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.

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.

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.

Study of Different Variants of Mo Enzyme crARC and the Interaction with Its Partners crCytb5-R and crCytb5-1.

The mARC (mitochondrial Amidoxime Reducing Component) proteins are recently discovered molybdenum (Mo) Cofactor containing enzymes. They are involved in the reduction of several N-hydroxylated compounds (NHC) and nitrite. Some NHC are prodrugs containing an amidoxime structure or mutagens such as 6-hydroxylaminopurine (HAP). We have studied this protein in the green alga Chlamydomonas reinhardtii (crARC). Interestingly, all the ARC proteins need the reducing power supplied by other proteins. It is known that crARC requires a cytochrome b5 (crCytb5-1) and a cytochrome b5 reductase (crCytb5-R) that form an electron transport chain from NADH to the substrates. Here, we have investigated NHC reduction by crARC, the interaction with its partners and the function of important conserved amino acids. Interactions among crARC, crCytb5-1 and crCytb5-R have been studied by size-exclusion chromatography. A protein complex between crARC, crCytb5-1 and crCytb5-R was identified. Twelve conserved crARC amino acids have been substituted by alanine by in vitro mutagenesis. We have determined that the amino acids D182, F210 and R276 are essential for NHC reduction activity, R276 is important and F210 is critical for the Mo Cofactor chelation. Finally, the crARC C-termini were shown to be involved in protein aggregation or oligomerization.

THB1, a truncated hemoglobin, modulates nitric oxide levels and nitrate reductase activity.

Hemoglobins are ubiquitous proteins that sense, store and transport oxygen, but the physiological processes in which they are implicated is currently expanding. Recent examples of previously unknown hemoglobin functions, which include scavenging of the signaling molecule nitric oxide (NO), illustrate how the implication of hemoglobins in different cell signaling processes is only starting to be unraveled. The extent and diversity of the hemoglobin protein family suggest that hemoglobins have diverged and have potentially evolved specialized functions in certain organisms. A unique model organism to study this functional diversity at the cellular level is the green alga Chlamydomonas reinhardtii because, among other reasons, it contains an unusually high number of a particular type of hemoglobins known as truncated hemoglobins (THB1-THB12). Here, we reveal a cell signaling function for a truncated hemoglobin of Chlamydomonas that affects the nitrogen assimilation pathway by simultaneously modulating NO levels and nitrate reductase (NR) activity. First, we found that THB1 and THB2 expression is modulated by the nitrogen source and depends on NIT2, a transcription factor required for nitrate assimilation genes expression. Furthermore, THB1 is highly expressed in the presence of NO and is able to convert NO into nitrate in vitro. Finally, THB1 is maintained on its active and reduced form by NR, and in vivo lower expression of THB1 results in increased NR activity. Thus, THB1 plays a dual role in NO detoxification and in the modulation of NR activity. This mechanism can partly explain how NO inhibits NR post-translationally.

A dual system formed by the ARC and NR molybdoenzymes mediates nitrite-dependent NO production in Chlamydomonas.

Nitric oxide (NO) is a relevant signal molecule involved in many plant processes. However, the mechanisms and proteins responsible for its synthesis are scarcely known. In most photosynthetic organisms NO synthases have not been identified, and Nitrate Reductase (NR) has been proposed as the main enzymatic NO source, a process that in vitro is also catalysed by other molybdoenzymes. By studying transcriptional regulation, enzyme approaches, activity assays with in vitro purified proteins and in vivo and in vitro NO determinations, we have addressed the role of NR and Amidoxime Reducing Component (ARC) in the NO synthesis process. N\R and ARC were intimately related both at transcriptional and activity level. Thus, arc mutants showed high NIA1 (NR gene) expression and NR activity. Conversely, mutants without active NR displayed an increased ARC expression in nitrite medium. Our results with nia1 and arc mutants and with purified enzymes support that ARC catalyses the NO production from nitrite taking electrons from NR and not from Cytb5-1/Cytb5-Reductase, the component partners previously described for ARC (proposed as NOFNiR, Nitric Oxide-Forming Nitrite Reductase). This NR-ARC dual system would be able to produce NO in the presence of nitrate, condition under which NR is unable to do it.

