Dynamics of Nitrogen-regulated Gene Expression Reveals a Reciprocal Relationship between Cell Growth Rate and Nitrogen Catabolism
ABSTRACT: Cell growth rate is regulated in response to resource availability including the abundance, and molecular form, of essential nutrients. In the model eukaryotic cell, Saccharomyces cerevisiae (budding yeast), the molecular form of environmental nitrogen impacts both cell growth rate and mRNA expression. Disentangling causal relationships between nitrogen availability, cell growth rate and differential gene expression poses a considerable challenge. Using experimental control of cell growth rate using chemostats, we studied the effect of variation in environmental nitrogen on differential gene expression. We find that the primary determinant of nitrogen-regulated gene expression is nitrogen abundance whereas variation in nitrogen source affects the expression of only a small number of transcripts with highly specialized functions. To study the dynamics of nitrogen-responsive gene expression we perturbed steady-state nitrogen-limited chemostat cultures by addition of either proline or glutamine. Addition of either proline or glutamine to cells growing in nitrogen-limited chemostats results in repression of the nitrogen catabolite repression (NCR) regulon consistent with nitrogen abundance, and not nitrogen source, being the primary determinant of nitrogen-regulated gene expression. We find that a transition from nitrogen-limited to nitrogen-replete conditions is accompanied by rapid induction of transcripts required for protein translation. We identified a reciprocal relationship between specific regulons required for protein translation (RP and RiBi) and the NCR regulon. Using mathematical modeling we find evidence that cells adopt a metabolically inefficient growth mode during this transition. By means of high resolution time series analysis we find evidence that rapid, and potentially accelerated, mRNA degradation plays an important role in remodeling gene expression programs in response to change in environmental nitrogen. We propose that the evolutionarily conserved TORC1 signaling pathway orchestrates the balance between protein translation and assimilation of nitrogen sources at the transcriptional level to optimize rates of cell proliferation. A total of of 102 samples were analyzed in different nitrogen-limited conditions using chemostats in both steady-state and dynamic conditions. A common reference obtained from an ammonium-limited chemostat growing at a dilution rate of 0.12/hr was used for all two color hybridization experiments.
Project description:Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae grown with six different nitrogen sources were subjected to transcriptome analysis. The use of chemostats enabled an analysis of nitrogen-source-dependent transcriptional regulation at a fixed specific growth rate. A selection of preferred (ammonium and asparagine) and non-preferred (leucine, phenylalanine, methionine and proline) nitrogen sources was investigated. For each nitrogen source, distinct sets of genes were induced or repressed relative to the other five nitrogen sources. A total number of 131 of such ‘signature transcripts’ were identified in this study. In addition to signature transcripts, genes were identified that showed a transcriptional co-response to two or more of the six nitrogen sources. For example, 33 genes were transcriptionally up-regulated in leucine-, phenylalanine- and methionine-grown cultures, which was partly attributed to the involvement of common enzymes in the dissimilation of these amino acids. In addition to specific transcriptional responses elicited by individual nitrogen sources, their impact on global regulatory mechanisms such as nitrogen catabolite repression (NCR) could be monitored. NCR-sensitive gene expression in the chemostat cultures showed that, ammonia and asparagine were ‘rich’ nitrogen sources. By this criterion, leucine, proline and methionine were ‘poor’ nitrogen sources and phenylalanine showed an ‘intermediate’ NCR response. Keywords: Response to growth on various nitrogen source transcriptome Chemostat based transcriptomics study. Each growth condition was performed in triplicate.
Project description:The eukaryotic translation factor eIF5A, originally identified as an initiation factor, was later shown to promote translation elongation of iterated proline sequences. Using a combination of ribosome profiling and in vitro biochemistry, we report a much broader role for eIF5A in elongation and uncover a substantial function for eIF5A in termination. Ribosome profiling of an eIF5A-depleted strain reveals a global elongation defect, with abundant ribosomes stalling at many sequences, not limited to proline stretches. Our data also show accumulation at stop codons and in the 3’-UTR, suggesting a global defect in termination in the absence of eIF5A. Using an in vitro reconstituted translation system, we find that eIF5A strongly promotes the translation of novel stalling sequences and increases the rate of peptidyl-tRNA hydrolysis more than 17-fold. We conclude that eIF5A functions broadly in elongation and termination, rationalizing its great cellular abundance and essential nature. Overall design: 8 biological samples are included in the study (8 ribosome footprinting samples). These include wild-type and eIF5A depletion strains (with biological replicates). Also included are ribosome footprint profiling after high salt treatment.
