Genetic variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) populations from Latin America is associated with variations in susceptibility to Bacillus thuringiensis cry toxins

Bacillus thuringiensis strains isolated from Latin American soil samples that showed toxicity against three Spodoptera frugiperda populations from different geographical areas (Mexico, Colombia, and Brazil) were characterized on the basis of their insecticidal activity, crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parasporal crystals, plasmid profiles, and cry gene content. We found that the different S. frugiperda populations display different susceptibilities to the selected B. thuringiensis strains and also to pure preparations of Cry1B, Cry1C, and Cry1D toxins. Binding assays performed with pure toxin demonstrated that the differences in the toxin binding capacities of these insect populations correlated with the observed differences in susceptibility to the three Cry toxins analyzed. Finally, the genetic variability of the three insect populations was analyzed by random amplification of polymorphic DNA-PCR, which showed significant genetic diversity among the three S. frugiperda populations analyzed. The data presented here show that the genetic variability of S. frugiperda populations should be carefully considered in the development of insect pest control strategies, including the deployment of genetically modified maize in different geographical regions.     

Jasmonic acid signalling and herbivore resistance traits constrain regrowth after herbivore attack in Nicotiana attenuata

Because traits conferring resistance on herbivores can reduce fitness-associated traits, trade-offs may occur between tolerance and resistance responses. We examined these trade-offs in genotypes of Nicotiana attenuata that were transformed to silence trypsin proteinase inhibitor (TPI) production (AS-Natpi), an antiherbivore defense associated with (14%) reductions in seed production, and the jasmonate signal cascade that elicits these defenses (AS-Nalox3), by measuring stalk and axillary branch growth and seed production after two defoliation regimes and Manduca sexta larval attack to bottom or middle and top stalk leaves. Larval attack and defoliation at middle and top leaves depressed seed production and increased axillary branching more than at bottom leaves. AS-Nalox3 and AS-Natpi plants produced significantly longer (two- to fourfold) branches than did wild-type (WT) plants, results that are consistent with resource-based trade-offs between resistance and regrowth. Methyl jasmonate (MeJA) treatment of AS-Nalox3 plants restored WT branch growth, suggesting that jasmonic acid (JA) signalling suppresses regrowth and contributes to apical dominance. These results are consistent with the existence of JA- and resource-mediated trade-offs between regrowth and herbivore resistance traits.     

Derivation of a growth hormone gene cassette for goat by mutagenesis of the corresponding bovine construct and its expression in Pichia pastoris

Recombinant bovine growth hormone (rbGH), a 191-aa polypeptide that affects animal growth and lactation, has been used for several years to increase milk production in dairy cattle. It has also been used in goats (Capra hircus) instead of their own hormone (chGH), which is still not available in the market. Since both hormones differ in only one amino acid residue, a strategy based on PCR mediated site-directed mutagenesis, was used to convert the bGH expression cassette harbored by an integration plasmid for Pichia pastoris into a chGH. Transformation by homologous recombination of Pichia pastoris GS115 strain with the linearized new plasmid resulted in transformants that, upon fermentation and induction with methanol, secreted a band with the expected size and immunoreactivity for GH. Production of total proteins secreted into culture medium (50 ml) was 20 µg/ml, of which 60% was chGH as judged by densitometry in SDS-PAGE. Its biological activity was confirmed in vitro when 3T3 pre-adipocytes exposed to the induced culture medium differentiated into adipocytes in cell culture.     

Parental food conditions affect sex-specific embryo mortality in the yellow-legged gull (Larus michahellis)

Different mortality of males and females during early post-hatching development in sexually size-dimorphic bird species is usually attributed to different nutritional requirements of the sexes, because mortality is mostly biassed toward the larger sex. We investigated whether sex-specific embryo mortality in the yellow-legged gull (Larus michahellis), a size-dimorphic seabird, depends on parental condition. To test this, we experimentally modified parental nutritional conditions by supplementary feeding of yellow-legged gulls during egg formation, to evaluate sex-biassed environmental sensitivity of gull embryos. We found that eggs were larger in supplemented clutches, but egg size did not affect embryo survival. Survival of male gull embryos was more related to parental food conditions than was survival of female embryos. Survival of male embryos in supplemented clutches was greater than in unsupplemented clutches whereas survival of female embryos was similar in both groups. Because size at hatching was similar in both sexes our results suggest that male phenotype disadvantage is not exclusively linked to the energy demands of size-dimorphic development at the embryo stage.     

The cbiS gene of the archaeon Methanopyrus kandleri AV19 encodes a bifunctional enzyme with adenosylcobinamide amidohydrolase and alpha-ribazole-phosphate phosphatase activities

Here we report the initial biochemical characterization of the bifunctional alpha-ribazole-P (alpha-RP) phosphatase, adenosylcobinamide (AdoCbi) amidohydrolase CbiS enzyme from the hyperthermophilic methanogenic archaeon Methanopyrus kandleri AV19. The cbiS gene encodes a 39-kDa protein with two distinct segments, one of which is homologous to the AdoCbi amidohydrolase (CbiZ, EC 3.5.1.90) enzyme and the other of which is homologous to the recently discovered archaeal alpha-RP phosphatase (CobZ, EC 3.1.3.73) enzyme. CbiS function restored AdoCbi salvaging and alpha-RP phosphatase activity in strains of the bacterium Salmonella enterica where either step was blocked. The two halves of the cbiS genes retained their function in vivo when they were cloned separately. The CbiS enzyme was overproduced in Escherichia coli and was isolated to >95% homogeneity. High-performance liquid chromatography, UV-visible spectroscopy, and mass spectroscopy established alpha-ribazole and cobyric acid as the products of the phosphatase and amidohydrolase reactions, respectively. Reasons why the CbiZ and CobZ enzymes are fused in some archaea are discussed.     

