Nitrogen-depleted Conditions Research Articles

Identification and characterization of mutation in the RNase T1 expression-sensitive strain of : Evidence for altered ambient response resulting in transportation of the secretory protein to vacuoles

We previously reported a genetic analysis of the growth-inhibitory effect caused by the overexpression of the Aspergillus oryzae rntA gene, encoding RNase T1 (Ribonuclease T1), in Saccharomyces cerevisiae. Subsequently, rns (ribonuclease T1 sensitive) mutants with mutations in the rns1 (DSL1), rns2 (UMP1), and rns3 (SEC17) genes, were identified. In the present study, rns4 (VPS32/SNF7) gene mutation was identified by complementation of tunicamycin sensitivity. While the rns4 mutant exhibited sensitivity to ambient stress conditions (200 mM CaCl(2), 1M NaCl and pH 8.0), genome-wide expression analysis revealed a similar pattern of genes up-regulated as was observed under nitrogen depletion condition by Gasch et al. [Mol. Biol. Cell 11 (2000) 4241]. Notably, the genes participating in autophagy (ATG4 and ATG8), the genes encoding a vacuolar protease (PRB1), vacuolar protease inhibitors (PAI3, PBI2 and TFS1) and YHR138c (a PBI2 homolog) were up-regulated in the rns4 mutant. Interestingly, the RNase T1*-GFP fusion protein (*inactive form) expressed in the rns4 mutant strain localized at the ER and vacuole under both stress or no-stress conditions. In contrast, the RNase T1*-GFP fusion protein expressed in the wild-type strain could not be detected under no-stress conditions, however, a stress-dependent localization of the fusion protein was observed at the vacuole. Since, the rns4 mutant exhibited a partial starvation-like response in spite of a rich ambient environment, leading to transportation of the secretory protein to the vacuole and accumulation in the endoplasmic reticulum, the present findings implicate a novel role for Rns4/Vps32 in proper response and adaptation to ambient conditions.

Microautophagy and macropexophagy may occur simultaneously in Hansenula polymorpha

We subjected methanol-grown cells of wild type Hansenula polymorpha simultaneously to nitrogen depletion and excess glucose conditions. Both treatments induce the degradation of peroxisomes, either selective (via excess glucose) or non-selective (via nitrogen limitation). Our combined data strongly suggest that both processes occur simultaneously under these conditions. The implications of these findings on studies of autophagy and related transport pathways to the vacuole in yeast are discussed.

Differential expression and localization of Mn and Fe superoxide dismutases in the heterocystous cyanobacterium Anabaena sp. strain PCC 7120.

Superoxide dismutases (Sods) play very important roles in preventing oxidative damages in aerobic organisms. The nitrogen-fixing heterocystous cyanobacterium Anabaena sp. strain PCC 7120 has two Sod-encoding genes: a sodB, encoding a soluble iron-containing Sod (FeSod), and a sodA, encoding a manganese-containing Sod (MnSod). The FeSod was purified and characterized. A recombinant FeSod was also obtained by overproduction in Escherichia coli. Immunoblot study of the FeSod during induction of heterocyst differentiation showed that the cells produced six- to eightfold more FeSod 8 h after a shift from a nitrogen-replete condition to a nitrogen-depleted condition. However, the amount of FeSod protein in filaments with mature heterocysts was the same as that in filaments grown with combined nitrogen. Superoxide anion-generating chemicals such as methyl viologen did not induce upregulation of the sodB gene expression. The predicted preprotein of the sodA gene has a leader peptide and a motif for membrane attachment at the N terminus of the mature protein. Activity staining after gel electrophoresis of the purified thylakoid membranes showed that most of the MnSod in Anabaena sp. strain PCC 7120 was located on thylakoids toward the lumenal side. Expression of the sodA gene in E. coli shows that the leader peptide was required for its activity and the membrane localization of the MnSod. Northern hybridization detected one 0.82-kb transcript of sodA. The sodA gene was upregulated by methyl viologen, whereas its amount was unchanged during heterocyst differentiation. Immunoblotting and activity staining showed that isolated heterocysts contained a lower but still significant amount of FeSod, suggesting that its function is required in heterocysts. No MnSod was observed in isolated heterocysts. These results show that the two different Sod proteins have differentiated roles in Anabaena sp. strain PCC 7120.

