Effects of Ammonium and Non-Ammonium Salt Additions on Methane Oxidation by Methylosinus trichosporium OB3b and Maine Forest Soils

Additions of ammonium and non-ammonium salts inhibit atmospheric methane consumption by soil at salt concentrations that do not significantly affect the soil water potential. The response of soils to non-ammonium salts has previously raised questions about the mechanism of ammonium inhibition. Results presented here show that inhibition of methane consumption by non-ammonium salts can be explained in part by ion-exchange reactions: cations desorb ammonium, with the level of desorption varying as a function of both the cation and anion added; differential desorption results in differential inhibition levels. Differences in the extent of inhibition among ammonium salts can also be explained in part by the effects of anions on ammonium exchange. In contrast, only minimal effects of cations and anions are observed in liquid cultures of Methylosinus trichosporium OB3b. The comparable level of inhibition by equinormal concentrations of NH₄Cl and (NH₄)₂SO₄ and the insensitivity of salt inhibition to increasing methane concentrations (from 10 to 100 ppm) are of particular interest, since both of these patterns are in contrast to results for soils. The greater inhibition of methane consumption for NH₄Cl than (NH₄)₂SO₄ in soils can be attributed to increased ammonium adsorption by sulfate; increasing inhibition by non-ammonium salts with increasing methane concentrations can be attributed to desorbed ammonium and a physiological mechanism proposed previously for pure cultures.     

Mechanistic Analysis of Ammonium Inhibition of Atmospheric Methane Consumption in Forest Soils

Methane consumption by forest soil was studied in situ and in vitro with respect to responses to nitrogen additions at atmospheric and elevated methane concentrations. Methane concentrations in intact soil decreased continuously from atmospheric levels at the surface to 0.5 ppm at a depth of 14 cm. The consumption rate of atmospheric methane in soils, however, was highest in the 4- to 8-cm depth interval (2.9 nmol per g of dry soil per day), with much lower activities below and above this zone. In contrast, extractable ammonium and nitrate concentrations were highest in the surface layer (0 to 2 cm; 22 and 1.6 μmol per g of dry soil, respectively), as was potential ammonium-oxidizing activity (19 nmol per g of dry soil per day). The difference in zonation between ammonium oxidation and methane consumption suggested that ammonia-oxidizing bacteria did not contribute significantly to atmospheric methane consumption. Exogenous ammonium inhibited methane consumption in situ and in vitro, but the pattern of inhibition did not conform to expectations based on simple competition between ammonia and methane for methane monooxygenase. The extent of ammonium inhibition increased with increasing methane concentration. Inhibition by a single ammonium addition remained constant over a period of 39 days. In addition, nitrite, the end product of methanotrophic ammonia oxidation, was a more effective inhibitor of methane consumption than ammonium. Factors that stimulated ammonium oxidation in soil, e.g., elevated methane concentrations and the availability of cosubstrates such as formate, methanol, or β-hydroxybutyrate, enhanced ammonium inhibition of methane oxidation, probably as a result of enhanced nitrite production.     

Toxicity of Bacillus thuringiensis var. israelensis Crystals to Aedes aegypti Larvae: Carbonate Reversal

The toxicity of purified Bacillus thuringiensis var. israelensis crystals to larvae of Aedes aegypti could be reversed 100-fold by levels of K₂CO₃ as low as 0.15%.     

UPTAKE AND METABOLISM OF 5-METHYL-TETRAHYDROFOLIC ACID BY RAT BRAIN SLICES

DNA-dependent DNA polymerases were partially purified from nuclei of cells from the occipital lobe of human brain. The purification procedure included successive DEAE-cellulose and phosphocellulose column chromatography, gel filtration and sucrose density gradient centrifugation steps. Four enzymes corresponding to DNA polymerases-α, β, γ, and terminal deoxynucleotidyl transferase were found. Brain DNA polymerases could be differentiated from one another by size, template preferences and sensitivity to sulfhydryl blocking agents.     