Project description:G. sulfurreducens (ATCC #51573) was obtained from the laboratory culture collection of Dr. Derek Lovley. Cells were grown under strict anaerobic conditions at 30 °C in chemostats, as previously described (for more information see Esteve-Núñez, A., M. M. Rothermich, M. Sharma, and D. R. Lovley. 2004. Growth of Geobacter sulfurreducens under nutrient-limiting conditions in continuous culture. Environ. Microbiol.:in press.), with acetate (5 mM) as the electron donor and fumarate (27.5 mM) as the electron acceptor. For growth in the absence of fixed nitrogen, the ammonium chloride (4.7 mM) was omitted from the medium and fumarate served as the electron acceptor. Therefore, cultures with ammonium chloride were limited by acetate while cultures without ammonium chloride were limited by nitrogen. Cultures were maintained at a dilution rate of 0.05 h-1 for 5 culture vessel volumes to ensure that cells were at steady-state prior to harvesting. Cells were harvested by centrifugation at 4 °C and the cell pellet was flash-frozen in liquid nitrogen and then stored at -80 °C prior to RNA extraction. Cells grown in the absence of ammonium had acetylene reduction rates of 0.012 nmol/hr compared to 0.003 nmol/hr in ammonium-grown cells, providing evidence that these cells were fixing nitrogen.. The steady-state concentration of cells in the nitrogen-fixing chemostats (0.178 mg/mL + 0.011; mean + standard deviation, n=3) was significantly lower than chemostats provided with ammonium (0.45 mg/mL + 0.007). Three cultures of acetate limited growth with fumarate as the electron acceptor and three cultures of nitrogen limited growth with fumarate as the electron acceptor were grown. Each culture vessel was harvested and extracted for total RNA separately to produce three biological replicates for this experiment.
Project description:Pyruvate fermentation pathway and energetics of Desulfovibrio alaskensis strain G20 under syntrophic coculture and fermentative monoculture conditions Expression data for Desulfovibrio alaskensis strain G20 grown in chemostats on pyruvate under respiratory conditions (sulfate-limited and pyruvate-limited monoculture, dilution rate 0.047 and 0.027 h-1), fermentative conditions (monoculture, dilution rate 0.036 h-1), and syntrophic conditions (coculture with Methanococcus maripaludis or Methanospirillum hungatei, dilution rate of 0.047 and 0.027 h-1) 2 replicates each for syntrophic coculture (M. maripaludis or M. hungatei pairing) and respiratory (sulfate- or pyruvate-limited) monoculture for both growth rates (0.027 and 0.047 h-1), and 4 replicates fermentative monoculture (gas flow rate through head space of bioreactor 10 ml/min (chemostats C91 and C93) or 1 ml/min (chemostats C92 and C94)
Project description:Methanococcus maripaludis is a methanogenic Archaea that conserves energy from molecular hydrogen to reduce carbon dioxide to methane. Chemostat grown cultures limited for hydrogen, phosphate, or leucine were compared to determine the regulatory response to hydrogen limitation. This was done by comparing hydrogen limited cultures to both leucine limited and phosphate limited cultures. Slow and rapid growing samples limited for either hydrogen or phosphate were compared to determine the regulatory effects of growth rate. Keywords: archaea, hydrogen, leucine, phosphate, nutrient limitation, growth rate, methanogen Overall design: Four biological replicates of hydrogen limited cells grown in chemostats were compared to four biological replicates of leucine limited and phosphate limited chemostat cultures. For growth rate comparisons, two sets were compared, each with three biological replicates of cultures grown at slow and rapid growth rates in chemostats. One set was limited by hydrogen availability at both growth rates, the other by phosphate availability. Each comparison was conducted with a dye swap and each contained two copies of the array, yielding 4 technical replicates for each biological replicate. This yielded a total of 16 replicates for the hydrogen versus leucine and hydrogen versus phosphate comparisons and 12 replicates each for the growth rate comparisons for both hydrogen and phosphate limitation. For growth rate comparisons under phosphate limited conditions a second scanning was done at lower gain set so that no spot reached saturation.
