[Mechanism of overproduction of secreted enzymes in the mycelial fungus Penicillium canescens]. / Mekhanizm superproduktsii sekretiruemykh fermentov u mitselial'nogo griba Penicillium canescens.

The fungus Penicillium canescens strain F178 (VKPM) and its niaD- mutant exhibited an increased capability of synthesizing extracellular enzymes beta-galactosidase (70-80 U/ml) and xylanase (100 U/ml). The synthesis was induced by arabinose and its catabolite, arabitol. A deficiency in arabitol dehydrogenase, leading to arabitol accumulation in the cell, was detected in the chain of reactions of arabinose catabolism. The increased synthesis of beta-galactosidase and xylanase in P. canescens is accounted for by (1) cellular accumulation of the inducer (arabitol) at low concentrations of arabinose in the medium and (2) prevalence of induction over repression.

A novel high efficiency, low maintenance, hydroponic system for synchronous growth and flowering of Arabidopsis thaliana.

BACKGROUND: Arabidopsis thaliana is now the model organism for genetic and molecular plant studies, but growing conditions may still impair the significance and reproducibility of the experimental strategies developed. Besides the use of phytotronic cabinets, controlling plant nutrition may be critical and could be achieved in hydroponics. The availability of such a system would also greatly facilitate studies dealing with root development. However, because of its small size and rosette growth habit, Arabidopsis is hardly grown in standard hydroponic devices and the systems described in the last years are still difficult to transpose at a large scale. Our aim was to design and optimize an up-scalable device that would be adaptable to any experimental conditions. RESULTS: An hydroponic system was designed for Arabidopsis, which is based on two units: a seed-holder and a 1-L tank with its cover. The original agar-containing seed-holder allows the plants to grow from sowing to seed set, without transplanting step and with minimal waste. The optimum nitrate supply was determined for vegetative growth, and the flowering response to photoperiod and vernalization was characterized to show the feasibility and reproducibility of experiments extending over the whole life cycle. How this equipment allowed to overcome experimental problems is illustrated by the analysis of developmental effects of nitrate reductase deficiency in nia1nia2 mutants. CONCLUSION: The hydroponic device described in this paper allows to drive small and large scale cultures of homogeneously growing Arabidopsis plants. Its major advantages are its flexibility, easy handling, fast maintenance and low cost. It should be suitable for many experimental purposes.

Retrotransposons of the Tnt1B family are mobile in Nicotiana plumbaginifolia and can induce alternative splicing of the host gene upon insertion.

Active retrotransposons have been identified in Nicotiana plumbaginifolia by their ability to disrupt the nitrate reductase gene in chlorate-resistant mutants selected from protoplast-derived cultures. In mutants E23 and F97, two independent insertions of Tnp2, a new retrotransposon closely related to the tobacco Tnt1 elements, were detected in the nitrate reductase gene. These two Tnp2 elements are members of the Tnt1B subfamily which shows that Tnt1B elements can be active and mutagenic in the N. plumbaginifolia genome. Furthermore, these results suggest that Tnt1B is the most active family of Tntl elements in N. plumbaginifolia, whereas in tobacco only members of the Tnt1A subfamily were found inserted in the nitrate reductase gene. The transcriptional regulations of Tnp2 and Tnt1A elements are most probably different due to non-conserved U3 regions. Our results thus support the hypothesis that different Nicotiana species contain different active Tntl subfamilies and that only one active Tntl subfamily might be maintained in each of these species. The Tnp2 insertion found in the F97 mutant was found to be spliced out of the nitrate reductase mRNA by activation of cryptic donor and acceptor sites in the nitrate reductase and the Tnp2 sequences respectively.

A reassessment of the genetic determinants, the effect of growth conditions and the availability of an electron donor on the nitrosating activity of Escherichia coli K-12.

