Growth and analysis of crystal forms of toxic shock syndrome toxin 1

Native toxic shock syndrom toxin 1 (TSST-1) purified from Staphylococcus aurius has been crystallized in four different forms. The highest resolution data (2.05 Å) was collected from orthorhombic crystals belonging to the space group C222₁. The unit cell dimension are a = 108.7 Å, b = 177.5 Å, c = 97.6 Å. Rotation function analysis of this from indicates that there is trimer of toxin molecules in the asymmetric unit with a local 3-fold axis parallel to the crystallographic c axis. Crystals of a double mutant of TSST-1 have been grown which has a single molecule in the asymmetric unit and diffract to 1.9 Å. The space group is P2₁ with unit cell parameters of a = 44.4 Å, b = 34.0 Å, c = 55.2 Å, β = 93.0°. © 1993 Wiley-Liss, Inc.     

Crystal structure of the hydroxylase component of methane monooxygenase from Methylosinus trichosporium OB3b.

Methane monooxygenase (MMO), found in aerobic methanotrophic bacteria, catalyzes the O2-dependent conversion of methane to methanol. The soluble form of the enzyme (sMMO) consists of three components: a reductase, a regulatory "B" component (MMOB), and a hydroxylase component (MMOH), which contains a hydroxo-bridged dinuclear iron cluster. Two genera of methanotrophs, termed Type X and Type II, which differ markedly in cellular and metabolic characteristics, are known to produce the sMMO. The structure of MMOH from the Type X methanotroph Methylococcus capsulatus Bath (MMO Bath) has been reported recently. Two different structures were found for the essential diiron cluster, depending upon the temperature at which the diffraction data were collected. In order to extend the structural studies to the Type II methanotrophs and to determine whether one of the two known MMOH structures is generally applicable to the MMOH family, we have determined the crystal structure of the MMOH from Type II Methylosinus trichosporium OB3b (MMO OB3b) in two crystal forms to 2.0 A resolution, respectively, both determined at 18 degrees C. The crystal forms differ in that MMOB was present during crystallization of the second form. Both crystal forms, however, yielded very similar results for the structure of the MMOH. Most of the major structural features of the MMOH Bath were also maintained with high fidelity. The two irons of the active site cluster of MMOH OB3b are bridged by two OH (or one OH and one H2O), as well as both carboxylate oxygens of Glu alpha 144. This bis-mu-hydroxo-bridged "diamond core" structure, with a short Fe-Fe distance of 2.99 A, is unique for the resting state of proteins containing analogous diiron clusters, and is very similar to the structure reported for the cluster from flash frozen (-160 degrees C) crystals of MMOH Bath, suggesting a common active site structure for the soluble MMOHs. The high-resolution structure of MMOH OB3b indicates 26 consecutive amino acid sequence differences in the beta chain when compared to the previously reported sequence inferred from the cloned gene. Fifteen additional sequence differences distributed randomly over the three chains were also observed, including D alpha 209E, a ligand of one of the irons.     

Molecular cloning of the Escherichia coli K-12 entACGBE genes

Abstract A 7-kb piece of Escherichia coli DNA that contains five genes ( entA, C, G, B and E ) required for the biosynthesis of the iron transport molecule enterochelin was isolated. A restriction map was constructed and proteins specified by the E. coli DNA were identified in mini- and maxicell systems. Plasmids containing portions of the entACGBE DNA generated by BAL31 digestion or restriction enzyme treatment were constructed; complementation studies done with these indicated that the five genes constitute an operon. The approximate site of the promoter was determined and the product of entE was tentatively identified as an M _r 63000 polypeptide.     

Coordinate regulation by iron of the synthesis of phenolate compounds and three outer membrane proteins in Escherichia coli.

