Cyanobacteria in Nitrogen-Fixing Symbioses

Chapter 2

Cyanobacteria in Nitrogen-Fixing Symbioses Edder D. Bustos-Díaz⁎,†, Francisco Barona-Gómez⁎, Angélica Cibrián-Jaramillo† ⁎

Evolution of Metabolic Diversity Laboratory, Langebio, Cinvestav-IPN, Guanajuato, Mexico, †Ecological and Evolutionary Genomics Laboratory, Langebio, Cinvestav-IPN, Guanajuato, Mexico

1. INTRODUCTION A reliable source of nitrogen is necessary for life. This element is crucial for the biosynthesis of biomolecules such as DNA. Despite being a major component of the atmosphere, obtaining nitrogen is often a problem for most living beings since the most common form of this element, N2, is biochemically inert. The majority of organisms rely on metabolizable forms of nitrogen, such as nitrates and nitrites, to grow. Atmospheric N2 can be converted to these bioactive forms in a chemical process called “nitrogen fixation,” which occurs naturally during the nitrogen cycle (Laneuville et al., 2018), albeit at such a slow rate that the quantities of this element required for sustaining life cannot be reached (Vitousek et al., 2002). The bulk of nitrogen fixation is therefore carried out by the biological activity of microorganisms, called diazotrophs (Newton, 2007). Diazotrophs fix nitrogen using an enzymatic reaction in which N2 is converted into ammonium, NH4 (Newton, 2007). These microorganisms can be found living freely (Reed et al., 2011), or as part of symbiotic relationships, in which a diazotroph is recruited by another organism to fix nitrogen usually in exchange of nourishment and protection, in mutually beneficial associations (Mylona et al., 1995). Among diazotrophs capable of symbiotic relationships, Cyanobacteria are remarkable. They are an ancestral lineage responsible for the generation of an oxygen-rich atmosphere 2.35 billion years ago (Schirrmeister et al., 2015); they formed the photosynthesis-based symbiosis that originated chloroplasts (McFadden, 2001); and continue to contribute to lifedepending processes such as maintaining the current ocean oxygen levels (Karl et al., 2012). Cyanobacteria display great symbiotic plasticity, as they are capable of forming symbioses with dramatically different hosts. In terrestrial systems and in contrast to well-known diazotrophic symbionts Rhizobium (Oldroyd et  al., 2011) and Frankia (Schwencke and Carú, 2001), their range of hosts is not limited to plants. Interestingly, cyanobacteria’s plant hosts usually belong to basal phyla of gymnosperms, angiosperms, and bryophytes, suggesting ancient relationships, and quite possibly, constituting the first examples of symbiotic associations to fix nitrogen. Furthermore, the ability of a single cyanobacterial strain to infect different plants suggests functional plasticity from the bacteria, but also that the hosts likely have conserved chemical signaling pathways to recruit cyanobionts, facilitating applications in agriculture (Gusev et al., 2002; Watanabe et al., 1977). Plant cyanobionts, or symbiotic cyanobacteria, are interesting from an evolutionary perspective and have important practical uses. Historically, cyanobacteria have been classified by their morphological characteristics in five groups, with the unicellular cyanobacteria distributed across groups I (Gloeothece, Synechococcus, Cyanothece), II (Myxosarcina, Xenococcus, Dermocarpa), and III (Lyngbya, Trichodesmium, Plectonema, Pseudanabaena); and filamentous, heterocyst-forming cyanobacteria contained in groups IV (Nostoc, Anabaena, Calothrix, Scytonema, Cylindrospermum, Nodularia, Tolypothrix, Trichormus) and V (Fischerella, Chlorogloeopsis) (Rippka et al., 1979, 2001). Cyanobacteria are currently classified based on single genetic markers or genome-wide orthologs by changing the tips of the phylogenetic trees (Dos Santos et al., 2012; Tomitani et al., 2006; Zehr et al., 1997). Their capacity to form symbiotic relationships is dispersed throughout their phylogeny, with some groups displaying evolutionary traits clearly associated with nitrogen fixation in the context of their interactions with other organisms (Zehr et al., 1997; Latysheva et al., 2012). Indeed, cyanobacteria are capable of forming a wide variety of symbiotic relationships with a multitude of hosts, each with their own peculiarities. Thus, symbiotic cyanobacteria are an important model to understand how symbioses that require nitrogen fixation originated. Questions like how these symbioses are formed, how they work, and what genetic, metabolic, and evolutionary changes affect cyanobionts and their hosts are unanswered. Cyanobacteria. https://doi.org/10.1016/B978-0-12-814667-5.00002-7 © 2019 Elsevier Inc. All rights reserved.

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30 Cyanobacteria

In this chapter we will provide an introduction to the genes required for nitrogen fixation, how they can be used to classify symbiotic cyanobacteria, and how they serve as a proxy to understand the biological mechanisms that cyanobacteria have evolved to deal with nitrogen fixation in the context of other metabolic processes, such as photosynthesis (Section 2). We will begin with obligate interactions distinguishing from inherited symbionts and captured symbionts (Section 3.1); and then the often less understood symbiotic relationships that are classified as facultative (Section 3.2). In each section we will mention the known genes, gene clusters, or genomic regions that are evolutionary innovations needed for fixing nitrogen, and deal with life in symbiosis. We will end with a brief summary of trends in heterocystous and nonheterocystous cyanobacteria (Section 4). It is clear from this review that although we have made progress in understanding the genomic signatures of symbiosis, in most relationships genome-level adaptations are not known or fully understood. The evolutionary and ecological history of many of the symbiotic associations we described in this chapter is only incipiently investigated, and even the classifications that are currently used (i.e., facultative and obligate), will soon require a revision based on a better understanding of genome evolution in cyanobacteria. We not only hope to add to the understanding of cyanobacteria symbiosis and the genomic processes that underlie them, but also to identify opportunities that could help advance the field.

