A Systems Biology Approach Uncovers Cellular Strategies Used by Methylobacterium extorquens AM1 During the Switch from Multi- to Single-Carbon Growth

When organisms experience environmental change, how does their metabolic network reset and adapt to the new condition? Methylobacterium extorquens is a bacterium capable of growth on both multi- and single-carbon compounds. These different modes of growth utilize dramatically different central metabolic pathways with limited pathway overlap.This study focused on the mechanisms of metabolic adaptation occurring during the transition from succinate growth (predicted to be energy-limited) to methanol growth (predicted to be reducing-power-limited), analyzing changes in carbon flux, gene expression, metabolites and enzymatic activities over time. Initially, cells experienced metabolic imbalance with excretion of metabolites, changes in nucleotide levels and cessation of cell growth. Though assimilatory pathways were induced rapidly, a transient block in carbon flow to biomass synthesis occurred, and enzymatic assays suggested methylene tetrahydrofolate dehydrogenase as one control point. This "downstream priming" mechanism ensures that significant carbon flux through these pathways does not occur until they are fully induced, precluding the buildup of toxic intermediates. Most metabolites that are required for growth on both carbon sources did not change significantly, even though transcripts and enzymatic activities required for their production changed radically, underscoring the concept of metabolic setpoints.This multi-level approach has resulted in new insights into the metabolic strategies carried out to effect this shift between two dramatically different modes of growth and identified a number of potential flux control and regulatory check points as a further step toward understanding metabolic adaptation and the cellular strategies employed to maintain metabolic setpoints

Functional Characterization of the Eukaryotic Cysteine Desulfurase Nfs1p from Saccharomyces cerevisiae

Previous studies have indicated that the essential protein Nfs1 performs a crucial role in cellular iron-sulfur (Fe/S) protein maturation. The protein is located predominantly in mitochondria, yet low amounts are present in cytosol and nucleus. Here we examined several aspects concerning the molecular function of yeast Nfs1p as a model protein. First, we demonstrated that purified Nfs1p facilitates the in vitro assembly of Fe/S proteins by using cysteine as its specific substrate. Thus, eukaryotic Nfs1 is a functional orthologue of the bacterial cysteine desulfurase IscS. Second, we showed that only the mitochondrial version but not the extramitochondrial version of Nfs1p is functional in generating cytosolic and nuclear Fe/S proteins. Mutation of the nuclear targeting signal of Nfs1p did not affect the maturation of cytosolic and nuclear Fe/S proteins, despite a severe growth defect under this condition. Nfs1p could not assemble an Fe/S cluster on the Isu scaffold proteins when they were located in the yeast cytosol. The lack of function of these central Fe/S cluster assembly components suggests that the maturation of extramitochondrial Fe/S protein does not involve functional copies of the mitochondrial Fe/S cluster assembly machinery in the yeast cytosol. Third, the extramitochondrial version of Nfs1p was shown to play a direct role in the thiomodification of tRNAs. Finally, we identified a highly conserved N-terminal {beta}-sheet of Nfs1p as a functionally essential part of the protein. The implication of these findings for the structural stability of Nfs1p and for its targeting mechanism to mitochondria and cytosol/nucleus will be discussed

Gene products and processes contributing to lanthanide homeostasis and methanol metabolism in \u3cem\u3eMethylorubrum extorquens\u3c/em\u3e AM1

Lanthanide elements have been recently recognized as “new life metals” yet much remains unknown regarding lanthanide acquisition and homeostasis. In Methylorubrum extorquens AM1, the periplasmic lanthanide-dependent methanol dehydrogenase XoxF1 produces formaldehyde, which is lethal if allowed to accumulate. This property enabled a transposon mutagenesis study and growth studies to confirm novel gene products required for XoxF1 function. The identified genes encode an MxaD homolog, an ABC-type transporter, an aminopeptidase, a putative homospermidine synthase, and two genes of unknown function annotated as orf6 and orf7. Lanthanide transport and trafficking genes were also identified. Growth and lanthanide uptake were measured using strains lacking individual lanthanide transport cluster genes, and transmission electron microscopy was used to visualize lanthanide localization. We corroborated previous reports that a TonB-ABC transport system is required for lanthanide incorporation to the cytoplasm. However, cells were able to acclimate over time and bypass the requirement for the TonB outer membrane transporter to allow expression of xoxF1 and growth. Transcriptional reporter fusions show that excess lanthanides repress the gene encoding the TonB-receptor. Using growth studies along with energy dispersive X-ray spectroscopy and transmission electron microscopy, we demonstrate that lanthanides are stored as cytoplasmic inclusions that resemble polyphosphate granules

