Control of the production of exo-β-N-acetylglucosaminidase by Bacillus subtilis B. Derepression during gluconeogenesis and initial stages of sporulation

1. The control of exo-beta-N-acetylglucosaminidase (EC 3.2.1.30) production by Bacillus subtilis B growing on a chemically defined medium was studied. 2. The enzyme was repressed during exponential growth by those carbon sources that enter the glycolytic pathway above the level of phosphoenolpyruvate. When exponential growth ceased as a result of low concentrations of the nitrogen, carbon or metal ion components of the medium, the enzyme was formed and its amount could be increased by the addition of cell-wall fragments as inducer. 3. The enzyme was de-repressed and could be induced during exponential growth on non-glycolytic compounds metabolized directly into pyruvate, acetyl-CoA or tricarboxylic acid cycle intermediates. 4. The major difference in the metabolism of the organism utilizing these two groups of compound was the existence of high activities of phosphoenolpyruvate carboxylase required for gluconeogenesis. 5. It is concluded that the de-repression of glucosaminidase occurs when the only principal change detected in the intermediary metabolism of the organism was the presence of high activities of phosphoenolpyruvate carboxylase. 6. When the organism was grown on media containing repressing compounds, the enzyme was only de-repressed on entry of the cells into the initial stages of sporulation, where phosphoenolpyruvate carboxylase activity, even in the presence of excess of glucose, increased in parallel with glucosaminidase, neutral proteinase and alkaline phosphatase activities. 7. These results suggest a strong link, at the level of the tricarboxylic acid cycle, between the control of phosphoenolpyruvate carboxylase and the control of the de-repression of glucosaminidase and sporulation.     

Poly(A) shortening and degradation of the 3'' A+U-rich sequences of human c-myc mRNA in a cell-free system.

The early steps in the degradation of human c-myc mRNA were investigated, using a previously described cell-free mRNA decay system. The first detectable step was poly(A) shortening, which generated a pool of oligoadenylated mRNA molecules. In contrast, the poly(A) of a stable mRNA, gamma globin, was not excised, even after prolonged incubation. The second step, degradation of oligoadenylated c-myc mRNA, generated decay products whose 3' termini were located within the A+U-rich portion of the 3' untranslated region. These products disappeared soon after they were formed, consistent with rapid degradation of the 3' region. In contrast, the 5' region, corresponding approximately to c-myc exon 1, was stable in vitro. The data indicate a sequential degradation pathway in which 3' region cleavages occur only after most or all of the poly(A) is removed. To account for rapid deadenylation, we suggest that the c-myc poly(A)-poly(A)-binding protein complex is readily dissociated, generating a protein-depleted poly(A) tract that is no longer resistant to nucleases.     

Self-reported risks for multiple-drug resistance among new tuberculosis cases: implications for drug susceptibility screening and treatment

Background

Multiple drug-resistance in new tuberculosis (TB) cases accounts for the majority of all multiple drug-resistant TB (MDR-TB) worldwide. Effective control requires determining which new TB patients should be tested for MDR disease, yet the effectiveness of global screening recommendations of high-risk groups is unknown.

Methods

Sixty MDR-TB cases with no history of previous TB treatment, 80 drug-sensitive TB and 80 community-based controls were recruited in Lima, Peru between August and December, 2008 to investigate whether recommended screening practices identify individuals presenting with MDR-TB. Odd ratios (OR) and 95% confidence intervals (CI) were calculated using logistic regression to study the association of potential risk factors with case/control variables.

Results

MDR-TB cases did not differ from drug-sensitive TB and community controls in rates of human immunodeficiency virus infection, reported hospital or prison visits in the 3 years prior to diagnosis. MDR-TB cases were more likely than drug-sensitive TB controls to have had a recent MDR-TB household contact (OR 4.66, (95% CI 1.56–13.87)); however, only 15 cases (28.3%) reported this exposure. In multivariate modeling, recent TB household contact, but not contact with an MDR-TB case, remained predictive of MDR-TB, OR 7.47, (95% CI 1.91–29.3). Living with a partner rather than parents was associated with a lower risk of MDR-TB, OR 0.15, (95% CI 0.04–0.51).

