Trichodesmium--a widespread marine cyanobacterium with unusual nitrogen fixation properties.

The last several decades have witnessed dramatic advances in unfolding the diversity and commonality of oceanic diazotrophs and their N2 -fixing potential. More recently, substantial progress in diazotrophic cell biology has provided a wealth of information on processes and mechanisms involved. The substantial contribution by the diazotrophic cyanobacterial genus Trichodesmium to the nitrogen influx of the global marine ecosystem is by now undisputable and of paramount ecological importance, while the underlying cellular and molecular regulatory physiology has only recently started to unfold. Here, we explore and summarize current knowledge, related to the optimization of its diazotrophic capacity, from genomics to ecophysiological processes, via, for example, cellular differentiation (diazocytes) and temporal regulations, and suggest cellular research avenues that now ought to be explored.

Introduction

Balann class="Chemical">cing of pan> class="Chemical">nitrogen (N) inputs and exports in global oceans requires substantial biogenic fixation of atmospheric nitrogen (N2). In this context, planktonic colony-forming cyanobacteria of the genus Trichodesmium are recognized as major players. Representatives within the genus have consistently been shown to be stable components of tropical and subtropical segments of the Atlantic, Pacific, and Indian Oceans where they may form enormous surface accumulations (‘blooms’) visible to the naked eye (see Capone & Carpenter, 1982; Capone ; Karl ; Tyrrell ; Davis & McGillicuddy, 2006; Westberry & Siegel, 2006; Carpenter & Capone, 2008). Trichodesmium contributes to sustaining marine life via active release of key nutrients, for example carbon and nitrogen, and upon death and decay, hence making this fully photoautotrophic genus a vital player in the biogeochemical cycling of basic elements in contemporary oceans (Carpenter & Capone, 2008). The global input via N2 fixation by Trichodesmium was initially estimated to amount to about 5 Tg N annually by Capone & Carpenter (1982), an estimate that by now has risen to about 60–80 Tg N annually (Capone ; Mahaffey ; Westberry & Siegel, 2006; Carpenter & Capone, 2008), which makes up a substantial part of the current estimate of global marine N2 fixation, 100–200 Tg N annually (Karl ).

As the n class="Chemical">N2-fixing enpan>zyme, pan> class="Chemical">nitrogenase, encoded by the nifHDK genes, is rapidly inactivated by O2, diazotrophic cyanobacteria either fix N2 at night (to avoid photosynthetically evolved oxygen) or differentiate a thick-walled, photosystem II-deficient heterocystous cell type to specifically sustain daytime N2 fixation (Kumar ). Members of the genus Trichodesmium fix N2 exclusively in the light (Dugdale ; Capone ), although the genus is affiliated to Section III filamentous cyanobacteria that are unable to form heterocysts and therefore expected to fix N2 (primarily) during the dark phase (see Bergman ). Knowledge has expanded dramatically in regard to the diazotrophic physiology and molecular biology of Trichodesmium, but there are still gaps related to its unique cell biology and overall behavior in an ecophysiological context. We here summarize our current knowledge by highlighting its diazotrophic peculiarities from various perspectives.

Speciation

n class="Species">Trichodesmium erythraeum was named by Ehrenpan>berg in 1830 after observing blooms that dispan> class="Chemical">colored the water at the Bay of Tor in the Red Sea (Ehrenberg, 1830). Jules Verne (1869) in'20 000 leagues under the sea' also mentions blooms in this Bay (Box 1). Two other species, T. thiebautii and T. hildebrandtii, were named by Gomont (1892), and Wille (1904) later described another three species, T. contortum, T. tenue, and T. radians. These species were re-examined in 1995 (Janson ) using specimens from the Indian Ocean, Caribbean, and Sargasso Seas. Ultrastructural arrangement of gas vesicles and glycogen clusters (carbon storage) were used as primary markers and separated the species into two clades: (i) T. tenue and T. erythraeum and (ii) T. thiebautii, T. hildebrandtii, and T. contortum. In 2001, a close relationship between Trichodesmium spp. (Lundgren ) and marine cyanobacterial members of the genus Katagnymene (K. pelagica and K. spiralis; Lemmermann, 1900) was discovered using phylogenetic analysis of nifH gene sequences. Using the more variable fragment of the hetR gene, and a few other genetic markers, as targets revealed that the two Katagnymene species in fact cluster within one of the two Trichodesmium clades (Orcutt ; Lundgren ). Despite the morphological differences, K. pelagica and K. spiralis were, in addition, found to be the same species (Lundgren ). Examining 21 cultivated isolates of Trichodesmium/Katagnymene, using genetic and morphological markers, Hynes ) verified the existence of two Trichodesmium clades and suggested that these may inhabit different ecological niches, based on different pigment characteristics.

Box 1

From Jules Verne, ‘20,000 leagues under the sea’ [translated by Lewis Mern class="Chemical">cier, (Verne, 1872)]:

‘Here it is, M. Aronnax. An class="Chemical">cpan> class="Chemical">cording to my idea, we must see in this appellation of the Red Sea a translation of the Hebrew word 'Edom’; and if the ancients gave it that name, it was on account of the particular colour of its waters.' ‘But up to this time I have seen nothing but transparent waves and without any particular colour.’ ‘Very likely; but as we advance to the bottom of the gulf, you will see this singular appearance. I remember seeing the Bay of Tor entirely red, like a sea of blood.’ ‘And you attribute this colour to the presence of a microscopic seaweed?’ ‘Yes.’ ‘So, Captain Nemo, it is not the first time you have overrun the Red Sea on board the Nautilus?’ ‘No, sir.’

However, a full revision of the genera n class="Species">Trichodesmium anpan>d Katagnpan>ymenpan>e is now warranpan>ted, as the latter also inn class="Chemical">cludes several freshwater species (T. iwanoffianum Nygaard, T. lacustre Klebahn, K. accurata Geitler, K. mucigera Compére and K. spirulinoides An; see Komárek & Anagnostidis, 2005), one of which, in addition, represents the ‘type strain’ of the genus Katagnymene. Sequencing of additional genomes within the Trichodesmuim genus is also needed if we are to fully comprehend the taxonomy and phylogeny of the genus.

