Genetic evidence for algal auxin production in Chlamydomonas and its role in algal-bacterial mutualism.

Interactions between algae and bacteria are ubiquitous and play fundamental roles in nutrient cycling and biomass production. Recent studies have shown that the plant auxin indole acetic acid (IAA) can mediate chemical crosstalk between algae and bacteria, resembling its role in plant-bacterial associations. Here, we report a mechanism for algal extracellular IAA production from L-tryptophan mediated by the enzyme L-amino acid oxidase (LAO1) in the model Chlamydomonas reinhardtii. High levels of IAA inhibit algal cell multiplication and chlorophyll degradation, and these inhibitory effects can be relieved by the presence of the plant-growth-promoting bacterium (PGPB) Methylobacterium aquaticum, whose growth is mutualistically enhanced by the presence of the alga. These findings reveal a complex interplay of microbial auxin production and degradation by algal-bacterial consortia and draws attention to potential ecophysiological roles of terrestrial microalgae and PGPB in association with land plants.

Chlamydomonas reinhardtii, a Reference Organism to Study Algal-Microbial Interactions: Why Can't They Be Friends?

The stability and harmony of ecological niches rely on intricate interactions between their members. During evolution, organisms have developed the ability to thrive in different environments, taking advantage of each other. Among these organisms, microalgae are a highly diverse and widely distributed group of major primary producers whose interactions with other organisms play essential roles in their habitats. Understanding the basis of these interactions is crucial to control and exploit these communities for ecological and biotechnological applications. The green microalga Chlamydomonas reinhardtii, a well-established model, is emerging as a model organism for studying a wide variety of microbial interactions with ecological and economic significance. In this review, we unite and discuss current knowledge that points to C. reinhardtii as a model organism for studying microbial interactions.

Nitrous Oxide Emissions from Nitrite Are Highly Dependent on Nitrate Reductase in the Microalga Chlamydomonas reinhardtii.

Nitrous oxide (N2O) is a powerful greenhouse gas and an ozone-depleting compound whose synthesis and release have traditionally been ascribed to bacteria and fungi. Although plants and microalgae have been proposed as N2O producers in recent decades, the proteins involved in this process have been only recently unveiled. In the green microalga Chlamydomonas reinhardtii, flavodiiron proteins (FLVs) and cytochrome P450 (CYP55) are two nitric oxide (NO) reductases responsible for N2O synthesis in the chloroplast and mitochondria, respectively. However, the molecular mechanisms feeding these NO reductases are unknown. In this work, we use cavity ring-down spectroscopy to monitor N2O and CO2 in cultures of nitrite reductase mutants, which cannot grow on nitrate or nitrite and exhibit enhanced N2O emissions. We show that these mutants constitute a very useful tool to study the rates and kinetics of N2O release under different conditions and the metabolism of this greenhouse gas. Our results indicate that N2O production, which was higher in the light than in the dark, requires nitrate reductase as the major provider of NO as substrate. Finally, we show that the presence of nitrate reductase impacts CO2 emissions in both light and dark conditions, and we discuss the role of NO in the balance between CO2 fixation and release.

Differential Phototactic Behavior of Closely Related Cyanobacterial Isolates from Yellowstone Hot Spring Biofilms.

Phototrophic biofilms in most environments experience major changes in light levels throughout a diel cycle. Phototaxis can be a useful strategy for optimizing light exposure under these conditions, but little is known about its role in cyanobacteria from thermal springs. We examined two closely related Synechococcus isolates (Synechococcus OS-A dominates at 60 to 65°C and OS-B' at 50 to 55°C) from outflows of Octopus Spring in Yellowstone National Park. Both isolates exhibited phototaxis and photokinesis in white light, but with differences in speed and motility bias. OS-B' exhibited phototaxis toward UVA, blue, green, and red wavelengths, while OS-A primarily exhibited phototaxis toward red and green. OS-A also exhibited negative phototaxis under certain conditions. The repertoires of photoreceptors and signal transduction elements in both isolates were quite different from those characterized in other unicellular cyanobacteria. These differences in the photoresponses between OS-A and OS-B' in conjunction with in situ observations indicate that phototactic strategies may be quite versatile and finely tuned to the light and local environment. IMPORTANCE Optimizing light absorption is of paramount importance to photosynthetic organisms. Some photosynthetic microbes have evolved a sophisticated process called phototaxis to move toward or away from a light source. In many hot springs in Yellowstone National Park, cyanobacteria thrive in thick, laminated biofilms or microbial mats, where small movements can result in large changes in light exposure. We quantified the light-dependent motility behaviors in isolates representing two of the most abundant and closely related cyanobacterial species from these springs. We found that they exhibited unexpected differences in their speed, directionality, and responses to different intensities or qualities of light. An examination of their genomes revealed several variations from well-studied phototaxis-related genes. Studying these recently isolated cyanobacteria reveals that diverse phototactic strategies can exist even among close relatives in the same environment. It also provides insights into the importance of phototaxis for growth and survival in microbial biofilm communities.

