Hierarchical modularity and the evolution of genetic interactomes across species.

To date, cross-species comparisons of genetic interactomes have been restricted to small or functionally related gene sets, limiting our ability to infer evolutionary trends. To facilitate a more comprehensive analysis, we constructed a genome-scale epistasis map (E-MAP) for the fission yeast Schizosaccharomyces pombe, providing phenotypic signatures for ~60% of the nonessential genome. Using these signatures, we generated a catalog of 297 functional modules, and we assigned function to 144 previously uncharacterized genes, including mRNA splicing and DNA damage checkpoint factors. Comparison with an integrated genetic interactome from the budding yeast Saccharomyces cerevisiae revealed a hierarchical model for the evolution of genetic interactions, with conservation highest within protein complexes, lower within biological processes, and lowest between distinct biological processes. Despite the large evolutionary distance and extensive rewiring of individual interactions, both networks retain conserved features and display similar levels of functional crosstalk between biological processes, suggesting general design principles of genetic interactomes.

Roles of the A and C sites in the manganese-specific activation of MntR.

The manganese transport regulator (MntR) represses the expression of genes involved in manganese uptake in Bacillus subtilis. It selectively responds to Mn(2+) and Cd(2+) over other divalent metal cations, including Fe(2+), Co(2+), and Zn(2+). Previous work has shown that MntR forms binuclear complexes with Mn(2+) or Cd(2+) at two binding sites, labeled A and C, that are separated by 4.4 Å. Zinc activates MntR poorly and binds only to the A site, forming a mononuclear complex. The difference in metal binding stoichiometry suggested a mechanism for selectivity in MntR. Larger metal cations are strongly activating because they can form the binuclear complex, while smaller metal ions cannot bind with the geometry needed to fully occupy both metal binding sites. To investigate this hypothesis, structures of MntR in complex with two other noncognate metal ions, Fe(2+) and Co(2+), have been determined. Each metal forms a mononuclear complex with MntR with the metal ion bound in the A site, supporting the conclusions drawn from the Zn(2+) complex. Additionally, we investigated two site-specific mutants of MntR, E11K and H77A, that contain substitutions of metal binding residues in the A site. While metal binding in each mutant is significantly altered relative to that of wild-type MntR, both mutants retain activity and selectivity for Mn(2+) in vitro and in vivo. That observation, coupled with previous studies, suggests that the A and C sites both contribute to the selectivity of MntR.

Characterization and structure of the manganese-responsive transcriptional regulator ScaR.

The streptococcal coaggregation regulator (ScaR) of Streptococcus gordonii is a manganese-dependent transcriptional regulator. When intracellular manganese concentrations become elevated, ScaR represses transcription of the scaCBA operon, which encodes a manganese uptake transporter. A member of the DtxR/MntR family of metalloregulators, ScaR shares sequence similarity with other family members, and many metal-binding residues are conserved. Here, we show that ScaR is an active dimer, with two dimers binding the 46 base pair scaC operator. Each ScaR subunit binds two manganese ions, and the protein is activated by a variety of other metal ions, including Cd(2+), Co(2+), and Ni(2+) but not Zn(2+). The crystal structure of apo-ScaR reveals a tertiary and quaternary structure similar to its homologue, the iron-responsive regulator DtxR. While each DtxR subunit binds a metal ion in two sites, labeled primary and ancillary, crystal structures of ScaR determined in the presence of Cd(2+) and Zn(2+) show only a single occupied metal-binding site that is novel to ScaR. The site analogous to the primary site in DtxR is unoccupied, and the ancillary site is absent from ScaR. Instead, metal ions bind to ScaR at a site labeled "secondary", which is composed of Glu80, Cys123, His125, and Asp160 and lies roughly 5 A away from where the ancillary site would be predicted to exist. This difference suggests that ScaR and its closely related homologues are activated by a mechanism distinct from that of either DtxR or MntR.

The conformations of the manganese transport regulator of Bacillus subtilis in its metal-free state.

The manganese transport regulator (MntR) from Bacillus subtilis binds cognate DNA sequences in response to elevated manganese concentrations. MntR functions as a homodimer that binds two manganese ions per subunit. Metal binding takes place at the interface of the two domains that comprise each MntR subunit: an N-terminal DNA-binding domain and a C-terminal dimerization domain. In order to elucidate the link between metal binding and activation, a crystallographic study of MntR in its metal-free state has been undertaken. Here we describe the structures of the native protein and a selenomethionine-containing variant, solved to 2.8 A. The two structures contain five crystallographically unique subunits of MntR, providing diverse views of the metal-free protein. In apo-MntR, as in the manganese complex, the dimer is formed by dyad-related C-terminal domains that provide a conserved structural core. Similarly, each DNA-binding domain largely retains the folded conformation found in metal bound forms of MntR. However, compared to metal-activated MntR, the DNA-binding domains move substantially with respect to the dimer interface in apo-MntR. Overlays of multiple apo-MntR structures indicate that there is a greater range of positioning allowed between N and C-terminal domains in the metal-free state and that the DNA-binding domains of the dimer are farther apart than in the activated complex. To further investigate the conformation of the DNA-binding domain of apo-MntR, a site-directed spin labeling experiment was performed on a mutant of MntR containing cysteine at residue 6. Consistent with the crystallographic results, EPR spectra of the spin-labeled mutant indicate that tertiary structure is conserved in the presence or absence of bound metals, though slightly greater flexibility is present in inactive forms of MntR.

