Characterization and molecular cloning of thermostable alpha-amylase from Streptomyces sp.To1

Summary A new thermophilic Streptomyces sp. TO1, isolated from Tunisian soil, produced a thermostable alpha-amylase and pullulanase. The gene encoding for the alpha-amylase activity was cloned into the multicopy cloning plasmid pLM1 using S. lividans ZX1 as host strain. The ZX1 / pLM1 strain has the same activity than the initial TO1 strain and about 25 fold higher activity than the ZX1 strain. This alpha-amylase has an optimum of pH and temperature at 6 and 70 °C respectively.     

A mutant cysteinyl-tRNA synthetase affecting timing of chromosomal replication initiation in B. subtilis and conferring resistance to a protein kinase C inhibitor.

A Bacillus subtilis mutant spnA95 was isolated as resistant at 30 degrees C to the protein kinase C (PKC) inhibitor, sphinganine, and temperature sensitive for growth. As deduced by flow cytometry measurements, the mutant has a 35% reduced initiation mass at permissive temperature, resulting in initiation of DNA replication much earlier in the cell cycle than in the wild type. This modification is accompanied by a change in cell size, as determined by phase-contrast microscopy and flow cytometry. Therefore, this strain displays the characteristics of a novel cell clock mutant. spnA is a newly identified gene in B.subtilis and was shown to encode a cysteinyl-tRNA synthetase. At non-permissive temperature, the mutant was defective in the synthesis of P70, a protein with several characteristics of PKC (a cysteine-rich protein). As one possibility, we propose that the altered timing of replication may be due to the reduced synthesis of specific cysteine-rich proteins normally involved in controlling chromosomal replication initiation in B. subtilis.     

Secreted recombinant P53 protein from Pichia pastoris is a useful antigen for detection of serum p53: autoantibody in patients with advanced colorectal adenocarcinoma

The detection of P53 alteration by serological method is easier to perform, does not require tumor tissues and is of interest for patients monitoring. In this study, we described the development of a home made ELISA test based on recombinant human P53 protein produced in Pichia pastoris and used as antigen for the detection of serum p53-Abs in colorectal carcinoma patients. The human P53 was secreted as a His-tagged protein by recombinant KM71 strain (Kα21) via the peptide signal α of the Saccharomyces cerevisiae mating type gene. The recombinant P53-His was able to detect p53-Abs in 23.4 % of patients. Serum p53-Abs correlated significantly with surgical treatment (P = 0.007), relapse during follow-up (P = 0.036), depth of invasion (P = 0.036) and the level of CA19-9 (P = 0.034). Survival analysis showed that patients negative for serum p53-Abs exhibited a prolonged disease free survival period (P log rank = 0.012). In conclusion, the secreted recombinant human P53-His produced in P. pastoris seems to be a useful antigen for detection of serum p53 Abs in patients with colorectal carcinoma.     

<Emphasis Type="Italic">Gr</Emphasis> and <Emphasis Type="Italic">hp</Emphasis>-<Emphasis Type="Italic">1</Emphasis> tomato mutants unveil unprecedented interactions between arbuscular mycorrhizal symbiosis and fruit ripening

Main conclusion

Systemic responses to an arbuscular mycorrhizal fungus reveal opposite phenological patterns in two tomato ripening mutants depending whether ethylene or light reception is involved. The availability of tomato ripening mutants has revealed many aspects of the genetics behind fleshy fruit ripening, plant hormones and light signal reception. Since previous analyses revealed that arbuscular mycorrhizal symbiosis influences tomato berry ripening, we wanted to test the hypothesis that an interplay might occur between root symbiosis and fruit ripening. With this aim, we screened seven tomato mutants affected in the ripening process for their responsiveness to the arbuscular mycorrhizal fungus Funneliformis mosseae. Following their phenological responses we selected two mutants for a deeper analysis: Green ripe (Gr), deficient in fruit ethylene perception and high-pigment-1 (hp-1), displaying enhanced light signal perception throughout the plant. We investigated the putative interactions between ripening processes, mycorrhizal establishment and systemic effects using biochemical and gene expression tools. Our experiments showed that both mutants, notwithstanding a normal mycorrhizal phenotype at root level, exhibit altered arbuscule functionality. Furthermore, in contrast to wild type, mycorrhization did not lead to a higher phosphate concentration in berries of both mutants. These results suggest that the mutations considered interfere with arbuscular mycorrhiza inducing systemic changes in plant phenology and fruits metabolism. We hypothesize a cross talk mechanism between AM and ripening processes that involves genes related to ethylene and light signaling.

