Single mutation to a sex pheromone receptor provides adaptive specificity between closely related moth species.

Sex pheromone communication, acting as a prezygotic barrier to mating, is believed to have contributed to the speciation of moths and butterflies in the order Lepidoptera. Five decades after the discovery of the first moth sex pheromone, little is known about the molecular mechanisms that underlie the evolution of pheromone communication between closely related species. Although Asian and European corn borers (ACB and ECB) can be interbred in the laboratory, they are behaviorally isolated from mating naturally by their responses to subtly different sex pheromone isomers, (E)-12- and (Z)-12-tetradecenyl acetate and (E)-11- and (Z)-11-tetradecenyl acetate (ACB: E12, Z12; ECB; E11, Z11). Male moth olfactory systems respond specifically to the pheromone blend produced by their conspecific females. In vitro, ECB(Z) odorant receptor 3 (OR3), a sex pheromone receptor expressed in male antennae, responds strongly to E11 but also generally to the Z11, E12, and Z12 pheromones. In contrast, we show that ACB OR3, a gene that has been subjected to positive selection (ω = 2.9), responds preferentially to the ACB E12 and Z12 pheromones. In Ostrinia species the amino acid residue corresponding to position 148 in transmembrane domain 3 of OR3 is alanine (A), except for ACB OR3 that has a threonine (T) in this position. Mutation of this residue from A to T alters the pheromone recognition pattern by selectively reducing the E11 response ∼14-fold. These results suggest that discrete mutations that narrow the specificity of more broadly responsive sex pheromone receptors may provide a mechanism that contributes to speciation.

Perchlorate reduction using free and encapsulated Azospira oryzae enzymes.

Existing methods for perchlorate remediation are hampered by the common co-occurrence of nitrate, which is structurally similar and a preferred electron acceptor. In this work, the potential for perchlorate removal using cell-free bacterial enzymes as biocatalysts was investigated using crude cell lysates and soluble protein fractions of Azospira oryzae PS, as well as soluble protein fractions encapsulated in lipid and polymer vesicles. The crude lysates showed activities between 41 700 to 54 400 U L(-1) (2.49 to 3.06 U mg(-1) total protein). Soluble protein fractions had activities of 15 400 to 29 900 U L(-1) (1.70 to 1.97 U mg(-1)) and still retained an average of 58.2% of their original activity after 23 days of storage at 4 °C under aerobic conditions. Perchlorate was removed by the soluble protein fraction at higher rates than nitrate. Importantly, perchlorate reduction occurred even in the presence of 500-fold excess nitrate. The soluble protein fraction retained its function after encapsulation in lipid or polymer vesicles, with activities of 13.8 to 70.7 U L(-1), in agreement with theoretical calculations accounting for the volume limitation of the vesicles. Further, encapsulation mitigated enzyme inactivation by proteinase K. Enzyme-based technologies could prove effective at perchlorate removal from water cocontaminated with nitrate or sulfate.

Use of the thyrocyte sodium iodide symporter as the basis for a perchlorate cell-based assay.

Perchlorates are strong oxidants widely employed in military and civilian energetic materials and recently have been scrutinized as persistent environmental pollutants. The perchlorate anion, ClO(4)(-), is a well-known and potent competitive inhibitor of iodide transport by the sodium iodide symporter (NIS) expressed in the basolateral membranes of thyroid follicular cells (thyrocytes). Iodide uptake by thyroid follicular cells is rapid and reproducible. The competitive radiotransporter assay in this study shows promise as a rapid and convenient method to assay for ClO(4)(-) in water samples at the nM level. This work describes the initial efforts to define the assay conditions that enhance NIS selectivity for ClO(4)(-). Experiments of 10 min co-incubation of ClO(4)(-) and (125)I(-) demonstrate a more significant effect on (125)I(-) transport, with a quantifiable ClO(4)(-) concentration range of 50 nM (5 ppb) to 2 microM (200 ppb), and IC(50) of 180 nM (18 ppb), nearly three-fold lower than previous reports. Since the IC(50) in our assay for other known competitor anions (SCN(-), ClO(3)(-), NO(3)(-)) remains unchanged from previous research, the increased sensitivity for ClO(4)(-) also produces a three-fold enhancement in selectivity. In addition to the possible applicability of the thyrocyte to the development of a cellular perchlorate biosensor, we propose that the high affinity of the NIS for ClO(4)(-) also creates the potential for exploiting this membrane protein as a selective, sensitive, and broadly applicable biomechanical mechanism for controlled movement and concentration of perchlorate.

Regulation of pyrG expression in Bacillus subtilis: CTP-regulated antitermination and reiterative transcription with pyrG templates in vitro.

The regulation of pyrG expression in a group of low GC Gram-positive bacteria was previously shown to be mediated by a novel form of transcription attenuation in which low levels of intracellular CTP induce reiterative addition of G residues at position +4 in the 5' end of the pyrG mRNA, which is encoded as pppGGGC. . . . The poly(G) sequences formed under these conditions act to prevent attenuation by base pairing with the C- and U-rich 5' strand of a downstream terminator stem-loop located in the pyrG leader. In this work we document the reconstitution of this regulatory system in vitro using only the native pyrG DNA template, RNA polymerase and appropriate concentrations of ribonucleotides. CTP-regulated reiterative transcription producing 5'-poly(G) tracts and regulation of transcription termination at the pyrG attenuator by CTP were demonstrated. Mutations in the native pyrG template that altered reiterative transcription and attenuation in vivo resulted in alternations in expression in the in vitro transcription system that were predicted by the mechanism described above. These findings provide strong experimental support for the proposed reiterative transcription/antitermination mechanism and confirm that no trans-acting regulatory protein is required for pyrG regulation.