Anaerobic degradation of 3-aminobenzoate by a newly isolated sulfate reducer and a methanogenic enrichment culture

A new rod-shaped, gram-negative, non-sporing sulfate reducer, strain mAB1, was enriched and isolated from marine sediment samples with 3-aminobenzoate as sole electron and carbon source. Strain mAB1 degraded 3-aminobenzoate completely to CO₂ and NH₃ with stoichiometric reduction of sulfate to sulfide. Cells contained carbon monoxide dehydrogenase, cytochromes, and sulfite reductase P582. Strain mAB1 degraded also benzoate, 4-aminobenzoate, hydroxybenzoates, and some aliphatic compounds. Besides sulfates, also sulfite was reduced with 3-aminobenzoate as electron donor, but not thiosulfate, sulfur, nitrate, or fumarate. The strain grew in sulfide-reduced mineral medium supplemented with 7 vitamins. Strain mAB1 was tentatively affiliated with the genus Desulfobacterium. Experiments with dense cell supsensions showed benzoate accumulation during 3-aminobenzoate degradation under conditions of sulfate limitation or cyanide inhibition. 3-Aminobenzoate was activated to 3-aminobenzoyl-CoA by cell extracts in the presence of ATP, coenzyme A, and Mg²⁺. Acitivity of 3-aminobenzoyl-CoA synthetase was 16 nmol per min and mg protein, with a K_M for 3-aminobenzoate lower than 50 M. Cell extract of 3-aminobenzoate-grown cells activated also 3-hydroxybenzoate (31.7 nmol per min and mg protein) and benzoate (2.3 nmol per min and mg protein), but not 2-aminobenzoate or 4-aminobenzoate. In the presence of NADH of NADPH, 3-aminobenzoyl-CoA was further metabolized to a not yet identified reduced product.Freshwater enrichments with 3-aminobenzoate in the absence of an extenal electron acceptor led to a stable methanogenic enrichment culture consisting of three types of bacteria. 3-Aminobenzoate was degraded completely to CO₂ and stoichiometric amounts of CH₄, with intermediary acetate accumulation.     

Differences in the nuclease sensitivity between the two alleles of the immunoglobulin kappa light chain genes in mouse liver and myeloma nuclei.

In mouse myeloma T the productive kappa light chain gene differs from its aberrantly rearranged allele in the patterns of DNAase I hypersensitive sites. In the region of the alleles where they are identical in sequence they have one site in common which lies 0.8 kb downstream of the coding region; but two sites upstream of and within the C gene segment (2) are found only on the non-productive allele. Within the region of different sequences both alleles have analogously located DNAase I hypersensitive sites; they lie 0.15 kb upstream of the respective leader segments and cover putative promoter sequences. Only one of the six DNAase I hypersensitive sites is also very sensitive towards micrococcal nuclease due to its particular DNA sequence. The non-rearranged gene studied in liver nuclei has no DNAase I hypersensitive sites but is preferentially cleaved in A/T rich regions.     

The integration of chloroplast protein targeting with plant developmental and stress responses

The plastids, including chloroplasts, are a group of interrelated organelles that confer photoautotrophic growth and the unique metabolic capabilities that are characteristic of plant systems. Plastid biogenesis relies on the expression, import, and assembly of thousands of nuclear encoded preproteins. Plastid proteomes undergo rapid remodeling in response to developmental and environmental signals to generate functionally distinct plastid types in specific cells and tissues. In this review, we will highlight the central role of the plastid protein import system in regulating and coordinating the import of functionally related sets of preproteins that are required for plastid-type transitions and maintenance.     