An expanding and flexible genome

n class="Species">Trichodesmium erythraeum IMS101 (from now onpan> pan> class="Species">Trichodesmium IMS101) was one of the first strains isolated into axenic cultures (Prufert-Bebout ) and still represents the only sequenced genome within the genus (http://genome.jgi-psf.org/trier/trier.home.html). The genome, which comprises 7.75 Mbp, is among the larger cyanobacterial genomes sequenced to date (Fig. 1a). A recent phylogenetic survey of 58 sequenced cyanobacterial genomes, based on a concatenated alignment of 285 protein orthologs, verified that Trichodesmium IMS101 is affiliated to a lineage composed of other filamentous nonheterocystous species within Oscillatoriales (Fig. 1b; Larsson ): the marine Lyngbya sp. PCC 8106 and two species within Arthrospira (A. platensis and A. maxima; previously denoted Spirulina). These are all ecologically successful and widespread inhabitants of marine waters and alkaline lakes, respectively. Recent analyses based on the 16S rRNA gene sequence give a similar clustering of Trichodesmium IMS101 (Schirrmeister ). Larsson ) also showed that the capacity to fix N2 within this Trichodesmium clade was lost in A. platensis and A. maxima, as also verified by Latysheva ), while retained in the deeper-branching Trichodesmium and Lyngbya sp. PCC 8106. Interestingly, these four lineages all possess hetR (Larsson ), a gene encoding a protease with a key function in cell differentiation (N2-fixing heterocysts and resting akinetes; see Kumar ), although they all lack these developmental capacities. Additionally, comparative genomic analyses show that several other gene orthologs involved in heterocyst differentiation are present in the Trichodesmium IMS101 genome (e.g. hetCF, patB) while others, not unexpectedly, are missing such as those involved in the deposition of the heterocyst outer envelope (e.g. hglCDE, hepB) (Table S1; El-Shehawy ; Larsson ). However, orthologous genes are not always functionally equivalent. For instance, the sepJ gene of Trichodesmium is missing a vital domain essential for filament integrity under nitrogen deprivation, although it fully complements a sepJ deletion mutant of Nostoc sp. PCC7120 when grown in the presence of combined nitrogen (Mariscal ). The nif gene operon of Trichodesmium IMS101 is conserved in a manner typical of some heterocystous cyanobacteria, although Trichodesmium lacks the large DNA insertion element present in the structural nifD gene of several of the heterocystous cyanobacteria, as well as the intergenic region between nifB and nifVZT/cysE (Fig. S1). These findings strengthen an evolutionary relationship between the genus Trichodesmium and the heterocystous clade, whereas distinct differences are also apparent, relationships now worth examining in greater detail.

Fig. 1

Phylogeny and genome properties of Trichodesmium IMS101. (a) Genome sizes and proportions of coding and noncoding nucleotides in genomes of organisms included in (b) and (c). Genomes are sorted by total size. The genome of Trichodesmium IMS101 is indicated by an arrow. (b) Maximum-likelihood phylogenetic tree based on a concatenated alignment of 285 single-copy orthologs. The tree is a subtree of a larger phylogeny of 58 cyanobacteria (see Larsson ). Specific phenotypes for cyanobacteria are shown by the colored boxes next to the tip labels. The clade containing Trichodesmium (order Oscillatoriales) is highlighted with blue branches. Thick and thin branches indicate bootstrap support values (200 replicates) of 100 and between 58 and 84, respectively. Bar, 0.4 expected substitutions per site. (c) Ancestral genome sizes (reconstructed by parsimony) in the phylogeny from b. Organism names are abbreviated (see below for full names). Contemporary genome sizes (Mbp) are shown in the right margin and at specific nodes in the tree. Organism abbreviations are as follows: Acam = Acaryochloris marina MBIC11017, Anav = Anabaena variabilis ATCC29413, Artm = Arthrospira maxima CS328, Artp = Arthrospira platensis str. Paraca, Crow = Crocosphaera watsonii WH8501, Cya0110 = Cyanothece sp. CCY0110, Cya51142 = Cyanothece sp. ATCC51142, Cya7424 = Cyanothece sp. PCC7424, Cya7425 = Cyanothece sp. PCC7425, Cya7822 = Cyanothece sp. PCC7822, Cya8801 = Cyanothece sp. PCC8801, Cya8802 = Cyanothece sp. PCC 8802, Cylr = Cylindrospermopsis raciborskii CS505, Lyns = Lyngbya sp. PCC 8106, Mica = Microcystis aeruginosa NIES 843, Micc = Microcoleus chthonoplastes PCC7420, NoAz = ‘Nostoc azollae’ 0708, Nods = Nodularia spumigena CCY9414, Nosp = Nostoc punctiforme PCC73102, Noss = Nostoc sp. PCC7120, Rapb = Raphidiopsis brookii D9, Scys6803 = Synechocystis sp. PCC6803, Syn7002 = Synechococcus sp. PCC7002, Thee = Thermosynechococcus elongatus, Trie = Trichodesmium erythraeum IMS101, Ucyn = cyanobacterium UCYN-A. The figures are adapted from Larsson ) with the author's permission.

Phylogeny and genome properties of n class="Species">Trichodesmium IMS101. (a) Genpan>ome sizes anpan>d proportionpan>s of pan> class="Chemical">coding and noncoding nucleotides in genomes of organisms included in (b) and (c). Genomes are sorted by total size. The genome of Trichodesmium IMS101 is indicated by an arrow. (b) Maximum-likelihood phylogenetic tree based on a concatenated alignment of 285 single-copy orthologs. The tree is a subtree of a larger phylogeny of 58 cyanobacteria (see Larsson ). Specific phenotypes for cyanobacteria are shown by the colored boxes next to the tip labels. The clade containing Trichodesmium (order Oscillatoriales) is highlighted with blue branches. Thick and thin branches indicate bootstrap support values (200 replicates) of 100 and between 58 and 84, respectively. Bar, 0.4 expected substitutions per site. (c) Ancestral genome sizes (reconstructed by parsimony) in the phylogeny from b. Organism names are abbreviated (see below for full names). Contemporary genome sizes (Mbp) are shown in the right margin and at specific nodes in the tree. Organism abbreviations are as follows: Acam = Acaryochloris marina MBIC11017, Anav = Anabaena variabilis ATCC29413, Artm = Arthrospira maxima CS328, Artp = Arthrospira platensis str. Paraca, Crow = Crocosphaera watsonii WH8501, Cya0110 = Cyanothece sp. CCY0110, Cya51142 = Cyanothece sp. ATCC51142, Cya7424 = Cyanothece sp. PCC7424, Cya7425 = Cyanothece sp. PCC7425, Cya7822 = Cyanothece sp. PCC7822, Cya8801 = Cyanothece sp. PCC8801, Cya8802 = Cyanothece sp. PCC 8802, Cylr = Cylindrospermopsis raciborskii CS505, Lyns = Lyngbya sp. PCC 8106, Mica = Microcystis aeruginosa NIES 843, Micc = Microcoleus chthonoplastes PCC7420, NoAz = ‘Nostoc azollae’ 0708, Nods = Nodularia spumigena CCY9414, Nosp = Nostoc punctiforme PCC73102, Noss = Nostoc sp. PCC7120, Rapb = Raphidiopsis brookii D9, Scys6803 = Synechocystis sp. PCC6803, Syn7002 = Synechococcus sp. PCC7002, Thee = Thermosynechococcus elongatus, Trie = Trichodesmium erythraeum IMS101, Ucyn = cyanobacterium UCYN-A. The figures are adapted from Larsson ) with the author's permission.

