Jean-Paul Aubert

Méthode de mesure des principales voies du métabolisme calcique chez l'homme

Abstract The method is based on the following techniques and theory: (1) Intravenous injection of a negligible weight of 45 CaCl 2 . (2) Determination of the specific activity of the serum, of the total activity in urine and stool and of the amount of ingested and excreted calcium. (3) Mathematical analysis of the curve showing the decrease of the specific activity of the serum calcium. Thus data are obtained about (a) the intestinal absorption of calcium present in food. (b) The quantity of calcium excreted by the intestinal tract, differentiated from unabsorbed calcium. (c) The rate of the physiological renewal processes of the skeleton through osteolysis and osteoblastosis. (d) The amount of readily exchangeable calcium, which is the calcium pool of the organism. This pool consists of three portions, which can be identified as clearly defined physiological entities, each having a different velocity constant of exchange with serum calcium. (e) The rate of exchange of serum calcium with these different portions of the pool. (f) The quantity of bone calcium undergoing recrystallisation. Examples are given of application of the method in man and the theory is verified in the rat. The accuracy of the described method is discussed and compared with the simplied method that is usually employed.

Interaction of the duplicated segment carried by Clostridium thermocellum cellulases with cellulosome components

The function of the non‐catalytic, duplicated segment found in C. thermocellum cellulases was investigated. Rabbit antibodies reacting with the duplicated segment of endoglucanase CelD cross‐reacted with a variety of cellulosome components ranging between 50 and 100 kDa. 125I‐labeled forms of CelD and of xylanase XynZ carrying the duplicated segment bound to a set of cellulosome proteins ranging between 66 and 250 kDa, particularly to the 250 kDa SL (or S1) subunit. 125I‐labeled forms of CelD and XynZ devoid of the duplicated segment failed to bind to any cellulosome protein. The duplicated segment appears thus to serve to anchor the various cellulosome subunits to the complex by binding to SL, which may be a scaffolding element of the cellulosome.

High activity of inclusion bodies formed in Escherichia coli overproducing Clostridium thermocellum endoglucanase D

The formation of cytoplasmic inclusion bodies by Escherichia coli overproducing Clostridium thermocellum endoglucanase D (EGD) was investigated. EGD was found in inclusion bodies as a 68 kDa form, whereas the size of the cytoplasmic form was 65 kDa. Upon solubilization with urea followed by dialysis, the 68 kDa form was converted to the 65 kDa species. Proteolysis occurred within the COOH‐terminal, reiterated region of the 68 kDa form, which is conserved among most C. thermocellum endoglucanase, but is not required for catalytic activity. The specific activity of the enzyme embedded in inclusion bodies was close to that of the purified protein. Thus, inclusion body formation does not involve denaturation of the catalytic domain of EGD, but more likely, the participation of the reiterated, conserved region in intermolecular interactions.

Involvement of separate domains of the cellulosomal protein S1 of Clostridium thermocellum in binding to cellulose and in anchoring of catalytic subunits to the cellulosome

Fragments of the 25OkDa SI subunit of the Clostridium thermocellum cellulosome were obtained by protease‐induced or spontaneous degradation. All detectable fragments, down to a mass of about 30 kDa, retained the ability to bind to 125I‐labelled endoglucanase CelD, one of the catalytic subunits of the cellulosome. Several fragments were able to bind both to cellulose and to CElD. However, some fragments that could still bind to CelD did not have the ability to bind to cellulose. Therefore, S1, a putative scaffolding protein of the cellulosome, is likely to carry two separate types of domains, one of which binds to cellulose, while the other type binds to the various catalytic subunits of the complex.

Purification and properties of the endoglucanase C of Clostridium thermocellum produced in Escherichia coli

The celC gene, which codes for a new endoglucanase of Clostridium thermocellum, termed endoglucanase C, was found to be expressed when cloned in Escherichia coli. The enzyme was purified to electrophoretic homogeneneity from E. coli and its biochemical properties were studied. It differs from the previously studied endoglucanases A and B. In particular, endoglucanase C displays features common to endo- and exoglucanases, since it had a high activity on carboxymethylcellulose and on p-nitrophenyl-beta-D-cellobioside where only the agluconic bond was split. In addition, the enzyme was able to release cellobiose units from G3, G4 and G5 cellodextrins. Endoglucanase C was characterized by Western blot in a culture supernatant from C. thermocellum grown on cellulose, using an antiserum raised against the enzyme produced by E. coli.

Nucleotide sequence of the cellulase gene celF of Clostridium thermocellum

The nucleotide sequence of the celF gene of Clostridium thermocellum was determined. The open reading frame extended over 2217 bp. The encoded 739-aa polypeptide, CelF, with a Mw = 82,015, was an endoglucanase with activity against carboxymethylcellulose. The N terminus showed a typical signal peptide, and a cleavage site after Ala-27 was predicted. From residues 28 to 470, the sequence of CelF was related to the catalytic domains of type E2 endoglucanases, with a strong homology to the endoglucanases CelZ of Clostridium stercorarium and CenB of Cellulomonas fimi. The catalytic region was followed by a 134-aa segment also present in C. stercorarium CelZ and in C. fimi CenB, and belonging to the family of non-catalytic, presumably cellulose-binding domains first identified in Bacillus subtilis endoglucanase. A 21-aa segment rich in Pro/Thr/Ser residues separated the putative cellulose-binding region from the COOH-terminal region, which contained two conserved stretches of 24 amino acids closely similar to those previously described in endoglucanases CelA, CelB, CelD, CelE, CelH and CelX, and xylanase XynZ of C. thermocellum.

