Enzymatic and metabolic diagnostic of nitrogen deficiency in Arabidopsis thaliana Wassileskija accession

Adaptation to steady-state low-nutrient availability was investigated by comparing the Wassileskija (WS) accession of Arabidopsis thaliana grown on 2 or 10 mM nitrate. Low nitrogen conditions led to a limited rosette biomass and seed yield. The latter was mainly due to reduced seed number, while seed weight was less affected. However, harvest index was lower in high nitrate compared with limited nitrate conditions. Under nitrogen-limiting conditions, nitrate reductase activity was decreased while glutamine synthetase activity was increased due to a higher accumulation of the cytosolic enzyme. The level of nitrogen remobilization to the seeds was higher under low nitrogen, and the vegetative parts of the plants remaining after seed production stored very low residual nitrogen. Through promoting nitrogen remobilization and recycling pathways, nitrogen limitation modified plant and seed compositions. Rosette leaves contained more sugars and less free amino acids when grown under nitrogen-limiting conditions. Compared with high nitrogen, the levels of proline, asparagine and glutamine were decreased. The seed amino acid composition reflected that of the rosette leaves, thus suggesting that phloem loading for seed filling was poorly selective. The major finding of this report was that together with decreasing biomass and yield, nitrogen limitation triggers large modifications in vegetative products and seed quality.     

Accumulation of non-superoxide anion reactive oxygen species mediates nitrogen-limited alcoholic fermentation by Saccharomyces cerevisiae

Throughout alcoholic fermentation, nitrogen depletion is one of the most important environmental stresses that can negatively affect the yeast metabolic activity and ultimately leads to fermentation arrest. Thus, the identification of the underlying effects and biomarkers of nitrogen limitation is valuable for controlling, and therefore optimizing, alcoholic fermentation. In this study, reactive oxygen species (ROS), plasma membrane integrity, and cell cycle were evaluated in a wine strain of Saccharomyces cerevisiae during alcoholic fermentation in nitrogen-limiting medium under anaerobic conditions. The results indicated that nitrogen limitation leads to an increase in ROS and that the superoxide anion is a minor component of the ROS, but there is increased activity of both Sod2p and Cta1p. Associated with these effects was a decrease in plasma membrane integrity and a persistent cell cycle arrest at G(0)/G(1) phases. Moreover, under these conditions it appears that autophagy, evaluated by ATG8 expression, is induced, suggesting that this mechanism is essential for cell survival but does not prevent the cell cycle arrest observed in slow fermentation. Conversely, nitrogen refeeding allowed cells to reenter cell cycle by decreasing ROS generation and autophagy. Altogether, the results provide new insights on the understanding of wine fermentations under nitrogen-limiting conditions and further indicate that ROS accumulation, evaluated by the MitoTracker Red dye CM-H(2)XRos, and plasma membrane integrity could be useful as predictive markers of fermentation problems.     

Differential protein expression in Colletotrichum acutatum: changes associated with reactive oxygen species and nitrogen starvation implicated in pathogenicity on strawberry

The cellular outcome of changes in nitrogen availability in the context of development and early stages of pathogenicity was studied by quantitative analysis of two-dimensional gel electrophoresis of Colletotrichum acutatum infecting strawberry. Significant alterations occurred in the abundance of proteins synthesized during appressorium formation under nitrogen-limiting conditions compared with a complete nutrient supply. Proteins that were up- or down-regulated were involved in energy metabolism, nitrogen and amino acid metabolism, protein synthesis and degradation, response to stress and reactive oxygen scavenging. Members belonging to the reactive oxygen species (ROS) scavenger machinery, superoxide dismutase and glutathione peroxidase, were up-regulated at the appressorium formation stage, as well as under nitrogen-limiting conditions relative to growth with a complete nutrient supply, whereas abundance of bifunctional catalase was up-regulated predominantly at the appressorium formation stage. Fungal ROS were detected within germinating conidia during host pre-penetration, penetration and colonization stages, accompanied by plant ROS, which were abundant in the apoplastic space. Application of exogenous antioxidants quenched ROS production and reduced the frequency of appressorium formation. Up-regulation in metabolic activity was detected during appressorium formation and nutrient deficiency compared with growth under complete nutrient supply. Enhanced levels of proteins related to the glyoxylate cycle and lipid metabolism (malate dehydrogenase, formate dehydrogenase and acetyl-CoA acetyltransferase) were observed at the appressorium formation stage, in contrast to down-regulation of isocitrate dehydrogenase. The present study demonstrates that appressoria formation processes, occurring under nutritional deprivation, are accompanied by metabolic shifts, and that ROS production is an early fungal response that may modulate initial stages of pathogen development.     

