Biochemistry of Hemolysin Toxin Activation by Fatty Acylation: Characterization of an Internal Protein Acyltransferase

Hemolysin toxin produced and secreted by pathogenic Escherichia coli is one of a family of cytolytic, structurally homologous protein toxins known as RTX (repeats in toxin) toxins. RTX toxins are products of a gene cluster, CABD . The A gene product, nontoxic hemolysin (proHlyA) is made toxic by post-translational fatty acylation of two internal lysine residues. HlyC, C gene product, is essential for acylation, and acyl-acyl carrier protein (ACP) is the acyl donor. HlyB and HlyD are involved in secretion of the toxin. HlyC was thought to serve as an internal protein acyltransferase and remained uncharacterized until now. ProHlyA and HlyC were separately subcloned, expressed, and purified, and acyl-ACPs with diverse radioactive acyl groups were synthesized. With these proteins, the conversion of proHlyA to HlyA by acyltransfer was assayed. Acyl-ACP was the obligate acyl donor. Acyltransfer was catalyzed by HlyC monomer, and an acyl-enzyme intermediate was detected and shown to catalyze the reverse reaction. The reaction mechanism was examined by steady state kinetics, and the nature of inhibitions by reaction products was determined. The kinetic mechanism of the internal protein acylation was compatible with an uni uni iso uni uni ping pong with isomerization of the F form of the enzyme. Clues to the chemical mechanism for the acyltransferase were elucidated by both chemical modification studies and site directed mutagenesis of the enzyme. Chemical modification experiments ruled out any critical cysteines, serines, and lysine residues, but suggested a role for histidine(s) and tyrosine(s) in acyltransferase function. In order to examine the function of specific residues and possibly corroborate the chemical findings, site directed mutagenesis studies of the acyltransferase were employed. Seventeen residues that were conserved among 13 different RTX toxin acyltransferases were individually mutated, and the respective HlyCs expressed, and characterized. Residues that were critical for acyltransferase function included Gly 11, His 23, Tyr 70, and Gly 85. As with chemical modification data, mutagenesis ruled out any conserved, essential, cysteines or serines critical for HlyC acyltransferase activity

Scholarship Reconsidered: What Boyer\u27s Proposal Means For Communication: A Response

Comments on the interpretation of Ernest Boyer regarding scholarship. Concept of Boyer\u27s reinterpretation of scholarship; Impact of the Boyer report on universities; Concerns on the redefinition of scholarship in terms of the Communication discipline

Resolved Spectroscopy of the T8.5 and Y0-0.5 Binary WISEPC J121756.91+162640.2AB

We present 0.9 - 2.5 um resolved spectra for the ultracool binary WISEPC J121756.91+162640.2AB. The system consists of a pair of brown dwarfs that straddles the currently defined T/Y spectral type boundary. We use synthetic spectra generated by model atmospheres that include chloride and sulfide clouds (Morley et al.), the distance to the system (Dupuy & Kraus), and the radius of each component based on evolutionary models (Saumon & Marley) to determine a probable range of physical properties for the binary. The effective temperature of the T8.5 primary is 550 - 600 K, and that of the Y0 - Y0.5 secondary is 450 K. The atmospheres of both components are either free of clouds or have extremely thin cloud layers. We find that the masses of the primary and secondary are 30 and 22 M_Jup, respectively, and that the age of the system is 4 - 8 Gyr. This age is consistent with astrometric measurements (Dupuy & Kraus) that show that the system has kinematics intermediate between those of the thin and thick disks of the Galaxy. An older age is also consistent with an indication by the H - K colors that the system is slightly metal-poor.Comment: 21 pages which include 6 Figures and 3 Tables. Accepted on November 8 2013 for publication in Ap

Germination responses of a dry sclerophyll forest soil-stored seedbank to fire related cues

Fire is an integral component of many ecosystems worldwide. Many plant species require fire-related cues, primarily heat and smoke, to trigger germination. Despite the importance of this process, the responses of many Australian species to these cues are unknown. Without this knowledge fire management strategies may be developed that are inappropriate for individual species and vegetation communities. In this study we examined the responses of a dry sclerophyll forest seed bank to heat and smoke germination cues. Analysis was possible for 48 taxa within the soil seedbank with 34 of these showing a response to one or both of the germination cues. 10 species responded to the heat treatment, 11 species responded to the smoke treatment and 13 species responded to both the heat and smoke treatments. Germination cues acted independently for all species considered. Results in this study were consistent with published reports for most species, although some differences were seen at the species and genus level. The study highlights the importance of fire-related cues in enhancing germination of a large proportion of the species occurring in dry sclerophyll forests