Chloroplast HCF101 is a scaffold protein for [4Fe-4S] cluster assembly

Oxygen-evolving chloroplasts possess their own iron-sulfur cluster assembly proteins including members of the SUF (sulfur mobilization) and the NFU family. Recently, the chloroplast protein HCF101 (high chlorophyll fluorescence 101) has been shown to be essential for the accumulation of the membrane complex Photosystem I and the soluble ferredoxin-thioredoxin reductases, both containing [4Fe-4S] clusters. The protein belongs to the FSC-NTPase ([4Fe-4S]-cluster-containing P-loop NTPase) superfamily, several members of which play a crucial role in Fe/S cluster biosynthesis. Although the C-terminal ISC-binding site, conserved in other members of the FSC-NTPase family, is not present in chloroplast HCF101 homologues using Mössbauer and EPR spectroscopy, we provide evidence that HCF101 binds a [4Fe-4S] cluster. 55Fe incorporation studies of mitochondrially targeted HCF101 in Saccharomyces cerevisiae confirmed the assembly of an Fe/S cluster in HCF101 in an Nfs1-dependent manner. Site-directed mutagenesis identified three HCF101-specific cysteine residues required for assembly and/or stability of the cluster. We further demonstrate that the reconstituted cluster is transiently bound and can be transferred from HCF101 to a [4Fe-4S] apoprotein. Together, our findings suggest that HCF101 may serve as a chloroplast scaffold protein that specifically assembles [4Fe-4S] clusters and transfers them to the chloroplast membrane and soluble target proteins

Supplemental Information 3: Ribosomal protein S3 protein alignment

Annually, half of all plant-derived carbon is added to soil where it is microbially respired to CO2. However, understanding of the microbiology of this process is limited because most culture-independent methods cannot link metabolic processes to the organisms present, and this link to causative agents is necessary to predict the results of perturbations on the system. We collected soil samples at two sub-root depths (10–20 cm and 30–40 cm) before and after a rainfall-driven nutrient perturbation event in a Northern California grassland that experiences a Mediterranean climate. From ten samples, we reconstructed 198 metagenome-assembled genomes that represent all major phylotypes. We also quantified 6,835 proteins and 175 metabolites and showed that after the rain event the concentrations of many sugars and amino acids approach zero at the base of the soil profile. Unexpectedly, the genomes of novel members of the Gemmatimonadetes and Candidate Phylum Rokubacteria phyla encode pathways for methylotrophy. We infer that these abundant organisms contribute substantially to carbon turnover in the soil, given that methylotrophy proteins were among the most abundant proteins in the proteome. Previously undescribed Bathyarchaeota and Thermoplasmatales archaea are abundant in deeper soil horizons and are inferred to contribute appreciably to aromatic amino acid degradation. Many of the other bacteria appear to breakdown other components of plant biomass, as evidenced by the prevalence of various sugar and amino acid transporters and corresponding hydrolyzing machinery in the proteome. Overall, our work provides organism-resolved insight into the spatial distribution of bacteria and archaea whose activities combine to degrade plant-derived organics, limiting the transport of methanol, amino acids and sugars into underlying weathered rock. The new insights into the soil carbon cycle during an intense period of carbon turnover, including biogeochemical roles to previously little known soil microbes, were made possible via the combination of metagenomics, proteomics, and metabolomics

