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Proline (Pro) is one of the most accumulated osmolytes in salinity and water deficit conditions in plants. In the present study, we measured the Pro content, the activity and the expression level of delta 1-pyrroline-5-carboxylate synthetase (P5CS: gamma-glutamyl kinase, EC 2.7.2.11 and glutamate-5-semialdehyde dehydrogenase, EC 1.2.1.41), a key regulatory enzyme involved in the biosynthesis of Pro, in cactus pear (Opuntia streptacantha) subjected to 6, 9 and 11 days of salt stress. Treatment with NaCl of O. streptacantha young plants resulted in a decrease in the cladode thickness and root length, and in a significant and gradual accumulation of Pro in young cladodes, in a time- and concentration-dependent manner. P5CS activity, studied as gamma-glutamyl kinase, was reduced at all times as a consequence of salt treatment, except at the sixth day at 75 and 150mM of NaCl, where a slight increase was observed. We isolated an open reading frame (ORF) fragment of p5cs gene. The deduced amino acid sequence of the P5CS protein exhibited 90.4% of identity with the P5CS protein from Mesembryanthemum crystallinum. RT-PCR analysis revealed that the Osp5cs gene of O. streptacantha was induced by salt stress at 9 and 11 days of treatment. Furthermore, ABA-induced Osp5cs gene expression was observed in cladodes of cactus pear young plants. We observed an evident correlation between the transcript up-regulation and the Pro accumulation under salt stress; however, these results do not parallel with the changes in P5CS enzymatic activity. This Pro accumulation might function as an osmolyte for the intracellular osmotic adjustment and might be playing a critical role in protecting photosynthetic activity in O. streptacantha plants under salt stress.

A novel N-acetylglutamate synthase/kinase bifunctional enzyme of arginine biosynthesis that was homologous to vertebrate N-acetylglutamate synthases was identified in Xanthomonas campestris. The protein was overexpressed, purified and crystallized. The crystals belong to the hexagonal space group P6(2)22, with unit-cell parameters a = b = 134.60, c = 192.11 A, and diffract to about 3.0 A resolution. Selenomethionine-substituted recombinant protein was produced and selenomethionine substitution was verified by mass spectroscopy. Multiple anomalous dispersion (MAD) data were collected at three wavelengths at SER-CAT, Advanced Photon Source, Argonne National Laboratory. Structure determination is under way using the MAD phasing method.

Glutamate-5-kinase (G5K) catalyzes the first step of proline (and, in mammals, ornithine) biosynthesis. It is a key regulatory point of these routes, since it is the subject of feedback allosteric inhibition by proline or ornithine. The Escherichia coli gene (proB) for G5K was cloned in pET22, overexpressed in E. coli, purified in a few steps in high yield to 95% homogeneity in the highly active proline-inhibitable form and was shown by cross-linking to be a tetramer. It was crystallized by the hanging-drop vapour-diffusion method at 294 K in the presence of ADP, MgCl(2) and L-glutamate using 1.6 M MgSO(4), 0.1 M KCl in 0.1 M MES pH 6.5 as the crystallization solution. The tetragonal bipyramid-shaped crystals diffracted to 2.5 A resolution using synchrotron radiation. The crystals belong to space group P4(1(3))2(1)2, with unit-cell parameters a = b = 101.1, c = 178.6 A, and contain two monomers in the asymmetric unit, with 58% solvent content.

Oenococcus oeni, the main species which induces malolactic fermentation in wine, uses arginine via the arginine deiminase (ADI) pathway. Using degenerated primers, two specific probes, one for ornithine transcarbamoylase (OTC) and the other for carbamate kinase (CK), were synthesized. These made it possible to clone and sequence a cluster containing genes encoding ADI (arcA), OTC (arcB) and CK (arcC). In addition, sequence analysis upstream of the arcA gene revealed the presence of an open reading frame (orf229) whose 3'-end was only 101 bp-distant from the start codon of the arcA gene and showed similarity with members of the FNR (regulation for fumarate and nitrate reduction) and CRP (cAMP receptor protein) family of transcriptional regulators. Moreover, a putative binding site for such regulators lies in the promoter region of the arcA gene. Induction of the arc cluster by arginine was studied first at the enzymatic level. The activities of the three enzymes strongly increased when cells were grown in the presence of the amino acid. In addition, the influence of arginine on gene transcription was monitored by RT-PCR (reverse transcriptase-polymerase chain reaction). Expression of the three arc genes, and particularly that of arcA, was positively affected by arginine supplementation and thus confirmed the enzymatic results. Moreover, transcription of the putative CRP-like gene orf229 was also stimulated by arginine. These data suggest that the protein encoded by orf229 could be a CRP-like regulator involved in the metabolism of O. oeni.