Effect of irradiance on growth, photosynthesis, pigment content and nutrient consumption in dense cultures of Rhodomonas salina (Wislouch) (Cryptophyceae)

The effects of different incident irradiances on growth, photosynthesis, pigment content and nutrient uptake were studied in dense cultures of the cryptophyte Rhodomonas salina (Wislouch) Hill y Wetherbee. Cells were grown for ten days at different incident irradiances: 11, 33, 75, 260 and 320 µmol photon m-2s-1. The average pigment content (phycoerythrin and chlorophyll a) was lower when cells were grown at high irradiances (260 and 320 µmol photon m-2s-1) compared with cells grown under low irradiances (11, 33 and 75 µmol photon m-2s-1).The maximum photosynthetic rate (Pmax) was maximal at 75 µmol photon m-2s-1 , decreasing considerably at the highest irradiance in which apparent photosynthetic efficiency per cell and per chlorophyll a unit (αcell and αchla) also decreased. Results showed that dense cultures of R. salina grew optimally in a range between 75 and 260 µmol photon m-2s-1 with optimal photosynthetic performance at 75 µmol photon m-2s-1. Nitrogen and phosphorus depletion occurred as a consequence of the exponential growth, except under the lowest and the highest irradiances (11 and 320 µmol photon m-2s-1, respectively). Associated to N-limitation, phycoerythrin (PE) and chlorophyll a decreased considerably. It is suggested that R.salina responds to nitrogen-depleted conditions by mobilizing nitrogen from PE, this behaviour being modelled by a relationship between external nitrogen availability and PE mobilization.

Effect of food quality on carbon and nitrogen growth efficiency in the copepod Acartia tonsa

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 235:147-156 (2002) - doi:10.3354/meps235147 Effect of food quality on carbon and nitrogen growth efficiency in the copepod Acartia tonsa Ruth H. Jones1, Kevin J. Flynn1,*, Thomas R. Anderson2 1Ecology Research Unit, School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, United Kingdom 2George Deacon Division, Southampton Oceanography Centre, European Way, Southampton SO14 3ZH, United Kingdom *Corresponding author. E-mail: k.j.flynn@swansea.ac.uk ABSTRACT: Populations of the copepod Acartia tonsa were fed a mixture of algal prey (diatom Thalassiosira weissflogii, prymnesiophyte Emiliania huxleyi, dinoflagellate Aureodinium [Gymnodium] pigmentosum) supplied at saturating concentrations, grown under either nitrogen-sufficient or nitrogen-deplete conditions, in order to study the impact of food quality on production and development throughout the life cycle of the copepod. Changes in predator population structure and biomass were recorded, along with consumption of each of the algal groups, permitting C and N growth efficiencies to be estimated. There was a clear difference in the Acartia tonsa population structure when fed N sufficient or N-deplete prey, with those fed N-deplete prey slower to develop and reproduce and laying fewer eggs. Algal nutrient status affected selectivity between the diatom and dinoflagellate, the latter being favoured under nutrient-deplete conditions, perhaps in part because their C:N ratio was less susceptible to altered nutrient status. There was no clear difference in the N growth efficiency (N GE, typically 5%) between N-sufficient and N-deplete prey, but certainly efficiency did not increase with N-deplete prey. C growth efficiency (C-GE) declined from 5 to 2% with N-deplete prey. However, while the ratio of N-GE:C-GE was clearly different between N-sufficient (1) and N-deplete (2.5) treatments, actual growth efficiencies increased with time during the progression to later life history stages, culminating in highest efficiencies during active egg production. Caution should be exercised in assigning GE and predation rates in models incorporating zooplankton feeding on prey of variable nutrient status; these parameters are not constants and GE estimates from egg production experiments are likely to significantly overestimate efficiencies over the whole copepod life cycle. KEY WORDS: Copepods · Food quality · Nutrient status · C:N · Growth efficiency · Population · Life cycle Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 235. Online publication date: June 19, 2002 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2002 Inter-Research.