Another notable feature of the n class="Species">Trichodesmium IMS101 genpan>ome is its pan> class="Chemical">comparatively low coding capacity (Larsson ). With c. 40% noncoding DNA, it holds one of the lowest coding percentages among all to date sequenced genomes of cyanobacteria (Fig. 1a) and other bacteria (Hou & Lin, 2009). The intergenic sequences within the Trichodesmium IMS101 genome (459-bp median length) are also relatively large for cyanobacteria (14.5- to 231-bp median intergenic length in 39 other finished cyanobacterial genomes). The reason for these large and presumably noncoding intergenic spacers in the Trichodesmium IMS101 genome is unknown. It is, however, interesting to note that among the 58 genomes examined, the genome of Trichodesmium IMS101 is one of a few in which the genome is currently expanding in size, as is also the case for the genomes of the limnic Microcystis aeruginosa NIES 843 and the marine Acaryochloris marina MBIC11017 (Fig. 1c; Larsson ). This suggests that the genome of Trichodesmium IMS101 is in an expanding dynamic state, in contrast to the shrinking genomes of the unicellular marine genera Synechococcus and Prochlorococcus (with genomes < 2 Mbp; Palenik ; Kettler ), genera which to a large extent share the same tropical/subtropical marine habitat as Trichodesmium. Based on these data, it is suggested that different strategies are used to cope with the various constraints enforced by these oligotrophic oceans (Larsson ). Trichodesmium may use a strategy to flexibly adapt by incorporating functions and capacities when needed (e.g. via horizontal gene transfer, HGT) and maintain gene duplications (in-paralogs) affecting c. 10% of all genes in the Trichodesmium IMS101 genome, as a mechanism to promote genome expansion and organismal adaptations (Swingley ; Treangen & Rocha, 2011). One example of HGT in Trichodesmium IMS101 is the acquisition of long eukaryotic triglyceride collagen protein fibers that may sustain Trichodesmium colony formation (Layton ). Paralogs and horizontally gained genes that do not provide a fitness advantage will undergo inactivation (sequence divergence and loss of function) and eventually be lost. Indeed, 21% of in-paralogs in Trichodesmium IMS101 appear to have been subject to inactivation and are now present only as pseudogenes within the genome (Larsson ), thereby contributing to the abundance of noncoding nucleotides (Fig. 1a). Considering that bacterial genomes are subject to a deletion bias (Mira ), the large noncoding proportion of the Trichodesmium IMS101 genome which cannot be attributed to remnants of previously functional genes (pseudogenes) is enigmatic. However, it is possible that at least parts of these intergenic regions contain as yet nonannotated genes or small RNAs (Hewson ; Shi ). It appears that the unicellular marine cyanobacteria with reduced genomes (e.g. Prochlorococcus spp. in Clade II) use an opposite strategy to compete for life-space and survival, that is maintain a large surface-to-volume ratio and fewer genes, a strategy recently characterized as ‘cryptic escape’ (Yooseph ).

The diazocytes – separation in space

A redun class="Chemical">ced pan> class="Chemical">oxygen environment is a prerequisite for effective N2 fixation activity in Trichodesmium as in all other bacteria. Both transcription of nif genes and biosynthesis of the nitrogenase enzyme complex have been found to be sensitive to oxygen inactivation (Zehr ; Staal ). Ever since the pioneering studies by Dugdale and co-workers (Dugdale ), a compelling research area has therefore been to elucidate how Trichodesmium reconciles oxygenic photosynthesis and oxygenophobic N2 fixation within its ‘heterocyst-free’ physiology. Initially, N2 fixation was proposed to be limited to low-oxygen or anaerobic regions in the center of Trichodesmium colonies (Paerl & Bebaut, 1988). However, later, it became apparent that colony formation was not a prerequisite as ‘free’ trichomes, the dominant form in Trichodesmium laboratory cultures, are also able to fix N2. Attention then switched to the structural differences along the Trichodesmium trichomes that early on were observed using light microscopy, recognized as ‘nongranulated’ or ‘lighter’ cell regions in parts of the trichomes (Carpenter & Price, 1976; Bryceson & Fay, 1981; Li & Lee, 1990), in both single trichomes, and colony-associated trichomes. This cellular arrangement occurs in both natural populations and cultures of Trichodesmium IMS101 (Lin ; El-Shehawy ). On average, ∼ 15% of the total cell population of the trichomes may be described as less granulated or lighter (more transparent). These cells are arranged in strings or ‘zones’ composed of ∼ 2–30 cells, but are not always obvious in LM (Fig. 2a). However, when stained (e.g. with Lugol's solution; Fig. 2c), each trichome harbors typically 1–2 such zones per trichome, but up to four zones have been observed in longer trichomes (El-Shehawy ). The nongranulated appearance is caused by a diminished number and/or size of subcellular structures such as cyanophycin granules, gas vacuoles, and polyphosphate granules (Fig. 2c and e), while additional membranes are synthesized (Fredriksson & Bergman, 1997).

