Analysis of expressed sequence tags from subtracted and unsubtracted Ctenocephalides felis hindgut and Malpighian tubule cDNA libraries.

Insect hindgut and Malpighian tubule (HMT) tissues regulate the contents of the haemolymph through the excretion of waste products and the specific reabsorption of nutrients. As such, they perform a role that is essential for survival and may contain molecular targets for insect control strategies. In order to discover genes expressed in the HMT tissues of the cat flea, Ctenocephalides felis, expressed sequence tags (ESTs) were generated from an unsubtracted HMT cDNA library and from a subtracted HMT cDNA library that had been enriched for HMT-specific cDNAs. A total of 4844 ESTs were analysed from both libraries: 3657 from the subtracted library and 1187 from the unsubtracted library. Of the 1418 distinct ESTs identified from both libraries, 953 had significant similarity to other sequences reported in the GenBank database. A comparison of the results from the two libraries confirmed that the percentages of genes likely to be involved with metabolism, cell structure, and digestion were reduced by the subtraction procedure, whereas genes likely to be involved with ion transport were enriched. Analysis of the prevalence of three individual cDNAs in each library revealed that the actin cDNA was reduced in the subtracted library whereas the cDNAs encoding allantoinase and a peritrophin-like protein were greatly enriched in the subtracted library. Northern blot analysis demonstrated that the actin cDNA was expressed in both the HMT and carcass tissues, whereas the allantoinase and peritrophin-like cDNAs were detected exclusively in the HMT tissues. In total, 97 distinct ESTs that appear to encode proteins involved with ion transport were analysed. Some of these proteins may be directly involved with diuresis or the specific reabsorption of salts and nutrients, and thus may be potential molecular targets for flea control strategies.

Isoprene biosynthesis in Bacillus subtilis via the methylerythritol phosphate pathway.

Isoprene (2-methyl-1,3-butadiene), an abundant natural product of unknown function in plants, has recently been found to be one of the major volatiles formed by Bacillus subtilis. To understand the metabolic origins of isoprene in B. subtilis, we used (13)C- and (2)H-labeling methods with GC-MS analysis of released isoprene. The results indicate that, in this bacterium, isoprene is not formed by the mevalonate pathway or from catabolism of leucine, but, as in plant systems, it is a product of the methylerythritol phosphate pathway of isoprenoid synthesis. This work supports the idea that B. subtilis could be used as a microbial model for studying the biochemistry of isoprene formation.

Acetone formation in the Vibrio family: a new pathway for bacterial leucine catabolism.

There is current interest in biological sources of acetone, a volatile organic compound that impacts atmospheric chemistry. Here, we determined that leucine-dependent acetone formation is widespread in the Vibrionaceae. Sixteen Vibrio isolates, two Listonella species, and two Photobacterium angustum isolates produced acetone in the presence of L-leucine. Shewanella isolates produced much less acetone. Growth of Vibrio splendidus and P. angustum in a fermentor with controlled aeration revealed that acetone was produced after a lag in late logarithmic or stationary phase of growth, depending on the medium, and was not derived from acetoacetate by nonenzymatic decarboxylation in the medium. L-Leucine, but not D-leucine, was converted to acetone with a stoichiometry of approximately 0.61 mol of acetone per mol of L-leucine. Testing various potential leucine catabolites as precursors of acetone showed that only alpha-ketoisocaproate was efficiently converted by whole cells to acetone. Acetone production was blocked by a nitrogen atmosphere but not by electron transport inhibitors, suggesting that an oxygen-dependent reaction is required for leucine catabolism. Metabolic labeling with deuterated (isopropyl-d(7))-L-leucine revealed that the isopropyl carbons give rise to acetone with full retention of deuterium in each methyl group. These results suggest the operation of a new catabolic pathway for leucine in vibrios that is distinct from the 3-hydroxy-3-methylglutaryl-coenzyme A pathway seen in pseudomonads.

Three distinct phases of isoprene formation during growth and sporulation of Bacillus subtilis.

During growth on a glucose-tryptone medium, Bacillus subtilis 6051 (Marburg strain) exhibited three phases of isoprene (2-methyl-1, 3-butadiene) formation, corresponding to (i) glucose catabolism and secretion of acetoin, (ii) catabolism of acetoin, and (iii) the early stages of sporulation. These results establish an experimental system for studying the biological role of isoprene formation.