Mechanism for nitrogen isotope fractionation during ammonium assimilation by Escherichia coli K12

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of National Academy of Sciences for personal use, not for redistribution. The definitive version was published in Proceedings of the National Academy of Sciences of the United States of America 110 (2013): 8696-8701, doi:10.1073/pnas.1216683110.Organisms that use ammonium as the sole nitrogen source discriminate between [15N] and [14N] ammonium. This selectivity leaves an isotopic signature in their biomass that depends on the external concentration of ammonium. To dissect how differences in discrimination arise molecularly, we examined a wild-type (WT) strain of Escherichia coli K12 and mutant strains with lesions affecting ammonium-assimilatory proteins. We used isotope ratio mass spectrometry (MS) to assess the nitrogen isotopic composition of cell material when the strains were grown in batch culture at either high or low external concentrations of NH3 (achieved by controlling total NH4Cl and pH of the medium). At high NH3 (≥0.89 µM), discrimination against the heavy isotope by the WT strain (−19.2‰) can be accounted for by the equilibrium isotope effect for dissociation of NH4+ to NH3 + H+. NH3 equilibrates across the cytoplasmic membrane, and glutamine synthetase does not manifest an isotope effect in vivo. At low NH3 (≤0.18 µM), discrimination reflects an isotope effect for the NH4+ channel AmtB (−14.1‰). By making E. coli dependent on the low-affinity ammonium-assimilatory pathway, we determined that biosynthetic glutamate dehydrogenase has an inverse isotope effect in vivo (+8.8‰). Likewise, by making unmediated diffusion of NH3 across the cytoplasmic membrane rate-limiting for cell growth in a mutant strain lacking AmtB, we could deduce an in vivo isotope effect for transport of NH3 across the membrane (−10.9‰). The paper presents the raw data from which our conclusions were drawn and discusses the assumptions underlying them.This work was supported by NIH grant GM38361 to S.K. J.M.H. thanks WHOI for support as an emeritus scientist.2013-11-0

Increased incidence of traffic accidents in Toxoplasma-infected military drivers and protective effect RhD molecule revealed by a large-scale prospective cohort study

<p>Abstract</p> <p>Background</p> <p>Latent toxoplasmosis, protozoan parasitosis with prevalence rates from 20 to 60% in most populations, is known to impair reaction times in infected subjects, which results, for example, in a higher risk of traffic accidents in subjects with this life-long infection. Two recent studies have reported that RhD-positive subjects, especially RhD heterozygotes, are protected against latent toxoplasmosis-induced impairment of reaction times. In the present study we searched for increased incidence of traffic accidents and for protective effect of RhD positivity in 3890 military drivers.</p> <p>Methods</p> <p>Male draftees who attended the Central Military Hospital in Prague for regular entrance psychological examinations between 2000 and 2003 were tested for <it>Toxoplasma </it>infection and RhD phenotype at the beginning of their 1 to1.5-year compulsory military service. Subsequently, the data on <it>Toxoplasma </it>infection and RhD phenotype were matched with those on traffic accidents from military police records and the effects of RhD phenotype and <it>Toxoplasma </it>infection on probability of traffic accident was estimated with logistic regression.</p> <p>Results</p> <p>We confirmed, using for the first time a prospective cohort study design, increased risk of traffic accidents in <it>Toxoplasma</it>-infected subjects and demonstrated a strong protective effect of RhD positivity against the risk of traffic accidents posed by latent toxoplasmosis. Our results show that RhD-negative subjects with high titers of anti-<it>Toxoplasma </it>antibodies had a probability of a traffic accident of about 16.7%, i.e. a more than six times higher rate than <it>Toxoplasma</it>-free or RhD-positive subjects.</p> <p>Conclusion</p> <p>Our results showed that a common infection by <it>Toxoplasma gondii </it>could have strong impact on the probability of traffic accident in RhD negative subjects. The observed effects could provide not only a clue to the long-standing evolutionary enigma of the origin of RhD polymorphism in humans (the effect of balancing selection), but might also be the missing piece in the puzzle of the physiological function of the RhD molecule.</p

Multiple controls affect arsenite oxidase gene expression in Herminiimonas arsenicoxydans