Fig. 2

Morphological characteristics of Trichodesmium trichomes, with emphasis on cell differentiation and their nitrogenase containing cell type, the diazocytes. (a) A light micrograph depicting a dark pigmented colony consisting of longitudinally arranged trichomes of a newly isolated strain, T. erythraeum TNZ0801. Scale bar, 25 μm. (b) The DNA distribution in cells of a Trichodesmium IMS101 trichome visualized after staining with the dye 4′,6-diamidino-2-phenylindole (DAPI), fluorescing blue. Note the DNA presence in all cells, the centrally located diazocyte-like zone (marked) being recognized as they are devoid of the yellow/green fluorescent granules representing polyphosphate storage. Scale bar, 20 μm. (c) Trichomes of Trichodesmium IMS101 stained with Lugol's solution. Note several lighter-stained central diazocyte zones, in which catabolic carbon metabolism has degraded the Lugol-stainable stored carbon supplies. Scale bar 20 μm. (d) Fluorescence in situ immunolocalization of NifH into groups of adjacent cells, diazocytes, in central areas of intact trichomes of Trichodesmium IMS101. The NifH protein is detected as a blue fluorescence due to a secondary anti-NifH-antibody coupled to a blue-fluorescing chromophore. Scale bar, 10 μm. (e) Transmission electron micrograph depicting a longitudinally sectioned trichome of Trichodesmium IMS101. Note the more homogenous zone of cells, representing diazocytes between the arrows. Arrowheads point to ongoing cell division (the formation of division septa) in two of the diazocytes. Scale bar, 20 μm.

Morphological pan> class="Chemical">characteristics of Trichodesmium trichomes, with emphasis on cell differentiation and their nitrogenase containing cell type, the diazocytes. (a) A light micrograph depicting a dark pigmented colony consisting of longitudinally arranged trichomes of a newly isolated strain, T. erythraeum TNZ0801. Scale bar, 25 μm. (b) The DNA distribution in cells of a Trichodesmium IMS101 trichome visualized after staining with the dye 4′,6-diamidino-2-phenylindole (DAPI), fluorescing blue. Note the DNA presence in all cells, the centrally located diazocyte-like zone (marked) being recognized as they are devoid of the yellow/green fluorescent granules representing polyphosphate storage. Scale bar, 20 μm. (c) Trichomes of Trichodesmium IMS101 stained with Lugol's solution. Note several lighter-stained central diazocyte zones, in which catabolic carbon metabolism has degraded the Lugol-stainable stored carbon supplies. Scale bar 20 μm. (d) Fluorescence in situ immunolocalization of NifH into groups of adjacent cells, diazocytes, in central areas of intact trichomes of Trichodesmium IMS101. The NifH protein is detected as a blue fluorescence due to a secondary anti-NifH-antibody coupled to a blue-fluorescing chromophore. Scale bar, 10 μm. (e) Transmission electron micrograph depicting a longitudinally sectioned trichome of Trichodesmium IMS101. Note the more homogenous zone of cells, representing diazocytes between the arrows. Arrowheads point to ongoing cell division (the formation of division septa) in two of the diazocytes. Scale bar, 20 μm.

The ‘lighter’ n class="Chemical">cells were in 1991 (Bergmanpan> & pan> class="Chemical">Carpenter, 1991) proven to be the nitrogenase enzyme containing cells in Trichodesmium (Fig. 2d). The existence of a ‘spatial’ nitrogenase sequestration mechanism in a nonheterocystous cyanobacterium was thereby proven. The cells were subsequently termed diazocytes: di (two) azo (nitrogen) cyte (cell) (Fredriksson & Bergman, 1997). The exclusive localization of nitrogenase in diazocytes was corroborated by immuno-TEM (sectioned trichomes) and immuno-LM (whole-mount intact trichomes; Fig. 2d) analyses of both cultured and natural populations from the Indian, Pacific, and Atlantic Oceans, using a battery of antibodies (Bergman & Carpenter, 1991; Bergman ; Janson ; Fredriksson & Bergman, 1997; Berman-Frank ), including also one monoclonal anti-Trichodesmium IMS101-NifH antibody (targeting the smaller Fe protein subunit; Zehr ; Bergman ). The frequency of the diazocytes is lower at dawn and increased toward noon and is negatively regulated by the presence of combined nitrogen (Fredriksson & Bergman, 1995; Lin ; Sandh , 11). Indeed, theoretical models suggest that a spatial separation of processes (such as nitrogen fixation and photosynthesis) favors biomass production compared to temporal separation (Rosetti & Bagheri, 2012) as, for instance, in unicellular cyanobacteria (Bergman ). As a few other studies have suggested that the nitrogenase enzyme is present in all cells within the trichomes (Paerl ; Ohki, 2008; Orcutt ), a variation in cellular localization may exist depending on different environmental conditions or species examined. Using 15N and Nano-SIMS, Finzi-Hart ) observed that the fixed N is rapidly distributed into the majority of cells along Trichodesmium trichomes, although cells in the center showed a lower 15N label, a pattern that may suggest a zone of diazocytes. A lower 15N label is also typical for heterocysts analyzed by Nano-SIMS due to a most rapid transfer of fixed nitrogen out of these cells (Popa ; Ploug ).

It was ren class="Chemical">cenpan>tly shownpan> that, as for the differenpan>tiationpan> of heterocysts, removal of combined nitrogen from the growth medium is the sole and sufficient mean needed to elicit the development of the centrally located nitrogenase containing diazocytes (Fig. 2d) in Trichodesmium IMS101 (Sandh ). The fact that the development of diazocytes takes between 8–27 h and that changes in cellular ultrastructure precede the expression of the nitrogenase enzyme (Sandh ) strongly argues for a genetically based developmental background. Phylogenetically, the nonheterocystous Trichodesmium clade is a sister group to the heterocystous clade (Fig. 1b) and Trichodesmium shares several genomic and behavioral features with this clade (see above, below and Table S1). For instance, some heterocystous cyanobacteria (Anabaena sp.) first develop strings or subsets of adjacent proheterocysts upon nitrogen deprivation, while only the central cell develops into a mature heterocyst and the other regresses into vegetative cells (Wilcox ). The similarity of the patterning of proheterocysts and diazocytes hints that several early regulatory elements may be shared in the nitrogen-regulated pathways of heterocystous genera and Trichodesmium. Diazocytes may during evolution have ‘frozen’ at this more minimalistic initial stage (Fig. 3), as a full differentiation is disadvantageous for the conditions offered in oceans (Staal ; : Stal, 2009). In contrast to heterocysts, the diazocytes are not terminally differentiated cells and retain their ability to divide (Fig. 2e; Fredriksson & Bergman, 1995), which may also contribute to their more flexible life style and allow the diazocytes (Trichodesmium) to more easily/rapidly adapt to prevailing conditions. To what extent Trichodesmium and heterocystous cyanobacteria share additional regulatory mechanisms that govern nitrogen deprivation signaling and pattern formation is now of great interest to be resolved.

