Corrected sequence of the mannitol (mtl) operon in Escherichia coli

The previously published sequences of the operator-promoter region of the mannitol operon of Escherichia coli and of the mtlD gene have been found to contain a number of errors. The major conclusions reported previously were correct, but additionally it is now clear that a C-terminal portion of mannitol-1-phosphate dehydrogenase (the mtlD gene product) exhibits significant sequence identity with an amino-terminal region of human liver fructose-6-phosphate-2-kinase:fructose-2,6-bisphosphatase.     

Correlation of Zn2+ content with aflatoxin content of corn.

Forty-nine samples from the 1983 Virginia corn harvest were analyzed for aflatoxin, zinc, copper, iron, and manganese content. Values (mean +/- standard deviation) were as follows: aflatoxin, 117 +/- 360 micrograms/kg; zinc, 22.5 +/- 3.4 mg/kg; copper, 2.27 +/- 0.56 mg/kg; iron, 40.8 +/- 18.7 mg/kg; and manganese, 5.1 +/- 1.1 mg/kg. Aflatoxin levels positively correlated with zinc (Spearman correlation coefficient, 0.385; P less than 0.006) and copper levels (Spearman correlation coefficient, 0.573; P less than 0.0001). Based on biochemical data in the literature, we believe that the correlation with zinc is important and that there may be a cause-and-effect relationship between zinc levels in corn and aflatoxin levels which are produced upon infection with Aspergillus flavus or A. parasiticus. Control of aflatoxin contamination in field corn by decreasing the zinc levels may be feasible, but no methods to decrease zinc levels are currently available.     

Stages of tubulin assembly and disassembly studied by time-resolved synchrotron X-ray scattering

The structural transitions occurring during the assembly and disassembly of pig brain microtubule protein were investigated by time-resolved X-ray scattering using synchrotron radiation. The reactions were introduced by a slow temperature scan (2 deg.C/min) from 0 °C to 37 °C and back. Several structurally distinct states could be resolved during one cycle of assembly/disassembly. During the temperature rise, one observes four main phases: prenucleation events, microtubule nucleation, growth, and postassembly events.Heating from 0 °C to 22 °C results in a biphasic breakdown of rings and other aggregates, while the apparent mean diameter increases from 38 to 41 nm. Parallel time-resolved electron microscopic observations suggest that the initial solution contains several types of aggregates, mostly double concentric and single rings, but also rod-like particles, clusters of rings and other aggregates. All of these tend to break down with increasing temperature. Double concentric rings seem to dissociate into large and small single rings before both types of rings break down into protofilament fragments and tubulin subunits. From the breakdown products, associations of several protofilament fragments are formed, which are important for initiating microtubule nucleation. Assembly of nuclei begins around 22 °C. Microtubule elongation takes place between 25 and 30 °C. They grow mainly by addition of tubulin subunits but not via rings.During the reverse temperature scan, microtubules shorten by the release of subunits and/or small protofilament fragments from their ends. The degree of disassembly is strongly increased below 22 °C. Below about 10 °C rings are reformed, probably from the fragments, but their final number is much less than initially.Conditions that prevent microtubule nucleation such as GDP or Ca²⁺ also stabilize rings, even at 37 °C. Thus, rings are viewed as storage aggregates of tubulin and microtubule associated proteins, whose breakdown is a prerequisite for microtubule formation, and whose reformation is independent of microtubule breakdown.The midpoints of microtubule growth and breakdown differ by about 12 deg.C so that the system shows hysteresis-like behavior. It is dependent on microtubule formation and is not seen when the temperature is cycled below that required for nucleation. Thus, even during a slow temperature scan, microtubule assembly is kinetically limited by nucleation. By contrast, depolymerization proceeds close to equilibrium.The radius of gyration of the tubulin heterodimers is 3.1 nm. The weight average diameter of rings in cold solutions is 38 nm, that of microtubules is 24.5 nm.At radiation dose rates of about 100 rad/s. radiation damage is of minor importance, as judged by the criterion of polymerizability. Total doses of up to 500,000 rad can be applied.Some concepts of analyzing time-resolved X-ray scattering data are presented. They make use of the fact that the scattering intensities vary continuously both with scattering angle and time. Cross-correlation of different regions of the pattern, and comparison of their temperature derivatives, reveals structural transitions not seen by other techniques.     

