Alberto Quesada

PCR-identification of a Nicotiana plumbaginifolia cDNA homologous to the high-affinity nitrate transporters of the crnA family.

A family of high-affinity nitrate transporters has been identified in Aspergillus nidulans and Chlamydomonas reinhardtii, and recently homologues of this family have been cloned from a higher plant (barley). Based on six of the peptide sequences most strongly conserved between the barley and C. reinhardtii polypeptides, a set of degenerate primers was designed to permit amplification of the corresponding genes from other plant species. The utility of these primers was demonstrated by RT-PCR with cDNA made from poly(A)+ RNA from barley, C. reinhardtii and Nicotiana plumbaginifolia. A PCR fragment amplified from N. plumbaginifolia was used as probe to isolate a full-length cDNA clone which encodes a protein, NRT2;1Np, that is closely related to the previously isolated crnA homologue from barley. Genomic Southern blots indicated that there are only 1 or 2 members of the Nrt2 gene family in N. plumbaginifolia. Northern blotting showed that the Nrt2 transcripts are most strongly expressed in roots. The effects of external treatments with different N sources showed that the regulation of the Nrt2 gene(s) is very similar to that reported for nitrate reductase and nitrite reductase genes: their expression was strongly induced by nitrate but was repressed when reduced forms of N were supplied to the roots.

Five nitrate assimilation-related loci are clustered in Chlamydomonas reinhardtii

Three overlapping clones covering a Chlarnydomonas reinhardtii genomic region of about 32 kb appear to contain five genes potentially involved in nitrate assimilation in addition to the nitrate reductase structural locus nit-1. These new loci produced transcripts of 2.8, 2.2, 1.8 and 1.7 kb in nitrate-induced wild-type cells that, like the 3.4 kb transcript of nit-1, were undetectable in cells grown in ammonium. In addition, in a mutant defective at the regulatory locus, nit-2 for nitrate assimilation, which does not express the nit-1 gene transcript, accumulation of the four other transcripts was also blocked. They have been named nar (nitrate assimilation related) genes. The nar-1 and nar-2 loci are transcribed in the same orientation as nit-1. The nar-3 and nar-4 loci are transcribed divergently from nit-1. DNA and RNA sequences from both nar-3 and nar-4 cross-hybridized with each other indicating that they share similar sequences. Four nitrate assimilation-deficient mutants (C2, D2, F6 and G1) were characterized. These mutants lack nar transcripts and have major deletions and/or rearrangements in the nar gene cluster. In contrast to other nitrate reductase-deficient mutants and to wild type, deletion mutants and the regulatory mutant nit-2 were incapable of accumulating intracellular nitrate. Two of the mutants in which expression of all of the nar loci did not occur, C2 and D2, grew in nitrite medium and showed wild-type levels of both nitrite uptake and nitrite reductase activities. Thus the nar loci cannot be required for nitrite assimilation. Mutants F6 and G1 were unable to grow in either nitrite- or nitrate-containing medium, and lacked nitrate reductase, nitrite reductase, nitrate uptake and nitrite uptake activities. The inability to assimilate nitrite co-segregated with nit-1 in crosses between these mutants and wild type. These results indicate that a complex gene cluster responsible for the assimilation of nitrate has been identified in C. reinhardtii, and that, in addition, at least one locus necessary for nitrite assimilation is genetically linked to this cluster.

Expression of nitrate assimilation related genes in Chlamydomonas reinhardtii

The mRNA accumulation pattern of the Chlamydomonas reinhardtii nitrate assimilation-related gene cluster has been elucidated. In ammonium-grown wild-type cells, nit-1 (nitrate reductase, NR), nar-1, nar-2 and nar-3 (nitrate transporter) genes showed very similar kinetics of expression when transferred to nitrate medium. Transcripts of all these genes accumulated transiently in ammonium-grown wild-type cells after a one-hour incubation in nitrogen-free medium, and practically disappeared at about 2 hours. Mutant strains lacking functional nitrate reductase showed similar accumulation kinetics of these transcripts during both nitrate induction and derepression in nitrogen-free media. In contrast to the other nar transcripts, that nar-4, a gene sharing similar sequences with nar-3, accumulated in small amounts in wild-type cells, and only increased after a long nitrate induction period. Nitrate and light showed a strong positive effect on the accumulation of nit-1 gene transcripts. Acetate as a carbon source allowed accumulation of nit-1 mRNA in the dark, indicating the existence of interactions between light and carbon metabolism in nit-1 gene expression. Our data strongly suggest that NR negatively autoregulates its own expression and that of nar genes.

