Disruptions in valine degradation affect seed development and germination in Arabidopsis.

We have functionally characterized the role of two putative mitochondrial enzymes in valine degradation using insertional mutants. Prior to this study, the relationship between branched-chain amino acid degradation (named for leucine, valine and isoleucine) and seed development was limited to leucine catabolism. Using a reverse genetics approach, we show that disruptions in the mitochondrial valine degradation pathway affect seed development and germination in Arabidopsis thaliana. A null mutant of 3-hydroxyisobutyryl-CoA hydrolase (CHY4, At4g31810) resulted in an embryo lethal phenotype, while a null mutant of methylmalonate semialdehyde dehydrogenase (MMSD, At2g14170) resulted in seeds with wrinkled coats, decreased storage reserves, elevated valine and leucine, and reduced germination rates. These data highlight the unique contributions CHY4 and MMSD make to the overall growth and viability of plants. It also increases our knowledge of the role branched-chain amino acid catabolism plays in seed development and amino acid homeostasis.

Benzoate- and Salicylate-Tolerant Strains of Escherichia coli K-12 Lose Antibiotic Resistance during Laboratory Evolution.

Escherichia coli K-12 W3110 grows in the presence of membrane-permeant organic acids that can depress cytoplasmic pH and accumulate in the cytoplasm. We conducted experimental evolution by daily diluting cultures in increasing concentrations of benzoic acid (up to 20 mM) buffered at external pH 6.5, a pH at which permeant acids concentrate in the cytoplasm. By 2,000 generations, clones isolated from evolving populations showed increasing tolerance to benzoate but were sensitive to chloramphenicol and tetracycline. Sixteen clones grew to stationary phase in 20 mM benzoate, whereas the ancestral strain W3110 peaked and declined. Similar growth occurred in 10 mM salicylate. Benzoate-evolved strains grew like W3110 in the absence of benzoate, in media buffered at pH 4.8, pH 7.0, or pH 9.0, or in 20 mM acetate or sorbate at pH 6.5. Genomes of 16 strains revealed over 100 mutations, including single-nucleotide polymorphisms (SNPs), large deletions, and insertion knockouts. Most strains acquired deletions in the benzoate-induced multiple antibiotic resistance (Mar) regulon or in associated regulators such as rob and cpxA, as well as the multidrug resistance (MDR) efflux pumps emrA, emrY, and mdtA Strains also lost or downregulated the Gad acid fitness regulon. In 5 mM benzoate or in 2 mM salicylate (2-hydroxybenzoate), most strains showed increased sensitivity to the antibiotics chloramphenicol and tetracycline; some strains were more sensitive than a marA knockout strain. Thus, our benzoate-evolved strains may reveal additional unknown drug resistance components. Benzoate or salicylate selection pressure may cause general loss of MDR genes and regulators. IMPORTANCE: Benzoate is a common food preservative, and salicylate is the primary active metabolite of aspirin. In the gut microbiome, genetic adaptation to salicylate may involve loss or downregulation of inducible multidrug resistance systems. This discovery implies that aspirin therapy may modulate the human gut microbiome to favor salicylate tolerance at the expense of drug resistance. Similar aspirin-associated loss of drug resistance might occur in bacterial pathogens found in arterial plaques.

Digital Image Analysis for DETCHIP® Code Determination.

DETECHIP® is a molecular sensing array used for identification of a large variety of substances. Previous methodology for the analysis of DETECHIP® used human vision to distinguish color changes induced by the presence of the analyte of interest. This paper describes several analysis techniques using digital images of DETECHIP®. Both a digital camera and flatbed desktop photo scanner were used to obtain Jpeg images. Color information within these digital images was obtained through the measurement of red-green-blue (RGB) values using software such as GIMP, Photoshop and ImageJ. Several different techniques were used to evaluate these color changes. It was determined that the flatbed scanner produced in the clearest and more reproducible images. Furthermore, codes obtained using a macro written for use within ImageJ showed improved consistency versus pervious methods.