A putative hydrogenase (hydA) gene of Clostridium perfringens encodes a protein with strong identity to Clostridium pasteurianum hydrogenase I. Disruption of the hydA gene abolished H(2) productivity, confirming its function. A putative butyrate kinase gene (buk) is adjacent to the hydA gene. When cultures were grown in medium with glucose, 1.8-kb hydA and 2.1-kb buk transcripts and a 3. 9-kb transcript hybridized with both hydA and buk-probe were detectable in all the exponential growth phases. In medium without glucose, these transcripts were decreased rapidly after the mid-exponential phase. These results suggest that the transcription of these two genes is probably regulated by a similar mechanism in response to glucose availability.

The arginine dihydrolase pathway in Giardia intestinalis produces energy via the carbamate kinase (CBK, ATP:carbamate phosphotransferase, EC 2.7.2.2) reaction. Characterisation of the CBK gene from the Portland 1 strain indicated that it is located on either chromosome 3 or 4, does not appear to contain introns and is expressed in both the trophozoite and early cyst stages. Heterologous expression of CBK in Escherichia coli, using the pQE-30 expression system (QIAGEN), enabled a one-step purification of the recombinant enzyme via affinity chromatography. The expressed protein was identified by enzyme assay and mass spectrometry. The native and recombinant forms of the enzyme have similar physical properties and the recombinant enzyme appears to be active as the homodimer.

Integrational plasmid technology has been used to disrupt metabolic pathways leading to acetate and butyrate formation in Clostridium acetobutylicum ATCC 824. Non-replicative plasmid constructs, containing either clostridial phosphotransacetylase (pta) or butyrate kinase (buk) gene fragments, were integrated into homologous regions on the chromosome. Integration was assumed to occur by a Campbell-like mechanism, inactivating either pta or buk. Inactivation of the pta gene reduced phosphotransacetylase and acetate kinase activity and significantly decreased acetate production. Inactivation of the buk gene reduced butyrate kinase activity, significantly decreased butyrate production and increased butanol production.

Fermentative growth via the arginine deiminase pathway is mediated by the enzymes arginine deiminase, carbamate kinase, and catabolic ornithine transcarbamylase and by a membrane-bound arginine-ornithine antiporter. Recently we reported the characterization of catabolic ornithine transcarbamylase and the corresponding gene, arcB, from Halobacterium salinarium (formerly Halobacterium halobium). Upstream of the arcB gene, three additional open reading frames with halobacterial codon usage were found. They were identified as the arcC gene coding for carbamate kinase, the arcA gene coding for arginine deiminase, and a gene, tentatively termed arcR, coding for a putative regulatory protein. The identification of the arcC and arcA genes was verified, respectively, by heterologous expression of the enzyme in Haloferax volcanii and by protein isolation and N-terminal sequence determination of three peptides. The gene order arcRACB differs from the gene order arcDABC in Pseudomonas aeruginosa, the only other organism for which sequence information is available. Transcripts from H. salinarium cultures grown fermentatively or aerobically were characterized by Northern (RNA) blot and primer extension analyses. It was determined (i) that monocistronic transcripts corresponding to the four open reading frames exist and that there are three polycistronic transcripts, (ii) that the level of induction during fermentative growth differs for the various transcripts, and (iii) that upstream of the putative transcriptional start sites for the three structural genes there are sequences with similarities to the halobacterial consensus promoter. The data indicate that expression of the arc gene cluster and its regulation differ in H. salinarium and P. aeruginosa.

N-Acetylglutamate kinase (AGK) and N-acetyl-gamma-glutamyl-phosphate reductase (AGPR) function as two separate mitochondrial enzymes, but are encoded by a single nuclear gene in several fungi. The Neurospora crassa arg-6 gene encoding these enzymes has been cloned and sequenced, and the enzymes responsible for processing the polyprotein precursor have been identified. The 871-amino acid precursor contains a normal N-terminal mitochondrial targeting sequence, an internal connecting region (approximately 200 amino acids) upstream of the distal reductase domain, and coding regions with N-terminal amino acid sequences identical with those of purified N-acetylglutamate kinase and N-acetyl-gamma-glutamyl-phosphate reductase. Sequence comparisons of the coding regions indicate high levels of conservation between prokaryotic and fungal proteins. Regions suggesting ancestral relationships to N-acetylglutamate synthase and aspartate beta-semialdehyde dehydrogenase have been identified. Both the N-terminal targeting sequence and the connecting region contain consensus sites for cleavage by the mitochondrial processing peptidase and processing enhancing protein. In vitro processing assays with intact mitochondria, solubilized mitochondria, and purified enzymes have shown that the mitochondrial processing peptidase and processing enhancing protein cleave not only the N-terminal mitochondrial targeting sequence but also process the polyprotein precursor into the two mature enzymes.