EFFECTS OF IRON AND LIGHT STRESS ON THE BIOCHEMICAL COMPOSITION OF ANTARCTIC PHAEOCYSTIS SP. (PRYMNESIOPHYCEAE). I. INTRACELLULAR DMSP CONCENTRATIONS

Iron is essential for phytoplankton growth, as it is involved in many metabolic processes. It controls photosynthesis as well as many enzymatic processes. As such, iron affects the cell's energy supply and contributes to the assimilation of carbon and nitrogen. To determine whether iron limitation would result in energy stress or induced nitrogen deficiency, an Antarctic Phaeocystis sp. (Prymnesiophyceae) strain was studied for its biochemical composition, with the main emphasis on intracellular production of dimethylsulfoniopropionate (DMSP). DMSP is suggested to replace nitrogen containing solutes under conditions of nitrogen deficiency. Batch cultures of Antarctic Phaeocystis sp. were grown under iron-rich and iron-poor conditions and simultaneously subjected to high and low light intensities. Iron depletion induced chlorosis and suppressed growth rates as well as the maximum yield of the cultures; these effects were reinforced by low light intensities. Cell volumes were strongly reduced under iron-limited conditions. However, this reduction in cell volume was accompanied by a reduced DMSP content only in cultures experiencing low light intensities. Under high light conditions, no reduction of DMSP was observed; hence, intracellular DMSP concentrations increased. These observations are discussed relative to carbon and nitrogen metabolism and the biosynthetic pathway of DMSP. It is argued that under high light, low iron conditions, the cells were bordering on nitrogen deficiency induced by iron limitation, whereas under low light, low iron conditions, the cells were energy limited resulting in overall suppressed metabolic rates. Between treatments, DMSP to chlorophyll- a ratios varied by a factor of 5, demonstrating the dependence of this parameter on the physiological state of the cell.     

Evaluation of the role of sigma B in Mycobacterium smegmatis

The alternate sigma factor, sigB, is known to play a crucial role in maintaining the stationary phase in mycobacteria. In this communication, we have studied the proteomics of Mycobacterium smegmatis mc(2)155 and its two derivatives, one of which has a disrupted sigB gene and the other, PMVSigB, which contains a multicopy plasmid containing sigB. We have identified by two-dimensional gel analyses, several proteins that are over-expressed in PMVSigB compared to mc(2)155. These proteins are either stress proteins or participate actively in different metabolic pathways of the organisms. On the other hand, when sigB deleted mycobacteria were grown until the stationary phase and its two-dimensional protein profile was compared to that of mc(2)155, few DNA binding proteins were found to be up-regulated. We have shown recently that upon over-expressing sigB, the cell surface glycopeptidolipids of M. smegmatis are hyperglycosylated, a situation similar to what was observed for nutritionally starved bacteria. Gene expression profile through quantitative PCR presented here identified a Rhamnosyltransferase responsible for this hyperglycosylation.     

Genome scale portrait of cAMP-receptor protein (CRP) regulons in mycobacteria points to their role in pathogenesis

cAMP Receptor Protein (CRP)/Fumarate Nitrate Reductase Regulator (FNR) family proteins are ubiquitous regulators of cell stress in eubacteria. These proteins are commonly associated with maintenance of intracellular oxygen levels, redox-state, oxidative and nitrosative stresses, and extreme temperature conditions by regulating expression of target genes that contain regulatory cognate DNA elements. We describe the use of informatics enabled comparative genomics to identify novel genes under the control of CRP regulator in Mycobacterium tuberculosis (M.tb). An inventory of CRP regulated genes and their operon context in important mycobacterial species such as M. leprae, M. avium subsp. paratuberculosis and M. smegmatis and several common genes within this genus including the important cellular functions, mainly, cell-wall biogenesis, cAMP signaling and metabolism associated with such regulons were identified. Our results provide a possible theoretical framework for better understanding of the stress response in mycobacteria. The conservation of the CRP regulated genes in pathogenic mycobacteria, as opposed to non-pathogenic ones, highlights the importance of CRP-regulated genes in pathogenesis.     

