Fermentative degradation of monohydroxybenzoates by defined syntrophic cocultures

From anaerobic freshwater enrichment cultures with 3-hydroxybenzoate as sole substrate, a slightly curved rod-shaped bacterium was isolated in coculture with Desulfovibrio vulgaris as hydrogen scavenger. The new isolate degraded only 3-hydroxybenzoate or benzoate, and depended on syntrophic cooperation with a hydrogenoxidizing methanogen or sulfate reducer. 3-Hydroxybenzoate was degraded via reductive dehydroxylation to benzoate. With 2-hydroxybenzoate (salicylate), short coccoid rods were enriched from anaerobic freshwater mud samples, and were isolated in defined coculture with D. vulgaris. This isolate also fermented 3-hydroxybenzoate or benzoate in obligate syntrophy with a hydrogen-oxidizing anaerobe. The new isolates were both Gram-negative, non-sporeforming strict anaerobes. They fermented hydroxybenzoate or benzoate to acetate, CO₂, and, presumably, hydrogen which was oxidized by the syntrophic partner organism. With hydroxybenzoates, but not with benzoate, Acetobacterium woodii could also serve as syntrophic partner. Other substrates such as sugars, alcohols, fatty or amino acids were not fermented. External electron acceptors such as sulfate, sulfite, nitrate, or fumarate were not reduced. In enrichment cultures with 4-hydroxybenzoate, decarboxylation to phenol was the initial step in degradation which finally led to acetate, methane and CO₂.     

Kinetic characterization of early intermediates in the folding of E. coli tryptophan-synthase beta 2 subunit

This report describes the use of fluorescence energy transfer between an intrinsic energy donor (tryptophan 177) and two chemically added acceptors to study intermediates in the folding of the beta 2 subunit of E. coli tryptophan-synthase. Two early folding steps are thus identified and characterized. One is very rapid (its rate constant at 12 degrees C is 0.02 sec-1) and corresponds to the folding of the N-terminal domain into a structure whose overall features approximate well those of the native domain. The second step is somewhat slower (its rate constant at 12 degrees C is 0.008 sec-1) and involves a conformational rearrangement of the N-terminal domain brought about by the interactions between the N- and C-terminal domains within a monomeric beta chain. This brings to five the number of intermediates which have been identified and ordered on the folding pathway of the dimeric beta 2 subunit.     

Toxoplasma gondii: decreased resistance to intracellular bacteria in mice

The effect of sublethal inocula of Toxoplasma gondii on the course of listeriosis and salmonellosis in mice was investigated. Intravenous injection of T. gondii 24 hr after inoculation of Listeria monocytogenes increased mortality from 16% (L. monocytogenes alone) to 68% (L. monocytogenes + T. gondii) (P less than 0.001). Multiplication of L. monocytogenes in spleens also was increased significantly in mice given T. gondii. By 3 days after infection, mice that had received T. gondii and L. monocytogenes had approximately 10 times the number of L. monocytogenes per spleen compared to mice receiving L. monocytogenes alone. Similarly, mortality and the number of bacteria in spleens were increased in mice injected with Salmonella typhimurium and then inoculated with T. gondii. An in vitro assay of macrophage listeriacidal activity was used to investigate the mechanism of this decreased resistance. Peritoneal macrophages from mice injected with T. gondii were less bactericidal than macrophages from uninfected mice. Delayed hypersensitivity responses to L. monocytogenes antigen were markedly suppressed in mice injected with T. gondii. T. gondii infection appears to suppress both macrophage and T-lymphocyte function and may result in decreased resistance to infections caused by intracellular bacteria.     

Isolation of a brain peptide identical to the intestinal PHI (peptide HI)

The isolation of a brain peptide identical to the intestinal peptide PHI (peptide HI) is described. The peptide was isolated from porcine brain extract using a chemical assay method based on its C-terminal isoleucine amide structure. The complete amino acid sequence of the peptide was found to be: His-Ala-Asp-Gly-Val-Phe-Thr-Ser-Asp-Phe-Ser-Arg-Leu-Leu-Gly-Gln-Leu-Ser-Ala- Lys-Lys-Tyr-Leu-Glu-Ser-Leu-Ile-NH2. This sequence is identical to the intestinal peptide thus demonstrating PHI to be a brain-gut peptide. The role of PHI in the central nervous system as a neurotransmitter or neuromodulator is discussed.     

Bacterioplankton in the acidified Lake Gårdsjön

Total bacterial numbers in different strata of lake water and in inlet and outlet streams have been recorded during a yearly cycle. A calculated mean cell volume of 0.342 µm² has then been used to estimate bacterial biomass in the lake. Change of biomass during the year was substantial and the range was from about 0.1 g · m^–3 to about 1.0–1.2 g · m^–3. The seasonal development included a spring-early summer increase followed by a decrease to the minimum in July–August. Correlation between epi- and hypolimnion was high and in both strata two dominant autumn peaks in biomass appeared. With the exception of the last autumn peak the development of bacterial biomass was closely related to development of phytoplankton biomass and production.     

