Rhizobitoxine inhibition of hydrogenase synthesis in free-living Bradyrhizobium japonicum.

Rhizobitoxine produced by Bradyrhizobium species strongly prevented derepression of hydrogenase expression in free-living Bradyrhizobium japonicum, although the toxin had no effect on the activity of cells which had already synthesized hydrogenase protein. Dihydrorhizobitoxine, a structural analog of rhizobitoxine, proved to be a less potent inhibitor of hydrogenase derepression. Rhizobitoxine did not cause cell death at a concentration sufficient to eliminate hydrogenase expression. The large subunit of hydrogenase was not detectable with antibody after derepression in the presence of rhizobitoxine. The general pattern of proteins synthesized from 14C-labeled amino acids during derepression was not significantly different in the presence or absence of rhizobitoxine. These results indicated that rhizobitoxine inhibited hydrogenase synthesis in free-living B. japonicum. Cystathionine and methionine strongly prevented the inhibition of hydrogenase derepression by rhizobitoxine, suggesting that the inhibition involves the level of sulfur-containing amino acids in the cell.     

Evidence that the GCN2 protein kinase regulates reinitiation by yeast ribosomes.

The yeast gene GCN4 produces an mRNA that has a long 5' 'untranslated' region containing four small open reading frames (ORFs) preceding the protein coding frame. This configuration suppresses the rate by which GCN4 protein is synthesized. However, translational derepression of the GCN4 mRNA occurs when yeast cells are grown under conditions of amino acid limitation. Such translational derepression requires the GCN2 protein kinase and the presence of the 5' most proximal ORF. In this study we show that a functional coupling between the translation of the first ORF and the amount of the GCN2 protein is responsible for the translational derepression of the GCN4 mRNA. Our evidence suggests that this coupling involves an increase in the ability of 40S ribosomal subunits that have translated the first frame to resume scanning and reinitiate translation at a downstream AUG independently of the base sequence in the intervening region.     

Plasma-membrane phospholipid unsaturation affects expression of the general amino-acid permease in Saccharomyces cerevisiae Y185

Saccharomyces cerevisiae Y185, enriched in linoleyl residues and incubated for up to 4 h in derepression buffer, more rapidly acquired general amino-acid permease (GAP) activity, as measured by the rate of accumulation of L-alanine, compared with organisms enriched in oleyl residues. A GAP-less mutant incubated under the same conditions did not acquire further L-alanine-accumulating ability, irrespective of the nature of the fatty-acyl enrichment. During derepression, KT values for the GAP were virtually identical irrespective of the fatty-acyl enrichment, but Vmax values were greater for linoleyl residue-enriched organisms, particularly after 1 h in derepression buffer. During incubation in derepression buffer, organisms with either fatty-acyl enrichment did not differ in the size of the amino-N pool, the concentration of L-alanine in that pool, rates of protein synthesis and glucose fermentation, or rate and extent of incorporation of label from H2 32PO-4. Under conditions used to measure rates of L-alanine accumulation, organisms with either enrichment showed no evidence of metabolism of accumulated L-alanine.     

Enzymatic lesions in methionine mutants of Aspergillus nidulans: role and regulation of an alternative pathway for cysteine and methionine synthesis.

In Aspergillus nidulans the pathway involving cystathionine formation is the main one for homocysteine synthesis. Mutants lacking cystathionine gamma-synthase or beta-cystathionase are auxotrophs suppressible by: (i) mutations in the main pathway of cysteine synthesis (cysA1, cysB1, and cysC1), (ii) mutations causing stimulation of cysteine catabolism (su101), and (iii) mutations in a presumed regulatory gene (suAmeth). A relative shortage of cysteine in the first group of suppressors causes a derepression of homocysteine synthase, the enzyme involved in the alternative pathway of homocysteine synthesis. A similar derepression is observed in the suAmeth strain. Homocysteine synthesized by this pathway serves as precursor for cysteine and methionine synthesis. A mutant with altered homocysteine synthase is a prototroph, indicating that this enzyme is not essential for the fungus.     