CND41, a chloroplast DNA-binding protease, is involved in Rubisco degradation

CND41 is a 41kDa DNA-binding protein with a basic pl isolated from chloroplast nucleoids of photomixotrophically cultured tobacco cells. CND41 has a transit peptide of 120 amino acids and a mature protein of 382 amino acids. The deduced amino acid sequence indicated that CND41 has active domain of aspartic protease. The purified CND41 had strong proteolytic activity at acidic pHs (pH2-4) (Murakami et al. FEBS Lett. 468:15(2000)). Furthermore, the fact that CND41 degraded denatured Rubisco at physiological pH suggests that it might be involved in Rubisco degradation. To confirm this idea in vivo, we examined the effect of nitrogen-depletion on the Rubisco degradation both in control (WT) and CND41 antisense (R22) tobacco. Analysis of total proteins and Rubisco clearly showed that those contents in WT deceased with leaf age, especially under nitrogen depletion condition, whereas those in R22 were rather constant through whole plant even under nitrogen-depletion. Western blot analysis also showed several bands of Rubisco-degradation products in young R22 leaves. These results indicated that CND41 plays an important role in the in vivo degradation of Rubisco both in young and senescent leaves.

High hopanoid/total lipids ratio in Frankia mycelia is not related to the nitrogen status.

Vesicles are specific Frankia structures which are produced under nitrogen-limiting culture conditions. Hopanoids are the most abundant lipids in these vesicles and are believed to protect the nitrogenase against oxygen. The amounts and quality of each hopanoid were estimated in different Frankia strains cultivated under nitrogen-depleted and nitrogen-replete conditions in order to detect a possible variation. Studied Frankia strains nodulating Eleagnus were phylogenetically characterized by analysis of the nifD-K intergenic region as closely related to genomic species 4 and 5. Phylogenetically different strains belonging to three infectivity groups were cultivated in the same medium with and without nitrogen source for 10 d before hopanoid content analysis by HPLC. Four hopanoids together accounted for 23-87% and 15-87% of the total lipids under nitrogen-replete and nitrogen-depleted culture conditions, respectively. Two of the hopanoids found, bacteriohopanetetrols and their phenylacetic acid esters, have previously been described in Frankia Two new hopanoids, moretan-29-ol and a bacteriohopanetetrol propionate, have also been identified. The moretan-29-ol and bacteriohopanetetrols were found to be the most abundant hopanoids whereas the bacteriohopanetetrol propionate and phenylacetates were present at a concentration close to the limit of detection. The ratio of (bacteriohopanetetrols + moretan-29-ol)/(total lipids) varied in most of the strains between nitrogen-depleted and nitrogen-replete culture conditions. In most of the strains, the hopanoid content was found to be slightly higher under nitrogen-replete conditions than under nitrogen-depleted conditions. These results suggest that remobilization, rather than neosynthesis of hopanoids, is implicated in vesicle formation in Frankia under nitrogen-depleted conditions.

Molecular cloning and sequencing of the sodB gene from a heterocystous cyanobacterium Anabaena sp. PCC 7120

Superoxide dismutase (Sod) plays an important role in all aerobic organisms. The sodB gene of a heterocystous cyanobacterium Anabaena sp. PCC 7120 was cloned and sequenced. The Sod protein is predicted to have 199 amino acids and a molecular mass of 22.5 kDa. Sequence comparison among SodB from cyanobacteria and chloroplasts revealed that the sodB gene indeed encodes an iron-Sod. Northern blot analysis showed that the sodB gene of Anabaena sp. PCC 7120 is transcribed as a single gene and its expression was up-regulated when the cells were subjected to a shift from a nitrogen repletion condition to a nitrogen depletion condition.