2.  CYANOBACTERIAL NITROGEN FIXATION, A BIOLOGICAL CONVERSION BASED ON NITROGENASES To make sense of the genomic features of nitrogen fixing cyanobacterial symbioses we will first review how this process works. Biological nitrogen conversion from N2 to NH4 is catalyzed by an enzyme, the nitrogenase (Latysheva et al., 2012; Boyd and Peters, 2013), which is oxygen sensitive. There are three types of nitrogenases: Nif (molybdenum-dependent), Vnf (vanadium-dependent), and Anf (iron-dependent), all of them phylogenetically related (Boyd and Peters, 2013). Nif nitrogenase is formed by the catalytic proteins encoded in the genes nifH, nifD, and nifK. The Vnf nitrogenase requires the protein products of four genes called vnfH, vnfD, vnfG, and vnfK, whereas the Anf nitrogenase consist of the products of anfH, anfD, anfG, and anfK genes. Virtually every diazotrophic organism uses a set of these enzymatic genes (Dos Santos et al., 2012; Boyd and Peters, 2013). Of the three nitrogenases, the molybdenum-dependent enzymes are the most used, due to its catalytic efficiency (Seefeldt et al., 2009). The other two nitrogenases are considered as auxiliary, or secondary (Dos Santos et al., 2012). Because of this, the so-called principal nitrogenase, or Nif, is the most studied both at the genomic and proteomic levels. Its importance is such that organisms without the nif genes are usually not considered diazotrophs (Dos Santos et al., 2012). To have a functional molybdenum-dependent nitrogenase (henceforth referred simply as nitrogenase) the genes encoding proteins with catalytic activity are needed. For instance, between the regulatory, catalytic, and auxiliary genes, the cyanobacterial nif operon is thought to contain 16 genes (i.e., nifHDKTEXWZ genes), eight of which seem to be indispensable (Latysheva et al., 2012). This is similar to what has been found in other diazotrophic organisms and is consistent with the idea that all nitrogenase genes came from a common ancestor (Zehr et al., 1997; Latysheva et al., 2012). The phylogenetic analysis of these nitrogenase sequences shows that they can be divided into two large groups: nitrogenases that belong to heterocystous cyanobacteria and that belong nonheterocystous cyanobacteria, both are able to perform photosynthesis (Zehr et al., 1997). On one hand, heterocystous cyanobacteria from groups IV and V form a monophyletic group, and are thought to have a common origin (Tomitani et al., 2006). On the other hand, nitrogenases of nonheterocystous cyanobacteria cannot be clustered, and the ability to fix nitrogen is scattered throughout the phylogeny (Zehr et al., 1997). This is believed to be caused by numerous losses of function, rather than by horizontal gene transfer of the nif operon between species, although this can also occur sporadically (Zehr et al., 1997; Latysheva et al., 2012). In addition to these two types, there are also strictly symbiotic cyanobacterial species that do not have a functional photosystem and therefore they are exclusively dedicated to fixing nitrogen (Zehr et al., 2008). However, these are extreme cases, and it is hypothesized that they represent a transition state between a symbiotic organism and an organelle. The distinction between heterocystous and nonheterocystous cyanobacteria is important as it determines how nitrogenase activity is isolated from oxygen (Rippka et  al., 2001). For instance, heterocystous cyanobacteria separate photosynthesis and nitrogen fixation by a physical barrier, allowing nitrogen fixation to be carried out by heterocysts, in which the photosystem II is inactivated. Heterocysts are also equipped with a double cellular membrane to restrict oxygen entry (Kumar et al., 2010). In photosynthetically active cells of the well-studied Anabaena (Nostoc) sp. PCC 7120 (Kaneko et al., 2001; Buikema and Haselkorn, 1991), nifD (coding for the catalytic component of the nitrogenase), fdxN (coding for a ferrodoxin-like protein located within the nif region), and hupL (coding for the large subunit of an uptake hydrogenase that recycles the H2 produced during nitrogen fixation) genes are inactivated by terminal DNA insertions of 11, 55, and 10.5 kbp respectively (Mulligan and Haselkorn, 1989; Golden et al., 1991). When the cell commits to forming an heterocyst, these terminal regions are excised, via recombination of flanking repetitive sequences on both sides of the insertions, a necessary step to make the nif operon transcriptionally active (Mulligan and Haselkorn, 1989).

Cyanobacteria in Nitrogen-Fixing Symbioses Chapter | 2  31

Nonheterocystous cyanobacteria, in contrast, separate nitrogen fixation from photosynthesis by a temporal barrier (Stöckel et al., 2008), restricting nitrogen fixation to low-light periods, and photosynthesizing during the high-light periods (Toepel et al., 2008; Bandyopadhyay et al., 2013). Therefore, nitrogenase synthesis is restricted to the dark period. During this period, cyanobacteria also lower the levels of intracellular oxygen and photosynthetic activity to a minimum, using a biochemical pathway known as the Mehler reaction, increasing cellular respiration and synthesizing cyanoglobin, an oxygen scavenger protein (Stöckel et al., 2008; Toepel et al., 2008; Bandyopadhyay et al., 2013). The genomic arrangement of genes within the nif operon is more streamlined in these organisms than in heterocystous cyanobacteria (Welsh et al., 2008); many nonheterocystous organisms have just one nitrogenase synthesis cluster whose expression can be controlled by cyanobacterial nitrogen fixation regulators, such as cnfR (Tsujimoto et al., 2014). Conservation of the basic regulatory system implies that regulation of nitrogen fixation in nonheterocystous cyanobacteria occurs at posttranscriptional level (Toepel et  al., 2008). A more detailed explanation of these symbiotic relationships will be provided in the following section.

3.  CYANOBACTERIA IN SYMBIOSIS, AN ANCIENT AND EVOLVING HISTORY OF INTERACTIONS All symbiotic organisms can currently be divided into two categories: facultative and obligate symbionts (Sachs et  al., 2011). Facultative symbionts are capable of living in their natural habitats without a symbiotic partner, while obligate symbionts cannot. In this section, driven by the availability of genome sequences for cyanobacterial symbionts, we focus on what can be learned from these data. The relevant biological and genomic information for all the symbiotic interactions that have been reviewed herein is provided in detail in Table 1 and summarized in Table 2.

