A hydrogen-evolving enzyme is present in Frankia sp. R43.

The ability to evolve hydrogen using methyl viologen as an electron donor was assayed in the nitrogen-fixing actinomycetes Frankia sp. R43 and Frankia sp. KB5. To further examine the nature of hydrogen-evolving enzymes that may be present in these organisms immunological studies were performed. Under anaerobic conditions (both nitrogen-limiting and nitrogen-containing) Frankia sp. R43 but not Frankia sp. KB5 evolved hydrogen,which was not linked to NAD-reducing activity. Immunological analysis of total protein from Frankia sp. R43 and Frankia sp. KB5 using an antiserum raised against Ralstonia eutropha HoxF, recognized an antigen in Frankia sp. R43 but not in Frankia sp. KB5. Immunogold labeling using antibodies raised against the R. eutropha HoxH recognized sites in both hyphae and vesicles of Frankia sp. R43, but not in Frankia sp. KB5. Based on these physiological and immunological findings, we conclude that Frankia sp. R43 has a hydrogen-evolving hydrogenase.

Biochemical and enzymological properties of the polyhydroxybutyrate synthase from the extremely halophilic archaeon strain 56.

Some members of the archaebacterial family Halobacteriaceae have been determined to accumulate polyhydroxyalkanoate (PHA) and poly(3-hydroxybutyrate) (PHB). The extremely halophilic archaebacterium strain 56 is capable of accumulating large amounts of PHB. Since measurements of enzyme activities related to archaebacterial PHB biosynthesis have never been achieved, we investigated the enzymology of PHB biosynthesis in strain 56. Crude extracts of strain 56 cultivated under accumulating conditions showed PHB synthase activity, whereas neither beta-ketothiolase nor NADH/NADPH-dependent acetoacetyl-CoA reductase activity was detectable. An 80-kDa protein, cross-reacting with the anti-PHB synthase antibodies raised against the PHB synthase from Ralstonia eutropha, was identified in the crude extract and was strongly enriched by purification of PHB granules. The granule-associated PHB synthase was enzymologically characterized. Enzyme kinetics showed a specific activity of about 4.6U/mg and Hill plot analysis revealed a K(0.5) of 56 microM with (R)-3-hydroxybutyryl-CoA employed as substrate. A Hill coefficient of 1.75 indicated that the PHB synthase exhibited positive cooperativity. The thioesters 3-hydroxyvaleryl-CoA, 4-hydroxybutyryl-CoA, and 3-hydroxydecanoyl-CoA were not accepted as substrates. Moreover, the PHB synthase was found to be competitively inhibited by CoA, showing an IC(50) of 160 microM. The PHB synthase was stable up to 60 degrees C and still exhibited about 90% of the maximum enzyme activity, which was obtained at 40 degrees C. In contrast to the soluble PHB synthase, the granule-bound PHB synthase was almost independent of the salt concentration. The PHB synthase could not be released from the PHB granules, indicating a covalent attachment to the PHB core. This is the first description of an archaebacterial PHA synthase.

Rapid identification of Ralstonia eutropha strain CH34 using monoclonal antibodies.

Ralstonia eutropha strain CH34 (formerly Alcaligenes eutrophus CH34) is an aerobic Gram-negative and facultative chemolithotrophic bacteria with plasmid-bound resistance to heavy metals. The presence of Ralstonia eutropha strain CH34 is an indication of environmental heavy metals pollution. The major purpose of this work was to produce monoclonal antibodies (MAbs) against the metal transport outer membrane proteins. In this way, bacteria outer membranes, grown with or without iron, were purified. The electrophoretic pattern of the outer membrane revealed that, in iron starvation conditions, at least four proteins were overexpressed. These outer membranes were used to immunize mice to produce MAbs. About 200 hybridomas were tested by enzyme-linked immunoadsorbent assay (ELISA). Most of these hybridomas exhibited cross reactions with Escherichia coli and Klebsiella aerogenes. Two hybridomas, AE5/7 and AE5/9, produced MAbs that detected specifically Ralstonia eutropha strain CH34. Analysis by Western blotting showed that these MAbs recognized a protein with a molecular weight of about 41 kDa. Moreover, the presence of the two megaplasmids was required for the full expression of the 41-kDa protein, as demonstrated by screening of the derivatives strains by ELISA. These MAbs could be used for a specific and rapid detection of Ralstonia eutropha strain CH34, using direct immunological methods.