     

New insights into the biogenesis of the cell envelope of corynebacteria: identification and functional characterization of five new mycoloyltransferase genes in Corynebacterium glutamicum

Mycolic acids, the major lipid constituents of Corynebacterineae, play an essential role in maintaining the integrity of the bacterial cell envelope. We have previously characterized a corynebacterial mycoloyltransferase (PS1) homologous in its N-terminal part to the three known mycobacterial mycoloyltransferases, the so-called fibronectin-binding proteins A, B and C. The genomes of Corynebacterium glutamicum (ATCC13032 and CGL2005) and Corynebacterium diphtheriae were explored for the occurrence of other putative corynebacterial mycoloyltransferase-encoding genes (cmyt). In addition to csp1 (renamed cmytA), five new cmyt genes (cmytB-F) were identified in the two strains of C. glutamicum and three cmyt genes in C. diphtheriae. In silico analysis showed that each of the putative cMyts contains the esterase domain, including the three key amino acids necessary for the catalysis. In C. glutamicum CGL2005 cmytE is a pseudogene. The four new cmyt genes were disrupted in this strain and overexpressed in the inactivated strains. Quantitative analyses of the mycolate content of all these mutants demonstrated that each of the new cMyt-defective strains, except cMytC, accumulated trehalose monocorynomycolate and exhibited a lower content of covalently bound corynomycolate than did the parent strain. For each mutant, the mycolate content was fully restored by complementation with the corresponding wild-type gene. Finally, complementation of the cmytA-inactivated mutant by the individual new cmyt genes established the existence of two classes of mycoloyltransferases in corynebacteria.     

Are there health risks for travel among Tunisians? What type of medical care should be assured?

The present work aims to assess the importance of the international travels of Tunisians, their destinations, the risks they face as well as the means to reduce them. Therefore, data related to the period from 1989 to 1998, were collected from official organisms involved in this area. Results revealed an increase of the number of Tunisians travelling outside the country of 23% between 1989 and 1998, with a mean progress of 5.7% per year since 1993. Main destinations were Maghrebin countries (65.7%), Europe (30%) and Middle East (4%). The number of persons leaving to high risk destinations remains difficult to assess and shows the importance of implementing a surveillance system to insure an adequate medical follow-up of this category of travellers. The structures actually involved in the health management of Tunisian travellers have a weak recruitment which demonstrates the importance to strengthen the sensitization of general practitioners to this subject and the promotion of the immunization ward of "Institut Pasteur" to a national centre of travelling medicine.     

Variation in volatiles of Astragalus gombiformis Pomel

The essential oils obtained by hydrodistillation (Clevenger apparatus) from aerial parts of Astragalus gombiformis were analysed by gas chromatography coupled with mass spectrometry (GC/MS). This study showed that the A. gombiformis essential oils are complex mixtures of important natural compounds, which varied qualitatively and quantitatively between cultivated and wild plants and between phenological stages of development. All analysed oils are characterized by the constant presence of phytol, 6,10,14-trimethyl-2-pentadecanone, 4-terpineol, and gamma-terpinene. This study is the first report on the chemical composition of essential oils from A. gombiformis and indicates that these oils should be more studied.     

Genetic diversity of nine faba bean (Vicia faba L.) populations revealed by isozyme markers

Seven isozyme systems (Sod, 6-Pgd, Me, Est, Skdh, Fdh and Gdh) representing nine loci were used to study the genetic diversity of nine faba bean populations. Seven loci revealed polymorphic bands and showed the same quaternary structure as that found in several species. They revealed a high number of phenotypes. Indeed, from 3 to 9 phenotypes per locus were investigated in this study. The percentage of polymorphic loci (P = 59.3 %) was higher than that mentioned in the autogamous species (P = 20.3 %) and less than the optimum (P=96 %) indicated for allogamous plants. Total genetic diversity (H _T) and within population genetic diversity (H _S) were estimated with the isozyme markers. The contribution of among population genetic diversity (D _ST) to total genetic diversity was 22%. Enzyme markers pointed out an average inbreeding level for whole population (F _IT) and within population (F _IS). Within population genetic diversity represents 78% of total diversity. Intra-population genetic diversity (H _S = 0.206) was ranged with the respect of allogamous species and was clearly higher than that of among population genetic diversity (D _ST = 0.057) indicating an out-crossing predominance in the studied populations. The expected heterozygosity was higher than that observed heterozygosity at the allogamous species was confirmed in this study. Although, the mean estimated gene flow was less than 1(Nm=0.814), the dendrogram based on Nei’s genetic distance of the 9 populations using UPGMA method showed some genetic drift between populations.