3.1  Obligate Cyanobacteria Symbionts Obligate symbionts are extremely well adapted to live with their hosts, maintaining their symbiosis through generations (Sachs et  al., 2011). This high degree of specialization can, if given enough time, lead to phylogenetic separation, or branching, of the symbiotic species from its sister groups (Sachs et al., 2011; Medina and Sachs, 2010). The genomes of these organisms tend to be smaller than their free-living counterparts, a reduction that is often accompanied by loss of gene function due to mutations (Sachs et al., 2011). This does not affect genes important for symbiosis, like the nif operon, which is highly conserved. Along with this, symbiotic genomes tend to have higher AT percentage in comparison to their freeliving counterparts (Rocha and Danchin, 2002). Cyanobacterial symbionts show these characteristics at various degrees, depending on the nature of the relationship between the cyanobacterial symbiont and its host. Obligate symbionts can be further subdivided into (i) inherited symbionts and (ii) captured symbionts (Sachs et al., 2011). Inherited symbionts are transferred from host to host via vertical transmission, but once into their hosts they can remain physically separated from them. In contrast, captured symbionts are obligate symbionts found in intracellular symbiotic relationships. In these, the cyanobiont is located inside its hosts, and are transmitted when the host’s cells divide (Sachs et al., 2011). The molecular mechanisms that control the synchronous division of host and symbionts are varied and complex (Kistner and Parniske, 2002) and will not be reviewed here. Examples of these two types of obligate symbionts are provided next.

3.1.1  Obligate Inherited Symbionts Inherited symbionts are those organisms passed from the host to its offspring. These symbiotic organisms are, therefore, well adapted to their host since they are intimately related to them. Some of the typical characteristics of these symbionts are the contraction of their genomes, caused by the loss of unnecessary genes, commonly those related to the free-living state, as well as their multiple adaptations to the niche represented by their hosts. Azolla-Trichormus azollae: In the symbiosis between the aquatic fern Azolla and the filamentous cyanobacteria Trichormus azollae (formerly known as Nostoc or Anabaena azollae) (Baker et al., 2003), the cyanobiont is inherited from the parent plant, as it is attached to the fern’s spores (Rasmussen and Johansson, 2002). When the plant reaches maturity, Trichormus cells locate on a leaf lobe cavity in which it fixes nitrogen (Peters, 1991). Inside this cavity, the percentage of heterocysts rise to 25%–30% and the whole cyanobiont becomes photosynthetically inactive (Peters, 1991). This cavity, however, is not only inhabited by Trichormus but also other cyanobacterial species, most likely facultative symbionts, and even noncyanobacterial species, have been found (Peters and Meeks, 1989). The putative cyanobionts, however, have the characteristic features of inherited symbionts.

Cyanobacterial Symbiont

Host

Symbiosis

Group

Order

Species

Group

Species/Order

Type

Location

Genomes

References

I

Chroococcales

Not identified

Cnidaria

Montastraea cavernosa

–

–

No

Lesser et al. (2004)

I

Chroococcales

Candidatus Atelocyanobacterium thalassa (UCYN-A)

Haptophytes

Braarudosphaera bigelowii

O

Intracellular

2 isolates

Zehr et al. (2017) and Tripp et al. (2010)

I

Chroococcales

UCYN-B

Haptophytes

Not identified

O

Intracellular

No

Thompson et al. (2014)

I

Chroococcales

Spheroid bodies

Bacillariophyta

Epithemia turgida

O

Intracellular

1 isolate

Nakayama et al. (2014)

I

Synechococcales

Synechococcus sp.

Protista

Solenicola setigera

–

–

No

Buck and Bentham (1998)

I

Synechococcales

Candidatus Synechococcus spongiarum

Porifera

30 orders

–

–

No

Konstantinou et al. (2018)

I

Synechococcales

Synechococcus spp.

Porifera

46 orders

–

–

No

Konstantinou et al. (2018)

I

Synechococcales

Candidatus Synechococcus sporangium

Porifera

Theonella swinhoei

–

–

1 isolate

Burgsdorf et al. (2015)

I

Synechococcales

Candidatus Synechococcus sporangium

Porifera

Ircinia variabilis

–

–

1 isolate

Burgsdorf et al. (2015)

I

Synechococcales

Candidatus Synechococcus sporangium

Porifera

Aplysina aerophoba

–

–

1 isolate

Burgsdorf et al. (2015)

I

Synechococcales

Candidatus Synechococcus sporangium

Porifera

Carteriospongia foliascens

–

–

1 isolate

Gao et al. (2014)

I

Synechococcales

Prochlorococcus spp.

Porifera

15 orders

–

–

No

Konstantinou et al. (2018), and references within

I

Synechococcales

Similar to Aphanocapsa raspaigella

Porifera

11 orders

–

–

No

Konstantinou et al. (2018), and references within

I

Synechococcales

Synechocystis spp.

Porifera

10 orders

–

–

No

Konstantinou et al. (2018), and references within

I

Synechococcales

Leptolyngbya spp.

Porifera

7 orders

–

–

No

Konstantinou et al. (2018), and references within

I

Synechococcales

Cyanobium spp.

Porifera

Petrosia ficiformis

–

–

No

Konstantinou et al. (2018), and references within

32 Cyanobacteria

TABLE 1  Cyanobacteria in Symbiosis Involving Nitrogen Fixation

Synechococcales

Halomicronema metazoicum

Porifera

Petrosia ficiformis

–

–

No

Konstantinou et al. (2018), and references within

I

Synechococcales

Unclassified Schizotrichaceae

Porifera

Aplysina aerophoba

–

–

No

Konstantinou et al. (2018), and references within

I

Synechococcales

Pseudanabaena cf. persicina

Porifera

Axinella damicornis

–

–

No

Konstantinou et al. (2018), and references within

II

Pleurocapsales

Myxosarcina sp.

Porifera

Terpios hoshinota

–

–

No

Konstantinou et al. (2018), and references within

II

Pleurocapsales

Xenococcus sp.

Porifera

Ircinia variabilis

–

–

No

Konstantinou et al. (2018), and references within

III

Oscillatoriales

Oscillatoria spongeliae

Porifera

13 orders

–

–

No

Konstantinou et al. (2018), and references within

III

Oscillatoriales

Spheroid bodies

Bacillariophyta

Rhopalodia gibba

O

I

1 isolate

Kneip et al. (2008)

IV

Nostocales

Richelia intracellularis

Bacillariophyta

Hemiaulus hauckii Rhizosolenia spp.