Molecular cloning, sequencing and expression in Escherichia coli of the poly(3-hydroxyalkanoate) synthesis genes from Alcaligenes latus DSM1124

Fragments of chromosomal DNA from Alcaligenes latus DSM1124 were cloned into Escherichia coli and transformants were screened for poly( d(−)-3-hydroxybutyrate) [P(3HB)] production during excess carbon supply. A plasmid harboring a 5.5-kb insert of A. latus DNA was isolated from a P(3HB)-producing bacterial colony. The insert was partially sequenced and three major open reading frames (ORFs) were found, representing the PHA synthase ( phaC), β-ketothiolase ( phaA) and acetoacetyl-CoA reductase ( phaB) genes. They show striking homology to the Ralstonia eutropha (formerly Alcaligenes eutrophus) phaC (71%), phaA (77%) and phaB (80%) genes, and are organized in the same way. The only major difference is the replacement of 560 nucleotides by 160 non-homologous nucleotides in the 5′ region of phaC in A. latus. The phaC ORF lacks 29 amino acids at the N-terminus, compared to that of R. eutropha, and starts with a GTG codon. The transcription start points of the operon were determined. P(3HB) production of recombinant E. coli strains harboring the pha operons of A. latus DSM1124 or R. eutropha H16 was investigated. Both operons gave rise to less than 5% P(3HB) formation during exponential growth. At the end of the growth phase, the P(3HB) content reached approximately 20% of cell dry mass. Under nitrogen-depleted conditions, the A. latus pha genes gave rise to 50–52% P(3HB), compared to 33–38% for the R. eutropha pha genes. No NADH oxidase activity was detectable in A. latus, indicating an impaired respiratory pathway and a dependence on PHA synthesis for storing reduction equivalents during growth.

Characterization of HetR protein turnover in Anabaena sp. PCC 7120.

The hetR gene plays an important role in heterocyst development and pattern formation in heterocystous cyanobacteria. The hetR gene from Anabaena sp. PCC 7120 was overexpressed in Escherichia coli. Antibodies raised against the recombinant HetR protein (rHetR) were used to characterize metabolism of the HetR of Anabaena sp. PCC 7120 in vivo. HetR was present at a low level when Anabaena sp. PCC 7120 was grown in the presence of combined nitrogen. Shifting from nitrogen repletion conditions to nitrogen depletion conditions led to a two fold increase of HetR in total cell extracts, and most of HetR was located in heterocysts. The amount of HetR in total cellular extracts increased rapidly after shifting to nitrogen depletion conditions and reached a maximum level 3 h after the shift. Isoelectrofocusing electrophoresis revealed that the native HetR had a more acidic isoelectric point than did rHetR. After combined nitrogen was added to the nitrogen-depleted cultures, the degradation of HetR depended on culture conditions: before heterocysts were fully developed, HetR was rapidly degraded; after heterocysts were fully developed, HetR was degraded much more slowly. The distribution of HetR in other species of cyanobacteria was also studied.

Promoters controlling expression of the alternative nitrogenase and the molybdenum uptake system in Rhodobacter capsulatus are activated by NtrC, independent of sigma54, and repressed by molybdenum.

The alternative nitrogenase of Rhodobacter capsulatus is expressed only under conditions of nitrogen and molybdenum depletion. The analysis of anfA-lacZ fusions demonstrated that this dual control occurred at the level of transcription of anfA, which encodes a transcriptional activator specific for the alternative nitrogenase. The anfA promoter was found to be activated under nitrogen-limiting conditions by NtrC in a sigma54-independent manner. In addition, anfA transcription was repressed by traces of molybdenum. This molybdenum-dependent repression of anfA was released in R. capsulatus mutants carrying either lesions in the high-affinity molybdenum uptake system (modABCD) or a double deletion of mopA and mopB, two genes encoding molybdenum-pterin-binding proteins. The expression of the molybdenum transport system itself was shown to be negatively regulated by molybdenum and, unexpectedly, to be also regulated by NtrC. This finding is in line with the presence of two tandemly arranged DNA motifs located in front of the R. capsulatus mopA-modABCD operon, which are homologous to R. capsulatus NtrC binding sites. Mapping of the transcriptional initiation sites of mopA and anfA revealed promoter sequences exhibiting significant homology to each other but no homology to known prokaryotic promoters. In addition, a conserved DNA sequence of dyad symmetry overlapping the transcriptional initiation sites of mopA and anfA was found. Deletions within this element resulted in molybdenum-independent expression of anfA, indicating that this DNA sequence may be the target of MopA/MopB-mediated repression.