Fig. 3

A potential evolutionary scenarium for the development of the diazocytic phenotype in Trichodesmium. (1) An ancient nonheterocystous filamentous cyanobacterium, a forerunner of Nostocales and Oscillatoriales (Fig. 1b and c), under nitrogen deprivation conditions develop strings of proheterocysts (as in Anabaena-type spp.; Wilcox ). (2) The majority of the proheterocysts revert back into vegetative cells, while one continus the evolution into a thick-walled heterocyst, the dominating nitrogen-fixing phenotype in limnic and terrestrial ecosystems today. (3) In oceans, this proheterocystous phenotype may have been retained and evolved into the strings of diazocytes we see in Trichodesmium spp. today, while the closest relatives (Fig. 1b) either fix nitrogen in the dark (Lyngbya spp.; e.g. Lundgren ) or have lost the capacity to fix nitrogen (Arthrospira spp.; Larsson ; Latysheva ).

A potential evolutionary sn class="Chemical">cenpan>arium for the developmenpan>t of the diazocytic phenotype in Trichodesmium. (1) An ancient nonheterocystous filamentous cyanobacterium, a forerunner of Nostocales and Oscillatoriales (Fig. 1b and c), under nitrogen deprivation conditions develop strings of proheterocysts (as in Anabaena-type spp.; Wilcox ). (2) The majority of the proheterocysts revert back into vegetative cells, while one continus the evolution into a thick-walled heterocyst, the dominating nitrogen-fixing phenotype in limnic and terrestrial ecosystems today. (3) In oceans, this proheterocystous phenotype may have been retained and evolved into the strings of diazocytes we see in Trichodesmium spp. today, while the closest relatives (Fig. 1b) either fix nitrogen in the dark (Lyngbya spp.; e.g. Lundgren ) or have lost the capacity to fix nitrogen (Arthrospira spp.; Larsson ; Latysheva ).

Diazotrophy – separation in time

The timing of the n class="Chemical">N2 fixationpan> physiology of pan> class="Species">Trichodesmium is governed by the circadian clock (Chen , 1998; Dong & Golden, 2008). This regulates the transcription of the nif genes, the daily de novo synthesis of the subunits of the nitrogenase enzyme (NifHDK), a post-translational modification of NifH (Capone ; Zehr ; Chen ), and the supply of appropriate levels of energy and reducing equivalents necessary for N2 fixation activity (Staal ).

Henn class="Chemical">ce, it appears that pan> class="Species">Trichodesmium spp. not only separates N2 fixation physically from net oxygen evolution via the development of a special cell type (diazocytes), although this may be the major protective mechanism, but also separates these two incompatible processes temporarily (Berman-Frank ), although in a more subtle way than in other nonheterocystous cyanobacteria (Bergman ). For instance, in contrast to in the latter and in concert with heterocystous cyanobacteria, nitrogenase activity in Trichodesmium operates within the day/light phase of the diel cycle, however, at a period around mid-day when the oxygen production is lowered and oxygen-scavenging mechanisms enhanced (respiration/Mehler reaction). This ‘mid-day depression’ in photosynthetic oxygen evolution is manifested as a lower quantum yield (∼ 50%) and a low or negative net O2 evolution (Berman-Frank ). Chlorophyll fluorescence kinetic microscopy at the single-cell level has also revealed flexible temporal and spatial switching between high fluorescence states (within a row of cells) and recovery states during subsequent non-N2-fixing periods in Trichodesmium IMS101 (Küpper ). This rapid sequential switching within type I cells (functional PSII activity and enhanced Mehler reaction; potentially being the diazocytes) would allow diazotrophy even in cells lacking thick cell walls, may be orchestrated by rearrangements of the phycobilisomes between PSI and PSII (Küpper ; Andresen ), and may constitute the part of Trichodesmium's nitrogenase-protecting mechanism.

The increase in Mehler reapan> class="Chemical">ction (Kana, 1993; Milligan ) and dark respiratory activity during the diazotrophic periods (Carpenter & Roenneberg, 1995; Kranz ) is supported by the enhanced levels of the respiratory enzyme cytochrome c oxidase detected using proteomic analyses on N2-fixing cultures of Trichodesmium IMS101 (Sandh ) and the enhanced levels of this enzyme specifically in the diazocytes, detected using immunogold localization in natural Trichodesmium populations (Bergman ). In addition, Bryceson & Fay (1981) described zones of cells with higher tetrazolium salt depositions within specific areas of the trichomes, which support a reducing environment within the diazocytes. Although the cellular localization is not yet resolved, recent reports on novel carotenoids with strong antioxidant activity in Trichodesmium (Kelman ) are also interesting in this context and worth examining further.

In addition to a lower net n class="Chemical">oxygen evolutionpan>, the pan> class="Chemical">CO2 fixation is also lowered at mid-day (Berman-Frank ) as recently verified using Nano-SIMS (Finzi-Hart ). Earlier 14C labeling experiments also demonstrated a lower CO2 incorporation in central parts of the trichomes (Paerl, 1996). The decreased CO2 fixation impacts the subcellular storage of carbon (glycogen) and the general carbon metabolism. For instance, carbohydrate storages are degrading (Fig. 2c; El-Shehawy ; Sandh ), and recent proteomic analysis verifies a shift toward a catabolic carbon metabolism under diazotrophy, that is, a down-regulation of enzymes involved in glycogen storage and an up-regulation of enzymes involved in central carbon metabolism (Sandh ). Diazotrophic conditions also led to enhanced levels of proteins involved in the biosynthesis of reducing equivalents (NADPH; through the oxidative pentose phosphate pathway) and the generation of a micro-oxic environment, consistent with increased respiratory activities (Sandh ) and oxygen levels at mid-day.

n class="Species">Trichodesmium IMS101 also prapan> class="Chemical">ctices a light/dark (day/night) separation of other basic cellular processes. While the highly energy-demanding processes, such as CO2 fixation and diazotrophy, take place in light/day phase, cell division and diazocyte development are more pronounced in the dark/night phase (Chen ; Sandh ). Temporal separation of similar processes on a diel basis has previously been observed in marine unicellular cyanobacteria (Holtzendorff , 2002; Stockel ; Shi ). The circadian clock governs many of these processes and is entrained by the cellular ATP/ADP ratio, which in turn is governed by photosynthesis (Rust ). Taken together, current data suggest that besides the development of diazocytes, some more subtle physiological mechanisms may act in concert to optimize diazotrophy in Trichodesmium.