O

I

5 isolates

Hilton et al. (2013) and Parks et al. (2017)

IV

Nostocales

Calothrix rhizosoleniae

Bacillariophyta

Chaetoceros spp.

F

E

1 isolate

Foster et al. (2010)

IV

Nostocales

Nostoc spp.

Ascomycota

All Peltigera Lobaria pulmonaria

O

E

6 isolates

Rikkinen (2017) and Gagunashvili et al. (2018)

IV

Nostocales

Nostoc spp.

Bryophyta

All Blasia All Cavicularia

F

E

4 isolates

Warshan et al. (2018) and Adams and Duggan (2008), and references within

IV

Nostocales

Nostoc spp.

Bryophyta

All Anthocerotae

F

E

No

Adams and Duggan (2008), and references within

IV

Nostocales

Nostoc spp.

Bryophyta

Hypnales

F

E

5 isolates

Warshan et al. (2018)

IV

Nostocales

Nostoc spp.

Bryophyta

Sphagnum lindebergii Sphagnum riparium

F

E

No

Adams and Duggan (2008), and references within

IV

Nostocales

Trichormus azollae

Pteridophyte

All Azolla

O

E

1 isolate

Ran et al. (2010)

IV

Nostocales

Nostoc punctiforme

Gymno

Macrozamia spp.

F

E

1 isolate

Moraes et al. (2017)

IV

Nostocales

“Cyanobacterium”

Gymno

Dioon edule Dioon caputoi

F

E

2 isolates

Gutierrez-Garcia et al. (2018)

IV

Nostocales

Nostoc cycadae

Gymno

Cycas revoluta

F

E

1 isolate

Kanesaki et al., (2018)

IV

Nostocales

Nostoc spp.

Angio

All Gunneraceae

F

I

No

Bergman and Osborne (2002)

“–” indicates that the nature of the symbiosis has not been characterized. O is obligate and F is facultative; E is extracellular, and I is intracellular.

Cyanobacteria in Nitrogen-Fixing Symbioses Chapter | 2  33

I

34 Cyanobacteria

TABLE 2  Genome Length and AT Content of Sequenced Symbiotic Cyanobacteria Taxonomic Group

Cyanobiont

Length (Mbp)

AT %

I

Candidatus Synechococcus sporangium SP3

2.2

39.1

I

Candidatus Synechococcus sporangium 142

2.5

41.3

I

Candidatus Synechococcus sporangium 15L

2.3

40.8

I

Candidatus Synechococcus sporangium SH4

1.9

36.9

I

Candidatus Atelocyanobacterium thalassa isolate ALOHA

1.4

68.9

I

Candidatus Atelocyanobacterium thalassa isolate SIO64986

1.4

69

I

Spheroid body of Rhopalodia gibba

2.6

62.8a

I

Spheroid body of Epithemia turgida isolate EtSB

2.79

66.6

IV

Richelia intracellularis HH01

3.2

66.3

IV

Richelia intracellularis HM01

2.2

66.2

IV

Richelia intracellularis UBA3481

3.2

66.5

IV

Richelia intracellularis UBA7409

3

66.4

IV

Richelia intracellularis RC01

5.4

66.8

IV

Trichormus (formerly Nostoc) azollae 0708

5.49

61.7

IV

Calothrix rhizosoleniae SC01

5.9

60.5

IV

Nostoc sp. "Lobaria pulmonaria cyanobiont"

7.34

58.4

IV

Nostoc sp. "Peltigera malacea cyanobiont"

8.52

58.4

IV

Nostoc sp. "Peltigera membranacea cyanobiont" N6

8.9

58.6

IV

Nostoc sp. "Peltigera membranacea cyanobiont" 210A

8.33

58.3

IV

Nostoc sp. "Peltigera membranacea cyanobiont" 213

8.33

58.6

IV

Nostoc sp. "Peltigera membranacea cyanobiont" 232

9.16

58.4

IV

Nostoc sp. Moss2

9.16

58

IV

Nostoc sp. Moss3

9.10

59

IV

Nostoc sp. Moss4

8.97

59

IV

Nostoc sp. Moss5

7.24

59

IV

Nostoc sp. Moss6

7.14

59

IV

Nostoc sp. KVJ2

8.69

59

IV

Nostoc sp. KVJ10

10.39

59

IV

Nostoc sp. KVJ20

9.18

59

IV

Nostoc sp. KVS11

8.30

59

IV

Nostoc punctiforme ATCC 29133

9.06

58.6

IV

“Cyanobacterium” RF31YmG

9.18

58

IV

“Cyanobacterium” 106C

8.6

58.7

IV

“Cyanobacterium” T09

8.2

58.5

IV

Nostoc cycadae WK-1

6.99

59.6

a

Estimated based on Kneip et al. (2008).