Chlamydomonas†NZF1, a tandem-repeated zinc finger factor involved in nitrate signalling by controlling the regulatory gene NIT2.

The Chlamydomonas reinhardtii NIT2 gene plays a central role in nitrate assimilation, thus, nit2 mutants are not able to sense or to use nitrate for growth. NIT2 protein is an RWP-RK-type transcriptional factor related to nodule inception (Nin, NLP) proteins from plants. NIT2 expression is down-regulated in ammonium and up-regulated under nitrogen deprivation. However, intracellular nitrate is required to activate NIT2 for subsequent expression of NIA1 and other nitrate assimilation genes. In this work, mutants defective in nitrate sensing have been studied. The identification of genomic regions affected allows proposing putative loci/genes for nitrate signalling in the alga. Among them, a CrNZF1 (Nitrate Zinc Finger 1) that encodes a tandem zinc finger protein CCCH-type. In the nzf1 mutant, the expression of the regulatory gene NIT2 is decreased and also that of nitrate assimilation genes. In this mutant, polyadenylated forms of NIT2 with different lengths could be detected, whereas in the wild type there appeared preferentially the longest forms. CrNZF1 is proposed to regulate NIT2 polyadenylation and thus nitrate signalling and nitrate-dependent growth in the alga.

Algae and humans share a molybdate transporter.

Almost all living organisms need to obtain molybdenum from the external medium to achieve essential processes for life. Activity of important enzymes such as sulfite oxidase, aldehyde oxidase, xanthine dehydrogenase, and nitrate reductase is strictly dependent on the presence of Mo in its active site. Cells take up Mo in the form of the oxianion molybdate, but the molecular nature of the transporters is still not well known in eukaryotes. MOT1 is the first molybdate transporter identified in plant-type eukaryotic organisms, but it is absent in animal genomes. Here we report a molybdate transporter different from the MOT1 family, encoded by the Chlamydomonas reinhardtii gene MoT2, that is also present in animals including humans. The knockdown of CrMoT2 transcription leads to the deficiency of molybdate uptake activity in Chlamydomonas. In addition, heterologous expression in Saccharomyces cerevisiae of MoT2 genes from Chlamydomonas and humans support the functionality of both proteins as molybdate transporters. Characterization of CrMOT2 and HsMOT2 activities showed an apparent Km of about 550 nM that, though higher than the Km reported for MOT1, still corresponds to high affinity systems. CrMoT2 transcription is activated when extracellular molybdate concentration is low but in contrast to MoT1 is not activated by nitrate. Analysis of protein databases revealed the presence of four motifs present in all the proteins with high similarity to MOT2, that label a previously undescribed family of proteins probably related to molybdate transport. Our results open the way toward the understanding of molybdate transport as part of molybdenum homeostasis and Moco biosynthesis in animals.

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 and Microbacterium forte sp. nov., a mutualistic association that favors sustainable hydrogen production.

A naturally occurring multispecies bacterial community composed of Bacillus cereus and two novel bacteria (Microbacterium forte sp. nov. and Stenotrophomonas goyi sp. nov.) has been identified from a contaminated culture of the microalga Chlamydomonas reinhardtii. When incubated in mannitol- and yeast extract-containing medium, this bacterial community can promote and sustain algal hydrogen production up to 313 mL H2·L-1 for 17 days and 163.5 mL H2·L-1 for 25 days in high-cell (76.7 µg·mL-1 of initial chlorophyll) and low-cell density (10 µg·mL-1 of initial chlorophyll) algal cultures, respectively. In low-cell density algal cultures, hydrogen production was compatible with algal growth (reaching up to 60 µg·mL-1 of chlorophyll). Among the bacterial community, M. forte sp. nov. was the sole responsible for the improvement in hydrogen production. However, algal growth was not observed in the Chlamydomonas-M. forte sp. nov. consortium during hydrogen-producing conditions (hypoxia), suggesting that the presence of B. cereus and S. goyi sp. nov. could be crucial to support the algal growth during hypoxia. Still, under non­hydrogen producing conditions (aerobiosis) the Chlamydomonas-M. forte sp. nov. consortium allowed algal growth (up to 40 µg·mL-1 of chlorophyll) and long-term algal viability (>45 days). The genome sequence and growth tests of M. forte sp. nov. have revealed that this bacterium is auxotroph for biotin and thiamine and unable to use sulfate as sulfur source; it requires S-reduced forms such as cysteine and methionine. Cocultures of Chlamydomonas reinhardtii and M. forte sp. nov. established a mutualistic association: the alga complemented the nutrient deficiencies of the bacterium, while the bacterium released ammonium (0.19 mM·day-1) and acetic acid (0.15 mM·day-1) for the alga. This work offers a promising avenue for photohydrogen production concomitant with algal biomass generation using nutrients not suitable for mixotrophic algal growth.

