Automated precolumn fluorescence labelling by carbodiimide activation of N-acetylaspartate and N-acetylaspartylglutamate applied to an HPLC brain tissue analysis.

An automated method is described to couple carboxyl-containing metabolites to the fluorophore 2-aminoanthracene in aqueous solution (containing 75% methanol) in the presence of N,N-dicyclohexylcarbodiimide. The reaction was optimized for N-acetylaspartate (N-Ac-Asp) and N-acetylaspartylglutamate (N-Ac-Asp-Glu). The reactions occurred within 5 min at room temperature in the presence of 0.5-2 mM HCl. At concentrations of electrolytes exceeding 10 mM the coupling reaction became suboptimal. Derivatization was performed in a commercial precolumn derivatization unit. Additional tubing was needed to provide the reagents prior to reversed-phase HPLC and fluorescence detection. The assay is linear over at least three orders of magnitude; as little as 1 pmol could reproducibly be assayed in 100 micrograms wet weight brain tissue extracted with a mixture of methanol and 4 mM HCl (9:1, v/v). N-Ac-Asp and N-Ac-Asp-Glu levels in several brain regions and spinal cord were similar to those so far reported. The compounds could not be detected in peripheral tissue. The advantages, prospects and limitations of the present approach over existing methods to estimate water-soluble carboxylic acids is discussed.     

Molecular Fate of Heterologous Bacterial DNA in Competent BACILLUS SUBTILIS. I. Processing of B. PUMILUS and B. LICHENIFORMIS DNA in B. SUBTILIS

Competent Bacillus subtilis cells were exposed to radioactive and density labeled donor DNA extracted from B. pumilus and B. licheniformis. The DNA from these strains hybridized with B. subtilis DNA in vitro at a rate of 24% and 11%, respectively. After entry the vast majority of heterologous DNA was found at the single-strand DNA position in CsCl gradients, and was gradually degraded during incubation. Much less donor DNA than expected from the hybridization values participated in the formation of the donorrecipient complex (DRC). By subjecting the heterologous DRC to sonication and alkaline CsCl gradient centrifugation, it was established that the DRC consisted of three components: (1) recipient DNA in which breakdown products of donor DNA were incorporated through DNA synthesis, (2) recipient DNA in which donor DNA was covalently integrated and (3) recipient DNA in which the donor moiety was not covalently integrated.     

Ultraviolet inactivation and excision-repair in Bacillus subtilis. I. Construction and characterization of a transformable eightfold auxotrophic strain and two ultraviolet-sensitive derivatives

An eightfold auxotrophic strain of Bacillus subtilis was constructed. It was about equally well transformable for all markers. When this strain was used as recipient in transformation, single marker transformation frequencies of 0.5–2.0% were obtained. The markers were located relatively to each other using marker frequency analysis. Two UV-sensitive derivatives, equally well transformable as the parental multiple auxotroph, were isolated. One was highly sensitive to UV irradiation, was host cell reactivation-negative and did not show DNA breakdown or recovery of DNA synthesis after exposure to UV. Using UV-inactivated transforming DNA, this strain's transformability was strongly reduced as compared with both the UV-resistant parental strain and the other, moderately UV-sensitive, strain.     

Ultraviolet inactivation and excision-repair in Bacillus subtilis. II. Differential inactivation and differential repair of transforming markers

Ultraviolet inactivation of transforming Bacillus subtilis markers was studied with the aid of an eightfold auxotrophic recipient and its excision-repair-deficient derivative. The results allow the following conclusions. (i) Wild-type B. subtilis cells are able to repair approx. 80% of the UV-induced lesions causing inactivation of transforming activity in UV-sensitive recipients; (ii) Saturating amounts of donor DNA increase the apparent marker sensitivities. This phenomenon is most pronounced in transformation of UV-sensitive recipients; (iii) various markers are inactivated to different degrees, both when assayed on the wild-type as well as on the UV-sensitive strain; (iv) Various markers are repaired to different degrees in the wild-type recipient.     