a

Cyanobacteria in Nitrogen-Fixing Symbioses Chapter | 2  35

From the various strains that have been isolated and cultivated from different Azolla plants, only the genome of one of these bacteria has been sequenced, Trichormus (formerly Nostoc) azollae 0708 (Ran et al., 2010). The genome of this strain is small (5.49 Mbp) and has a relatively high AT percentage (61.7%). Additionally, the percentage of coding genes is fairly low, with just 52% of the entire genome predicted to code for functional genes (Meeks et al., 1988). This loss of function has affected mostly the synthesis of natural products or specialized metabolites, signal transduction mechanisms, and DNA replication, recombination, and repair systems (Ran et al., 2010). While the nif operon in other filamentous cyanobacteria is interrupted by excision sequences in vegetative cells, Trichormus lack these elements in the genes fdxN and nifD (Meeks et al., 1988). Due to this, Trichormus mutants that cannot differentiate heterocysts can express nitrogenase in vegetative cells (Meeks et al., 1988). Overall, the analysis of Trichormus azollae 0708 reveals a specialization of its genome to adapt to its unique ecological niche provided by the leaf lobe cavity of its fern host. Fungi-Nostoc: Various fungi, principally from the phylum Ascomycota, can form symbiotic relationships with cyanobacteria. Most of these symbioses, called bipartite cyanolichens, are formed between the fungus (phycobiont) and photosynthetic cyanobacteria or algae (photobiont). However, some of these symbioses can also involve a third cyanobacterial partner, whose role is to fix nitrogen (cyanobiont), giving place to tripartite cyanolichens (Rikkinen, 2017; Honegger, 1991). In these symbioses, the symbiotic partner, or partners, are transmitted through the development of the new thalli that sprang from propagules. Inside them, the cyanobiont is found in the cephalodia, in which nitrogen fixation activity is carried out (Honegger, 1991; Rai, 2002). Similar to the Azolla symbiosis, the cyanolichen’s cephalodia is also inhabited by different cyanobacterial species (Honegger, 1991). While the symbiosis between mycobiont and photobionts appears to be strictly obligate (Stocker-Wörgötter and Hager, 2008), the symbiosis between mycobiont and cyanobiont cannot be readily classified as obligate, as some of the symbiotic strains isolated from cyanolichens have physiological (Ahmadjian, 1993) and genomic characteristic (Gagunashvili and Andrésson, 2018) more akin to the ones exhibited by facultative symbionts. Many strains of the putative cyanobiont have been isolated and cultivated. Taxonomic identification of cyanobiont strains by analysis of the 16 s subunit of the ribosomal RNA (rRNA) gene revealed that these cyanobacteria belong to Nostocales (O'Brien et al., 2005). This was confirmed when two complete genomes, along with four draft genomes, were obtained (Gagunashvili and Andrésson, 2018). The two complete genomes, Nostoc sp. N6 and Nostoc sp. "Lobaria pulmonaria cyanobiont" were obtained from the bipartite cyanolichen Peltigera membranacea and the tripartite cyanolichen Lobaria pulmonaria, respectively. Three of the draft genomes, Nostoc sp. 210A, Nostoc sp. 213 and Nostoc sp. 232, were obtained from P. membranacea, while the remaining one, Nostoc sp. "Peltigera malacea cyanobiont," was obtained from Peltigera malacea. The genome size of these organisms ranged from 7.34 to 9.16 Mb, with an average of 8.43 Mb, and a AT percentage of 58%. Nostoc sp. N6 has one chromosome (8.2 Mb) and 10 small replicons, seven circular and three linear. This strain has a functional nif operon, with two excision elements in its nifD gene (7.5 and 29 kb), and one in its fdxN gene (36.7 kb). Nostoc sp. "Lobaria pulmonaria cyanobiont" has one chromosome (7.06 Mb) and four circular replicons. This strain also has a functional nif operon, but with just one excision element in its nifD gene (22.8 kb). Both genomes have the hupL gene. The smaller genome size of Nostoc sp. "Lobaria pulmonaria cyanobiont," as well as its high degree of pseudogenization and slower growth rate indicate that this cyanobiont isolated from a tripartite cyanolichen has a more obligate lifestyle. Nostoc sp. N6, on the contrary, seems to form a facultative bipartite symbiosis. Porifera-Cyanobacteria: Cyanobacterial species found in symbiosis with sponges are highly diverse, including Candidatus Synechococcus spongiarum, Synechococcus spp., Prochlorococcus spp., as well as other organisms similar to Aphanocapsa raspaigella, Synechocystis spp., Oscillatoria spongeliae, Leptolyngbya spp. These symbioses are distributed throughout sponges, with members of 22 orders capable of forming associations with cyanobacteria (Konstantinou et al., 2018). There are four draft genomes of cyanobacterial species in association with sponges (Gao et al., 2014; Burgsdorf et al., 2015). These genomes are small, with median predicted length of 2.2 Mbp, with a slightly low AT percentage with an average of 60.4% (Burgsdorf et al., 2015). Since their genomes are reduced, and evidence of vertical transmission of the cyanobiont has been found, we situate the Porifera-Cyanobacteria symbiosis in this section. However, not much is known about these symbioses, and it remains unknown if the cyanobiont fixes nitrogen as part of the symbiotic partnership.

3.1.2  Obligate Captured Symbionts The genomic characteristics of captured symbionts include an extreme reduction in their genomes, loss of several metabolic functions through gene mutation, and a total dependency of their host. It is thought that the symbiosis between ancient plants and CO2 fixing cyanobacteria was of this type (Martin et al., 2002). The continued and irreversible loss of autonomy of the cyanobacterial partner in that symbiosis ultimately transformed the symbiotic cyanobacteria into chloroplasts (Martin et al., 2002; Raven and Allen, 2003). The symbiotic associations found in this category seem to be approaching the same

