We propose the use of Desulfovibrio desulfuricans ND132 as a model species for understanding the mechanism of microbial Hg methylation. Strain ND132 is an anaerobic dissimilatory sulfate-reducing bacterium (DSRB), isolated from estuarine mid-Chesapeake Bay sediments. It was chosen for study because of its exceptionally high rates of Hg methylation in culture and its metabolic similarity to the lost strain D. desulfuricans LS, the only organism for which methylation pathways have been partially defined. Strain ND132 is an incomplete oxidizer of short-chain fatty acids. It is capable of respiratory growth using fumarate as an electron acceptor, supporting growth without sulfide production. We used enriched stable Hg isotopes to show that ND132 simultaneously produces and degrades methylmercury (MeHg) during growth but does not produce elemental Hg. MeHg produced by cells is mainly excreted, and no MeHg is produced in spent medium. Mass balances for Hg and MeHg during the growth of cultures, including the distribution between filterable and particulate phases, illustrate how medium chemistry and growth phase dramatically affect Hg solubility and availability for methylation. The available information on Hg methylation among strains in the genus Desulfovibrio is summarized, and we present methylation rates for several previously untested species. About 50% of Desulfovibrio strains tested to date have the ability to produce MeHg. Importantly, the ability to produce MeHg is constitutive and does not confer Hg resistance. A 16S rRNA-based alignment of the genus Desulfovibrio allows the very preliminary assessment that there may be some evolutionary basis for the ability to produce MeHg within this genus.

Although gamma-aminobutyric acid (GABA)A receptor alpha subunits are important for benzodiazepine (BZD) binding and GABA-current potentiation by BZDs, the presence of a gamma subunit is required for high affinity BZD effects. To determine which regions unique to the gamma2S subunit confer BZD binding and potentiation, we generated chimeric protein combinations of rat gamma2S and alpha1 subunits using a modified protocol to target crossover events to the amino-terminal extracellular region of the subunits. Several chimeras with full open reading frames were constructed and placed into vectors for either voltage-clamp experiments in Xenopus laevis oocytes or radioligand binding experiments in human embryonic kidney 293 cells. Chimeras (chi) containing at least the amino-terminal 161 amino acids of gamma2S bound BZDs with wild-type affinity when coexpressed with alpha1 and beta2 subunits. Further analysis of the gamma2S binding site region uncovered two areas, gamma2S K41-W82 and gamma2S R114-D161, that together are necessary and sufficient for high affinity BZD binding. Surprisingly, although the 161-amino acid residue amino terminus of the gamma2S subunit is sufficient for high affinity BZD binding, it is not sufficient for efficient allosteric coupling of the GABA and BZD binding sites, as demonstrated by reduced diazepam potentiation of the GABA-gated current and GABA potentiation of [3H]flunitrazepam binding. Thus, by using gamma/alpha chimeras, we identified two gamma2 subunit regions required for BZD binding that are distinct from domain or domains responsible for allosteric coupling of the BZD and GABA binding sites.

Several structural subclasses of ligands bind to the benzodiazepine (BZD) binding site of the GABA A receptor. Previous studies from this laboratory have suggested that imidazobenzodiazepines (i-BZDs, e.g., Ro 15-1788) require domains in the BZD binding site for high-affinity binding that are distinct from the requirements of classic BZDs (e.g., flunitrazepam). Here, we used systematic mutagenesis and the substituted cysteine accessibility method to map the recognition domain of i-BZDs near two residues implicated in BZD binding, ␥ 2 A79 and ␥ 2 T81. Both classic BZDs and i-BZDs protect cysteines substituted at ␥ 2 A79 and ␥ 2 T81 from covalent modification, suggesting that these ligands may occupy common volumetric spaces during binding. However, the binding of i-BZDs is more sensitive to mutations at ␥ 2 A79 than classic BZDs or BZDs that lack a 3Ј-imidazo substituent (e.g., midazolam). The effect that ␥ 2 A79 mutagenesis has on the binding affinities of a series of structurally rigid i-BZDs is related to the volume of the 3Ј-imidazo substituents. Furthermore, larger amino acid side chains introduced at ␥ 2 A79 cause correspondingly larger decreases in the binding affinities of i-BZDs with bulky 3Ј substituents. These data are consistent with a model in which ␥ 2 A79 lines a subsite within the BZD binding pocket that accommodates the 3Ј substituent of i-BZDs. In agreement with our experimental data, computer-assisted docking of Ro 15-4513 into a molecular model of the BZD binding site positions the 3Ј-imidazo substituent of Ro 15-4513 near ␥ 2 A79.Benzodiazepines (BZDs) are therapeutic agents commonly used in the treatment of anxiety, sleeplessness, and epilepsy (Doble and Martin, 1996). BZDs exert their anxiolytic, hypnotic, and anticonvulsant effects by interacting with a unique modulatory site on the GABA A receptor, the main effector of neuronal inhibition within the central nervous system (Hevers and Lü ddens, 1998). The BZD binding site is on the extracellular surface of the GABA A receptor at an interface formed by the ␣ and ␥ subunits (Smith and Olsen, 1995;Sigel and Buhr, 1997). Several studies have identified residues on both the ␣ subunit (Duncalfe et al

The imidazopyridine zolpidem (Ambien)

Desulfovibrio desulfuricans strain ND132 is an anaerobic sulfate-reducing bacterium (SRB) capable of producing methylmercury (MeHg), a potent human neurotoxin. The mechanism of methylation by this and other organisms is unknown. We present the 3.8-Mb genome sequence to provide further insight into microbial mercury methylation.