Adaptation to nutrient stress

The n class="Chemical">nitrogen fixed in pan> class="Species">Trichodesmium is, as in other cyanobacteria, assimilated via the glutamine synthetase–glutamate synthase (GS-GOGAT) pathway, and as in heterocysts, the GS protein levels are higher in the diazocytes (Carpenter ) to prevent feedback inhibition of the nitrogenase activity by the accumulation of the ammonia produced. Likewise, externally administrated sources of nitrogen negatively affect the expression of the nif genes, the synthesis of the nitrogenase enzyme, the nitrogenase activity, and diazocyte abundance in Trichodesmium (Ohki ; Lin ; Mulholland ; El-Shehawy ; Holl & Montoya, 2005; Sandh ). As in other cyanobacteria (Herrero ), when subject to N deprivation, there is a significant upshift in the cellular C : N ratio in Trichodesmium (Kranz ), which may be a signal for enhanced transcription by the transcription factor NtcA (Table S1) of N-regulated genes. However, Trichodesmium appears to be flexible in this context, being able to fix N2 in the presence of low concentrations of dissolved inorganic and organic nitrogen (Holl & Montoya, 2005), and the transcript of ntcA is not exclusively regulated by the availability of, for example, ammonium (Post ), which suggests that our knowledge in this area is still limited.

n class="Chemical">Phosphorus anpan>d pan> class="Chemical">iron are critical nutrients restricting growth and N2 fixation in today's oceans (Sanudo-Wilhelmy ; Mills ; Sohm ; Moore ). To overcome P limitations in oligotrophic waters, the Trichodesmium colonies migrate vertically in the water column to scavenge P and other nutrients using a buoyancy-regulating mechanism. This is provided by the pronounced gas vacuoles of Trichodesmium, which can withstand pressures down to depth of about 100–200 m (the highest known; Kromkamp & Walsby, 1992). In the upper euphotic zone, the colonies capture and store carbon and nitrogen (as glycogen and cyanophycin granules; Romans ), and with this ‘ballast’, the colonies sink into deeper waters where P species may be acquired (Romans ; Villareal & Carpenter, 2003; White ; Hewson ). As the cellular ballast is metabolized in the deeper darker waters, the subsequently lighter colonies return to the euphotic zone to again capture light energy.

n class="Species">Trichodesmium is also knpan>ownpan> to adjust to periods of low P bioavailability by adopting high pan> class="Chemical">cellular N : P ratios (White ) in part by a substitution of its phospholipids by non-P membrane lipids (Van Mooy ). Phosphorus uptake is also maximized via the uptake of both inorganic and organic phosphorous species (Stihl ; Fu ; Dyhrman ; Orchard ; Beversdorf ; White ). Enzymes hydrolyzing phosphoesters (alkaline phosphatase; Stihl ; Orchard ) and phosphonates (e.g. phosphonate hydrolase; Dyhrman ) to yield phosphate have been identified in Trichodesmium. However, the globally widespread T. thiebautii lacks one of the alkaline phosphatase–encoding genes, phoA (Orchard ). It has been shown that colonial alkaline phosphatase activities in Trichodesmium may rather be further enhanced by quorum-sensing signals (acylated homoserine lactones) released from colony-associated microorganisms (Van Mooy ). The various phosphate pools available can be utilized either individually or in combination to sustain growth and N2 fixation (White ). Any ‘luxury’ uptake of phosphate is stored in subcellular structures (polyphosphate granules), which are common in natural Trichodesmium populations (Romans ). As increased polyphosphate storages have been found in phosphate-starved cells (Orchard ), the regulation of these subcellular structures in Trichodesmium is enigmatic. The variability among Trichodesmium species in relation to P-uptake genes (Orchard ) also raises the question of niche differentiation and calls for further investigation.

n class="Chemical">Iron is a pivotal pan> class="Chemical">cofactor in a number of cellular processes, such as photosynthesis, N2 fixation (in nitrogenase), and oxygen scavenging (Kustka ; Shi ). The high iron content of Trichodesmium cells suggests an efficient uptake and detainment capacity of iron and may even make Trichodesmium colonies ecologically valuable sources of this otherwise poorly soluble element in oligotrophic oceans (Kustka ; Whittaker ). No obvious genes in the Trichodesmium genome seem to encode for siderophores (Chappell & Webb, 2010), while the genes for transporters of siderophore-bound Fe3+ and Fe2+ and for enzymes related to cellular storage of iron are present (Castruita ; Chappell & Webb, 2010). However, the involvement of siderophores in enhancing the uptake of low iron concentrations in Trichodesmium colonies has been reported (Achilles ). The current hypothesis is that these siderophores are synthesized by associated microorganisms that indirectly facilitate the uptake of iron by Trichodesmium (Achilles ). On the other hand, a recent study showed that such siderophore-bound iron is rather consumed by the bacteria than by the Trichodesmium cells (Roe ). Another option is that the Trichodesmium colony formation per se facilitates the capture of enough particulate iron from, for example, eolian dust depositions to feed the colonies (Rubin ).

n class="Chemical">Iron depletionpan> is knpan>ownpan> to lead to a depan> class="Chemical">crease in the frequency of diazocytes (Berman-Frank ; Küpper ) and to a down-regulation of N2 fixation, while photosynthetic capacities are maintained (Shi ; Brown ; Küpper ). Iron limitation may also elicit a switch in the phycobiliproteins being used (Küpper ). Other cyanobacteria have also been shown to replace ferredoxin with the iron-free flavodoxin (Sandmann ). Monitoring isiB transcription, encoding a flavodoxin, has suggested that Trichodesmium populations in the Atlantic Ocean are rarely, or not at all, iron limited, while those in the Pacific Ocean are (Chappell ). However, the expression of the two fld genes, encoding flavodoxins, in Trichodesmium IMS101 is also regulated by N availability and growth stage (Lin ; Chappell & Webb, 2010; Sandh ), and their proposed use as iron limitation ‘markers’ may be questioned. Rather, the enhanced idiA transcription and IdiA levels noted under iron limitation may be a more suitable marker for iron stress in Trichodesmium (Webb ; Chappell & Webb, 2010). As a strong up-regulation of Dps, yet another protein related to iron acquisition, was observed on the transfer of Trichodesmium to diazotrophic conditions (N stress), the role of Dps and other proteins related to iron acquisition and metabolism now also needs attention.