The biosynthesis of the low-molecular-weight iron carrier enterochelin and of three outer membrane polypeptides appears to be coordinately regulated by the amount of cell-associated iron in Escherichia coli K-12. Measurements of iron acquisition made throughout the growth cycle in iron-deficient media indicate that a very rapid accumulation of iron occurs in the first 2 h of growth; there is comparatively little iron uptake during exponential growth, which results in a gradual decrease in the cellular iron content with each generation. When this level falls below 400 ng of iron per mg (dry weight) of cells, there is a simultaneous onset of synthesis of the three outer membrane polypeptides and of enterochelin. This coordinate regulation was also observed in cells able to transport iron actively using only citrate as an iron-carrier.     

Effect of Metalaxyl on the incidence and severity of Pearl millet downy mildew disease in Northern Nigeria

Pearl millet downy mildew (DM) incidence, severity and yield losses of two pearl millet varieties (local and improved) due to the disease were determined in the field. Significant differences in the disease incidence and severity were recorded in the plots sown with metalaxyl-treated seeds and those sown with non-treated seeds, indicating the efficacy of the fungicide on the fungus. Yield losses due to non-treatment of seeds with metalaxyl was 40.88 and 45.39% in a local variety and 43.00 and 18.60% in an improved variety in the 2000 and 2001 cropping seasons respectively. Significant differences between plots sown with metalaxyl-treated and those sown with non-treated seeds were obtained for other yield components such as 1000-grains weight, panicle length and weight.     

Gene expression and the evolution of phenotypic diversity in social wasps

Background  

Organisms are capable of developing different phenotypes by altering the genes they express. This phenotypic plasticity provides a means for species to respond effectively to environmental conditions. One of the most dramatic examples of phenotypic plasticity occurs in the highly social hymenopteran insects (ants, social bees, and social wasps), where distinct castes and sexes all arise from the same genes. To elucidate how variation in patterns of gene expression affects phenotypic variation, we conducted a study to simultaneously address the influence of developmental stage, sex, and caste on patterns of gene expression in Vespula wasps. Furthermore, we compared the patterns found in this species to those found in other taxa in order to investigate how variation in gene expression leads to phenotypic evolution.     

Tests of induction in mice by acute and chronic ionizing radiation and ethylnitrosourea of dominant mutations that cause the more common skeletal anomalies

Assessment-of-Dominant-Damage (ADD) experiments explored induction by proven specific-locus mutagens of dominant mutations that cause skeletal anomalies, cataracts, and stunted growth in offspring of mutagenized male mice. The data set reported here includes 6134 offspring. Mutagenic treatments included 600 R (i.e., approximately 6 Gy) of X-rays delivered in about 7 min, 600 R of gamma rays delivered over about 110 days, and four weekly intraperitoneal (i.p.) injections of 77.5 mg/kg of ethylnitrosourea (ENU). The results reported in this paper are restricted to mutations induced in stem-cell spermatogonia and to the 34 more common skeletal anomalies (i.e., those found in 0.5% or more of the control offspring). Mutation induction was demonstrated for eight anomalies in the acute X-ray experiment and for 17 anomalies (including those same eight anomalies) in the ENU experiment. In spite of the surprisingly high mutation rates found for these treatments, there was no hint of any induction of such dominant mutations by 600 R of chronic gamma radiation. Our results suggest that several anomalies related to variation in the sacralization pattern may be particularly useful for revealing induction of dominant mutations.     

Molecular basis for control of conjugation by bacterial pheromone and inhibitor peptides

In many bacteria expression of lateral gene transfer and of virulence factors is controlled by cell-cell signalling systems. Molecular interactions of microbial signal molecules with their cognate receptors are not well understood. For the Enterococcus faecalis conjugative plasmid pCF10, the PrgX protein serves as a molecular switch controlling expression of conjugation and virulence genes encoded by the plasmid. The induction state of a pCF10-carrying donor cell is determined by the ratio of two signalling peptides, cCF10 pheromone and iCF10 inhibitor. Recent analysis of PrgX/cCF10 interactions suggests a mechanism for conversion to the induced state. However, the means by which iCF10 peptide antagonizes cCF10 activity is unclear, and it has been suggested that inhibitor peptides block import of pheromone peptides. We now show that both of these peptides interact with the same binding pocket of PrgX, but they differentially alter the conformation of the protein and its oligomerization state, resulting in opposing biological activities.