Activation of the urease of Schizosaccharomyces pombe by the UreF accessory protein from soybean.

Plant orthologs of the bacterial urease accessory genes ureD and ureF, which are required for the insertion of the nickel ion at the active site, have been isolated from soybean ( Glycine max L. Merr.), tomato ( Lycopersicon esculentum) and Arabidopsis thaliana. The functionality of soybean UreD and UreF was tested by measuring their ability to complement urease-negative mutants of Schizosaccharomyces pombe, a eukaryote which produces a "plant-like" urease of ~90 kDa. The S. pombe ure4 mutant was complemented by a 12-kb fragment of S. pombe genomic DNA, which was shown by PCR to contain a putative ureD gene. However, ure4 was not complemented by a UreD cDNA soybean, expressed under the control of a strong promoter. In contrast, an S. pombe ure3 mutation was complemented by both a 10-kb fragment of S. pombe DNA containing ureF and the UreF cDNA from soybean. Soybean Eu2 is a candidate urease accessory gene; its product cooperates with the Eu3 protein in activating apourease in vitro. However, the sequences of UreD and UreF transcripts from two eu2/eu2 mutants, recovered as RT-PCR products, revealed no mutational alteration, suggesting that Eu2 encodes neither UreD nor UreF.

Terminal-repeat retrotransposons in miniature (TRIM) are involved in restructuring plant genomes.

A new group of long terminal repeats (LTR) retrotransposons, termed terminal-repeat retrotransposons in miniature (TRIM), are described that are present in both monocotyledonous and dicotyledonous plant. TRIM elements have terminal direct repeat sequences between approximately 100 and 250 bp in length that encompass an internal domain of approximately 100-300 bp. The internal domain contains primer binding site and polypurine tract motifs but lacks the coding domains required for mobility. Thus TRIM elements are not capable of autonomous transposition and probably require the help of mobility-related proteins encoded by other retrotransposons. The structural organization of TRIM elements suggests an evolutionary relationship to either LTR retrotransposons or retroviruses. The past mobility of TRIM elements is indicated by the presence of flanking 5-bp direct repeats found typically at LTR retrotransposon insertion sites, the high degree of sequence conservation between elements from different genomic locations, and the identification of related to empty sites (RESites). TRIM elements seem to be involved actively in the restructuring of plant genomes, affecting the promoter, coding region and intron-exon structure of genes. In solanaceous species and maize, TRIM elements provided target sites for further retrotransposon insertions. In Arabidopsis, evidence is provided that the TRIM element also can be involved in the transduction of host genes.

Functional characterisation of urease accessory protein G (ureG) from potato.

The activation of the nickel metalloenzyme urease is a complex process. In bacteria, several urease accessory proteins are essential for incorporation of nickel into the active centre of urease. Comparatively little is known about the activation process and the proteins involved in plants. We cloned five different cDNAs encoding isoforms of urease accessory protein G (ureG) in potato. The 5'-coding region of these cDNAs is highly polymorphic within Solanum tuberosum ssp. tuberosum, containing mainly a simple sequence repeat encoding histidine and aspartate. Mapping on an ultrahigh-density map of the potato genome and Southern blot analysis showed that the isoforms arise from allelic differences of a single-copy gene which was located on chromosome 2. Expression analysis at the mRNA and protein levels indicated the presence of ureG in almost all tissues examined, consistent with the ubiquitous expression of urease. An attempt to correlate urease activity with ureG expression levels in different tissues was made. Allelic copies of ureG were expressed in a tissue-specific manner. UreG from potato and the Klebsiella aerogenes urease operon defective in bacterial ureG were co-expressed in Escherichia coli. The plant gene complements the K. aerogenes ureG mutation, demonstrating that it encodes a urease accessory protein and indicating a structural conservation between the plant and the bacterial urease activation complexes.

In-gel detection of urease with nitroblue tetrazolium and quantification of the enzyme from different crop plants using the indophenol reaction.

Two methods for measurement of urease activity are described and demonstrated on extracts from several crop plants. With an in-gel staining method based on the principle of Fishbein's noninhibitory stain for urease as little as 25 microU of jackbean urease can be detected within 2 h following electrophoresis. A comparison with published in-gel staining methods shows that the sensitivity is improved by at least two orders of magnitude. The second method allows quantification of urease activity from small amounts of plant material without the need for special laboratory equipment. It employs the detection of ammonium by the indophenol reaction. To eliminate reducing agents which are often necessary to maintain urease activity during extraction, but which interfere with ammonium detection, a simple spin-column procedure is used. The quantification of less than 5 mU/ml extract is possible.

The Chlamydomonas reinhardtii MoCo carrier protein is multimeric and stabilizes molybdopterin cofactor in a molybdate charged form.

In Chlamydomonas reinhardtii, molybdopterin cofactor (MoCo) able to reconstitute active nitrate reductase (NR) with apoenzyme from the Neurospora crassa mutant nit-1 was found mostly bound to a carrier protein (CP). This protein is scarce in the algal free extracts and has been purified 520-fold. MoCoCP is a protein of 64 kDa with subunits of 16.5 kDa and an isoelectric point of 4.5. In contrast to free MoCo, MoCo bound to CP was remarkably protected against inactivation under both aerobic conditions and basic pH. MocoCP transferred active MoCo to apoNR in vitro without addition of molybdate, though reconstituted activity was 20% higher in the presence of molybdate. Incubation with tungstate specifically inhibited MoCoCP activity but had no effect on the activity of free MoCo released from milk xanthine oxidase. MoCoCP did not charge molybdate unless in the presence of N. crassa extracts. Our data support that MoCoCP stabilizes MoCo in an active form charged with molybdate to provide MoCo to apomolybdoenzymes.

Microbial photodegradation of aminoarenes. Metabolism of 2-amino-4-nitrophenol by Rhodobacter capsulatus.

The phototrophic bacterium Rhodobacter capsulatus photoreduces 2,4-dinitrophenol to 2-amino-4-nitrophenol, which is further metabolized by an aerobic pathway that is also light-dependent. The catabolism of 2-amino-4-nitrophenol requires O2 and the presence of alternative carbon (C) and nitrogen (N) sources, preferably acetate and ammonium. Rhodobacter capsulatus B10, a bacterium unable to assimilate nitrate, releases negligible amounts of nitrite when growing with 2-amino-4-nitrophenol, thus suggesting that an oxygenase, nitrite-producing activity is not involved in the metabolization of the compound. The diazotrophic growth of R. capsulatus increases in the presence of 2-amino-4-nitrophenol, but growth with ammonium is clearly inhibited by the compound. Mutant strains of R. capsulatus B10, which are affected in nifHDK, nifR1, or nifR4 genes, unable to fix dinitrogen, do not grow with 2-amino-4-nitrophenol as the sole N source. This indicates that the compound cannot be used as a N source. The nif mutants degrade 2-amino-4-nitrophenol to the same extent as the wild-type in the presence of ammonium. The compound is not used as a C source by the bacterium, either. Aromatic stable intermediates, such as 2,4-diaminophenol or 4-nitrocatechol, are not detectable in microaerobic cultures of R. capsulatus growing with 2,4-dinitrophenol or 2-amino-4-nitrophenol.