Impact on the ecosystem

The n class="Species">Trichodesmium abunpan>danpan>pan> class="Chemical">ce is roughly limited to waters warmer than 20 °C, and temperature tolerance for growth and N2 fixation in cultured strains of Trichodesmium (T. erythraeum IMS101, T. erythraeum GBRTRLI101 and T. tenue H94) ranges from 20 to 34 °C, with optimal temperatures being 24–30 °C depending on species and other growth conditions (Breitbarth ; Chappell & Webb, 2010). However, Trichodesmium-like cyanobacteria (nifH and hetR phylogenies) were recently reported in Arctic waters suggesting wider temperature limits (Díez ). Indeed, the very spotty nature of surface blooms of Trichodesmium does not represent the entire population in the ecosystem monitored and illustrates the difficulty in estimating the full global distribution of Trichodesmium via bloom registrations. In spite of this limitation, monitoring such blooms via remote sensing (via the SeaWiFS satellite; Subramaniam ) verified that Trichodesmium blooms occur roughly between 20˚N and 20˚S in the eastern Pacific Ocean and that patches may occur even toward 40˚N and 40˚S in the Atlantic and the western Pacific and Indian Oceans (1998 and 2003; Westberry & Siegel, 2006).

Besides n class="Species">Trichodesmium, numerous unpan>ipan> class="Chemical">cellular cyanobacteria share the same marine aquatic environment, notably the small-celled non-N2-fixing genera Prochlorococcus and Synechococcus (cell diameter ∼ 1 μm, genome sizes of ∼ 2 Mbp; Partensky ; Scanlan ), but also several N2-fixing unicellular cyanobacteria (Zehr ; Montoya ; Moisander ). Among the latter are representatives of the marine genera Cyanothece, Crocosphaera, and N2-fixing cyanobacteria of the ‘group A’ nifH phylotype (e.g. UCYN-A). These diazotrophic unicellular cyanobacteria may show a broader temperature tolerance (15–30 °C) than Trichodesmium, and some have been recovered from waters with detectable nitrate concentrations (Langlois ). Observed community shifts from filamentous cyanobacteria in surface waters to unicellular cyanobacteria and/or heterotrophic bacteria in deeper waters (Langlois ) may also suggest different ecological niche occupancies. Although estimates of the relative contribution to the total biogenic N2 fixation in oceans by unicellular cyanobacteria (and heterotrophic bacteria) are increasing (Halm ; Sohm ,b; Turk ; Zehr & Kudela, 2011), the role of Trichodesmium as a C and N source in the world's oceans is still profound. For instance, Trichodesmium may account for up to 50% of the nifH genes in the North Atlantic Ocean (0°N – 42°N and 67°W – 13°W; Langlois ), and Trichodesmium/Katagnymene represent up to 106 nifH genes per liter, while the unicellular cyanobacteria were represented by 105 nifH genes per liter and proteobacteria by 104 nifH genes per liter (Rijkenberg ). Tyrrell ) reported an even higher colony abundance of Trichodesmium in the tropical Atlantic Ocean (0–15oN and 20oW), and a Trichodesmium surface bloom covered about 100 000 km2 in the Arabian Sea (Capone ).

For still unknown reasons, large segments of the n class="Species">Trichodesmium populationpan> are suddenpan>ly trapped at the surfapan> class="Chemical">ce forming easily observed pigmented layers of dying and decomposing cells (‘blooms’) (Capone ). Such decomposing blooms function as gigantic ‘fertilizer heaps’ releasing large quantities of carbon, nitrogen, and other nutrients for the benefit of nondiazotrophic and heterotrophic biota in the surrounding water bodies. The cause of this destructive ‘bloom’ phenomenon is unknown, while the involvement of viral infections (Hewson ) and/or autocatalyzed cell death processes (Berman-Frank ) have been proposed, but the question is still open. The nitrogen fixed by Trichodesmium may, in addition, enter marine food webs via grazing by tunicates, copepods, and fish (Roman, 1978; Bryceson, 1980; Carpenter, 1983; Oneil & Roman, 1994; Eberl & Carpenter, 2007). Nitrogen isotope ratios in zooplankton in the North Atlantic Ocean strongly indicate that N2 fixation is a major source of nitrogen for the marine zooplankton community (Montoya ). This is at the same time unexpected, as the Trichodesmium toxin production has been inferred as a predator-deterring function (Layton ) as well as a cause of death of several eukaryotic organisms, notably the copepod Acartia tonsa (Guo & Tester, 1994), several species of fish (Endean ), and pearl oysters (Negri ). Trichodesmium is now known to release different secondary metabolites, such as toxins, including the lipophilic chlorinated trichotoxin (Schock ), the palytoxin causing clupeotoxism in humans via fish (Kerbrat ), and the neurotoxin β-N-methylamino-L-alanine (Cox ), the latter also found in diazotrophic bloom-forming cyanobacteria in the Baltic Sea (Jonasson ). The ecological function of these toxins is still unknown.

Yet another factor that may play a role besides the mere pan> class="Chemical">cell number of an organism is their cell size/cell volume. As illustrated in Fig. 4, the size of Trichodesmium cells (approximate sizes given) makes the cellular volumes of this organism ‘gigantic’ compared to, for instance, cells of the unicellular genus Prochlorococcus and the diazotrophic genus Croccosphaera. As about 10–20 cells in each Trichodesmium filament are diazocytes (filled with NifH; Fig. 2b) and a large fraction of the nitrogen fixed may be released (as dissolved organic nitrogen or ammonium) from actively growing Trichodesmium populations (Capone ; Glibert & Bronk, 1994; Mulholland & Capone, 2001; Mulholland ; Mulholland, 2007), each Trichodesmium cell and filament may be viewed as a highly important source of new nitrogen for all nondiazotrophic small cells (unicellular cyanobacteria and bacteria) when sharing similar N-depleted aquatic marine environments.