Mating in the heterothallic haploid yeast Clavispora opuntiae, with special reference to mating type imbalances in local populations

Mating was studied in the haploid, heterothallic yeast Clavispora opuntiae to assess the importance of nutritional, genetic, and other factors that may favour mating and recombination. Local populations of this yeast generally exhibit dramatic inequalities in mating type distributions, suggesting that mating is rare in nature even though most isolates mate freely in the laboratory. The absence of assimilable nitrogen is prerequisite to mating competence, presumably by causing G1 arrest. Maximum mating competence is found in cells entering stationary phase in nitrogen-limited media. Unlike the vast majority of mating yeasts, C. opuntiae does not appear to produce diffusible mating factors (sex pheromones), and mating-competent cells do not undergo sexual agglutination. Pairwise cell contact appears to be the only signal that triggers the sexual process in this case. In order to determine if mating type imbalances in nature are caused by reduced fertility of 'consanguine' crosses, meiotic recombination was measured in pairs of strains that varied in their genetic distances as indicated by restriction mapping. That hypothesis was rejected, as recombination efficiency decreased with increasing genetic distance. We conclude that the rarity of mating in local populations is exacerbated by the stringent physical (pairwise cell contact) and nutritional (nitrogen depletion) conditions that will allow mating to proceed. Parallels are drawn with mating patterns observed in Clavispora lusitaniae.

Mating pheromone-like diffusible factor released by Schizosaccharomyces pombe

We demonstrate that a diffusible factor is secreted by h cells of the fission yeast Schizosaccharomyces pombe, whose mating pheromones have not been described. This factor, tentatively named the h-factor, affects hS. pombe cells and induces their elongation under nitrogen-depleted conditions. Circumstantial evidence suggests its physiological significance in the mating process. Despite their sterility, hras1 cells secrete this factor. However, hras1 cells have apparently lost the ability to respond to it. This may suggest that the gene product of S. pombe ras1, a homologue of mammalian ras oncogenes, is involved in the mechanism for responding to mating pheromones.

Production of extracellular carbohydrate in cultures of Ankistrodesmus densus Kors. (Chlorophyceae)

An extracellular polysacchande, released into the culture medium by Ankistrodesmus densus is shown to be a product of active photosynthesis rather than diagenesis. Although produced in the exponential phase, its accumulation is most pronounced in the stationary phase under conditions of nitrogen depletion. It is a glycan, containing residues of galactose, mannose, glucose, xylose, arabinose and a trace of rhamnose.

Induction of nitrate reductase under conditions of nitrogen depletion

Induction of nitrate reductase under conditions of nitrogen depletion

Photoreactivation of ultraviolet-induced suppression of division in Chilomonas paramecium

The two phases of division-suppression found after UV-irradiation of Chilomonas paramecium populations were examined for photoreactivability. The second, or delayed, phase was readily photoreactivated whether the tests were run on cultures growing in complete medium, on nitrogen-depleted cells in nitrogen-deficient medium, or nitrogen-depleted cells returned to complete medium. Photoreactivability declined as cells progressed toward division, and could not be demonstrated if PR exposure was performed later than a few hours before the first post-UV division. Photoreactivability was prolonged when division was prevented by nitrogen deprivation. The initial, or immediate, phase of division-suppression was also photoreactivable, but this, for unknown reasons, could only be demonstrated if lag was prolonged by nitrogen-deprivation. These results may be interpreted as indicating that both phases of the division-suppression are the result of UV-damage to DNA, and that there is retention of photoreactivating enzyme complement even under conditions of nitrogen depletion. It is noted, however, that UV may produce photoreactivable lesions in cell constituents other than DNA, and that these may also account for either or both of the two phases of division-suppression.