Development of a sensitive chemiluminometric assay for the detection of beta-galactosidase in permeabilized coliform bacteria and comparison with fluorometry and colorimetry.

We developed a chemiluminometric assay of beta-galactosidase in coliform bacteria, using a phenylgalactose-substituted 1,2-dioxetane derivative as a substrate. Permeabilization of cells is required to ensure the efficient cellular uptake of this compound. By this method, one coliform seeded in 100 ml of sterile water can be detected after a 6- to 9-h propagation phase followed by a 45-min enzyme assay in the presence of polymyxin B. Compared with fluorometry and colorimetry, chemiluminometry afforded 4- and 1,000-fold increases in sensitivity and 1- and 6-h increases in the speed of detection, respectively.     

Maize endosperm ADP-glucose pyrophosphorylase SHRUNKEN2 and BRITTLE2 subunit interactions

ADP-glucose pyrophosphorylase (AGP) represents a key regulatory step in polysaccharide synthesis in organisms ranging from bacteria to plants. Higher plant AGPs are complex in nature and are heterotetramers consisting of two similar but distinct subunits. How the subunits are assembled into enzymatically active polymers is not yet understood. Here, we address this issue by using naturally occurring null mutants of the Shrunken2 (Sh2) and Brittle2 (Bt2) loci of maize as well as the yeast two-hybrid expression system. In the absence of the maize endosperm large AGP subunit (SH2), the BT2 subunit remains as a monomer in the developing endosperm. In contrast, the SH2 protein, in the absence of BT2, is found in a complex of 100 kD. A direct interaction between SH2 and BT2 was proven when they were both expressed in yeast. Several motifs are essential for SH2:BT2 interaction because truncations removing the N or C terminus of either subunit eliminate SH2:BT2 interactions. Analysis of subunit interaction mutants (sim) also identified motifs essential for protein interactions.     

Cis-and all-trans-canthaxanthin levels in fairy shrimps

The carotenoid composition of cysts of seven species of fairy shrimps (Anostraca) was studied using high-performance liquid chromatography. Canthaxanthin was detected as the major pigment, but an important and often predominant fraction of it occurred in the cis-configuration, which is consistent with previous findings in Artemia cysts. Cis-canthaxanthin rapidly disappeared in nauplii of Thamnocephalus platyurus through conversion to all-trans-canthaxanthin and was preferably localized in the abdominal section carrying the genital segment of females, unlike in mature male animals. In contrast, the intense orange-red colour of the thoracopods and the cercopods in both sexes was only due to all-trans-canthaxanthin.     

Profiling and Quantitation of Bacterial Carotenoids by Liquid Chromatography and Photodiode Array Detection

An analytical method for the profiling and quantitative determination of carotenoids in bacteria is described. Exhaustive extraction of the pigments from four selected bacterial strains required treatment of the cells with potassium hydroxide or liquefied phenol or both before the addition of the extracting solvent (methanol or diethyl ether). The carotenoids in the extracts were separated by nonaqueous reversed-phase liquid chromatography in conjunction with photodiode array absorption detection. The identity of a peak was considered definitive only when both its retention time and absorption spectrum, before and after chemical reactions, matched those of a reference component. In the absence of the latter, most peaks could be tentatively identified. Two examples illustrate how in the analysis of pigmented bacteria errors may result from using nonchromatographic procedures or liquid chromatographic methods lacking sufficient criteria for peak identification. Carotenoids of interest were determined quantitatively when the authentic reference substance was available or, alternatively, were determined semiquantitatively.     

Reinvestigation of Brevibacterium sp. Strain KY-4313 as a Source of Canthaxanthin

The hydrocarbon-utilizing Brevibacterium sp. strain KY-4313 was reevaluated for its potential to produce canthaxanthin, a carotenoid pigment of strong commercial interest. Three approaches were used to optimize the canthaxanthin yield from this organism, i.e., the preparation of mutants, the addition of supposedly carotenogenic chemicals to the growth medium, and growth promotion. Following treatment of the parent strain with N-nitrosomethylurea, a presumed mutant was isolated which showed a 32% increase in cellular canthaxanthin content. No effective carotenogenic chemicals were found in connection with hydrocarbon fermentations, in which mainly growth promotion through periodic medium renewal proved conducive to enhanced pigment production. Carotenogenesis could be stimulated in brain heart infusion broth by adding alcohols or retinol. Improved growth in this medium was generally not associated with higher canthaxanthin yields. Both superior growth and pigment levels were obtained in a newly designed medium based on fumaric acid-molasses. The maximum yields of canthaxanthin in shake flasks were (in milligrams per liter) 4.2 (brain heart infusion broth plus propanol-zinc sulfate), 3.6 (hydrocarbon medium), and 9.3 (fumaric acid-molasses), which represent a significant improvement over the originally reported optimal result (1 mg/liter). The corresponding yields of echinenone, the direct precursor of canthaxanthin, were 1.2, 1.6, and 2.3 mg/liter, respectively. Two-liter hydrocarbon batch fermentations involving medium renewal maximally produced 7.2 mg of canthaxanthin and 3.7 mg of echinenone per liter.     

Qualitative and quantitative changes in the carotenoids during development of the brine shrimp Artemia

In order to study the biological fate of all-trans- and cis-canthaxanthin in the brine shrimp Artemia, a quantitative method was developed for the determination of both carotenoids and their metabolic precursors in encysted embryos (cysts), nauplii, whole animals, organs, and subcellular fractions. This method is based on nonaqueous reversed-phase chromatography, two new exhaustive extraction procedures, and the determination of proteins in the extracted residue. Hydration of Artemia cysts caused a reversible conversion of part of the all-trans- to cis-canthaxanthin. During further pre-emergence embryonic development, there was little change in the levels of both isomers. After hatching of cysts, cis-canthaxanthin was progressively isomerized to the all-trans form, while the total (all-trans + cis) canthaxanthin to protein ratio tended to remain constant. Cis-canthaxanthin rapidly became undetectable in animals fed on algae and reappeared in females at an advanced stage of the reproductive cycle. All-trans-canthaxanthin remained present during the whole Artemia life cycle in addition to its metabolic precursors echinenone and beta-carotene. The carotenoid distribution in organs and subcellular fractions indicated high affinity of cis-canthaxanthin for the female reproductive system, oocytes in general, and yolk in particular. A role for cis-canthaxanthin is suggested at an early developmental stage, i.e., in cysts, before hatching.     

Effect of diet on the photoperiodic induction of diapause in three species of predatory mite,Amblyseius potentillae,A. cucumeris andTyphlodromus pyri

The predatory mitesAmblyseius potentillae, A. cucumeris andTyphlodromus pyri entered diapause in response to a short-day photoperiodic regime, when they were reared on pollen of the ice plant,Dorotheanthus bellidiformis. With pollen of the broad bean,Vicia faba, as food, however, diapause was virtually absent inA. potentillae andA. cucumeris under the same short-day regime, but full diapause was found inT. pyri. The importance of carotenoids for the photoperiodic response in these predatory mites is discussed.