Effect of unsaturated fatty acids on the biosynthesis of glucose-repressed enzymes in yeast

In anaerobically glucose-grown yeast isocitrate lyase (EC 4.1.3.1.), malate synthase (EC 4.1.3.2.) and malate dehydrogenase (EC 1.1.1.37.) are repressed by glucose. 24 h cultures still contain 0.3–0.4% glucose in the medium, which is enough to completely repress these activities. Aeration of these cells, in buffer containing acetate, initiates the formation of the three enzymes. Within 16 h, the specific activities of these enzymes increase about 140, 120 and 70-fold, respectively. Glucose-6-phosphate dehydrogenase activity was not altered. When the yeast was grown anaerobically, but with a supplement of an unsaturated fatty acid in the medium, synthesis of the three enzymes was much faster and the specific activities after 16 h of derepression were considerably higher. A relationship exists between the number of double bonds in the unsaturated fatty acid molecule and its capability to stimulate enzyme synthesis: linolenic acid is more effective than linoleic acid, which, in turn, is much more effective than oleic acid. Increasing periods of aeration with glucose of anaerobically grown cells prior to derepression results in an increasing stimulation of enzyme synthesis on subsequent derepression. Anaerobic incubation of yeast in the presence of an unsaturated fatty acid in advance to derepression also increased the velocity of enzyme formation. It is suggested that during the aeration period with glucose and during anaerobic incubation with an unsaturated fatty acid a more active protein synthesizing apparatus was formed.     

Solid-state NMR studies of Klebsiella pneumoniae grown under nitrogen-fixing conditions

The carbon and nitrogen metabolism of Klebsiella pneumoniae M5a1 has been characterized using 13C and 15N labeling with detection by cross-polarization magic-angle spinning solid-state NMR. Cells grown on ammonium typically require some 20 h to derepress fully for nitrogenase when transferred to medium devoid of any source of fixed nitrogen. We have established that during this period some cellular proteins are catabolized with the liberated nitrogen being used for the synthesis of purines needed for formation of ribosomal RNA. The 20-h derepression period can be shortened to 6 h by the introduction of fixed nitrogen in certain specific forms. Serine is the most successful agent we have examined for shortening the derepression period and glycine among the least successful. We attribute this difference to the advantage of serine over glycine in providing both specific and nonspecific carbon and nitrogen sources for complete purine synthesis. These determinations were made by tracing the metabolism of 13C- and 15N-labeled chemical bonds from the 2 amino acids during derepression.     

Regulation of horizontal gene transfer in Bacillus subtilis by activation of a conserved site-specific protease

The mobile genetic element ICEBs1 is an integrative and conjugative element (a conjugative transposon) found in Bacillus subtilis. The RecA-dependent SOS response and the RapI-PhrI cell sensory system activate ICEBs1 gene expression by stimulating cleavage of ImmR, the ICEBs1 immunity repressor, by the protease ImmA. We found that increasing the amount of wild-type ImmA in vivo caused partial derepression of ICEBs1 gene expression. However, during RapI-mediated derepression of ICEBs1 gene expression, ImmA levels did not detectably increase, indicating that RapI likely activates the protease ImmA by increasing its specific activity. We also isolated and characterized mutations in immA (immA(h)) that cause partial derepression of ICEBs1 gene expression in the absence of inducing signals. We obtained two types of immA(h) mutations: one type caused increased amounts of the mutant proteins in vivo but no detectable effect on specific activity in vitro; the other type had no detectable effect on the amount of the mutant protein in vivo but caused increased specific activity of the protein (as measured in vitro). Together, these findings indicate that derepression of ICEBs1 gene expression is likely caused by an increase in the specific activity of ImmA. Homologs of ImmA and ImmR are found in many mobile genetic elements, so the mechanisms that regulate ImmA-mediated cleavage of ImmR may be widely conserved.     

Translational control of alpha-amylase gene expression in Aspergillus awamori

Regulation of alpha-amylase gene expression in Aspergillus awamori was studied by analyzing the enzyme activity levels, rate of protein synthesis, and alpha-amylase-specific mRNA levels under various conditions of growth. alpha-Amylase synthesis was sensitive to catabolite repression as glucose repressed its synthesis by about fourfold. The stimulation of alpha-amylase synthesis in the presence of its substrate starch was shown to be due to derepression rather than induction as the enzyme was synthesized at similar rates in both starch and starvation media. Repression and derepression of enzyme synthesis was found to be mediated at the translational level. The cellular levels of alpha-amylase-specific mRNA as measured by an in vitro translation assay system, were almost identical under all conditions of enzyme synthesis. Relative in vivo and in vitro alpha-amylase mRNA template activities suggest that alpha-amylase mRNA is translated much more efficiently during the derepression than under the conditions of repressed synthesis.     

Carbon-nitrogen requirements for the expression of nitrogenase activity in cultured Parasponia-Rhizobium strain ANU 289

Nutritional factors controlling derepression of nitrogenase activity in Parasponia-Rhizobium strain ANU 289 were studied in stationary and agitated liquid cultures. Altering type and/or concentrations of the constituents of the derepression medium in respect of carbon and nitrogen sources influenced both derepression kinetics as well as the maximal level of activity. Hexose sugars and disaccharides stimulated nitrogenase activity three to six-fold compared to pentose sugars. Activity was also modulated by combining sugars with some organic acids such as succinate, fumarate and pyruvate but not with others (e.g. -ketoglutarate, malate, malonate). Of the range of nitrogen sources tested, either casamino acids (at 0.05%, but not at 0.1%), glutamate, proline or to a lesser extent histidine (each at 5 mM N) supported significant derepression of nitrogenase activity. Notably glutamine, urea, alanine, ammonium sulfate, nitrate, nitrite (each at 5 mM N) and yeast extract (0.05%) failed to derepress or support nitrogenase activity. Ammonium (5 mM) abolished established nitrogenase activity of rapidly agitated cultures within 15 h after addition. This inhibitory effect was alleviated by the addition of methionine sulfoximime (10 mM). Thus, in view of strong glutamine effects, ammonium repression appears to be mediated by glutamine and not by ammonium itself.Abbreviations HEPES [4-(2-hydroxyethyl)-1-piperazine-ethane; sulfonic acid] - MOPS [3-(N-morpholino) propane sulphonic acid] - MSX Methionine sulfoximine     

Catabolite repression and derepression of arylsulfatase synthesis in Klebsiella aerogenes

When a mutant (Mao(-)) of Klebsiella aerogenes lacking an enzyme for tyramine degradation (monoamine oxidase) was grown with d-xylose as a carbon source, arylsulfatase was repressed by inorganic sulfate and repression was relieved by tyramine. When the cells were grown on glucose, tyramine failed to derepress the arylsulfatase synthesis. When grown with methionine as the sole sulfur source, the enzyme was synthesized irrespective of the carbon source used. Addition of cyclic adenosine monophosphate overcame the catabolite repression of synthesis of the derepressed enzyme caused by tyramine. Uptake of tyramine was not affected by the carbon source. We isolated a mutant strain in which derepression of arylsulfatase synthesis by tyramine occurred even in the presence of glucose and inorganic sulfate. This strain also produced beta-galactosidase in the presence of an inducer and glucose. These results, and those on other mutant strains in which tyramine cannot derepress enzyme synthesis, strongly suggest that a protein factor regulated by catabolite repression is involved in the derepression of arylsulfatase synthesis by tyramine.     