Hybridization of DNA from methanogenic bacteria with nitrogenase structural genes (nifHDK)

SummaryUsing the Southern hybridization technique, homologies were examined between restricted DNA of four methanogenic bacteria (Methanobacterium ivanovi, Methanobacterium thermoautotrophicum, Methanococcus voltae, Methanosarcina barkeri) and the nif (nitrogen fixation) genes of Klebsiella pneumoniae and Anabaena strain 7120. With K. pneumoniae probes, no hybridization was observed with nifA, nifNE, and nifJ but positive results were obtained with the nifHDK genes coding for nitrogenase. Homology was detected, in the four strains, with K. pneumoniae and Anabaena nifH probes. In M. voltae and M. ivanovi, the homology found with nifH was estimated to be about 70% and a weaker hybridization was observed also with nifD and nifK. In M. voltae, the sequence homologous to nifH was found on a 3.0 kbp HindIII fragment and sequences homologous to nifD and nifK on a 3.8 kbp HindIII fragment. The 3.0 kbp fragment was cloned and the region homologous to nifH was localized more precisely. When this fragment was used as a probe against other DNAs, it behaved as a K. pneumoniae and Anabaena nifH probe. The results suggest that the structural genes for nitrogenase may be present in archaebacteria and raise interesting questions regarding their evolution.

Nitrogenase of Sesbania rhizobium strain ORS571: purification, properties and switch-off by ammonia

Summary: Nitrogenase from the Rhizobium strain ORS571, which forms both root and stem nodules on the tropical plant Sesbania rostrata, was purified from free-living diazotrophically grown organisms. The enzyme complex was resolved into two protein components resembling those obtained from other diazotrophs. Both components were purified to homogeneity as judged by SDS-gel electrophoresis. Component 1, a Mo-Fe protein, had a M r of 219000 and contained 1·2 g-atoms Mo mol-1, 22·5 g-atoms Fe mol-1and 20·5 g-atoms acid-labile S mol-1. It consisted of two types of subunit of M r 56000 and 59000. The specific activity [nmol product formed min-1(mg protein)-1] of component 1, when assayed in the presence of an optimum concentration of component 2 (molar ratio 1:40), was 1250 for acetylene reduction and 1090 for hydrogen evolution. Component 2, an Fe protein, had a M r of 74000 and contained 3·1 g-atoms Fe mol-1and 3·1 g-atoms acid-labile S mol-1. It consisted of a single type of subunit of M r 36000. The specific activity of component 2, when assayed in the presence of an optimum concentration of component 1 (molar ratio 1:1) was 1700 for acetylene reduction and 1305 for hydrogen evolution. Nitrogenase activity of strain ORS571 was subject to ‘switch-off’ when ammonia was added to a N2-fixing culture. This effect was independent of protein synthesis and was reversible. Nitrogenase components 1 and 2 isolated from ‘switched-off’ organisms were purified to homogeneity. Both components had the same mobility on SDS-gels as those isolated from active cultures. ‘Switch-off’ resulted in a decrease in the specific activity of component 2 from 1700 to 280. Component 1 remained fully active. The addition of Mn2+to a crude extract containing inactivated nitrogenase did not restore activity.

Construction of new yeast vectors and cloning of the nif (nitrogen fixation) gene cluster of Klebsiella pneumoniae in yeast

SummaryTwo vectors, termed pG63.11 (7.6 Kb) and pHCG3 (9.6 Kb), suitable for yeast transformation have been constructed. The pHCG3 vector has cosmid properties. Both vectors contain a single 3.3 Kb EcoRI-HindIII fragment of yeast origin which carries the yeast URA3 gene (1.1 Kb) and the origin of replication of the 2 µm plasmid (2.2 Kb). They confer ampicillin resistance and they contain 5 unique EcoRI,HpaI,HindIII,BamHI and SalI restriction sites. Cosmid pHCG3 was used to clone the nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae carried by twoHindIII fragments of 17 and 26 Kb, respectively. The resulting cosmid, termed pGPC875 (53 Kb) which conferred a Nif+ phenotype to Escherichia coli, was introduced in yeast by transformation. No acetylene reduction activity was detectable in the transformants. However it was shown that the entire information for nitrogen fixation can be replicated and maintained intact in yeast for more than 50 generations of growth.

Mapping of mRNA encoding endoglucanase A from Clostridium thermocellum

SummaryThe size and location of the 5′ end of celA mRNA encoding endoglucanase A of Clostridium thermocellum were investigated in C. thermocellum and in an Escherichia coli clone that carries and expresses the celA gene. In E. coli, the 5′ end of celA mRNA was located 134 bp upstream from the initiation codon and 10 bp downstream from a sequence homologous to the consensus sequence of E. coli σ70 and Bacillus subtilis σ43 (formerly σ55) vegetative promoters. In C. thermocellum, a minor transcript appeared to start from the same site, but a major species started 57 bp upstream from the coding sequence. The 5′ end of this mRNA was preceded by a sequence reminiscent of B. subtilis σ28 vegetative promoters. In both organisms, the size of the transcript suggested that celA belongs to a monocistronic unit of transcription.