Control of acetyl-coenzyme A synthetase (AcsA) activity by acetylation/deacetylation without NAD(+) involvement in Bacillus subtilis

Posttranslational modification is an efficient mechanism for controlling the activity of structural proteins, gene expression regulators, and enzymes in response to rapidly changing physiological conditions. Here we report in vitro and in vivo evidence that the acuABC operon of the gram-positive soil bacterium Bacillus subtilis encodes a protein acetyltransferase (AcuA) and a protein deacetylase (AcuC), which may control the activity of acetyl-coenzyme A (CoA) synthetase (AMP-forming, AcsA) in this bacterium. Results from in vitro experiments using purified proteins show that AcsA is a substrate for the acetyl-CoA-dependent AcuA acetyltransferase. Mass spectrometry analysis of a tryptic digest of acetylated AcsA (AcsA(Ac)) identified residue Lys549 as the sole modification site in the protein. Unlike sirtuins, the AcuC protein did not require NAD(+) as cosubstrate to deacetylate AcsA(Ac). The function of the putative AcuB protein remains unknown.     

Studies of the CobA-type ATP:Co(I)rrinoid adenosyltransferase enzyme of Methanosarcina mazei strain Go1

Although methanogenic archaea use B(12) extensively as a methyl carrier for methanogenesis, little is known about B(12) metabolism in these prokaryotes or any other archaea. To improve our understanding of how B(12) metabolism differs between bacteria and archaea, the gene encoding the ATP:co(I)rrinoid adenosyltransferase in Methanosarcina mazei strain G?1 (open reading frame MM3138, referred to as cobA(Mm) here) was cloned and used to restore coenzyme B(12) synthesis in a Salmonella enterica strain lacking the housekeeping CobA enzyme. cobA(Mm) protein was purified and its initial biochemical analysis performed. In vitro, the activity is enhanced 2.5-fold by the addition of Ca(2+) ions, but the activity was not enhanced by Mg(2+) and, unlike the S. enterica CobA enzyme, it was >50% inhibited by Mn(2+). The CobA(Mm) enzyme had a K(m)(ATP) of 3 microM and a K(m)(HOCbl) of 1 microM. Unlike the S. enterica enzyme, CobA(Mm) used cobalamin (Cbl) as a substrate better than cobinamide (Cbi; a Cbl precursor); the beta phosphate of ATP was required for binding to the enzyme. A striking difference between CobA(Se) and CobA(Mm) was the use of ADP as a substrate by CobA(Mm), suggesting an important role for the gamma phosphate of ATP in binding. The results from (31)P-nuclear magnetic resonance spectroscopy experiments showed that triphosphate (PPP(i)) is the reaction by-product; no cleavage of PPP(i) was observed, and the enzyme was only slightly inhibited by pyrophosphate (PP(i)). The data suggested substantial variations in ATP binding and probably corrinoid binding between CobA(Se) and CobA(Mm) enzymes.     

Mutation in a "tesB-like" hydroxyacyl-coenzyme A-specific thioesterase gene causes hyperproduction of extracellular polyhydroxyalkanoates by Alcanivorax borkumensis SK2

A novel mutant of the marine oil-degrading bacterium Alcanivorax borkumensis SK2, containing a mini-Tn5 transposon disrupting a "tesB-like" acyl-coenzyme A (CoA) thioesterase gene, was found to hyperproduce polyhydroxyalkanoates (PHA), resulting in the extracellular deposition of this biotechnologically important polymer when grown on alkanes. The tesB-like gene encodes a distinct novel enzyme activity, which acts exclusively on hydroxylated acyl-CoAs and thus represents a hydroxyacyl-CoA-specific thioesterase. Inactivation of this enzyme results in the rechanneling of CoA-activated hydroxylated fatty acids, the cellular intermediates of alkane degradation, towards PHA production. These findings may open up new avenues for the development of simplified biotechnological processes for the production of PHA as a raw material for the production of bioplastics.     

Modeling product formation in anaerobic mixed culture fermentations

The anaerobic conversion of organic matter to fermentation products is an important biotechnological process. The prediction of the fermentation products is until now a complicated issue for mixed cultures. A modeling approach is presented here as an effort to develop a methodology for modeling fermentative mixed culture systems. To illustrate this methodology, a steady-state metabolic model was developed for prediction of product formation in mixed culture fermentations as a function of the environmental conditions. The model predicts product formation from glucose as a function of the hydrogen partial pressure (P(H2)), reactor pH, and substrate concentration. The model treats the mixed culture as a single virtual microorganism catalyzing the most common fermentative pathways, producing ethanol, acetate, propionate, butyrate, lactate, hydrogen, carbon dioxide, and biomass. The product spectrum is obtained by maximizing the biomass growth yield which is limited by catabolic energy production. The optimization is constrained by mass balances and thermodynamics of the bioreactions involved. Energetic implications of concentration gradients across the cytoplasmic membrane are considered and transport processes are associated with metabolic energy exchange to model the pH effect. Preliminary results confirmed qualitatively the anticipated behavior of the system at variable pH and P(H2) values. A shift from acetate to butyrate as main product when either P(H2) increases and/or pH decreases is predicted as well as ethanol formation at lower pH values. Future work aims at extension of the model and structural validation with experimental data.