Retrotransposition facilitated the establishment of a primary plastid in the thecate amoeba Paulinella.

The evolution of eukaryotic life was predicated on the development of organelles such as mitochondria and plastids. During this complex process of organellogenesis, the host cell and the engulfed prokaryote became genetically codependent, with the integration of genes from the endosymbiont into the host nuclear genome and subsequent gene loss from the endosymbiont. This process required that horizontally transferred genes become active and properly regulated despite inherent differences in genetic features between donor (endosymbiont) and recipient (host). Although this genetic reorganization is considered critical for early stages of organellogenesis, we have little knowledge about the mechanisms governing this process. The photosynthetic amoeba Paulinella micropora offers a unique opportunity to study early evolutionary events associated with organellogenesis and primary endosymbiosis. This amoeba harbors a "chromatophore," a nascent photosynthetic organelle derived from a relatively recent cyanobacterial association (∼120 million years ago) that is independent of the evolution of primary plastids in plants (initiated ∼1.5 billion years ago). Analysis of the genome and transcriptome of Paulinella revealed that retrotransposition of endosymbiont-derived nuclear genes was critical for their domestication in the host. These retrocopied genes involved in photoprotection in cyanobacteria became expanded gene families and were "rewired," acquiring light-responsive regulatory elements that function in the host. The establishment of host control of endosymbiont-derived genes likely enabled the cell to withstand photo-oxidative stress generated by oxygenic photosynthesis in the nascent organelle. These results provide insights into the genetic mechanisms and evolutionary pressures that facilitated the metabolic integration of the host­endosymbiont association and sustained the evolution of a photosynthetic organelle.

Why is primary endosymbiosis so rare?

Endosymbiosis is a relationship between two organisms wherein one cell resides inside the other. This affiliation, when stable and beneficial for the 'host' cell, can result in massive genetic innovation with the foremost examples being the evolution of eukaryotic organelles, the mitochondria and plastids. Despite its critical evolutionary role, there is limited knowledge about how endosymbiosis is initially established and how host-endosymbiont biology is integrated. Here, we explore this issue, using as our model the rhizarian amoeba Paulinella, which represents an independent case of primary plastid origin that occurred c. 120 million yr ago. We propose the 'chassis and engine' model that provides a theoretical framework for understanding primary plastid endosymbiosis, potentially explaining why it is so rare.

Chlamydomonas reinhardtii, an Algal Model in the Nitrogen Cycle.

Nitrogen (N) is an essential constituent of all living organisms and the main limiting macronutrient. Even when dinitrogen gas is the most abundant form of N, it can only be used by fixing bacteria but is inaccessible to most organisms, algae among them. Algae preferentially use ammonium (NH4+) and nitrate (NO3-) for growth, and the reactions for their conversion into amino acids (N assimilation) constitute an important part of the nitrogen cycle by primary producers. Recently, it was claimed that algae are also involved in denitrification, because of the production of nitric oxide (NO), a signal molecule, which is also a substrate of NO reductases to produce nitrous oxide (N2O), a potent greenhouse gas. This review is focused on the microalga Chlamydomonas reinhardtii as an algal model and its participation in different reactions of the N cycle. Emphasis will be paid to new actors, such as putative genes involved in NO and N2O production and their occurrence in other algae genomes. Furthermore, algae/bacteria mutualism will be considered in terms of expanding the N cycle to ammonification and N fixation, which are based on the exchange of carbon and nitrogen between the two organisms.

Identification of the MAPK Cascade and its Relationship with Nitrogen Metabolism in the Green Alga Chlamydomonas reinhardtii.

The mitogen activated protein kinases (MAPKs) form part of a signaling cascade through phosphorylation reactions conserved in all eukaryotic organisms. The MAPK cascades are mainly composed by three proteins, MAPKKKs, MAPKKs and MAPKs. Some signals induce MAPKKK-mediated phosphorylation and activation of MAPKK that phosphorylate and activate MAPK. Afterward, MAPKs can act either in the cytoplasm or be imported into the nucleus to activate other proteins or transcription factors. In the green microalga Chlamydomonas reinhardtii the pathway for nitrogen (N) assimilation is well characterized, yet its regulation still has many unknown features. Nitric oxide (NO) is a fundamental signal molecule for N regulation, where nitrate reductase (NR) plays a central role in its synthesis. The MAPK cascades could be regulating N assimilation, since it has been described that the phosphorylation of NR by MAPK6 promotes NO production in Arabidopsis thaliana. We have identified the proteins involved in the MAPK cascades in Chlamydomonas reinhardtii, finding 17 MAPKs, 2 MAPKKs and 108 MAPKKKs (11 MEKK-, 94 RAF- and 3 ZIK-type) that have been structurally and phylogenetically characterized. The genetic expressions of MAPKs and the MAPKK were slightly regulated by N. However, the genetic expressions of MAPKKKs RAF14 and RAF79 showed a very strong repression by ammonium, which suggests that they may have a key role in the regulation of N assimilation, encouraging to further analyze in detail the role of MAPK cascades in the regulation of N metabolism.