<p>Abstract</p> <p>Background</p> <p>Both the speciation and toxicity of arsenic are affected by bacterial transformations, i.e. oxidation, reduction or methylation. These transformations have a major impact on environmental contamination and more particularly on arsenic contamination of drinking water. <it>Herminiimonas arsenicoxydans </it>has been isolated from an arsenic- contaminated environment and has developed various mechanisms for coping with arsenic, including the oxidation of As(III) to As(V) as a detoxification mechanism.</p> <p>Results</p> <p>In the present study, a differential transcriptome analysis was used to identify genes, including arsenite oxidase encoding genes, involved in the response of <it>H. arsenicoxydans </it>to As(III). To get insight into the molecular mechanisms of this enzyme activity, a Tn<it>5 </it>transposon mutagenesis was performed. Transposon insertions resulting in a lack of arsenite oxidase activity disrupted <it>aoxR </it>and <it>aoxS </it>genes, showing that the <it>aox </it>operon transcription is regulated by the AoxRS two-component system. Remarkably, transposon insertions were also identified in <it>rpoN </it>coding for the alternative N sigma factor (σ⁵⁴) of RNA polymerase and in <it>dnaJ </it>coding for the Hsp70 co-chaperone. Western blotting with anti-AoxB antibodies and quantitative RT-PCR experiments allowed us to demonstrate that the <it>rpoN </it>and <it>dnaJ </it>gene products are involved in the control of arsenite oxidase gene expression. Finally, the transcriptional start site of the <it>aoxAB </it>operon was determined using rapid amplification of cDNA ends (RACE) and a putative -12/-24 σ⁵⁴-dependent promoter motif was identified upstream of <it>aoxAB </it>coding sequences.</p> <p>Conclusion</p> <p>These results reveal the existence of novel molecular regulatory processes governing arsenite oxidase expression in <it>H. arsenicoxydans</it>. These data are summarized in a model that functionally integrates arsenite oxidation in the adaptive response to As(III) in this microorganism.</p

Using Genomic Sequencing for Classical Genetics in E. coli K12

We here develop computational methods to facilitate use of 454 whole genome shotgun sequencing to identify mutations in Escherichia coli K12. We had Roche sequence eight related strains derived as spontaneous mutants in a background without a whole genome sequence. They provided difference tables based on assembling each genome to reference strain E. coli MG1655 (NC_000913). Due to the evolutionary distance to MG1655, these contained a large number of both false negatives and positives. By manual analysis of the dataset, we detected all the known mutations (24 at nine locations) and identified and genetically confirmed new mutations necessary and sufficient for the phenotypes we had selected in four strains. We then had Roche assemble contigs de novo, which we further assembled to full-length pseudomolecules based on synteny with MG1655. This hybrid method facilitated detection of insertion mutations and allowed annotation from MG1655. After removing one genome with less than the optimal 20- to 30-fold sequence coverage, we identified 544 putative polymorphisms that included all of the known and selected mutations apart from insertions. Finally, we detected seven new mutations in a total of only 41 candidates by comparing single genomes to composite data for the remaining six and using a ranking system to penalize homopolymer sequencing and misassembly errors. An additional benefit of the analysis is a table of differences between MG1655 and a physiologically robust E. coli wild-type strain NCM3722. Both projects were greatly facilitated by use of comparative genomics tools in the CoGe software package (http://genomevolution.org/)

Achieving Optimal Growth through Product Feedback Inhibition in Metabolism

Recent evidence suggests that the metabolism of some organisms, such as Escherichia coli, is remarkably efficient, producing close to the maximum amount of biomass per unit of nutrient consumed. This observation raises the question of what regulatory mechanisms enable such efficiency. Here, we propose that simple product-feedback inhibition by itself is capable of leading to such optimality. We analyze several representative metabolic modules—starting from a linear pathway and advancing to a bidirectional pathway and metabolic cycle, and finally to integration of two different nutrient inputs. In each case, our mathematical analysis shows that product-feedback inhibition is not only homeostatic but also, with appropriate feedback connections, can minimize futile cycling and optimize fluxes. However, the effectiveness of simple product-feedback inhibition comes at the cost of high levels of some metabolite pools, potentially associated with toxicity and osmotic imbalance. These large metabolite pool sizes can be restricted if feedback inhibition is ultrasensitive. Indeed, the multi-layer regulation of metabolism by control of enzyme expression, enzyme covalent modification, and allostery is expected to result in such ultrasensitive feedbacks. To experimentally test whether the qualitative predictions from our analysis of feedback inhibition apply to metabolic modules beyond linear pathways, we examine the case of nitrogen assimilation in E. coli, which involves both nutrient integration and a metabolic cycle. We find that the feedback regulation scheme suggested by our mathematical analysis closely aligns with the actual regulation of the network and is sufficient to explain much of the dynamical behavior of relevant metabolite pool sizes in nutrient-switching experiments

Comparative analyses imply that the enigmatic sigma factor 54 is a central controller of the bacterial exterior