Anaerobic, but not aerobic, cultures of Escherichia coli K-12 catalysed the rapid nitrosation of the model substrate 2,3-diaminonaphthalene when incubated with nitrite. Formate and lactate were effective electron donors for the nitrosation reaction, which was inhibited by nitrate. Optimal growth conditions for the expression of nitrosation activity by various strains and mutants were determined. Highest activities were found with bacteria that had been grown anaerobically in a minimal medium rather than in Lennox broth, with glycerol and fumarate rather than glucose as the main carbon and energy source, and in the presence of a low concentration of nitrate. Bacteria harvested in the early exponential phase were more active than those harvested in later stages of growth. Well-characterized mutants defective in the synthesis of one or more anaerobically induced electron transfer chains were screened for nitrosation activity under these optimal growth conditions: only the respiratory nitrate reductase encoded by the narGHJI operon was implicated as a major contributor to nitrosation activity. Due to the limited sensitivity of the assays currently available, a minor contribution from the two alternative nitrate reductases or even other molybdoproteins could not be excluded. The role of formate in nitrosation was complex and was clearly not limited simply to that of an electron donor in the bacterial reduction of nitrite to nitric oxide: at least two further, chemical roles were inferred. This extensive study of more than 400 independent cultures of E. coli K-12 and its derivatives resolved some, but not all, of the apparently conflicting data in the literature concerning nitrosation catalysed by enteric bacteria.

Genetic and biochemical analysis of intragenic complementation events among nitrate reductase apoenzyme-deficient mutants of Nicotiana plumbaginifolia.

Intragenic complementation has been observed between apoenzyme nitrate reductase-deficient mutants (nia) of Nicotiana plumbaginifolia. In vivo as in vitro, the NADH-nitrate reductase (NR) activity in plants heterozygous for two different nia alleles was lower than in the wild type plant, but the plants were able to grow on nitrate as a sole nitrogen source. NR activity, absent in extracts of homozygous nia mutants was restored by mixing extracts from two complementing nia mutants. These observations suggest that NR intragenic complementation results from either the formation of heteromeric NR or from the interaction between two modified enzymes. Complementation was only observed between mutants retaining different partial catalytic activities of the enzyme. Results are in agreement with molecular data suggesting the presence of three catalytic domains in the subunit of the enzyme.

Biochemical and genetic comparison of two nitrate reductase-deficient pea mutants disturbed in the cofactor.

Two nitrate reductase (NaR)-deficient mutants of pea (Pisum sativum L.), E1 and A300, both disturbed in the molybdenum cofactor function and isolated, respectively, from cv Rondo and cv Juneau, were tested for allelism and were compared in biochemical and growth characteristics. The F1 plants of the cross E1 X A300 possessed NaR and xanthine dehydrogenase (XDH) activities comparable to those of the wild types, indicating that these mutants belong to different complementation groups, representing two different loci. Therefore, mutant E1 represents, besides mutant A300 and the allelic mutants A317 and A334, a third locus governing NaR and is assigned the gene destignation nar 3. In comparison with the wild types, cytochrome c reductase activity was increased in both mutants. The mutants had different cytochrome c reductase distribution patterns, indicating that mutant A300 could be disturbed in the ability to dimerize NaR apoprotein monomers, and mutant E1 in the catalytic function of the molybdenum cofactor. In growth characteristics studied, A300 did not differ from the wild types, whereas fully grown leaves of mutant E1 became necrotic in soil and in liquid media containing nitrate.

In vivo complementation analysis of nitrate reductase-deficient mutants in Chlamydomonas reinhardtii.

In vivo complementation between different wild and mutant strains defective for nitrate assimilation has been performed by isolating diploid strains from the appropriate crosses. Twenty-two diploids homozygous or heterozygous with respect to nitrate reduction and able to grow on nitrate medium were obtained and their diploid character demonstrated from analyses of mating type, cell volume, nuclear size and progeny of crosses with haploid wild-type. All diploids were assayed for overall- and terminal-nitrate reductase (NR) activity and for the occurrence of the NR-diaphorase subunit. Data on NR activities in heterozygotes carrying mutation(s) in structural gene(s) (nit-1 or nit-1a, nit-1b) agree with the heteromultimeric nature of the enzyme complex previously described (Franco et al. (1984) EMBO J 3: 1403-1407), and indicate that subunits are exchangeable to form hybrid enzymes. In addition, in vitro complementation tests with mutant nit-1 of C. reinhardtii indicate that this mutant has defective NR-diaphorase subunits but intact terminal subunits. Super-repression caused by the mutant allele nit-2 is suppressed by the wild allele in heterozygotes, which suggests a positive control by the nit-2 product on structural gene(s) transcription. Mutant alleles of genes for the biosynthesis of molybdenum-containing cofactor, either nit-4 or nit-5 and nit-6, were recessive in diploids carrying them. The mutant allele of nit-3, from strain 307, was codominant in all heterozygotes suggesting that nit-3 codes for a protein whose activity is limiting for the molybdenum-cofactor biosynthetic pathway.