Poor transformability with novr and eryr donor DNA of some mitomycin-C-sensitive strains of Haemophilus influenzae

Three mitomycin-C-sensitive (MC^s) strains of Haemophilus influenzae, being poorly transformable with DNA carrying the antibiotic resistence markers nov^r and ery^r, were further investigated to determine the cause of their poor transformability. After being genetically integrated into the mutant-recipient genome the donor marker is replicated at the same rate as in the wild type, indicating that recombination in the mutant strains is normal. In the mutants, designated T^d (transformation-deficient), the poor transformability for the nov^r and ery^r markers is due to the lack of phenotypic expression of the markers, because the strains are killed by concentrations of antibiotics normally used to select for nov^r and ery^r transformants. Since the strains exhibit extreme sensitivity both to deoxycholate and osmotic shock in the presence of EDTA, the increased sensitivity to antibiotics (including mitomycin-C) is probably caused by a change in the cell envelope. Although recombination in the mutant strains proceeds normally, the T^d mutation nevertheless decreases both the rate of inactivation and of integration of donor DNA.     

Molecular dissection of the C-terminal regulatory domain of the plant plasma membrane H+-ATPase AHA2: mapping of residues that when altered give rise to an activated enzyme.

The plasma membrane H+-ATPase is a proton pump belonging to the P-type ATPase superfamily and is important for nutrient acquisition in plants. The H+-ATPase is controlled by an autoinhibitory C-terminal regulatory domain and is activated by 14-3-3 proteins which bind to this part of the enzyme. Alanine-scanning mutagenesis through 87 consecutive amino acid residues was used to evaluate the role of the C-terminus in autoinhibition of the plasma membrane H+-ATPase AHA2 from Arabidopsis thaliana. Mutant enzymes were expressed in a strain of Saccharomyces cerevisiae with a defective endogenous H+-ATPase. The enzymes were characterized by their ability to promote growth in acidic conditions and to promote H+ extrusion from intact cells, both of which are measures of plasma membrane H+-ATPase activity, and were also characterized with respect to kinetic properties such as affinity for H+ and ATP. Residues that when altered lead to increased pump activity group together in two regions of the C-terminus. One region stretches from K863 to L885 and includes two residues (Q879 and R880) that are conserved between plant and fungal H+-ATPases. The other region, incorporating S904 to L919, is situated in an extension of the C-terminus unique to plant H+-ATPases. Alteration of residues in both regions led to increased binding of yeast 14-3-3 protein to the plasma membrane of transformed cells. Taken together, our data suggest that modification of residues in two regions of the C-terminal regulatory domain exposes a latent binding site for activatory 14-3-3 proteins.     

Different mechanisms for thermal inactivation of Bacillus subtilis signal peptidase mutants.

The type I signal peptidase SipS of Bacillus subtilis is of major importance for the processing of secretory precursor proteins. In the present studies, we have investigated possible mechanisms of thermal inactivation of five temperature-sensitive SipS mutants. The results demonstrate that two of these mutants, L74A and Y81A, are structurally stable but strongly impaired in catalytic activity at 48 degrees C, showing the (unprecedented) involvement of the conserved leucine 74 and tyrosine 81 residues in the catalytic reaction of type I signal peptidases. This conclusion is supported by the crystal structure of the homologous signal peptidase of Escherichia coli (Paetzel, M., Dalbey, R. E., and Strynadka, N. C. J. (1998) Nature 396, 186-190). In contrast, the SipS mutant proteins R84A, R84H, and D146A were inactivated by proteolytic degradation, indicating that the conserved arginine 84 and aspartic acid 146 residues are required to obtain a protease-resistant conformation. The cell wall-bound protease WprA was shown to be involved in the degradation of SipS D146A, which is in accord with the fact that SipS has a large extracytoplasmic domain. As WprA was not involved in the degradation of the SipS mutant proteins R84A and R84H, we conclude that multiple proteases are responsible for the thermal inactivation of temperature-sensitive SipS mutants.