Topical and Intralesional Immunotherapy for the Management of Basal Cell Carcinoma.

Basal Cell Carcinoma (BCC) is the most common type of cancer among the white population. Individuals with fair skin have an average lifetime risk of around 30% for developing BCC, and there is a noticeable upward trend in its incidence rate. The principal treatment objectives for BCC involve achieving the total excision of the tumor while maximizing the preservation of function and cosmesis. Surgery is considered the treatment of choice for BCC for two main reasons: it allows for the highest cure rates and facilitates histological control of resection margins. However, in the subgroup of patients with low-risk recurrence or medical contraindications for surgery, new non-surgical treatment alternatives can provide an excellent oncological and cosmetic outcome. An evident and justified instance of these local therapies occurred during the COVID-19 pandemic, a period when surgical interventions carried out in hospital settings were not a viable option.

Nitric oxide controls nitrate and ammonium assimilation in Chlamydomonas reinhardtii.

Nitrate and ammonium are major inorganic nitrogen sources for plants and algae. These compounds are assimilated by means of finely regulated processes at transcriptional and post-translational levels. In Chlamydomonas, the expression of several genes involved in high-affinity ammonium (AMT1.1, AMT1.2) and nitrate transport (NRT2.1) as well as nitrate reduction (NIA1) are downregulated by ammonium through a nitric oxide (NO)-dependent mechanism. At the post-translational level, nitrate/nitrite uptake and nitrate reductase (NR) are also inhibited by ammonium, but the mechanisms implicated in this regulation are scarcely known. In this work, the effect of NO on nitrate assimilation and the high-affinity ammonium uptake was addressed. NO inhibited the high-affinity uptake of ammonium and nitrate/nitrite, as well as the NR activity, in a reversible form. In contrast, nitrite reductase and glutamine synthetase activities were not affected. The in vivo and in vitro studies suggested that NR enzyme is inhibited by NO in a mediated process that requires the cell integrity. These data highlight a role of NO in inorganic nitrogen assimilation and suggest that this signalling molecule is an important regulator for the first steps of the pathway.

A soluble guanylate cyclase mediates negative signaling by ammonium on expression of nitrate reductase in Chlamydomonas.

Nitrate assimilation in plants and related organisms is a highly regulated and conserved pathway in which the enzyme nitrate reductase (NR) occupies a central position. Although some progress has been made in understanding the regulation of the protein, transcriptional regulation of the NR gene (NIA1) is poorly understood. This work describes a mechanism for the ammonium-mediated repression of NIA1. We report the characterization of a mutant defective in the repression of NIA1 and NR in response to ammonium and show that a gene (CYG56) coding for a nitric oxide (NO)-dependent guanylate cyclase (GC) was interrupted in this mutant. NO donors, cGMP analogs, a phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), and a calcium ionophore (A23187) repress the expression of NIA1 in Chlamydomonas reinhardtii wild-type cells and also repress the expression of other ammonium-sensitive genes. In addition, the GC inhibitors LY83,583 (6-anilino-5,8-quinolinedione) and ODQ (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one) release cells from ammonium repression. Intracellular NO and cGMP levels were increased in the presence of ammonium in wild-type cells. In the cyg56 mutant, NIA1 transcription was less sensitive to NO donors and A23187, but responded like the wild type to IBMX. Results presented here suggest that CYG56 participates in ammonium-mediated NIA1 repression through a pathway that involves NO, cGMP, and calcium and that similar mechanisms might be occurring in 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.

