Structural and functional insights into asymmetric enzymatic dehydration of alkenols.

The asymmetric dehydration of alcohols is an important process for the direct synthesis of alkenes. We report the structure and substrate specificity of the bifunctional linalool dehydratase isomerase (LinD) from the bacterium Castellaniella defragrans that catalyzes in nature the hydration of β-myrcene to linalool and the subsequent isomerization to geraniol. Enzymatic kinetic resolutions of truncated and elongated aromatic and aliphatic tertiary alcohols (C5-C15) that contain a specific signature motif demonstrate the broad substrate specificity of LinD.

Regulation of mprF by antisense RNA restores daptomycin susceptibility to daptomycin-resistant isolates of Staphylococcus aureus.

Mutations in mprF have been shown to result in reduced susceptibility to daptomycin and other cationic antibacterials. An mprF antisense-inducible plasmid was constructed and used to demonstrate that depletion of mprF can reestablish susceptibility to daptomycin. Inducing antisense to mprF also resulted in increased susceptibility to vancomycin and gentamicin but, paradoxically, decreased susceptibility to oxacillin.

Staphylococcus aureus TargetArray: comprehensive differential essential gene expression as a mechanistic tool to profile antibacterials.

The widespread emergence of antibiotic-resistant bacteria and a lack of new pharmaceutical development have catalyzed a need for new and innovative approaches for antibiotic drug discovery. One bottleneck in antibiotic discovery is the lack of a rapid and comprehensive method to identify compound mode of action (MOA). Since a hallmark of antibiotic action is as an inhibitor of essential cellular targets and processes, we identify a set of 308 essential genes in the clinically important pathogen Staphylococcus aureus.

Comparison of the essential cellular functions of the two murA genes of Bacillus anthracis.

Targeted antisense and gene replacement mutagenesis experiments demonstrate that only the murA1 gene and not the murA2 gene is required for the normal cellular growth of Bacillus anthracis. Antisense-based modulation of murA1 gene expression hypersensitizes cells to the MurA-specific antibiotic fosfomycin despite the normally high resistance of B. anthracis to this drug.

Evaluation of the metS and murB loci for antibiotic discovery using targeted antisense RNA expression analysis in Bacillus anthracis.

The biowarfare-relevant bacterial pathogen Bacillus anthracis contains two paralogs each of the metS and murB genes, which encode the important antibiotic target functions methionyl-tRNA synthetase and UDP-N-acetylenolpyruvoylglucosamine reductase, respectively. Empirical screens were conducted to detect and characterize gene fragments of each of these four genes that could cause growth reduction of B. anthracis when inducibly expressed from a plasmid-borne promoter.

Novel retinoic acid generating activities in the neural tube and heart identified by conditional rescue of Raldh2 null mutant mice.

Retinoid control of vertebrate development depends upon tissue-specific metabolism of retinol to retinoic acid (RA). The RA biosynthetic enzyme RALDH2 catalyzes much, but not all, RA production in mouse embryos, as revealed here with Raldh2 null mutants carrying an RA-responsive transgene. Targeted disruption of Raldh2 arrests development at midgestation and eliminates all RA synthesis except that associated with Raldh3 expression in the surface ectoderm of the eye field.

A genome-wide strategy for the identification of essential genes in Staphylococcus aureus.

To address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus.