Stable inheritance of shuttle vectors based on plasmid pIM13 in a mutant strain of Clostridium acetobutylicum.

New shuttle vectors for Clostridium acetobutylicum were constructed, using as replicons the Gram-positive plasmid pIM13, and derivatives of the Gram-negative plasmid pBR322, including pUC19. These vectors transformed C. acetobutylicum at a high frequency (up to 10(6) transformants per microgram DNA) by PEG-mediated protoplast transformation. A mutant host strain, NI-4082, was isolated on the basis of its ability to maintain plasmid pIM13 stably in the absence of selection pressure. The shuttle vectors showed no segregational or structural instability in this mutant strain. Moreover, the results suggested a relationship between segregational instability and the multimerization of pIM13 in C. acetobutylicum. The host/vector system described possessed all the properties required for efficient gene cloning in this species.     

On Circuit Functionality in Boolean Networks

It has been proved, for several classes of continuous and discrete dynamical systems, that the presence of a positive (resp. negative) circuit in the interaction graph of a system is a necessary condition for the presence of multiple stable states (resp. a cyclic attractor). A positive (resp. negative) circuit is said to be functional when it “generates” several stable states (resp. a cyclic attractor). However, there are no definite mathematical frameworks translating the underlying meaning of “generates.” Focusing on Boolean networks, we recall and propose some definitions concerning the notion of functionality along with associated mathematical results.     

Characterization of a methyl-specific restriction system in Clostridium acetobutylicum strain N1-4081

A type II restriction endonuclease, named CacI, was detected in Clostridium acetobutylicum strain N1-4081. CacI cleaved the tetranucleotide sequence [5' decreases -GATC-3']. The modification system consisted of the methylation of the adenine present in this sequence. CacI, an isoschizomer of MboI, is inactive on dam methylated substrates.     

Is Fe deficiency rather than P deficiency the cause of cluster root formation in Casuarina species?

When subjected, directly (through nutritional deficiencies) or indirectly (through alkaline constraints leading to such deficiencies) to nutrient deficiencies, certain plants respond by developing special root structures called cluster roots. This phenomenon can be considered as an ecophysiological response to a specific nutrient deficiency enabling plants to enhance nutrient uptake. Experiments conducted on an alkaline and an acid soil showed that Casuarina glauca (Sieber ex Spreng.) produced cluster roots only in the alkaline soil and not in the acid soil. In addition, iron (Fe) and phosphorus (P) deficiencies were examined separately or together to determine their effect on cluster root formation in C. glauca seedlings grown hydroponically. Results from experiments carried out on three Casuarina species (C. glauca, C. cunninghamiana Miq. and C. equisetifolia L.) indicated that Fe is involved in cluster root formation. In nutrient media lacking P but containing Fe, no cluster roots formed while seedlings receiving P and lacking Fe developed cluster roots. When incubated on chrome-azurol S-agar on blue plates (CAS assay), a technique used routinely to detect the production of siderophores by micro-organisms, the root system of Fe-deficient plants exhibited orange halos around cluster roots, indicating production of a ferric-chelating agent. It is concluded that the capacity of cluster roots of C. glauca to chelate Fe allows the plant to grow normally on alkaline soils.     

Recognition sequence of a new methyl-specific restriction system from Clostridium acetobutylicum strain ABKn8

A new type II restriction endonuclease, named Cac8I was detected in Clostridium acetobutylicum strain ABKn8. Cac8I cleaved the hexanucleotide sequence [5'-GCN decreases NGC-3'] and generated blunt ends. Up to now no isoschizomer of Cac8I has been described [corrected].     

Purification and characterization of SepII a new restriction endonuclease from Staphylococcus epidermidis

A Type II restriction enzyme SepII has been purified to apparent homogeneity from the gram-positive coccus, Staphylococcus epidermidis. The purification included an ammonium sulfate precipitation followed by Q-sepharose, heparin-sepharose and MonoQ column chromatography on an FPLC system. SDS-PAGE analysis showed a denatured molecular weight of 29 kDa. The effects of temperature, pH, NaCl, Mn(2+), Ca(2+), and Mg(2+) ion concentrations were studied to determine the optimal reaction conditions. The enzyme exhibits near maximal levels of activity between pH 8-10, at 10-20mM MgCl(2), 100-150 mM NaCl and 1mM DTT. The results also show that in NEB Buffer 3 the enzyme is active over a broad temperature range from 0 to 70 °C, and in the absence of DNA, enzyme thermostability is observed up to 50 °C for 20 min, while most of the original activity is conserved in 50% glycerol for weeks at room temperature. Single and double digestion in presence of commercial restriction enzymes of known DNA substrates (lambda, pBR322, pET21, pTrcHisB, pPB67) showed that the purified SepII recognized and cleaved the same site as EcoRV. Genomic DNA modification status was also determined.     

Characterization of restriction endonuclease BfrBI from Bacteroides fragilis strains BE3 and AIP 10006

A new type II restriction endonuclease, named BfrBI, was detected in two strains of Bacteroides fragilis, BE3 and AIP 10006 (NCTC 9343T). The enzyme BfrBI, an isoschizomer of NsiI and AvaIII, recognized the hexanucleotide sequence [5'-ATG decreases CAT-3'], with a cleavage site generating blunt ends.