Conclusion

Targeted drug susceptibility testing (DST) linked to reported MDR-TB contact or other high-risk exposures does not identify the majority of new TB cases with MDR disease in Lima where it is endemic. All new TB cases should be screened with DST to identify MDR patients. These findings are likely applicable to other regions with endemic MDR-TB.     

Stereochemical studies of D-glucal hydration by alpha-glucosidases and exo-alpha-glucanases: indications of plastic and conserved phases in catalysis by glycosylases

Alpha-Glucosidases from Aspergillus niger, pig serum, ungerminated rice, buckwheat, and sugar beet seeds (but not from brewers' yeast or honeybee) were found to catalyze the hydration of D-glucal. Each reactive alpha-glucosidase, incubated with D-glucal in D2O, was shown to protonate (deuteriate) this prochiral substrate from above its re face, i.e., from a direction opposite that assumed for protonating alpha-D-glucosidic substrates. At the same time, D-glucal hydration catalyzed by three of the alpha-glucosidases that acted rapidly enough in D2O to determine product configuration was found to yield 2-deoxy-D-glucose of the same specific (alpha-) configuration as the D-glucose produced from alpha-D-glucosidic substrates. These findings substantially extend those reported earlier for the hydration of D-glucal by one (Candida tropicalis) alpha-glucosidase preparation. Together with other recent results, they suggest that the process of catalysis by alpha-glucosidases (and perhaps glycosylases in general) may comprise two separate and separately controlled parts, namely, a "plastic" phase concerned with substrate protonation and a substrate-unrelated "conserved" phase concerned with the creation of product configuration. In contrast to the alpha-glucosidases, three "inverting" exo-alpha-glucanases (Arthrobacter globiformis glucodextranase; Rhizopus niveus and Paecilomyces varioti glucoamylase) were found to protonate D-glucal from below its si face. Further, whereas the catalysis of D-glucal hydration by the alpha-glucosidases was intensively inhibited by excess substrate, that promoted by the exo-glucanases showed no detectable substrate inhibition.     

Molecular dynamics study of the KcsA potassium channel

The structural, dynamical, and thermodynamic properties of a model potassium channel are studied using molecular dynamics simulations. We use the recently unveiled protein structure for the KcsA potassium channel from Streptomyces lividans. Total and free energy profiles of potassium and sodium ions reveal a considerable preference for the larger potassium ions. The selectivity of the channel arises from its ability to completely solvate the potassium ions, but not the smaller sodium ions. Self-diffusion of water within the narrow selectivity filter is found to be reduced by an order of magnitude from bulk levels, whereas the wider hydrophobic section of the pore maintains near-bulk self-diffusion. Simulations examining multiple ion configurations suggest a two-ion channel. Ion diffusion is found to be reduced to approximately (1)/(3) of bulk diffusion within the selectivity filter. The reduced ion mobility does not hinder the passage of ions, as permeation appears to be driven by Coulomb repulsion within this multiple ion channel.     

Resistance in sunflower and interaction with Bacillus thuringiensis for control of banded sunflower moth (Lepidoptera: Tortricidae)

Four sunflower accessions were compared with a susceptible check, hybrid '894', in the greenhouse to determine their resistance to the banded sunflower moth, Cochylis hospes Walsingham, and their interaction with Bacillus thuringiensis Berliner variety kurstaki. Antibiosis, expressed as lower larval weight, was detected in all of the accessions. In addition to being antibiotic, sunflower accession Ames 3291 was antixenotic to banded sunflower moth oviposition and exhibited an additional impact on larval weight when B. thuringiensis was applied. By itself, B. thuringiensis provided better control of banded sunflower moth than the resistance tested. However, banded sunflower moth-resistant sunflower would be a good option when B. thuringiensis or another insecticide is not applied, and it may prevent the economic threshold from being reached.     