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inevitable destiny, making them an interesting model to study organelle evolution. The symbiotic relationships found in this group are the following: Bacillariophyceae-Richelia intracellularis: The symbiotic relationship between diatoms Hemiaulus and Rhizosolenia, with filamentous cyanobacteria Richelia intracelllularis, are the best-known symbiosis of this group. In this symbiosis, the intracellular filaments (two to four filaments per diatom) are short and have a single terminal heterocyst. In the heterocyst, nitrogenase activity is high, and the synthesized ammonia disseminates into the host (Jahson et al., 1995; Zeev et al., 2008). When the diatom divides by binary fission, filaments of Richelia intracellularis are distributed among the newly formed hosts (Villareal, 1989). The genome of various strains of Richelia intacellularis has been sequenced. These strains include Richelia intracellularis HH01, Richelia intracellularis HM01, Richelia intracellularis UBA3481, Richelia intracellularis UBA7409, and Richelia intracellularis RC01 (Parks et al., 2017; Hilton et al., 2013). These genomes present a reduced size, the genome of Richelia intracellularis HH01 being the smallest of them, with a length of 3.2 Mbp. This strain also has a high AT percentage (66%), and a low percentage of functional genes (56%), which is concomitant with the loss of metabolic functions, including the ability to synthesize amino acids (Parks et al., 2017). The rest of the strains that have been sequenced present similar values. Interestingly, the nif operon of Richelia intracellularis HH01 does have excision sequences in its nifH gene. However, it lacks the necessary genes to take up simple nitrogenated compounds like urea, which forces it to fix nitrogen continuously (Parks et al., 2017). Haptophytes-Uncultivated unicellular cyanobacterium: This association has been hard to define, as the host of the socalled uncultivated unicellular cyanobacterium (UCYN), which are unicellular cyanobacteria thought to be located inside their host, has not been definitely found. In fact, different strains of UCYN seem to be associated with different species of unicellular algae (Thompson et al., 2012). To date, at least three species of this cyanobiont have been recognized, UCYN-A1, UCYN-A2 (Candidatus Atelocyanobacterium thalassa), and UCYN-B (Zehr et al., 2017). While UCYN-A1 and UCYN-A2’s nif operon could not be grouped with existent cyanobacterial groups, UCYN-B’s nif operon was found to be very similar to that present in Crocosphaera watsonii (Zehr et al., 2001). The phylogenetic relationship between UCYN-A1 and UCYN-A2 is unclear, so it is not known if these are ecotypes of the same species, different strains, or different species (Thompson et al., 2014). This distinction is important, since UCYN-A1 and UCYN-A2 appear to have different hosts (Thompson et al., 2014). While free-living unicellular cyanobacteria fix nitrogen during the night, Candidatus Atelocyanobacterium thalassa (C.A. thalassa) is capable of fixing nitrogen at a high rate during the day, which contrasts with UCYN-B (Zehr et al., 2001). The genome of Candidatus Atelocyanobacterium thalassa isolate ALOHA has been sequenced, showing some of the typical characteristics of an obligate symbiont, namely, an extremely small genome size (1.4 Mbp) and high AT percentage (68.9%). Contrary to other obligate symbionts, though, the ALOHA isolate had a high percentage of coding genes (94.17%). This was probably caused by the excision of most of the pseudogenes formed during prolonged symbiosis. This explanation is consistent with the lack of many important functions for life outside the host, like the photosystem II or TCA cycle. C.A. thalassa has also lost its ammonia transporters, which obligates it to obtain its nitrogen exclusively through fixation (Tripp et al., 2010; Bombar et al., 2014). For UCYN-A2, a draft genome has been released under the name Candidatus Atelocyanobacterium thalassa isolate SIO64986. It is similar to isolate ALOHA, with a very small genome (1.4 Mbp), high AT percentage (69%), but a smaller yet high percentage of coding DNA for a cyanobiont (79.3%) (Bombar et  al., 2014). It is clear, therefore, that C.A. thalassa isolates SIO64986 and ALOHA share genomic characteristics and content, which suggests a monophyletic origin (Thompson et al., 2014). The absence of essential functions in both genomes is indicative of their advanced state of decay (Zehr et al., 2017). With the irreversible loss of autonomy from the host, C.A. thalassa might represent the point of no return in the path to becoming an organelle. Rhopalodiaceae-spheroid bodies: The spheroid bodies found in Rhopalodia gibba and Epithemia turgida can barely be classified anymore as independent organisms (Nakayama et al., 2011; Trapp et al., 2012). As indicated by their name, these bodies lack clear independence from their host and can be practically labeled as proto-organelles. Phylogenetic analysis of spheroid bodies has shown that they have a cyanobacterial origin, closely related to Cyanothece (Nakayama et al., 2011). A genomic region of 140 kbp in the spheroid bodies of Rhopalodia gibba has been sequenced. This region contains the nif operon, yet although completely functional, has lost some genes, most importantly fdxN, when compared to the nif operon of its close relative Cyanothece sp. ATCC 51142. The obtained sequence has an AT percentage of 62.8%, and while the genome was not fully sequenced, it was estimated to have a length of 2.6 Mbp (Kneip et al., 2008). The genome of the spheroid body of Epithemia turgida isolate EtSB has also been sequenced (Nakayama et al., 2014), showing similar characteristics to C.A. thalassa ALOHA isolate (Zehr et al., 2017). It has an extremely reduced genome size (2.79 Mbp), high AT percentage (66.6%), and a high percentage of coding genes (88.7%) (Nakayama et al., 2014). However, the spheroid bodies lack almost every metabolically important pathway, including the Calvin cycle, and the

Cyanobacteria in Nitrogen-Fixing Symbioses Chapter | 2  37

photosystems I and II. What remains are the nif operon and ribosomal genes, along with hupL, which indicates that nitrogen fixation is done effectively. Nevertheless, the absence of metabolic genes makes spheroid bodies completely dependent on their host to survive (Nakayama et al., 2014).