Project description:Expression data for Desulfovibrio alaskensis strain G20 grown on lactate in sulfate-limited monoculture and syntrophic coculture with Methanococcus maripaludis in chemostats at a high growth rate of 0.047h-1 5 replicates of coculture and 3 replicates of sulfate-limited monoculture
Project description:Expression data for Desulfovibrio alaskensis strain G20 grown on lactate in sulfate-limited monoculture and syntrophic coculture with Methanococcus maripaludis or Methanospirillum hungatei in chemostats at a low growth rate of 0.027h-1. 7 samples of Desulfovibrio alaskensis strain G20 grown in syntrophic coculture on lactate with either Methanococcus maripaludis (4 replicates) or Methanospirillum hungatei (3 replicates), and 5 samples of sulfate-limited monoculture growth of strain G20 on lactate.
Project description:Our approach for the identification and quantification of growth control by the components of yeast cells relies on two complementary screens: In the first, growth rate is changed and the effect on the concentration of cell components is measured (Castrillo et al. 2007, Genome Biology, 6,4). In the second, the concentration of the components is changed (by reducing gene copy number) and the effect on growth rate is measured (Delneri et al., 2008, Nat Gen, 40, 113). The heterozygous deletant (hemizygote) library we use is composed of diploid mutants each lacking one copy of a different gene (Giaever et al., 2002, Nature, 418, 387). When such a pool of mutants is grown in competition, some will grow slower than the others due to their particular mutation. Over time, the proportion of these mutants in the population will decrease a phenotype termed `haploinsufficient. Conversely, some mutants will have a growth-rate advantage and so will increase their proportion in the population; we call these strains `haploproficient. Screening a pool of hemizygotes for their haploinsufficiency (HI)/haploproficiency (HP) phenotypes thus identifies cellular components that have high flux control (HFC) over growth rate. We found that genes that are major controllers of growth rate under nutrient-limitation are not, themselves, subject to growth rate control of their transcription. In this study we have investigated the generality of this rule, first by identifying HFC genes in turbidostat culture, where biomass concentration sets the rate of nutrient addition, growing at the maximum rate (mmax=0.32h-1) permitted by the complex defined medium (FPM) employed. And to study growth-rate effects, competition experiments were performed at higher growth rates (D=0.2h-1 and 0.3h-1) in nitrogen-limited chemostats and, to control for the effect of nitrogen limitation, in FPM using a chemostat at D=0.3h-1 (Pir et al., manuscript in preparation)
Project description:Nitrogen is essential for microbial growth and its importance is demonstrated by complex regulatory systems used to control the transport, assimilation and metabolism of nitrogen. To gain further insight into the response of mycobacteria to nitrogen limitation, we developed a nitrogen-limited chemostat and compared the transcriptional response of nitrogen-limited cells to nitrogen-replete cells in a continuous culture model at a constant growth rate (0.12 h-1) (td = 5.7 hrs) and 50% oxygen saturation.
Project description:G. sulfurreducens (ATCC #51573) was obtained from the laboratory culture collection of Dr. Derek Lovley. Cells were grown under strict anaerobic conditions at 30 °C in chemostats, (see Esteve-Núñez, A., M. M. Rothermich, M. Sharma, and D. R. Lovley. 2004. Growth of Geobacter sulfurreducens under nutrient-limiting conditions in continuous culture. Environ. Microbiol.:in press. for more information), with acetate (5 mM) as the electron donor and Fe(III) citrate (55 mM) or fumarate (27.5 mM) as the electron acceptor. Under these conditions acetate is the substrate limiting growth. Cultures were maintained at a dilution rate of 0.05 h-1 for 5 culture vessel volumes to ensure that cells were at steady-state prior to harvesting. Cells were harvested by centrifugation at 4 °C and the cell pellet was flash-frozen in liquid nitrogen and then stored at 80 °C prior to RNA extraction. Three cultures of acetate limited growth with fumarate as the electron acceptor and three cultures of acetate limited growth with Fe(III) as the electron acceptor were grown. Each culture vessel was harvested and extracted for total RNA separately to produce three biological replicates for this experiment.