen fixation and respiration in six different Cyanothece spp. strains in an effort to elucidate the underlying differences and similarities in these processes in unicellular diazotrophic strains with similar genotypic but varied ecological backgrounds. Our study reveals inherent differences in the regulation of these processes, which are likely controlled by strain-specific cellular signals. However, despite the differences in the patterns of nitrogenase activity, aerobic nitrogen fixation and hydrogen production was found to be a characteristic of this genus, with most members exhibiting nitrogenase-mediated hydrogen production at rates higher than any other wild-type cyanobacterial strain.