3.2  Facultative Cyanobacteria Symbionts Cyanobacterial species are capable of forming facultative symbiotic relationships with many organisms, especially plants. The host recruits facultative symbionts during the so-called infection stage from its surroundings. Once the cyanobacteria contacts the host, the symbiosis is established. To infect the host, symbionts must be capable of detecting and responding to chemical signals produced by the host (Meeks and Elhai, 2002). The majority of cyanobacterial facultative symbioses involve filamentous cyanobacteria, which are capable of developing hormogonia, or motile cells (Meeks et  al., 2002). Since many hosts produce, or are thought to produce, hormogonia-inducing factors (HIFs), the symbiont must have the necessary genetic equipment to produce the motile cell and detect the HIF gradient that will guide them to the host to form the symbiosis (Meeks and Elhai, 2002). The genes encoding this function, then, are crucial to separate strictly free-living symbionts from their symbiotic counterparts. The capacity to alternate between symbiotic and free-living lifestyles of facultative symbionts is reflected by an increase in their genome size, caused by the accumulation of the necessary genes to sustain both lifestyles. Other typical genomic characteristics of these symbionts are a high percentage of coding genes, as well as AT content (Medina and Sachs, 2010). Among the facultative symbioses we find the following: Chaetoceros-Calothrix rhizosoleniae: The exact mechanism of cyanobiont infection in the symbiosis between the diatom Chaetoceros and the filamentous cyanobacteria Calothrix rhizosoleniae is not known. However, this Calothrix species is believed to be an opportunistic symbiont, as suggested by its genetic traits. In this symbiotic system, the cyanobacteria are attached to the host, positioned alongside the diatom’s intercellular spaces, anchored to the host through its sturdier cells, the heterocysts (Villareal, 1992). It stands to reason that this contact point allows the exchange of fixed nitrogen. However, an exact description of this process, if true, is still lacking. The genome of Calothrix rhizosoleniae SC01 has 5.9 Mbp, an AT percentage of 60.5%, and 90.6% of its genes are predicted to be functional (Foster et al., 2010). C. rhizosoleniae SC01 has certain nif genes, nifH, and nifK, interrupted by insertion elements of at least 20 kbp (Hilton et al., 2013). While the genome is relatively small (just marginally bigger than the genome of the obligate symbiont Trichormus azollae 0708), the rest of its features are consistent with those of a facultative symbiont. It is interesting to note how different this symbiosis is, when compared to the previously mentioned diatom-cyanobacteria symbiosis. While the cyanobiont of Chaetoceros is located outside the cell, the cyanobionts of both Hemiaulus and Rhizosolenia are located intracellular. This changed the evolutionary history of these symbionts, as revealed by their genomes (Hilton et al., 2013). Bryophytes-Nostoc: Among Bryophytes, the liverworts Blasia and Calvicularia, along with hornworts Anthoceros, Phaeoceros, Notothylas, and Dendroceros, have well-defined symbiotic associations with cyanobacteria (Meeks, 2005; Adams and Duggan, 2008). The best-studied symbiosis of this group is Anthoceros punctatus-Nostoc. This symbiosis is formed when Nostoc infects the plant, which is attracted to its host after release of HIFs into the environment. Upon infection, the cyanobiont locates itself in slime cavities present on the gametophyte thallus. When Nostoc enters this region, the cavity closes itself to prevent further infection, creating the low oxygenic conditions necessary for enhanced nitrogen fixation. When the symbiosis is established, the quantity of heterocysts rises sharply, up to 45%– 50%, nitrogenase activity increases, and the photosystem shut down. Most of the nitrogen fixed by the cyanobiont (80%) is transferred to the host (Adams and Duggan, 2008), via an uncharacterized mechanism. There are nine genomes of Nostocales species associated with Bryophytes. Nostoc sp. Moss2, Moss3, and Moss4 were isolated from Pleurozium schreberi; Nostoc spp. Moss5, and Moss6 were isolated from Hylocomium splendens; and Nostoc spp. KVJ2, KVJ10, KVJ20, and KVS11 were isolated from Blasia pusilla (Warshan et al., 2018). The size of these genomes varied considerably, with ranges from 7.14 to 10.39 Mb, with an average of 8.69 Mb. The AT content of these genomes, on the contrary, seems more stable, with values in the range of 58% to 59%, with an average of 58.8%. The majority of these species, with the exceptions of Nostoc spp. Moss3, Moss4, Moss5, and Moss6, were able to form functional symbioses with Gunnera manicata (Warshan et al., 2018). Interestingly, the bryophyte Anthoceros punctatus was able to form a functional symbiosis with a cyanobacterium isolated from a Cycadophyta (Enderlin and Meeks, 1983). While there are no studies on the structure of the nif operon of these microorganisms, the smallest genomes of this group, Nostoc spp. Moss5 (7.24 Mb) and Moss6 (7.14 Mb), were phylogenetically grouped (Warshan et al., 2018), indicating that they may have similar evolutionary symbiotic histories. Cycadophyta-Nostoc: All of the known species of cycads can form a symbiosis with cyanobacteria. The symbiosis is physically located at specialized nodules called coralloid roots, which host the cyanobionts forming a ring called

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the “cyanobacterial zone.” (Costa and Lindblad, 2002) The method of infection is not known, but HIFs, along with phenolic compounds, are thought to play an important role in the infection and subsequent symbiosis with the cyanobiont (Lobakova et al., 2004). Once inside the coralloid root, nitrogen fixation increases, as well as the proportion of heterocysts (Ahern and Staff, 1994). The increase in the amount of heterocysts is controlled by the host, via chemical signaling, greatly increasing the yield of fixed nitrogen. The optimum percentage of heterocyst per filament seems to be around 60%. When this threshold is passed, the heterocysts separate themselves from the filament, becoming inactive (Norstog and Nicholls, 1997). Different cyanobacterial strains can be found in each nodule of the coralloid root, indicating that the symbiosis is not specific—at least taxonomically (Costa et al., 1999; Zheng et al., 2002), but it remains to be seen if there is selection toward a functional cyanobacterial community. It has been shown that nitrogen fixed by the cyanobiont is exported from this cyanobacterial zone to the meristem, from where it can be transported to the rest of the plant (Pate et al., 1988). Nostoc punctiforme ATCC 29133 (PCC 73102) was isolated from the coralloid roots of a Macrozamia spp. host (Meeks et al., 2001). The genome of this symbiont is remarkably big (9.06 Mbp), with regular AT percentage (58.6%) and a high percentage of coding DNA (81.5%) (Moraes et al., 2017). N. punctiforme, as expected for a facultative symbiont, is capable of living outside its putative host, since it has a fully functional photosystem, aside from a complete nif operon (Meeks et al., 2001; Moraes et al., 2017). In this operon, the gene nifD presents a longer insertion of an excision element than that found in other heterocystous species (Moraes et al., 2017). Nostoc punctiforme, however, does not have the excision elements commonly found at the fdxN and hupL genes (Lindberg et al., 2000). Whether these features are characteristic of facultative cyanobionts or are unique to this particular organism remains to be seen. Three more genomes, from strains that remain to be taxonomically characterized, have been obtained from coralloid roots of Mexican Dioon plants (Gutierrez-Garcia et  al., 2018). These include strains “Cyanobacterium” T09, “Cyanobacterium” RF31YmG, and “Cyanobacterium” 106c, which are closely related to Nostoc species. These strains were isolated from Dioon caputoi (T09) and Dioon edule (106c and RF31YmG), and similar to Nostoc punctiforme ATCC 29133, they have big genomes and medium AT percentages. Other symbiotic organisms, both closely related to Nostoc species and noncyanobacteria, were found to be present in the coralloid roots of these cycads (Gutierrez-Garcia et al., 2018), which is in agreement with a recent report of the Asian cycad Cycas bifida (Zheng et al., 2018). Finally, there is also a draft genome of a Nostoc symbiont isolated from a Japanese Cycas revoluta in 1977. The genome of this isolate, named Nostoc cycadae WK-1, had a length of 6.99 Mb, and a AT percentage of 59.6 % (Kanesaki et al., 2018). Gunnera-Nostoc: Similar to cycads, every Gunnera (40 species) form symbiotic associations with cyanobacteria (Bonnett and Silvester, 1981). This association is physically located in the stem, near the leaf base, in glands overflown with mucilage (Silvester and McNamara, 1976). The infection process is well documented and culminates with the penetration of cortical gland cells of Gunnera by the cyanobiont (Silvester and McNamara, 1976; Johansson and Bergman, 1992). Once there, the cyanobiont begins its continuous nitrogen fixation activity (Silvester and McNamara, 1976). Simultaneously, photosynthetic activity diminishes, and the symbiont differentiate more heterocysts per filament, up to 60%–80% (Johansson and Bergman, 1992). While the cyanobiont is located intracellularly, this relationship is not transmitted to seedling, which obligates every new generation to form the symbiosis de novo (Silvester and McNamara, 1976). This facultative symbiosis is the most effective among all of the symbioses reported to date, with up to 90% of the fixed nitrogen exported to the host, making Gunnera plants totally impervious to changes in external levels of nitrogen (Silvester et al., 1996). As with bryophytes, no Nostoc isolated from a Gunnera has been sequenced. However, it is known that different symbiotic strains exist and that the symbiosis can be artificially established with Nostoc punctiforme ATCC 29133 (PCC 73102). Indeed, it has been shown that cyanobionts from these plants, along with cyanobionts from Cycadophyta and Anthoceros, can match the ability of N. punctiforme ATCC 29133 to form symbiotic relationships with different hosts (Bonnett and Silvester, 1981).