Contains fulltext : 95738.pdf (publisher's version ) (Open Access)BACKGROUND: Sigma-54 is a central regulator in many pathogenic bacteria and has been linked to a multitude of cellular processes like nitrogen assimilation and important functional traits such as motility, virulence, and biofilm formation. Until now it has remained obscure whether these phenomena and the control by Sigma-54 share an underlying theme. RESULTS: We have uncovered the commonality by performing a range of comparative genome analyses. A) The presence of Sigma-54 and its associated activators was determined for all sequenced prokaryotes. We observed a phylum-dependent distribution that is suggestive of an evolutionary relationship between Sigma-54 and lipopolysaccharide and flagellar biosynthesis. B) All Sigma-54 activators were identified and annotated. The relation with phosphotransfer-mediated signaling (TCS and PTS) and the transport and assimilation of carboxylates and nitrogen containing metabolites was substantiated. C) The function annotations, that were represented within the genomic context of all genes encoding Sigma-54, its activators and its promoters, were analyzed for intra-phylum representation and inter-phylum conservation. Promoters were localized using a straightforward scoring strategy that was formulated to identify similar motifs. We found clear highly-represented and conserved genetic associations with genes that concern the transport and biosynthesis of the metabolic intermediates of exopolysaccharides, flagella, lipids, lipopolysaccharides, lipoproteins and peptidoglycan. CONCLUSION: Our analyses directly implicate Sigma-54 as a central player in the control over the processes that involve the physical interaction of an organism with its environment like in the colonization of a host (virulence) or the formation of biofilm

Fate of water pumped from underground and contributions to sea-level rise

The contributions from terrestrial water sources to sea-level rise, other than ice caps and glaciers, are highly uncertain and heavily debated.. Recent assessments indicate that groundwater depletion (GWD) may become the most important positive terrestrial contribution over the next 50 years, probably equal in magnitude to the current contributions from glaciers and ice caps6. However, the existing estimates assume that nearly 100% of groundwater extracted eventually ends up in the oceans. Owing to limited knowledge of the pathways and mechanisms governing the ultimate fate of pumped groundwater, the relative fraction of global GWD that contributes to sea-level rise remains unknown. Here, using a coupled climate–hydrological model simulation, we show that only 80% of GWD ends up in the ocean. An increase in runoff to the ocean accounts for roughly two-thirds, whereas the remainder results from the enhanced net flux of precipitation minus evaporation over the ocean, due to increased atmospheric vapour transport from the land to the ocean. The contribution of GWD to global sea-level rise amounted to 0.02 (±0.004) mm yr−1 in 1900 and increased to 0.27 (±0.04) mm yr−1 in 2000. This indicates that existing studies have substantially overestimated the contribution of GWD to global sea-level rise by a cumulative amount of at least 10 mm during the twentieth century and early twenty-first century. With other terrestrial water contributions included, we estimate the net terrestrial water contribution during the period 1993–2010 to be +0.12 (±0.04) mm yr−1, suggesting that the net terrestrial water contribution reported in the IPCC Fifth Assessment Report report is probably overestimated by a factor of three

Possible Link Between Irrigation in the U.S. High Plains and Increased Summer Streamflow in the Midwest

We have previously presented evidence that higher rates of evapotranspiration (ET) associated with irrigation in the U.S. High Plains has likely caused an increased downwind precipitation (P). July P over the Midwest increased by 20%-30% from the pre-irrigation period (1900-1950) to the post-irrigation (1950-2000) period. In this study, we test the hypothesis that the increased July P has had hydrologic consequences, possibly increasing groundwater storage and streamflow. Seasonal analyses of hydrologic variables over Illinois suggest that the water table and streamflow response lags P - ET by 1-2 months, indicating August and September as the months when the increased July P may be detected. We analyzed long-term observations of water table depth at 10 wells in Illinois and streamflow at 46 gauges in Illinois-Ohio basins. The Mann-Kendal test for trends suggests field significant increases in groundwater storage and streamflow in August-September over the period of irrigation expansion. Examination of soil moisture response to present-day above-normal July P suggests that the increased July P can reach the water table in normal to wet years. Mann-Kendall tests suggest that there has been no change in pan evaporation and atmospheric vapor pressure deficit. This implies that soil water availability is the driver of changes in ET, and the increased P may have possibly increased ET. Other studies in the literature give further evidence of increased ET due to increased P. By ruling out a reduction in ET, we suggest that the observed increase in groundwater storage and streamflow in the Midwest is linked to the increased July precipitation attributed to High Plains irrigation. We note that the increases in late summer streamflow are rather small when placed in the context of seasonal dynamics, but they are conceptually important in that they point to a different cause of change

Prokaryotic enhancer-binding proteins reflect eukaryote-like modularity: the puzzle of nitrogen regulatory protein C.

Eukaryotic enhancer-binding proteins are often modular in design in that they are composed of physically separable domains that can function independently of one another (18, 46, 55). Detailed analyses have identified regions involved in specific DNA recognition and others involved in activation of transcription. In fact, in many instances, combining a domain from one protein with a second domain from another has produced a chimeric protein that demonstrates the expected functional properties of each parent (3, 19, 21, 22). As outlined below, members of a family of prokaryotic enhancer-binding proteins are also modular in structure. However, one member of the family, the NTRC protein (nitrogen regulatory protein C; also called NRI) of enteric bacteria, is apparently an exception. Comparison of the sequence of NTRC with that of other activators and with the sequence and structure of the factor for inversion stimulation (FIS) reveals a likely explanation for the puzzling properties of NTRC