Molecular Aspects of Interferon Induction by Viruses

Virus-induced interferon formation depends on the presence within the cell of a viral ribonucleic acid. This RNA may either be double stranded or, in certain cases, single stranded. The double-stranded RNA can be derived from a virus, such as reovirus, which contains this type of RNA, or it may be synthesized within the cell using viral single-stranded RNA as a template. Single-stranded RNA must possess a stable configuration in solution to be active, and certain viral RNA molecules appear to be active for this reason. The presence of this RNA triggers a derepression event, which is probably nuclear, by an unknown mechanism, and this is followed by the production of an interferon messenger RNA and its translation. Little is known of the derepression event or the events that follow it.     

Effect of mitochondrial functions on synthesis of yeast cytochrome c

The effects of the mitochondrial protein synthesis inhibitor chloramphenicol and the mitochondrial F0 adenosine triphosphatase inhibitor oligomycin on the synthesis of nucleus-encoded cytochrome c protein were studied. Both inhibitors stimulated cytochrome c protein synthesis in the derepressed state (growth in media containing 2% raffinose) but had no effect on the synthesis of the cytochrome c protein in the repressed state (growth in media containing 5% glucose). Oligomycin uncoupled the synthesis of the apoprotein from its processing into the hemoprotein. Neither antibiotic had a significant effect on the rate of glucose repression of cytochrome protein synthesis. The kinetics of cytochrome c derepression and the effects of these two antibiotics on these kinetics were also studied. Cells were derepressed by transfer from glucose- to faffinose-containing media, and the rate of cytochrome c synthesis increased from the repressed to the derepressed level during the second hour of derepression. Chloramphenicol delayed this derepression, but after 5 h the rate of cytochrome c protein synthesis increased to twice the rate of synthesis in uninhibited cells. On the other hand, oligomycin inhibited derepression of cytochrome c. These results are discussed with respect to the effects of mitochondrial function in the derepressed and repressed states and during the processes of repression and derepression of cytochrome c.     

Differential regulation of protein biosynthesis at translational level in the fat body cells of adult Locusta migratoria

Vitellogenin (Vg) and lipophorin (Lp) are synthesized by the fat body of adult locust (Locusta migratoria) females. We have shown by an immunohistochemical technique that both proteins are produced in the same cells of the fat body. The rate of Vg synthesis was measured with the use of double immunoprecipitation of labeled proteins at oviposition and 24 h later. It was found that the rate of Vg synthesis declined significantly by the time of oviposition; however, 24 h later, it was raised to the highest possible level. The rate of Lp synthesis remained constant at both indicated points. The similar postlaying increase in the Vg synthesis rate was observed in the fat bodies of females treated by alpha-amanitin immediately after oviposition. The data provide evidence that Vg biosynthesis in L. migratoria is regulated by selective periodical repression and derepression of Vg mRNAs in the fat body cells but not by total inhibition and stimulation of protein-synthesizing machinery.     

Isolation and partial characterization of Rhodopseudomonas sphaeroides mutants defective in the regulation of ribulose bisphosphate carboxylase/oxygenase.

Several mutants of Rhodopseudomonas sphaeroides defective in the derepression of the enzyme ribulose 1,5-bisphosphate carboxylase have been isolated by using the unstable Tn5 vectors pJB4JI and pRK340. Transpositional insertion mutants obtained with pJB4JI were demonstrated to be incapable of increasing ribulose 1,5-bisphosphate carboxylase/oxygenase levels when grown on butyrate-bicarbonate medium or under conditions of carbon starvation, whereas the wild-type strain increased activity four- to eightfold. When the wild-type strain was starved for carbon in the presence of chloramphenicol, no derepression was observed. Crude extracts from mutant and wild-type strains had distinct and consistent differences in protein content as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chromatographic evidence indicated that mutants were defective in the regulation of only one of the two forms of ribulose 1,5-bisphosphate carboxylase/oxygenase synthesized by R. sphaeroides.