Cooperative Action of Rhizobium meliloti Nodulation and Infection Mutants during the Process of Forming Mixed Infected Alfalfa Nodules

Alfalfa plants co-inoculated with Rhizobium meliloti nodulation (Nod-) and infection mutants deficient in exopolysaccharide production (Inf-EPS-) formed mixed infected nodules that were capable of fixing atmospheric nitrogen. The formation of infected nodules was dependent on close contact between the inoculation partners. When the partners were separated by a filter, empty Fix- nodules were formed, suggesting that infection thread formation in alfalfa is dependent on signals from the nodulation and infection genes. In mixed infected nodules, both nodulation and infection mutants colonized the plant cells and differentiated into bacteroids. The formation of bacteroids was not dependent on cell-to-cell contact between the mutants. Immunogold/silver staining revealed that the ratio of the two mutants varied considerably in colonized plant cells following mixed inoculation. The introduction of an additional nif/fix mutation into one of the inoculation partners did not abolish nitrogen fixation in mixed infected nodules. The expression of nif D::lacZ fusions additionally demonstrated that mutations in the nodulation and infection genes did not prevent the nif genes from being expressed in the mutant bacteroids.     

Regulation of mannitol biosynthesis and degradation by Cryptococcus neoformans.

Cryptococcus neoformans, an encapsulated yeast that is an opportunistic pathogen of AIDS patients, produced and secreted mannitol when incubated with an appropriate carbon source. Glucose, fructose, and mannose were good growth substrates and were converted to mannitol. Maltose and xylose were good growth substrates but were not converted to mannitol. Cells of C. neoformans that were grown on a non-mannitol-generating carbon source, such as peptone or xylose, were able to convert glucose to mannitol only after a prolonged lag period in the presence of glucose. It was concluded that the enzymes of the mannitol biosynthetic pathway were not constitutively expressed but were induced in response to glucose or to a glucose metabolite. Enzymes required to catabolize mannitol, however, were constitutively expressed. The production of mannitol was inhibited by anaerobiosis, by the respiratory poison rotenone, and by polyethylenesulfonate, a specific inhibitor of fungal NADP-dependent dehydrogenases. When cells were incubated with deuterated glucose, the deuterium content of the mannitol produced was much lower than that of the glucose precursor, indicating that the glucose was diluted by an intracellular pool of an intermediate. We had previously shown that C. neoformans contains a large intracellular pool of glucose 6-phosphate, and we now conclude that this pool of glucose 6-phosphate is metabolically active.     

A potent specific inhibitor of 6-phosphogluconate dehydrogenase ofCryptococcus neoformans and of certain other fungal enzymes

A particular lot of the zwitterionic buffer, 2(N-morpholino) ethane sulfonic acid (MES), contained a contaminant that inhibited a number of fungal NADP-dependent dehydrogenases. Enzymes that were particularly sensitive include 6-phosphogluconate dehydrogenases fromCryptococcus neoformans andSchizophyllum commune and glucose-6-phosphate dehydrogenase fromSchizophyllum commune. A number of NADP-dependent dehydrogenases of animal origin were tested and all were completely insensitive to inhibition except for rat liver 6-phosphogluconate dehydrogenase, which was 10-fold less sensitive than theCryptococcal enzyme. The pattern of inhibition in all cases was linear competitive versus NADP. The inhibitor has been purified and identified as an ethylenesulfonic acid oligomer. This inhibitor holds promise as a model compound for the development of a specific antifungal agent.     

Purification and characterization of glucose-6-phosphate dehydrogenase from Aspergillus parasiticus

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was purified from mycelium of Aspergillus parasiticus (1-11-105 Whl). The enzyme had a molecular weight of 1.8 × 10⁵ and was composed of four subunits of apparently equal size. The substrate specificity was very strict, only glucose 6-phosphate and glucose being oxidized by NADP or thio-NADP. Zinc ion was a powerful inhibitor of the enzyme, inhibition being competitive with respect to glucose 6-phosphate, with K_i about 2.5 μm. Other divalent metal ions which also serve as inhibitors are nickel, cadmium, and cobalt. It is proposed that the stimulation of polyketide synthesis by zinc ion may be mediated in part by inhibition of glucose-6-phosphate dehydrogenase.