Factors associated with blood product requirements during the transoperative period in pediatric patients.

BACKGROUND: The efficiency of blood products (BP) requisition in elective non-cardiac surgeries is inherently complex. Moreover, it is aggravated in the pediatric population. This study aimed to identify the factors associated with using less than the requested BP during the transoperative period in pediatric patients undergoing elective non-cardiac surgery. METHODS: We conducted a cross-sectional comparative study including 320 patients undergoing elective non-cardiac surgery for whom BPs were requested. Low requirements were considered when less than 50% of the requested amount or no BPs were used, and high requirements when more than the requested amount was used. The Mann-Whitney's U test was applied for comparative analysis, and multiple logistic regression was used to adjust for factors associated with lower requirements. RESULTS: The median age of the patients was 3 years. From 320 patients, 68.1% (n = 218) received less than the requested amount of BP, while only 1.25% (n = 4) received more than the requested amount of BP. Factors associated with transfusion of less than the requested BPs were prolonged clotting time (odds ratio (OR) = 2.66) and anemia (OR = 0.43). CONCLUSIONS: Factors associated with lower than requested BP transfusion were prolonged clotting time and anemia.

INTRODUCCIÓN: La eficiencia de la solicitud de productos sanguíneos (PS) en las cirugías electivas no cardiacas es, de por sí, compleja. No obstante, se agrava para la población pediátrica. El objetivo de este estudio fue identificar los factores asociados con la utilización de una cantidad de PS menor a la solicitada durante el transoperatorio en pacientes pediátricos sometidos a cirugía electiva no cardiaca. MÉTODOS: Se realizó un estudio transversal comparativo donde se incluyeron 320 pacientes sometidos a cirugía electiva no cardiaca para quienes se solicitaron PS. Los requerimientos de hemoderivados se consideraron como menores cuando no se utilizaron o se utilizó menos del 50% de lo solicitado y como mayores cuando se utilizó una cantidad mayor a la solicitada. Se aplicó la prueba U de Mann-Whitney para el análisis comparativo y regresión logística múltiple para ajustar los factores asociados a la presencia de menores requerimientos. RESULTADOS: La mediana para la edad de los pacientes fue de 3 años. Se transfundió una cantidad de PS menor a la solicitada en el 68.1% (n = 218) de los pacientes, mientras que se transfundió una cantidad mayor a la solicitada solo en el 1.25% de los pacientes (n = 4). Los factores asociados con la transfusión de una cantidad de PS menor a la solicitada fueron tiempos de coagulación alargados (TCA) (razón de momios (RM) = 2.66) y anemia (RM = 0.43). CONCLUSIONES: Los factores asociados a una transfusión de PS inferior a la solicitada fueron el tiempo de coagulación prolongado y la anemia.

The Chlamydomonas reinhardtii molybdenum cofactor enzyme crARC has a Zn-dependent activity and protein partners similar to those of its human homologue.

The ARC (amidoxime reducing component) proteins are molybdenum cofactor (Moco) enzymes named hmARC1 and hmARC2 (human ARCs [hmARCs]) in humans and YcbX in Escherichia coli. They catalyze the reduction of a broad range of N-hydroxylated compounds (NHC) using reducing power supplied by other proteins. Some NHC are prodrugs or toxic compounds. YcbX contains a ferredoxin (Fd) domain and requires the NADPH flavin reductase CysJ to reduce NHC. In contrast, hmARCs lack the Fd domain and require a human cytochrome b5 (hCyt b5) and a human NADH Cyt b5 reductase (hCyt b5-R) to reduce NHC. The ARC proteins in the plant kingdom are uncharacterized. We demonstrate that Chlamydomonas reinhardtii mutants defective in Moco biosynthesis genes are sensitive to the NHC N(6)-hydroxylaminopurine (HAP). The Chlamydomonas reinhardtii ARC protein crARC has been purified and characterized. The six Chlamydomonas Fds were isolated, but none of them are required by crARC to reduce HAP. We have also purified and characterized five C. reinhardtii Cyt b5 (crCyt b5) and two flavin reductases, one that is NADPH dependent (crCysJ) and one that is NADH dependent (crCyt b5-R). The data show that crARC uses crCyt b5-1 and crCyt b5-R to reduce HAP. The crARC has a Zn-dependent activity, and the presence of Zn increases its V(max) more than 14-fold. In addition, all five cysteines of crARC were substituted by alanine, and we demonstrate that the fully conserved cysteine 252 is essential for both Moco binding and catalysis. Therefore, it is proposed that crARC belongs to the sulfite oxidase family of Moco enzymes.