Assessing biosynthetic potential of agricultural groundwater through metagenomic sequencing: A diverse anammox community dominates nitrate-rich groundwater.

BACKGROUND: Climate change produces extremes in both temperature and precipitation causing increased drought severity and increased reliance on groundwater resources. Agricultural practices, which rely on groundwater, are sensitive to but also sources of contaminants, including nitrate. How agricultural contamination drives groundwater geochemistry through microbial metabolism is poorly understood. METHODS: On an active cow dairy in the Central Valley of California, we sampled groundwater from three wells at depths of 4.3 m (two wells) and 100 m (one well) below ground surface (bgs) as well as an effluent surface water lagoon that fertilizes surrounding corn fields. We analyzed the samples for concentrations of solutes, heavy metals, and USDA pathogenic bacteria of the Escherichia coli and Enterococcus groups as part of a long term groundwater monitoring study. Whole metagenome shotgun sequencing and assembly revealed taxonomic composition and metabolic potential of the community. RESULTS: Elevated nitrate and dissolved organic carbon occurred at 4.3m but not at 100m bgs. Metagenomics confirmed chemical observations and revealed several Planctomycete genomes, including a new Brocadiaceae lineage and a likely Planctomycetes OM190, as well novel diversity and high abundance of nano-prokaryotes from the Candidate Phyla Radiation (CPR), the Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaea (DPANN) and the Thaumarchaeota, Aigarchaeota, Crenarchaeota, Korarchaeota (TACK) superphyla. Pathway analysis suggests community interactions based on complimentary primary metabolic pathways and abundant secondary metabolite operons encoding antimicrobials and quorum sensing systems. CONCLUSIONS: The metagenomes show strong resemblance to activated sludge communities from a nitrogen removal reactor at a wastewater treatment plant, suggesting that natural bioremediation occurs through microbial metabolism. Elevated nitrate and rich secondary metabolite biosynthetic capacity suggest incomplete remediation and the potential for novel pharmacologically active compounds.

The logic and design of analog-sensitive kinases and their small molecule inhibitors.

Analog-sensitive AS Kinase technology allows for rapid, reversible, and highly specific inhibition of individual engineered kinases in cells and in mouse models of human diseases. The technique consists of two parts: a kinase containing a space-creating mutation in the ATP-binding pocket and a bulky ATP-competitive small molecule inhibitor that complements the shape of the mutant ATP pocket. This strategy enables dissection of phospho-signaling pathways, elucidation of the physiological function of individual kinases, and characterization of the pharmacology of clinical-kinase inhibitors. Here, we present an overview of AS technology and describe a stepwise approach for generating AS Kinase mutants and identifying appropriate small molecule inhibitors. We also describe commonly encountered technical obstacles and provide strategies to overcome them.

Chemical genetics of rapamycin-insensitive TORC2 in S. cerevisiae.

Current approaches for identifying synergistic targets use cell culture models to see if the combined effect of clinically available drugs is better than predicted by their individual efficacy. New techniques are needed to systematically and rationally identify targets and pathways that may be synergistic targets. Here, we created a tool to screen and identify molecular targets that may synergize with new inhibitors of target of rapamycin (TOR), a conserved protein that is a major integrator of cell proliferation signals in the nutrient-signaling pathway. Although clinical results from TOR complex 1 (TORC1)-specific inhibition using rapamycin analogs have been disappointing, trials using inhibitors that also target TORC2 have been promising. To understand this increased therapeutic efficacy and to discover secondary targets for combination therapy, we engineered Tor2 in S. cerevisiae to accept an orthogonal inhibitor. We used this tool to create a chemical epistasis miniarray profile (ChE-MAP) by measuring interactions between the chemically inhibited Tor2 kinase and a diverse library of deletion mutants. The ChE-MAP identified known TOR components and distinguished between TORC1- and TORC2-dependent functions. The results showed a TORC2-specific interaction with the pentose phosphate pathway, a previously unappreciated TORC2 function that suggests a role for the complex in balancing the high energy demand required for ribosome biogenesis.

Structural basis for the metal-selective activation of the manganese transport regulator of Bacillus subtilis.

The manganese transport regulator (MntR) of Bacillus subtilis is activated by Mn(2+) to repress transcription of genes encoding transporters involved in the uptake of manganese. MntR is also strongly activated by cadmium, both in vivo and in vitro, but it is poorly activated by other metal cations, including calcium and zinc. The previously published MntR.Mn(2+) structure revealed a binuclear complex of manganese ions with a metal-metal separation of 3.3 A (herein designated the AB conformer). Analysis of four additional crystal forms of MntR.Mn(2+) reveals that the AB conformer is only observed in monoclinic crystals at 100 K, suggesting that this conformation may be stabilized by crystal packing forces. In contrast, monoclinic crystals analyzed at room temperature (at either pH 6.5 or pH 8.5), and a second hexagonal crystal form (analyzed at 100 K), all reveal the shift of one manganese ion by 2.5 A, thereby leading to a newly identified conformation (the AC conformer) with an internuclear distance of 4.4 A. Significantly, the cadmium and calcium complexes of MntR also contain binuclear complexes with a 4.4 A internuclear separation. In contrast, the zinc complex of MntR contains only one metal ion per subunit, in the A site. Isothermal titration calorimetry confirms the stoichiometry of Mn(2+), Cd(2+), and Zn(2+) binding to MntR. We propose that the specificity of MntR activation is tied to productive binding of metal ions at two sites; the A site appears to act as a selectivity filter, determining whether the B or C site will be occupied and thereby fully activate MntR.