Import pathways of chloroplast interior proteins and the outer-membrane protein OEP14 converge at Toc75

Most chloroplast outer-membrane proteins are synthesized at their mature size without cleavable targeting signals. Their insertion into the outer membrane is insensitive to thermolysin pretreatment of chloroplasts and does not require ATP. It has therefore been assumed that insertion of outer-membrane proteins proceeds through a different pathway from import into the interior of chloroplasts, which requires a thermolysin-sensitive translocon complex and ATP. Here, we show that a model outer-membrane protein, OEP14, competed with the import of a chloroplast interior protein, indicating that the two import pathways partially overlapped. Cross-linking studies showed that, during insertion, OEP14 was associated with Toc75, a thermolysin-resistant component of the outer-membrane protein-conducting channel that mediates the import of interior-targeted precursor proteins. Whereas almost no OEP14 inserted into protein-free liposomes, OEP14 inserted into proteoliposomes containing reconstituted Toc75 with a high efficiency. Taken together, our data indicate that Toc75 mediates OEP14 insertion, and therefore plays a dual role in the targeting of proteins to the outer envelope membrane and interior of chloroplasts.     

atToc159 is a selective transit peptide receptor for the import of nucleus-encoded chloroplast proteins

The members of the Toc159 family of GTPases act as the primary receptors for the import of nucleus-encoded preproteins into plastids. Toc159, the most abundant member of this family in chloroplasts, is required for chloroplast biogenesis (Bauer, J., K. Chen, A. Hiltbunner, E. Wehrli, M. Eugster, D. Schnell, and F. Kessler. 2000. Nature. 403:203-207) and has been shown to covalently cross-link to bound preproteins at the chloroplast surface (Ma, Y., A. Kouranov, S. LaSala, and D.J. Schnell. 1996. J. Cell Biol. 134:1-13; Perry, S.E., and K. Keegstra. 1994. Plant Cell. 6:93-105). These reports led to the hypothesis that Toc159 functions as a selective import receptor for preproteins that are required for chloroplast development. In this report, we provide evidence that Toc159 is required for the import of several highly expressed photosynthetic preproteins in vivo. Furthermore, we demonstrate that the cytoplasmic and recombinant forms of soluble Toc159 bind directly and selectively to the transit peptides of these representative photosynthetic preproteins, but not representative constitutively expressed plastid preproteins. These data support the function of Toc159 as a selective import receptor for the targeting of a set of preproteins required for chloroplast biogenesis.     

Budding of PPxY-containing rhabdoviruses is not dependent on host proteins TGS101 and VPS4A

Viral matrix proteins of several enveloped RNA viruses play important roles in virus assembly and budding and are by themselves able to bud from the cell surface in the form of lipid-enveloped, virus-like particles (VLPs). Three motifs (PT/SAP, PPxY, and YxxL) have been identified as late budding domains (L-domains) responsible for efficient budding. L-domains can functionally interact with cellular proteins involved in vacuolar sorting (VPS4A and TSG101) and endocytic pathways (Nedd4), suggesting involvement of these pathways in virus budding. Ebola virus VP40 has overlapping PTAP and PPEY motifs, which can functionally interact with TSG101 and Nedd4, respectively. As for vesicular stomatitis virus (VSV), a PPPY motif within M protein can interact with Nedd4. In addition, M protein has a PSAP sequence downstream of the PPPY motif, but the function of PSAP in budding is not clear. In this study, we compared L-domain functions between Ebola virus and VSV by constructing a chimeric M protein (M40), in which the PPPY motif of VSV M is replaced by the L domains of VP40. The budding efficiency of M40 was 10-fold higher than that of wild-type (wt) M protein. Overexpression of a dominant negative mutant of VPS4A or depletion of cellular TSG101 reduced the budding of only M40-containing VLPs but not that of wt M VLPs or live VSV. These findings suggest that the PSAP motif of M protein is not critical for budding and that there are fundamental differences between PTAP-containing viruses (Ebola virus and human immunodeficiency virus type 1) and PPPY-containing viruses (VSV and rabies virus) regarding their dependence on specific host factors for efficient budding.