The sarA locus in Staphylococcus aureus controls the expression of many virulence genes. The sarA regulatory molecule, SarA, is a 14.7-kDa protein (124 residues) that binds to the promoter region of target genes. Here we report the 2.6 Å-resolution x-ray crystal structure of the dimeric winged helix SarA protein, which differs from the published SarA structure dramatically. In the crystal packing, multiple dimers of SarA form a scaffold, possibly via divalent cations. Mutations of individual residues within the DNAbinding helix-turn-helix and the winged region as well as within the metal-binding pocket implicate basic residues R84 and R90 within the winged region to be critical in DNA binding, whereas acidic residues D88 and E89 (wing), D8 and E11 (metal-binding pocket), and cysteine 9 are essential for SarA function. These data suggest that the winged region of the winged helix protein participates in DNA binding and activation, whereas the putative divalent cation binding pocket is only involved in gene function.

Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. This reaction generates single carbon units for purine, thymidine, and methionine biosynthesis. The enzyme is a homotetramer comprising two obligate dimers and four pyridoxal phosphate-bound active sites. The mammalian enzyme is present in cells in both catalytically active and inactive forms. The inactive form is a ternary complex that results from the binding of glycine and 5-formyltetrahydrofolate polyglutamate, a slow tight-binding inhibitor. The crystal structure of a close analogue of the inactive form of murine cytoplasmic SHMT (cSHMT), lacking only the polyglutamate tail of the inhibitor, has been determined to 2.9 A resolution. This first structure of a ligand-bound mammalian SHMT allows identification of amino acid residues involved in substrate binding and catalysis. It also reveals that the two obligate dimers making up a tetramer are not equivalent; one can be described as "tight-binding" and the other as "loose-binding" for folate. Both active sites of the tight-binding dimer are occupied by 5-formyltetrahydrofolate (5-formylTHF), whose N5-formyl carbon is within 4 A of the glycine alpha-carbon of the glycine-pyridoxal phosphate complex; the complex appears to be primarily in its quinonoid form. In the loose-binding dimer, 5-formylTHF is present in only one of the active sites, and its N5-formyl carbon is 5 A from the glycine alpha-carbon. The pyridoxal phosphates appear to be primarily present as geminal diamine complexes, with bonds to both glycine and the active site lysine. This structure suggests that only two of the four catalytic sites on SHMT are catalytically competent and that the cSHMT-glycine-5-formylTHF ternary complex is an intermediate state analogue of the catalytic complex associated with serine and glycine interconversion.

Summary1. Climate extremes are expected to increase in frequency and magnitude as a consequence of global warming. 2. Managed permanent grasslands cover a large surface in Europe and contribute substantially to agricultural production. These managed plant communities are dominated by perennial clonal species. Their capacity to adapt to rapidly changing environmental conditions may be limited. 3. We hypothesize that those plant populations that have already been exposed to conditions that are expected to occur due to future climate change, particularly conditions that would be 'extreme' in the target area, are able to cope better with these conditions. 4. For a common-garden experiment we selected ecotypes (provenances as supported by accessions in seed banks) of important European grass species: Arrhenatherum elatius, Festuca pratensis, Holcus lanatus and Alopecurus pratensis. Southern target locations of ecotypes (populations) were identified based on climate model projections for the local site in Northern Bavaria, Germany. 5. In a controlled experiment, the plants were exposed to warming and extreme drought. Drought conditions(16-19 days, depending on the species) were imposed starting from the end of May in combination with and without an increase in the average temperature from May to September 2009 ( +1.5 K compared with control; +2.5 K compared with ambient conditions outside of the experimental units). 6. Ecotypes and drought manipulation had significant impacts on biomass production and tissue die-back. Significant interactions between ecotype and drought indicated a different drought tolerance of the ecotypes in some cases. The warming treatment yielded a less significant response. The local ecotype generally did not perform significantly worse than the presumably better-adapted southern ecotypes. 7. Synthesis. The selection of ecotypes that are adapted to more extreme climatic conditions could be an option for maintaining future ecosystem functioning in temperate managed grasslands, as was indicated by the clear differences between ecotypes in our experiment. Based on our data, however, performance cannot be predicted from climatic origin. Therefore, we recommend enhancing the genetic variability within populations of species in general.

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