The orientation of the magnetic axes of membrane-bound iron-sulfur clusters and a cytochrome b-559 in the green halophilic alga Dunaliella parva

Photosynthetic membrane fragments separated from whole cells of the green alga Dunaliella parva, were oriented by incorporation into multilayers on thin Mylar films. These partially dehydrated films were then examined by EPR spectroscopy for evidence of orientation of paramagnetic components. Five previously identified paramagnetic components, the reduced states of iron-sulfur clusters A and B, the intermediate acceptor X^?, the reduced Rieske iron-sulfur cluster, and oxidized cytochrome b-559, displayed EPR signals showing orientation. In addition, several previously unknown paramagnetic components were also observed to be oriented. Four components, previously characterized in spinach chloroplast preparations, the iron-sulfur clusters A and B, the intermediate acceptor X^?, and cytochrome b-559, were shown to be similar in the green alga, D. parva. The orientations of iron-sulfur clusters A and B, however, were determined unambiguously in this preparation; this was not possible in previous work with spinach. The heme plane orientation of cytochrome b-559 was found to be perpendicular to the membrane plane in agreement with the results in spinach preparations. A new photoinduced EPR signal with g values of 1.88, 1.97 and 2.12 was seen only in the oriented preparations and was indicative of a reduced iron-sulfur cluster with an orientation different from that of iron-sulfur cluster A or B. This suggests the existence of a previously unidentified acceptor in Photosystem I of green plants. These studies clearly show that the orientation of these components in bioenergetic membranes are conserved over a large span of evolutionary development and are, therefore, an important aspect of the mechanism of electron transfer.     

Similarity of EPR Signal IIf rise and P-680+ decay kinetics in Tris-washed chloroplast Photosystem II preparations as a function of pH

The rise time, of Signal II_f and the decay time of P-680⁺ have been measured kinetically as a function of pH by using EPR. The Photosystem II-enriched preparations which were used as samples were derived from spinach chloroplasts, and they evolved oxygen before Tris washing. The onset kinetics of Signal II_f are in agreement, within experimental error, with the fast component of the decay of an EPR signal attributable to P-680⁺. The signal II_f rise kinetics also show good agreement with published values of the pH dependence of the decay of P-680⁺ measured optically (Conjeaud, H. and Mathis, P. (1980) Biochim. Biophys. Acta 590, 353–359). These results are consistent with a model where the species Z (or D₁) responsible for Signal II_f is the immediate electron donor to P-680⁺ in tris-washed Photosystem II fragments.     

Arrangement of MP26 in lens junctional membranes: Analysis with proteases and antibodies

Summary The major membrane protein of the bovine lens fiber cell is a 26-kilodalton (kD) protein (MP26), which appears to be a component of the extensive junctional specializations found in these cells. To examine the arrangement of MP26 within the junctional membranes, various proteases were incubated with fiber cell membranes that had been isolated with or without urea and/or detergents. These membranes were analyzed with electron microscopy and SDS-PAGE to determine whether the junctional specializations or the proteins were altered by proteolysis. Microscopy revealed no obvious structural changes. Electrophoresis showed that chymotrypsin, papain, and trypsin degraded MP26 to 21–22 kD species. A variety of protease treatments, including overnight digestions, failed to generate additional proteolysis. Regions on MP26 which were sensitive to these three proteases overlapped. Smaller peptides were cleaved from MP26 with V8 protease and carboxypptidases A and B. Protein domains cleaved by these proteases also overlapped with regions sensitive to chymotrypsin, papain, and trypsin. Specific inhibition of the carboxypeptidases suggested that cleavage obtained with these preparations was not likely due to contaminating endoproteases. Since antibodies are not thought to readily penetrate the 2–3 nm extracellular gap in the fiber cell junctions, antibodies to MP26 were used to analyze the location of the protease-sensitive domains. Antisera were applied to control (26 kD) and proteolyzed (22 kD) membranes, with binding being evaluated by means of ELISA reactions on intact membranes. Antibody labeling was also done following SDS-PAGE and transfer to derivatized paper. Both assays showed a significant decrease in binding following proteolysis, with the 22 kD product showing no reaction with the anti-MP26 sera. These investigations suggest that MP26 is arranged with approximately fourfifths of the primary sequence “protected” by the lipid bilayer and the narrow extracellular gap. One-fifth of the molecule, including the C-terminus, appears to be exposed on the cytoplasmic side of the membrane.     

In vitro incorporation of 2′-deoxyadenosine and 3′-deoxyadenosine into yeast tRNAPhe using tRNA nucleotidyl transferase,and properties of tRNAPhe-C-C-2′dA and tRNAPhe-C-C-3′dA

2′-Deoxyadenosine and 3′-deoxyadenosine (cordycepin) can be incorporated into the 3′-terminal position of tRNA^Phe by tRNA nucleotidyl transferase. tRNA^Phe-C-C-2′dA and tRNA^Phe-C-C-3′dA, missing the cis-diol group at the 3′-terminal end are resistant to periodate oxidation and are not able to form borate complexes. In aminoacylation experiments only the tRNA^Phe-C-C-3′dA proved to be chargeable.     

Development of single blastomeres from four- and eight-cell mouse embryos fused into the enucleated half of a two-cell embryo

Single blastomeres from four- and eight-cell mouse embryos were fused into the enucleated halves of two-cell embryos, and the ability of these reconstituted embryos to develop in vitro and in vivo was examined. The proportion of these reconstituted embryos developing to blastocysts was 74% (60/81) when four-cell embryo blastomeres were used as nuclei donors and 31% (57/182) when eight-cell embryo blastomeres were used. Eight complete sets of the quadruplet-reconstituted embryos developed to blastocysts, and five live young (9%, 5/57) were obtained after transfer; however, none of the live young were clones. Although when using blastomeres from eight-cell embryos no complete set of eight developed to blastocysts, sextuplets were obtained. The blastocysts, however, failed to produce live young after transfer. In assessing the outgrowths, it was found that 43% of those derived from reconstituted embryos using blastomeres from four-cell embryos had an inner cell mass (ICM); however, outgrowths derived from reconstituted embryos using blastomeres from eight-cell embryos lacked an ICM. These results suggest that the genomes of four- and eight-cell nuclei introduced into the enucleated halves of two-cell embryos are reversed to support the development of the reconstituted embryo.