Horizontal 'gene drives' harness indigenous bacteria for bioremediation.

Engineering bacteria to clean-up oil spills is rapidly advancing but faces regulatory hurdles and environmental concerns. Here, we develop a new technology to harness indigenous soil microbial communities for bioremediation by flooding local populations with catabolic genes for petroleum hydrocarbon degradation. Overexpressing three enzymes (almA, xylE, p450cam) in Escherichia coli led to degradation of 60-99% of target hydrocarbon substrates. Mating experiments, fluorescence microscopy and TEM revealed indigenous bacteria could obtain these vectors from E. coli through several mechanisms of horizontal gene transfer (HGT), including conjugation and cytoplasmic exchange through nanotubes. Inoculating petroleum-polluted sediments with E. coli carrying the vector pSF-OXB15-p450camfusion showed that the E. coli cells died after five days but a variety of bacteria received and carried the vector for over 60 days after inoculation. Within 60 days, the total petroleum hydrocarbon content of the polluted soil was reduced by 46%. Pilot experiments show that vectors only persist in indigenous populations when under selection pressure, disappearing when this carbon source is removed. This approach to remediation could prime indigenous bacteria for degrading pollutants while providing minimal ecosystem disturbance.

Using Predefined M3(µ3-O) Clusters as Building Blocks for an Isostructural Series of Metal-Organic Frameworks.

Metal-organic frameworks (MOFs) have attracted much attention in the past decade owing to their unprecedented internal surface areas, tunable topologies, designable surfaces, and various potential applications. One bottleneck in the field regarding MOF synthesis is controlling the metal-containing secondary building unit (SBU) incorporated into the structure. In this work we report the synthesis and characterization of five trimeric [M3(µ3-O)(CH3CO2)6]x clusters (where M = Fe3+, Cr3+, Fe3+/Cr3+, Fe3+/Co2+, or Fe3+/Ni2+ and x = +1 or 0). The monocarboxylate capping ligand, acetate in this case, readily undergoes exchange with several difunctional counterparts, including 1,4-benzenedicarboxylic acid (H2-BDC) and biphenyl-4,4'-dicarboxylic acid (H2-BPDC), for the formation of an isostructural series of MOFs, several of which are newly reported (for M = Fe3+/Cr3+, Fe3+/Co2+, and Fe3+/Ni2+) and show excellent CO2 adsorption properties. In this report, a host of techniques including NMR, ICP, and ESI-MS are used to probe the ligand exchange process and composition of the SBUs, and XAS is used to monitor the Fe3+ and Cr3+ environment throughout the reactions, giving strong evidence that the clusters stay intact throughout the MOF synthesis. This work reveals that predefined SBUs is an effective means to create metal-substituted analogues of known frameworks. Further, CO adsorption and in situ IR are used to probe accessibility of the metals after solvent removal. We show for the first time that the incorporation of the neutral clusters, containing weaker Lewis acids like Ni2+ and Co2+, can promote the formation of open metal sites in the MOF frameworks, structural features known to enhance the binding energy of small guest molecules like CO2.

Structures and cytotoxic evaluation of new and known acyclic Ene-Ynes from an American Samoa Petrosia sp. Sponge.

Four new compounds, (-)-petrosynoic acids A-D (1-4), and five known congeners, pellynols A (5), C (6), D (7), F (8), and I (9), were isolated from a Petrosia sp. marine sponge collected in American Samoa. Isolation work was guided by cytotoxicity against human lung cancer cells (H460). The structures of the C31-C33 polyacetylenes (1-9) were determined on the basis of 1D- and 2D-NMR analysis, mass spectrometry, and comparison of specific rotation values. Compounds 1-9 were found to be broadly cytotoxic with limited selectivity for cancer cells, as they were all moderately active against the A2058 (melanoma), H522-T1 (lung), and H460 (lung) human cancer cell lines as well as IMR-90 quiescent human fibroblast cells.

Bacillus cereus iron uptake protein fishes out an unstable ferric citrate trimer.

Citrate is a common biomolecule that chelates Fe(III). Many bacteria and plants use ferric citrate to fulfill their nutritional requirement for iron. Only the Escherichia coli ferric citrate outer-membrane transport protein FecA has been characterized; little is known about other ferric citrate-binding proteins. Here we report a unique siderophore-binding protein from the gram-positive pathogenic bacterium Bacillus cereus that binds multinuclear ferric citrate complexes. We have demonstrated that B. cereus ATCC 14579 takes up (55)Fe radiolabeled ferric citrate and that a protein, BC_3466 [renamed FctC (ferric citrate-binding protein C)], binds ferric citrate. The dissociation constant (K(d)) of FctC at pH 7.4 with ferric citrate (molar ratio 1:50) is 2.6 nM. This is the tightest binding observed of any B. cereus siderophore-binding protein. Nano electrospray ionization-mass spectrometry (nano ESI-MS) analysis of FctC and ferric citrate complexes or citrate alone show that FctC binds diferric di-citrate, and triferric tricitrate, but does not bind ferric di-citrate, ferric monocitrate, or citrate alone. Significantly, the protein selectively binds triferric tricitrate even though this species is naturally present at very low equilibrium concentrations.

A previously undescribed pathway for pyrimidine catabolism.

The b1012 operon of Escherichia coli K-12, which is composed of seven unidentified ORFs, is one of the most highly expressed operons under control of nitrogen regulatory protein C. Examination of strains with lesions in this operon on Biolog Phenotype MicroArray (PM3) plates and subsequent growth tests indicated that they failed to use uridine or uracil as the sole nitrogen source and that the parental strain could use them at room temperature but not at 37 degrees C. A strain carrying an ntrB(Con) mutation, which elevates transcription of genes under nitrogen regulatory protein C control, could also grow on thymidine as the sole nitrogen source, whereas strains with lesions in the b1012 operon could not. Growth-yield experiments indicated that both nitrogens of uridine and thymidine were available. Studies with [(14)C]uridine indicated that a three-carbon waste product from the pyrimidine ring was excreted. After trimethylsilylation and gas chromatography, the waste product was identified by mass spectrometry as 3-hydroxypropionic acid. In agreement with this finding, 2-methyl-3-hydroxypropionic acid was released from thymidine. Both the number of available nitrogens and the waste products distinguished the pathway encoded by the b1012 operon from pyrimidine catabolic pathways described previously. We propose that the genes of this operon be named rutA-G for pyrimidine utilization. The product of the divergently transcribed gene, b1013, is a tetracycline repressor family regulator that controls transcription of the b1012 operon negatively.