From the Eukaryotic Molybdenum Cofactor Biosynthesis to the Moonlighting Enzyme mARC.

All eukaryotic molybdenum (Mo) enzymes contain in their active site a Mo Cofactor (Moco), which is formed by a tricyclic pyranopterin with a dithiolene chelating the Mo atom. Here, the eukaryotic Moco biosynthetic pathway and the eukaryotic Moco enzymes are overviewed, including nitrate reductase (NR), sulfite oxidase, xanthine oxidoreductase, aldehyde oxidase, and the last one discovered, the moonlighting enzyme mitochondrial Amidoxime Reducing Component (mARC). The mARC enzymes catalyze the reduction of hydroxylated compounds, mostly N-hydroxylated (NHC), but as well of nitrite to nitric oxide, a second messenger. mARC shows a broad spectrum of NHC as substrates, some are prodrugs containing an amidoxime structure, some are mutagens, such as 6-hydroxylaminepurine and some others, which most probably will be discovered soon. Interestingly, all known mARC need the reducing power supplied by different partners. For the NHC reduction, mARC uses cytochrome b5 and cytochrome b5 reductase, however for the nitrite reduction, plant mARC uses NR. Despite the functional importance of mARC enzymatic reactions, the structural mechanism of its Moco-mediated catalysis is starting to be revealed. We propose and compare the mARC catalytic mechanism of nitrite versus NHC reduction. By using the recently resolved structure of a prokaryotic MOSC enzyme, from the mARC protein family, we have modeled an in silico three-dimensional structure of a eukaryotic homologue.

OK, thanks! A new mutualism between Chlamydomonas and methylobacteria facilitates growth on amino acids and peptides.

Nitrogen is a key nutrient for land plants and phytoplankton in terrestrial and aquatic ecosystems. The model alga Chlamydomonas reinhardtii can grow efficiently on several inorganic nitrogen sources (e.g. ammonium, nitrate, nitrite) as well as many amino acids. In this study, we show that Chlamydomonas is unable to use proline, hydroxyproline and peptides that contain these amino acids. However, we discovered that algal growth on these substrates is supported in association with Methylobacterium spp., and that a mutualistic carbon-nitrogen metabolic exchange between Chlamydomonas and Methylobacterium spp. is established. Specifically, the mineralization of these amino acids and peptides by Methylobacterium spp. produces ammonium that can be assimilated by Chlamydomonas, and CO2 photosynthetically fixed by Chlamydomonas yields glycerol that can be assimilated by Methylobacterium. As Chlamydomonas is an algal ancestor to land plants and Methylobacterium is a plant growth-promoting bacterium, this new model of mutualism may facilitate insights into the ecology and evolution of plant-bacterial interactions and design principles of synthetic ecology.

How Chlamydomonas handles nitrate and the nitric oxide cycle.

The green alga Chlamydomonas is a valuable model system capable of assimilating different forms of nitrogen (N). Nitrate (NO3-) has a relevant role in plant-like organisms, first as a nitrogen source for growth and second as a signalling molecule. Several modules are necessary for Chlamydomonas to handle nitrate, including transporters, nitrate reductase (NR), nitrite reductase (NiR), GS/GOGAT enzymes for ammonium assimilation, and regulatory protein(s). Transporters provide a first step for influx/efflux, homeostasis, and sensing of nitrate; and NIT2 is the key transcription factor (RWP-RK) for mediating the nitrate-dependent activation of a number of genes. Here, we review how NR participates in the cycle NO3- →NO2- →NO →NO3-. NR uses the partner protein amidoxime-reducing component/nitric oxide-forming nitrite reductase (ARC/NOFNiR) for the conversion of nitrite (NO2-) into nitric oxide (NO). It also uses the truncated haemoglobin THB1 in the conversion of nitric oxide to nitrate. Nitric oxide is a negative signal for nitrate assimilation; it inhibits the activity and expression of high-affinity nitrate/nitrite transporters and NR. During this cycle, the positive signal of nitrate is transformed into the negative signal of nitric oxide, which can then be converted back into nitrate. Thus, NR is back in the spotlight as a strategic regulator of the nitric oxide cycle and the nitrate assimilation pathway.