Most polluted sites contain mixed waste. This is especially true of the U.S. Department of Energy (DOE) waste sites which hold a complex mixture of heavy metals, radionuclides, and organic solvents. ...In such environments enzymes that can remediate multiple pollutants are advantageous. We report here evolution of an enzyme, ChrR6 (formerly referred to as Y6), which shows a markedly enhanced capacity for remediating two of the most serious and prevalent DOE contaminants, chromate and uranyl. ChrR6 is a soluble enzyme and reduces chromate and uranyl intracellularly. Thus, the reduced product is at least partially sequestered and nucleated, minimizing the chances of reoxidation. Only one amino acid change, Tyr128Asn, was responsible for the observed improvement. We show here that ChrR6 makes Pseudomonas putida and Escherichia coli more efficient agents for bioremediation if the cellular permeability barrier to the metals is decreased.
Summary
Chromate Cr(VI) is a serious environmental pollutant, which is amenable to bacterial bioremediation. NfsA, the major oxygen‐insensitive nitroreductase of Escherichia coli, is a flavoprotein ...that is able to reduce chromate to less soluble and less toxic Cr(III). We show that this process involves single‐electron transfer, giving rise to a flavin semiquinone form of NfsA and Cr(V) as intermediates, which redox cycle, generating more reactive oxygen species (ROS) than a divalent chromate reducer, YieF. However, NfsA generates less ROS than a known one‐electron chromate reducer, lipoyl dehydrogenase (LpDH), suggesting that NfsA employs a mixture of uni‐ and di‐valent electron transfer steps. The presence of YieF, ChrR (another chromate reductase we previously characterized), or NfsA in an LpDH‐catalysed chromate reduction reaction decreased ROS generation by c. 65, 40, or 20%, respectively, suggesting that these enzymes can pre‐empt ROS generation by LpDH. We previously showed that ChrR protects Pseudomonas putida against chromate toxicity; here we show that NfsA or YieF overproduction can also increase the tolerance of E. coli to this compound.
Gene-directed enzyme prodrug therapy (GDEPT) aims to achieve highly selective tumor-cell killing through the use of tumor-tropic gene delivery vectors coupled with systemic administration of ...otherwise inert prodrugs. Nitroaromatic prodrugs such as CB1954 hold promise for GDEPT as they are readily reduced to potent DNA alkylating agents by bacterial nitroreductase enzymes (NTRs). Transfection with the
nfsB gene from
Escherichia coli can increase the sensitivity of tumor cells to CB1954 by greater than 1000-fold. However, poor catalytic efficiency limits the activation of CB1954 by NfsB at clinically relevant doses. A lack of flexible, high-throughput screening technology has hindered efforts to discover superior NTR candidates. Here we demonstrate how the SOS chromotest and complementary screening technologies can be used to evaluate novel enzymes that activate CB1954 and other bioreductive and/or genotoxic prodrugs. We identify the major
E. coli NTR, NfsA, as 10-fold more efficient than NfsB in activating CB1954 as purified protein (
k
cat/
K
m) and when over-expressed in an
E. coli
nfsA
−/
nfsB
− gene deleted strain. NfsA also confers sensitivity to CB1954 when expressed in HCT-116 human colon carcinoma cells, with similar efficiency to NfsB. In addition, we identify two novel
E. coli NTRs, AzoR and NemA, that have not previously been characterized in the context of nitroaromatic prodrug activation.
Climates of the past, such as the Last Glacial Maximum (LGM), give a useful insight into the response of atmosphere and ocean processes to various forcings that were different to the present day. The ...use of General Circulation Models aids in understanding past climates in combination with the available proxy data. In this study, a selection of models from the Paleoclimate Modelling Intercomparison Project was used to assess the changes in Southern Hemisphere atmospheric variability on seasonal to interannual time-scales. The evidence from two of the models suggests a seasonal deepening of the circumpolar trough (CPT) during the LGM relative to present day, with enhanced tropospheric westerlies in all seasons. The third model suggested there was a weakening of the CPT in all seasons except austral spring, where a strengthening was simulated. Finally, the work considers how the Southern Annular Mode may have been different in the LGM for the three models under consideration.
Summary
The single‐module non‐ribosomal peptide synthetase BpsA from Streptomyces lavendulae has the unique ability to autonomously synthesize a coloured product (indigoidine) from a single substrate ...(l‐glutamine), conditional upon activation by a 4′‐phosphopantetheinyl transferase (PPTase) partner. We show that bpsA can be expressed in an entD PPTase gene deleted mutant of Escherichia coli to yield a sensitive reporter strain for recovery of PPTase genes from metagenome libraries. We also show that recombinant bpsA constructs, generated by substitution of the native peptidyl carrier protein domain followed by directed evolution to restore function, can be used to increase the diversity of PPTase genes recovered from a sample. As PPTases are essential for activation of non‐ribosomal peptide synthetase and polyketide synthase enzymes, they are frequently associated with secondary metabolite gene clusters. Nearly half of the PPTases recovered in our screening of two small‐insert soil metagenome libraries were genetically linked to recognizable secondary metabolite biosynthetic genes, demonstrating that PPTase‐targeting functional screens can be used for efficient recovery of natural product gene clusters from metagenome libraries. The plasticity and portability of bpsA reporter genes can potentially be exploited to maximize recovery and expression of PPTase‐bearing clones in a wide range of hosts.
Engineering of enzymes to more efficiently activate genotoxic prodrugs holds great potential for improving anticancer gene or antibody therapies. We report the development of a new, GFP-based, ...high-throughput screening platform to enable engineering of prodrug-activating enzymes by directed evolution. By fusing an inducible SOS promoter to an engineered GFP reporter gene, we were able to measure levels of DNA damage in intact Escherichia coli and separate cell populations by fluorescence activating cell sorting (FACS). In two FACS iterations, we were able to achieve a 90 000-fold enrichment of a functional prodrug-activating nitroreductase from a null library background.
Tropical precipitation is caused by many processes that occur over a wide range of temporal and spatial scales. Such processes vary from local, diurnal convection driven by a destabilisation of the ...boundary layer to planetary‐scale systems that result in rainfall over many days. It is therefore important to assess whether general circulation models (GCMs) can represent these processes given that such models are routinely used to project future rainfall in the low latitudes. In this study, we evaluate the rainfall and circulation characteristics of ten GCMs from the Coupled Model Intercomparison Project Phase 5 (CMIP5) over northern Australia. This work shows that the diurnal cycle of the low‐level (925 hPa) flow around the heat low is represented well by the models but the timing of precipitation is not (triggered too early). There is also evidence that mid‐level synoptic systems that are responsible for initiating rain in the observations are also present in all of the models. Nevertheless, the biases in the modelled seasonal mean precipitation seem to be linked to the strength of both the meridional flow into northern Australia and the vertical mass flux. Furthermore, there is also evidence that the representation of convection in these models is likely contributing to both the precipitation and circulation errors over northern Australia.
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