4.  TRENDS IN CYANOBACTERIAL NITROGEN FIXATION SYMBIOSES We will briefly summarize the main observations of the extraordinary plasticity of symbiotic cyanobacteria. First, in heterocystous cyanobacteria, the presence of heterocysts allows these organisms to fix nitrogen continuously, unlike their nonheterocystous counterparts, whose nitrogen fixation activity is limited to a timeframe regulated in a circadian manner. As they are capable of fixing nitrogen continuously, heterocystous cyanobacteria were likely better suited to form symbioses and were easily recruited by their hosts. Even though there are a wide variety of symbioses within this group, the available information suggests that they all have adaptations related to their nif operon, as well as to other shared genome traits. For instance, in contrast to nonsymbiotic cyanobacteria, symbiotic strains tend toward less regulation of

Cyanobacteria in Nitrogen-Fixing Symbioses Chapter | 2  39

nitrogenase synthesis. The excision elements in fdxN, fdxN, nifD, and nifH of many symbiotic heterocystous cyanobacteria are absent, likely streamlining the nitrogen fixation process. Moreover, in the case of Trichormus azollae, the nitrogenase can be expressed outside the heterocyst when the cyanobacterium is incapable of forming these specialized cells (Meeks et al., 1988). In addition to the changes in the composition of the nif genes, a change in the copy of these genes can also be observed. While free-living heterocystous cyanobacteria have multiple copies of nif genes (Rice et al., 1982) symbiotic species are usually limited to a conserved, albeit unique, copy of the Mo‑nitrogenase gene. Only Nostoc punctiforme ATCC 29133 has two instances of nif sequences in its genome, one in a small cluster containing additional nifH, nifE, and nifN genes; and the other (another copy of nifH) totally isolated from any nif-related gene. The latter seems to be a remnant of a V‑nitrogenase gene system (Meeks et al., 2001). Overall, secondary nitrogenases seems to have been pruned from the genomes of symbiotic species, with the exception of a Nostoc in symbiosis with Peltigera, found in an arctic environment where molybdenum is scarce (Hodkinson et al., 2014). Second, nonheterocystous cyanobacteria seem to be limited to intracellular symbioses, where oxygen levels can be controlled by the host to allow continuous nitrogenase activity. As with heterocystous cyanobacteria, the dominant nitrogenase in these organisms is the Mo‑nitrogenase, and it is found once in the genome. However, nonheterocystous symbiotic species do not form a clade, indicating that these symbioses are isolated from each other and consistent with their obligate intracellular state. Since these species do not have excision sequences, like heterocystous cyanobacteria, nif genes of symbiotic and nonsymbiotic species are similar. The greatest differences between symbiotic and nonsymbiotic strains, then, seems to be the advanced state of genomic decay observed in the former, with the loss of the necessary genes to live outside the host. Alternatively, these organisms had an ancestral genome that is increased as facultative symbionts adapt to the outside and inner world of their host. It is likely a combination of mechanisms and evolutionary processes that can be tested with genomic tools. Also, the combined knowledge of both types of nitrogen-fixing cyanobacteria will hopefully guide a new and more comprehensive classification system of cyanobacteria in symbiosis, akin to the complexity of their biological interactions.

5. CONCLUSIONS Cyanobacteria can form different types of symbiosis with their phyla-rich hosts, making them a wellspring of information for the study of symbiotic nitrogen fixation evolution and origin, and in industrial and agricultural applications. Despite their importance, research of nitrogen fixing symbioses involving cyanobacteria is currently biased toward certain aspects of their biology, and although some species are well understood, others lack basic characterization. This is especially true at the genomic level, in which cyanobacteria remain an undersequenced phylum, and most of the sequenced cyanobacterial genomes to date belong to nonsymbiotic species. The examples provided in this chapter can be a guide to upcoming studies in cyanobacteria genome evolution. The postgenomic era provides tools to carry out such studies. In the framework of comparative evolution, we will be able gain a deeper understanding of cyanobacterial symbiotic diazotrophs from these cyanobacteria and their genomes and begin answering questions such as how these microorganisms evolve, and how they shaped—and still do—the Earth’s history.

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FURTHER READING Talley, S.N., Talley, B.J., Rains, D.W., 1977. Nitrogen fixation by Azolla in rice fields. In: Hollaender, A. (Ed.), Genetic Engineering for Nitrogen Fixation. Plenum Press, New York, pp. 259–281.