Metal Ion Binding and Conformational Transitions in Concanavalin A: A Structure-Function Study

Abstract

The affinity of the lectin Concanavalin A (Con A) for saccharides, and its requirement for metal ions such as Mn²⁺ and Ca²⁺, have been known for about 50 years. However the relationship between metal ion binding and the saccharide binding activity of Con A has only recently been examined in detail. Brown et al. (Biochemistry 16, 3883 (1977)) showed that Con A exists as a mixture of two conformational states: a “locked” form and an “unlocked” form. The unlocked form of the protein weakly binds metal ions and saccharide, and is the predominate conformation of demetallized Con A (apo-Con A) at equilibrium. The locked form binds two metal ions per monomer with the resulting complex(es) possessing full saccharide binding activity. Brown and coworkers measured the kinetics of the transition of the unlocked form to the fully metallized locked conformation containing Mn²⁺and Ca²⁺. They also demonstrated that Mn²⁺ alone could form a locked ternary complex with Con A, and that rapid removal of the ions resulted in a metastable form of apo-Con A in the locked conformation which slowly (hours at 25°C) reverted back to (predominantly) the unlocked conformation. The ability to form either conformation in the absence or presence of metal ions has thus allowed us to explore the relationship between metal ion binding and conformational transitions in Con A as determinants of the saccharide binding activity of the lectin.

Based on the kinetics of the transition of unlocked apo-Con A to fully metallized locked Con A, and X-ray crystallographic data, it appears that the transition between the two conformations of Con A involves a cis-trans isomerization of an Ala-Asp peptide bond in the backbone of the protein, near one of the two metal ion binding sites. The relatively large activation energy for the transition (～ 22 kcal M^?1) results in relatively slow interconversions between the conformations (from minutes to days), whereas the equilibria with metal ions and saccharide are rapid. Thus, many metastable complexes can be formed and a variety of transition pathways between the two conformations studied.

We have identified and characterized binary, ternary, and quaternary complexes of both conformations of Con A containing Mn²⁺ and saccharide, and have determined both metalion and saccharide dissociation constants for all of them, as well as equilibrium and kinetic values for the conformational transitions between them. The main finding is that saccharide binds very weakly (K_d～2 M) to unlocked apo-Con A and very tightly to the locked ternary Mn²⁺-Con A complex (K_d～ 10^?4 M). Saccharide binding increases along the various pathways connecting these two species in a nonadditive fashion. Thus, both conformation and metal ion binding determine the saccharide affinity of each complex, although the specificity of saccharide binding of the various species is maintained throughout.     

Fluoride inhibition of enolase: crystal structure and thermodynamics

Enolase is a dimeric metal-activated metalloenzyme which uses two magnesium ions per subunit: the strongly bound conformational ion and the catalytic ion that binds to the enzyme-substrate complex inducing catalysis. The crystal structure of the human neuronal enolase-Mg2F2P(i) complex (enolase fluoride/phosphate inhibitory complex, EFPIC) determined at 1.36 A resolution shows that the combination of anions effectively mimics an intermediate state in catalysis. The phosphate ion binds in the same site as the phosphate group of the substrate/product, 2-phospho-D-glycerate/phosphoenolpyruvate, and induces binding of the catalytic Mg2+ ion. One fluoride ion bridges the structural and catalytic magnesium ions while the other interacts with the structural magnesium ion and the ammonio groups of Lys 342 and Lys 393. These fluoride ion positions correspond closely to the positions of the oxygen atoms of the substrate's carboxylate moiety. To relate structural changes resulting from fluoride, phosphate, and magnesium ions binding to those that are induced by phosphate and magnesium ions alone, we also determined the structure of the human neuronal enolase-Mg2P(i) complex (enolase phosphate inhibitory complex, EPIC) at 1.92 A resolution. It shows the closed conformation in one subunit and a mixture of open and semiclosed conformations in the other. The EPFIC dimer is essentially symmetric while the EPIC dimer is asymmetric. Isothermal titration calorimetry data confirmed binding of four fluoride ions per dimer and yielded Kb values of 7.5 x 10(5) +/- 1.3 x 10(5), 1.2 x 10(5) +/- 0.2 x 10(5), 8.6 x 10(4) +/- 1.6 x 10(4), and 1.6 x 10(4) +/- 0.7 x 10(4) M(-1). The different binding constants indicate negative cooperativity between the subunits; the asymmetry of EPIC supports such an interpretation.