Analysis of Hypoxia and Hypoxia-Like States through Metabolite Profiling

In diverse organisms, adaptation to low oxygen (hypoxia) is mediated through complex gene expression changes that can, in part, be mimicked by exposure to metals such as cobalt. Although much is known about the transcriptional response to hypoxia and cobalt, little is known about the all-important cell metabolism effects that trigger these responses.Herein we use a low molecular weight metabolome profiling approach to identify classes of metabolites in yeast cells that are altered as a consequence of hypoxia or cobalt exposures. Key findings on metabolites were followed-up by measuring expression of relevant proteins and enzyme activities. We find that both hypoxia and cobalt result in a loss of essential sterols and unsaturated fatty acids, but the basis for these changes are disparate. While hypoxia can affect a variety of enzymatic steps requiring oxygen and heme, cobalt specifically interferes with diiron-oxo enzymatic steps for sterol synthesis and fatty acid desaturation. In addition to diiron-oxo enzymes, cobalt but not hypoxia results in loss of labile 4Fe-4S dehydratases in the mitochondria, but has no effect on homologous 4Fe-4S dehydratases in the cytosol. Most striking, hypoxia but not cobalt affected cellular pools of amino acids. Amino acids such as aromatics were elevated whereas leucine and methionine, essential to the strain used here, dramatically decreased due to hypoxia induced down-regulation of amino acid permeases.These studies underscore the notion that cobalt targets a specific class of iron proteins and provide the first evidence for hypoxia effects on amino acid regulation. This research illustrates the power of metabolite profiling for uncovering new adaptations to environmental stress

Systems Biology of the qa Gene Cluster in Neurospora crassa

An ensemble of genetic networks that describe how the model fungal system, Neurospora crassa, utilizes quinic acid (QA) as a sole carbon source has been identified previously. A genetic network for QA metabolism involves the genes, qa-1F and qa-1S, that encode a transcriptional activator and repressor, respectively and structural genes, qa-2, qa-3, qa-4, qa-x, and qa-y. By a series of 4 separate and independent, model-guided, microarray experiments a total of 50 genes are identified as QA-responsive and hypothesized to be under QA-1F control and/or the control of a second QA-responsive transcription factor (NCU03643) both in the fungal binuclear Zn(II)2Cys6 cluster family. QA-1F regulation is not sufficient to explain the quantitative variation in expression profiles of the 50 QA-responsive genes. QA-responsive genes include genes with products in 8 mutually connected metabolic pathways with 7 of them one step removed from the tricarboxylic (TCA) Cycle and with 7 of them one step removed from glycolysis: (1) starch and sucrose metabolism; (2) glycolysis/glucanogenesis; (3) TCA Cycle; (4) butanoate metabolism; (5) pyruvate metabolism; (6) aromatic amino acid and QA metabolism; (7) valine, leucine, and isoleucine degradation; and (8) transport of sugars and amino acids. Gene products both in aromatic amino acid and QA metabolism and transport show an immediate response to shift to QA, while genes with products in the remaining 7 metabolic modules generally show a delayed response to shift to QA. The additional QA-responsive cutinase transcription factor-1β (NCU03643) is found to have a delayed response to shift to QA. The series of microarray experiments are used to expand the previously identified genetic network describing the qa gene cluster to include all 50 QA-responsive genes including the second transcription factor (NCU03643). These studies illustrate new methodologies from systems biology to guide model-driven discoveries about a core metabolic network involving carbon and amino acid metabolism in N. crassa

On being angry and punitive: How anger alters perception of criminal intent

Previous research has demonstrated that anger increases the tendency to blame and punish others for harmful behaviors. This study investigated whether such attributions extend to judgments of criminal intent, and it examined the mechanisms by which anger influences punitiveness. In an experiment, angry, sad, and neutral participants read about an ambiguously criminal behavior. As hypothesized, angry participants judged the behavior as being more intentional and the perpetrator as having more causal control than did neutral participants, and they were more willing to punish the wrongdoer. Sadness did not have a demonstrable effect on judgments, indicating a specific role of anger rather than a general negative affect. Moreover, the effect of anger on punitiveness was mediated by perceived criminal intent but not by perceived causal control. Implications for legal judgments and theories of blame attribution are discussed