Fig. 4

Schematic illustration of approximate cell size differences of Trichodesmium species and some unicellular cyanobacteria. The approximate cell sizes and volumes of different species of Trichodesmium are compared to the cell volumes of representatives of the nondiazotrophic but ubiquitous unicellular cyanobacterial genus Prochlorococcus and the unicellular diazotrophic genus Croccosphaera. Note the many-fold larger volume of the Trichodesmium cells. Hatched line shows maximum cell sizes. Trichodesmium cell sizes are according to Janson ).

Schematipan> class="Chemical">c illustration of approximate cell size differences of Trichodesmium species and some unicellular cyanobacteria. The approximate cell sizes and volumes of different species of Trichodesmium are compared to the cell volumes of representatives of the nondiazotrophic but ubiquitous unicellular cyanobacterial genus Prochlorococcus and the unicellular diazotrophic genus Croccosphaera. Note the many-fold larger volume of the Trichodesmium cells. Hatched line shows maximum cell sizes. Trichodesmium cell sizes are according to Janson ).

Trichodesmium in future scenarios

The global importann class="Chemical">ce of pan> class="Species">Trichodesmium in oceanic biogeochemistry has triggered numerous studies mimicking future global warming scenarios. As mentioned above, the optimum temperature range for growth and nitrogen fixation of Trichodesmium is 24–30 °C (Breitbarth ; Chappell & Webb, 2010), but Trichodesmium also survives lower temperatures and darkness (White ; Breitbarth ). The ability to live at lower temperatures and in darkness is essential for their vertical migrations (White ) and may explain the occurrence of Trichodesmium in temperate (see LaRoche & Breitbarth, 2005) and potentially in cooler (Díez ) waters. Global warming will lead to increased stratification resulting in shallower mixed layers and increased irradiance. High light intensity (up to 1000 μE) stimulates growth, diazocyte abundance, and N2 fixation in Trichodesmium (Andresen ; Kranz ; Levitan ) and provokes changes in pigment composition (Andresen ), and correlates with increased O2 evolution and CO2 fixation (Kranz ). Higher light intensities also cause faster protein turnover (Andresen ) and an increase in RuBisC/O:PSII ratio (Brown ). The down-regulation in O2 production via PSII correlates with the earlier noted peaks in N2 fixation at noon (Berman-Frank ). Increased pCO2 levels will not only stimulate CO2 fixation, but also stimulate N2 fixation (dependent on carbon skeletons for sequestration of the ammonium produced) and growth in Trichodesmium (Hutchins ; Levitan , 2010; Ramos ; Kranz , 2010). One mechanism may be energy relocation from the costly carbon-concentrating mechanism (CCM; Badger ; Kranz ) toward CO2 and N2 fixation (Levitan ; Kranz ). This may in turn increase the release of the newly fixed N into the surrounding water body, thereby enhancing primary production of other organisms (Hutchins ). Hence, in a scenario of increased temperatures and CO2 concentrations in the world's oceans, the abundance of surface blooms of Trichodesmium is expected to increase (Breitbarth ; Hutchins ; Levitan ), unless other unforeseen natural factors provoke the opposite reaction. For instance, element colimitations, rather than single-element limitations, may regulate the growth and N2 fixation of natural Trichodesmium populations (Mills ; Hutchins ). Also, Garcia ) have shown that the positive effect of higher CO2 concentrations (on, e.g., N2 fixation) is primarily seen at lower light intensities, and Rijkenberg ) stress that the stimulations expected may be counteracted by decreased supply of nutrients from deeper waters as a consequence of enhanced stratification.

Conclusions and future outlooks

In 1968, Fay ) proposed that the redun class="Chemical">cing pan> class="Chemical">conditions in the cyanobacterial cell type recognized as ‘heterocysts’ could be the site for N2 fixation. In 1973, Fleming & Haselkorn (1973) were the first to isolate nitrogenase from such heterocysts. Hence, heterocysts have during four decades acted as the ‘consensus model’ for successful light-driven N2 fixation in cyanobacteria. In 1991, Bergman and Carpenter were the first to show that nitrogenase is localized in subsets or short strings of cells (diazocyte) in Trichodesmium. These are now recognized as a prerequisite for the light-driven N2 fixation in Trichodesmium. This developmental mechanism is combined with mechanisms temporarily lowering oxygen evolution and orchestrating energy-competing processes in a multifaceted fashion. Hence, Trichodesmium is ‘second-to-none’ among potent daytime N2 fixers, as is in particular evidenced by its great global ecological impact. However, many questions still remain. These include identification of genes/proteins that underpin the development of the diazocytes and their regulation, including mechanisms involved in the protection of the oxygen-sensitive nitrogenase. Because freshwater species exist within the (former) genus Katagnymene (see e.g. Komárek & Anagnostidis, 2005), an intriguing question is whether these are capable of developing diazocytes. Sequencing additional genomes within this globally important genus will allow comparative genomic analyses and potentially shed light on, for example, its uniquely low DNA coding proportion and the significance of its apparently expanding genome. Besides genomic analyses, other ‘omics’ and ‘meta-omics’ approaches (transcriptomics, proteomics, and metabolomics) need to be introduced if we are to comprehend the unique N2-fixing physiology of Trichodesmium at all organization levels. ‘Meta-omics’ may provide more accurate information pertaining to the genetic diversity and the role of Trichodesmium and associated microorganisms than cultures, which often represents a minor part of the total species radiation. Such data may also reveal mutual and potentially life-sustaining interplays between Trichodesmium and the numerous associated microorganisms and open exciting research avenues into microbial evolution and marine microbial interphylum interactions, some potentially of a symbiotic nature. Focus should also be given to proteins that are highly up-regulated under N2-fixing conditions (Hewson ; Sandh ). Another area in need of exploration is the identification of adaptive ecological strategies, and possible niche differentiations, used by natural Trichodesmium populations. Finally, evolutionary aspects related to the uniquely different diazotrophic behavior of the genus Trichodesmium and to its placement in the evolution of affiliated cyanobacteria with unusual diazotrophic behavior are yet other compelling research areas to explore.