Nitrogen Assimilation and its Regulation

Nitrogen is available in the environment in different redox states and in a wide variety of nitrogenous compounds. Algal cells are capable of assimilating nitrogen from many of these molecules into, ultimately, the amino group of glutamate. In this chapter, recent findings on nitrogen assimilation pathways in Chlamydomonas reinhardtii are reviewed. Specific transport systems are required to take up nitrogenous compounds, though most amino acids are deaminated extracellularly. Ammonium is the preferred nitrogen source. The presence of ammonium both represses and inhibits the utilization of alternative nitrogen compounds. Purines are assimilated phototrophically and catabolized to generate urea and then ammonium by the standard aerobic pathway that operates in other organisms. The nitrate assimilation pathway has been dissected at the molecular level and a useful collection of mutants is now available. The mutants are affected at various steps: nitrate/nitrite transport, nitrate and nitrite reduction, biosynthesis of molybdopterin cofactor, and regulation of the pathway. At least six genes which might encode structural elements for this pathway are clustered together within a 45 kb genomic region. They show coordinated regulation with a similar response to different nutritional or environmental effectors. Regulatory elements responsible for molecular mechanisms of control, including sensing of nitrate and ammonium and the transduction of these signals, are starting to be defined. More research is required for resolution of the details.

Clustering of the nitrite reductase gene and a light-regulated gene with nitrate assimilation loci in Chlamydomonas reinhardtii

Abstract. Two new loci have been found to be clustered with five other genes for the nitrate assimilation pathway in the Chlamydomonas reinhardtii genome. One gene, located close to the 3′-end of the high-affinity nitrate transporter (HANT) gene Nrt2;2, corresponds to the nitrite reductase (NiR) structural gene Nii1. This is supported by a number of experimental findings: (i) NiR-deficient mutants have lost Nii1 gene expression; (ii) Nii1 mRNA accumulation is co-regulated with the expression of other structural genes of the nitrate assimilation pathway; (iii) nitrite (nitrate) utilization ability is recovered in the NiR mutants by functional complementation with a wild-type Nii1 gene; (iv) the elucidated NII1 amino acid sequence is highly similar to that of the cyanobacterial and higher-plant enzyme, and contains the predicted domains for plastidic ferredoxin-NiRs. Thus, the mutant phenotype and the mRNA sequence and expression of the Nii1 gene have been unequivocally related. Accumulation of mRNA for the second locus identified, Lde1 (light-dependent expression), was not regulated by nitrogen, but like nitrate-assimilation clustered genes, its expression was down-regulated in the dark.

NADP-Malate Dehydrogenase from Unicellular Green Alga Chlamydomonas reinhardtii. A First Step toward Redox Regulation?

The determinants of the thioredoxin (TRX)-dependent redox regulation of the chloroplastic NADP-malate dehydrogenase (NADP-MDH) from the eukaryotic green alga Chlamydomonas reinhardtii have been investigated using site-directed mutagenesis. The results indicate that a single C-terminal disulfide is responsible for this regulation. The redox midpoint potential of this disulfide is less negative than that of the higher plant enzyme. The regulation is of an all-or-nothing type, lacking the fine-tuning provided by the second N-terminal disulfide found only in NADP-MDH from higher plants. The decreased stability of specific cysteine/alanine mutants is consistent with the presence of a structural disulfide formed by two cysteine residues that are not involved in regulation of activity. Measurements of the ability of C. reinhardtii thioredoxin f (TRX f) to activate wild-type and site-directed mutants of sorghum (Sorghum vulgare) NADP-MDH suggest that the algal TRX f has a redox midpoint potential that is less negative than most those of higher plant TRXs f. These results are discussed from an evolutionary point of view.

Essential Role of Cytochrome bd-Related Oxidase in Cyanide Resistance of Pseudomonas pseudoalcaligenes CECT5344†

ABSTRACT Pseudomonas pseudoalcaligenes CECT5344 grows in minimal medium containing cyanide as the sole nitrogen source. Under these conditions, an O2-dependent respiration highly resistant to cyanide was detected in cell extracts. The structural genes for the cyanide-resistant terminal oxidase, cioA and cioB, are clustered and encode the integral membrane proteins that correspond to subunits I and II of classical cytochrome bd, although the presence of heme d in the membrane could not be detected by difference spectra. The cio operon from P. pseudoalcaligenes presents a singular organization, starting upstream of cioAB by the coding sequence of a putative ferredoxin-dependent sulfite or nitrite reductase and spanning downstream two additional open reading frames that encode uncharacterized gene products. PCR amplifications of RNA (reverse transcription-PCR) indicated the cyanide-dependent up-regulation and cotranscription along the operon. The targeted disruption of cioA eliminates both the expression of the cyanide-stimulated respiratory activity and the growth with cyanide as the nitrogen source, which suggests a critical role of this cytochrome bd-related oxidase in the metabolism of cyanide by P. pseudoalcaligenes CECT5344.