Reduction of the amount of periplasmic hydrogenase in Desulfovibrio vulgaris (Hildenborough) with antisense RNA: direct evidence for an important role of this hydrogenase in lactate metabolism.

To establish the function of the periplasmic Fe-only hydrogenase in the anaerobic sulfate reducer Desulfovibrio vulgaris (Hildenborough), derivatives with a reduced content of this enzyme were constructed by introduction of a plasmid that directs the synthesis of antisense RNA complementary to hydrogenase mRNA. It was demonstrated that the antisense RNA technique allowed specific suppression of the synthesis of this hydrogenase in D. vulgaris by decreasing the amount of hydrogenase mRNA but did not result in the complete elimination of the enzyme, as is usual with most conventional mutagenesis techniques. The hydrogenase content in these antisense RNA-producing D. vulgaris clones was two- to threefold lower than in the parental strain when the strains were grown in batch cultures with lactate as a substrate and sulfate as a terminal electron acceptor. Under these conditions, several differences in growth parameters were measured between the hydrogenase-suppressed clones and wild-type D. vulgaris: growth rates of the clones decreased two- to threefold, and at excess lactate, growth yields were reduced by 20%. Furthermore, the amount of hydrogen measured in the culture headspaces was reduced three- to fivefold for the clones. These observations indicate that this hydrogenase has an important function during growth on lactate and is involved in hydrogen production from protons and electrons originating from at least one of the two oxidation reactions in the conversion of lactate to acetate. The implications for the energy metabolism of D. vulgaris are discussed.     

Hydrolysis and reduction of factor 390 by cell extracts of Methanobacterium thermoautotrophicum (strain delta H).

Cell extracts of Methanobacterium thermoautotrophicum (strain delta H) were found to perform a hydrogen-dependent reduction of factor 390 (F390), the 8-adenylyl derivative of coenzyme F420. Upon resolution of cell extracts, F390-reducing activity copurified with the coenzyme F420-dependent hydrogenase. This indicates that F390 serves as a substrate of that enzyme. Activity towards F390 was approximately 40-fold lower than that towards coenzyme F420 (0.12 and 5.2 mumol.min-1.mg of protein-1, respectively). In addition, cell extracts catalyzed the hydrolysis of F390 to AMP and coenzyme F420. This hydrolysis required the presence of thiols (6 mM) and much ionic strength (1 M KCl) and was reversibly inhibited by oxygen. The reaction proceeded optimally at pH 8.2 and was Mn dependent. Conditions for F390 hydrolysis in cell extracts are in many respects opposite to those previously described for F390 synthesis.     

Pathogen-induced proteins with inhibitory activity toward Phytophthora infestans.

A bioassay using Phytophthora infestans was developed to determine whether inhibitory proteins are induced in pathogen-inoculated plants. Using this bioassay, AP24, a 24-kilodalton protein causing lysis of sporangia and growth inhibition of P. infestans, was purified from tobacco plants inoculated with tobacco mosaic virus. Analysis of the N-terminal amino acid sequence identified AP24 as the thaumatin-like protein osmotin II. The sequence was also similar to NP24, the salt-induced protein from tomato. Subsequently, we purified a protein from tomato plants inoculated with P. infestans that had inhibitory activities identical to those of the tobacco AP24. The N-terminal amino acid sequence of this protein was also similar to those of osmotin and NP24. In general, both the tobacco and tomato AP24 caused lysis of sporangia at concentrations greater than 40 nanomolar and severely inhibited hyphal growth at concentrations greater than 400 nanomolar. Because both proteins were induced by pathogen inoculation, we discussed the possible involvement of these proteins as a plant defense mechanism.     

Purification of insect vitellogenin and vitellin by gel-immobilized ferric chelate.

Vitellogenin and vitellin of Manduca sexta and some other insect species were purified by immobilized metal ion affinity chromatography. Ferric ion was chosen as the immobilized metal ion. Agarose-bound carboxymethylpicolylamine was used as the chelating adsorbent for the ferric ion. Vitellogenin and vitellin, both phosphorylated lipoproteins, were shown to bind specifically to the iron. The general applicability of immobilized ferric ion affinity chromatography for the purification of insect vitellogenin and vitellin is suggested.     

The influence of coppicing on vegetation

In the Netherlands and adjacent countries on old (non-holocene) soils the relation was studied between high forest dominated by Fagus sylvatica, and coppice derived from it, with Quercus spp. and Betula spp. or Carpinus betulus. From place-names a shift Fagus > Quercus > Betula is found, caused by cutting and coppicing. An analysis of relevés of the vegetation yielded more detailed information.The occurrence and rejuvenation of Fagus and other climax tree species decrease, while most other trees and shrubs show an increase. The mean total number of species in 1396 relevés increases from 22.0 to 24.4. Decreasing species include widely distributed species as well as character species. Increasing species include shrubs, woodedge species of heathland or limestone grassland, and nitrophilous species.The light-indication according to Ellenberg increases considerably, most of all in the types on poor soils. Moisture seems hardly involved, but pH-indication is divergent: decreasing in acid types, increasing in alkaline types. For the latter erosion is held responsible. N-indication follows the same trend. All ecological shifts remain within the framework of each association concerned.The Sørensen index for floristic composition shows a mean of 68% correlation, of the same order as variants within one association. The ecological correlation is even higher. So coppice can be considered as management variants, not deserving an association status, as often was given in the past.     