Effect of rpoS gene knockout on the metabolism of Escherichia coli during exponential growth phase and early stationary phase based on gene expressions, enzyme activities and intracellular metabolite concentrations

The RNA polymerase sigma factor, encoded by rpoS gene, controls the expression of a large number of genes in Escherichia coli under stress conditions. The present study investigated the growth characteristics and metabolic pathways of rpoS gene knockout mutant of E. coli growing in LB media under aerobic condition. The analyses were made based on gene expressions obtained by DNA microarray and RT-PCR, enzyme activities and intracellular metabolite concentrations at the exponential and early stationary phases of growth. Although the glucose utilization pattern of the mutant was similar to the parent strain, the mutant failed to utilize acetate throughout the cultivation period. Microarray data indicated that the expression levels of several important genes of acetate metabolism such as acs, aceAB, cysDEK, fadR, etc. were significantly altered in the absence of rpoS gene. Interestingly, there was an increased activity of TCA cycle during the exponential growth phase, which was gradually diminished at the onset of stationary phase. Moreover, rpoS mutation had profound effect on the expression of several other genes of E. coli metabolic pathways that were not described earlier. The changes in the gene expressions, enzyme activities and intracellular metabolite concentrations of the rpoS mutant are discussed in details with reference to the major metabolic pathways of E. coli.     

Mycobacterium tuberculosis Is a Natural Ornithine Aminotransferase (rocD) Mutant and Depends on Rv2323c for Growth on Arginine

Mycobacterium tuberculosis (Mtb) possesses a genetic repertoire for metabolic pathways, which are specific and fit to its intracellular life style. Under in vitro conditions, Mtb is known to use arginine as a nitrogen source, but the metabolic pathways for arginine utilization have not been identified. Here we show that, in the presence of arginine, Mtb upregulates a gene cluster which includes an ornithine aminotransferase (rocD) and Rv2323c, a gene of unknown function. Isotopologue analysis by using ¹³C- or ¹⁵N-arginine revealed that in Mtb arginine is not only used as nitrogen source but also as carbon source for the formation of amino acids, in particular of proline. Surprisingly, rocD, which is widespread in other bacteria and is part of the classical arginase pathway turned out to be naturally deleted in Mtb, but not in non-tuberculous mycobacteria. Mtb lacking Rv2323c showed a growth defect on arginine, did not produce proline from arginine, and incorporated less nitrogen derived from arginine in its core nitrogen metabolism. We conclude that the highly induced pathway for arginine utilization in Mtb differs from that of other bacteria including non-tuberculous mycobacteria, probably reflecting a specific metabolic feature of intracellular Mtb.     

Comparison of mammalian cell entry operons of mycobacteria: in silico analysis and expression profiling

Mammalian cell entry (mce) operons, implicated in the entry of mycobacteria into host cells, are present in pathogenic and saprophytic species. It is likely that the genes in these operons have functions other than those required for entry into host cells. Using in silico analysis we have identified domains within the mce operons that might justify their occurrence in saprophytic species like Mycobacterium smegmatis. Our analysis identified in addition to the mce domain, the presence of the Ttg2B and Ttg2C domains, typical of proteins involved in transport. We have also analysed and compared the expression profile between mce operons of Mycobacterium tuberculosis, Mycobacterium bovis and M. smegmatis under different growth conditions. In case of M. smegmatis, each operon presented domain truncation for at least one gene. We observe differential expression among the operons in M. smegmatis growing under different culture conditions. Bacilli growing in nutritionally rich medium with aeration, only the mce4 operon was expressed while during stationary phase of a standing culture, all four mce operons were expressed. In M. bovis, in addition to the absence of the mce3 operon, several protein domains encoded by the other operons were truncated. We detected expression of the mce2 operon in the exponential and stationary growth phase, while the mce1 operon was only expressed in the stationary growth phase. Differential expression of mce operons and their redundancy in the genome of the majority members of mycobacteria are discussed in view of our results.     

Iron uptake processes in Mycobacterium vaccae R877R, a mycobacterium lacking mycobactin

The production of exochelins (MV) was established in Mycobacterium vaccae R877R under iron-deficient conditions in concentrations about five times greater than in Mycobacterium smegmatis. M. vaccae does not produce mycobactin nor is salicylic acid secreted into the medium. A simple method is described using 55Fe-labelled culture filtrates for assessing exochelin production and which would be applicable to other mycobacteria. One of the exochelins produced (MV3) is part of an active iron uptake system and another (MV1) is responsible for a passive uptake system. MV3 exochelin has similar chromatographic properties and biological activity to the major exochelin produced by M. smegmatis: iron uptake from MV3 exochelin was inhibited by dinitrophenol, NaN3 and HgCl2, and was judged to be an active transport process. This process was not inhibited by equimolar amounts of ferri-salicylate or ferri-citrate both of which could be used separately as sources of iron for the organism. Uptake from these latter sources was insensitive to metabolic inhibitors and uncouplers. The multiplicity of pathways for iron uptake in a single organism is discussed.