Chlamydomonas-Methylobacterium oryzae cooperation leads to increased biomass, nitrogen removal and hydrogen production.

In the context of algal wastewater bioremediation, this study has identified a novel consortium formed by the bacterium Methylobacterium oryzae and the microalga Chlamydomonas reinhardtii that greatly increase biomass generation (1.22 g L-1·d-1), inorganic nitrogen removal (>99%), and hydrogen production (33 mL·L-1) when incubated in media containing ethanol and methanol. The key metabolic aspect of this relationship relied on the bacterial oxidation of ethanol to acetate, which supported heterotrophic algal growth. However, in the bacterial monocultures the acetate accumulation inhibited bacterial growth. Moreover, in the absence of methanol, ethanol was an unsuitable carbon source and its incomplete oxidation to acetaldehyde had a toxic effect on both the alga and the bacterium. In cocultures, both alcohols were used as carbon sources by the bacteria, the inhibitory effects were overcome and both microorganisms mutually benefited. Potential biotechnological applications in wastewater treatment, biomass generation and hydrogen production are discussed.

Transcriptional regulation of CDP1 and CYG56 is required for proper NH4+ sensing in Chlamydomonas.

The assimilation of inorganic nitrogen is an essential process for all plant-like organisms. In the presence of ammonium and nitrate as nitrogen sources, Chlamydomonas reinhardtii preferentially assimilates ammonium and represses the nitrate assimilation pathway through an unknown mechanism that in part involves the guanylate cyclase CYG56. It is demonstrated that cells not only respond quantitatively to the NH(4)(+) signal but are also able to sense a balance between both nitrogen sources. This quantitative response was altered in a collection of mutants that were partially insensitive to NH(4)(+). In one of these mutants, reduced function of a gene named CDP1 encoding a cysteine domain-containing protein was genetically linked to NH(4)(+) insensitivity. Alteration of CYG56 or CDP1 transcription was detected in several mutants, and combined down-regulation of both genes seemed to enhance the incapacity to sense NH(4)(+) properly. These results suggest that transcriptional regulation of CYG56 and CDP1 are central and independent steps of the NH(4)(+) signalling pathway.

A high-affinity molybdate transporter in eukaryotes.

Molybdenum is an essential element for almost all living beings, which, in the form of a molybdopterin-cofactor, participates in the active site of enzymes involved in key reactions of carbon, nitrogen, and sulfur metabolism. This metal is taken up by cells in form of the oxyanion molybdate. Bacteria acquire molybdate by an ATP-binding-cassette (ABC) transport system in a widely studied process, but how eukaryotic cells take up molybdenum is unknown because molybdate transporters have not been identified so far. Here, we report a eukaryotic high-affinity molybdate transporter, encoded by the green alga Chlamydomonas reinhardtii gene MoT1. An antisense RNA strategy over the MoT1 gene showed that interference of the expression of this gene leads to the inhibition of molybdate transport activity and, in turn, of the Mo-containing enzyme nitrate reductase, indicating a function of MoT1 in molybdate transport. MOT1 functionality was also shown by heterologous expression in Saccharomyces cerevisiae. Molybdate uptake mediated by MOT1 showed a K(m) of approximately 6 nM, which is the range of the lowest K(m) values reported and was activated in the presence of nitrate. Analysis of deduced sequence from the putative protein coded by MoT1 showed motifs specifically conserved in similar proteins present in the databases, and defines a family of membrane proteins in both eukaryotes and prokaryotes probably involved in molybdate transport and distantly related to plant sulfate transporters SULTR. These findings represent an important step in the understanding of molybdate transport, a crucial process in eukaryotic cells.