Identification of the Escherichia coli murI gene, which is required for the biosynthesis of D-glutamic acid, a specific component of bacterial peptidoglycan.

The murI gene of Escherichia coli, whose inactivation results in the inability to form colonies in the absence of D-glutamic acid, was identified in the 90-min region of the chromosome. The complementation of an auxotrophic E. coli B/r strain by various DNA sources allowed us to clone a 2.5-kbp EcoRI chromosomal fragment carrying the murI gene into multicopy plasmids. The murI gene corresponds to a previously sequenced open reading frame, ORF1 (J. Brosius, T. J. Dull, D. D. Sleeter, and H. F. Noller. J. Bacteriol. 148:107-127, 1987), located between the btuB gene, encoding the vitamin B12 outer membrane receptor protein, and the rrnB operon, which contains the genes for 16S, 23S, and 5S rRNAs. The murI gene product is predicted to be a protein of 289 amino acids with a molecular weight of 31,500. Attempts to identify its enzymatic activity were unsuccessful. Cells altered in the murI gene accumulate UDP-N-acetylmuramyl-L-alanine to a high level when depleted of D-glutamic acid. Pools of precursors located downstream in the pathway are consequently depleted, and cell lysis finally occurs when the peptidoglycan content is 25% lower than that of normally growing cells.     

Murein-metabolizing enzymes from Escherichia coli: existence of a second lytic transglycosylase.

In addition to the soluble lytic transglycosylase, a murein-metabolizing enzyme with a molecular mass of 70 kDa (Slt70), Escherichia coli possesses a second lytic transglycosylase, which has been described as a membrane-bound lytic transglycosylase (Mlt; 35 kDa; EC 3.2.1.-). The mlt gene, which supposedly encodes Mlt, was cloned, and the complete nucleotide sequence was determined. The open reading frame, identified on a 1.7-kb SalI-PstI fragment, codes for a protein of 323 amino acids (M(r) = 37,410). Two transmembrane helices and one membrane-associated helix were predicted in the N-terminal half of the protein. Lysine and arginine residues represent up to 15% of the amino acids, resulting in a calculated isoelectric point of 10.0. The deduced primary structure did not show significant sequence similarity to Slt70 from E. coli. High-level expression of the presumed mlt gene was not paralleled by an increase in murein hydrolase activity. To clarify the identity of the second transglycosylase, we purified an enzyme with the specificity of a transglycosylase from an E. coli slt deletion strain. The completely soluble transglycosylase, with a molecular mass of approximately 35 kDa, was designated Slt35. Its determined 26 N-terminal amino acids showed similarity to a segment in the middle of the Slt70 primary structure. Polyclonal anti-Mlt antibodies, which had been used for the isolation of the mlt gene, were found to cross-react with Mlt as well as with Slt35, suggesting that the previously described Mlt preparation was contaminated with Slt35. We conclude that the second transglycosylase of E. coli is not a membrane-bound protein but rather is a soluble protein.     

Cloning and sequencing of IS1086, an Alcaligenes eutrophus insertion element related to IS30 and IS4351.

A new insertion sequence (IS), designated IS1086, was isolated from Alcaligenes eutrophus CH34 by being trapped in plasmid pJV240, which contains the Bacillus subtilis sacB and sacR genes. The 1,106-bp IS1086 element contains partially matched (22 of 28 bp) terminal-inverted repeats and a long open reading frame. Hybridization data suggest the presence of one copy of IS1086 in the strain CH34 heavy-metal resistance plasmid pMOL28 and at least two copies in its chromosome. Analysis of the IS1086 nucleotide sequence revealed striking homology with two other IS elements, IS30 and IS4351, suggesting that they are three close members in a family of phylogenetically related insertion sequences. One open reading frame of the Spiroplasma citri phage SpV1-R8A2 B was also found to be related to this IS family but to a lesser extent. Comparison of the G+C contents of IS30 and IS1086 revealed that they conform to their respective hosts (46 versus 50% for IS30 and Escherichia coli and 64.5% for IS1086 and A. eutrophus). The pressure on the AT/GC ratio led to a very different codon usage in these two closely related IS elements. Results suggesting that IS1086 transposition might be activated by some forms of stress are discussed.     

Effects on ADH activity and distribution, following selection for tolerance to ethanol in Drosophila melanogaster.

Strains of Drosophila melanogaster homozygous for either the AdhF or the AdhS allele were kept on food supplemented with ethanol for 20 generations. These strains (FE and SE) were tested for tolerance to ethanol and compared with control strains (FN and SN). The E strains showed increased tolerance to ethanol both in the adult and in the juvenile life stages. In adults the increase in tolerance was not accompanied by an increase in overall ADH activity. However, there were changes in the distribution of ADH over the body parts. Flies of the FE strain possessed significantly more ADH in the abdomen, compared with FN. Another set of FN and SN populations were started both on standard food and on ethanol food with reduced yeast concentrations. After 9 months ADH activities were determined in flies from these populations which had been placed on three different media: the food the populations had been kept on, regular food and regular food supplemented with ethanol. The phenotypic effects of yeast reduction on ADH activity were considerably, but longterm genetic effects were limited.