The Bacillus thuringiensis cyt genes for hemolytic endotoxins constitute a gene family.

In the same way that cry genes, coding for larvicidal delta endotoxins, constitute a large and diverse gene family, the cyt genes for hemolytic toxins seem to compose another set of highly related genes in Bacillus thuringiensis. Although the occurrence of Cyt hemolytic factors in B. thuringiensis has been typically associated with mosquitocidal strains, we have recently shown that cyt genes are also present in strains with different pathotypes; this is the case for the morrisoni subspecies, which includes strains biologically active against dipteran, lepidopteran, and coleopteran larvae. In addition, while one Cyt type of protein has been described in all of the mosquitocidal strains studied so far, the present study confirms that at least two Cyt toxins coexist in the more toxic antidipteran strains, such as B. thuringiensis subsp. israelensis and subsp. morrisoni PG14, and that this could also be the case for many others. In fact, PCR screening and Western blot analysis of 50 B. thuringiensis strains revealed that cyt2-related genes are present in all strains with known antidipteran activity, as well as in some others with different or unknown host ranges. Partial DNA sequences for several of these genes were determined, and protein sequence alignments revealed a high degree of conservation of the structural domains. These findings point to an important biological role for Cyt toxins in the final in vivo toxic activity of many B. thuringiensis strains.

Normal induction of the SOS response in Bacillus subtilis is prevented by the mutant repressor from phage phi 105cts23.

The presence of the phi 105cts23 mutant prophage in Bacillus subtilis induces a series of pleiotropic effects that could be ascribed to an anti-SOS activity. In order to circumvent the phage function responsible for this phenomenon, the cts23 mutant repressor was cloned and sequenced. The isolated repressor reduced the survival capacity of the host cells after mitomycin C or nalidixic acid treatments and lowered the spontaneous reversion frequency. When SOS induction kinetics were studied, low or null induction of the damage-inducible din22::LacZ fusion was observed. In contrast, the presence of the wild-type prophage amplified the SOS response. Sequencing of the mutant repressor revealed that the cts23 mutation is a T-->C transition affecting the 5' closest codon to one of the two reported DNA binding domains.

Identification and characterization of a previously undescribed cyt gene in Bacillus thuringiensis subsp. israelensis.

Mosquitocidal Bacillus thuringiensis strains show as a common feature the presence of toxic proteins with cytolytic and hemolytic activities, Cyt1Aa1 being the characteristic cytolytic toxin of Bacillus thuringiensis subsp. israelensis. We have detected the presence of another cyt gene in this subspecies, highly homologous to cyt2An1, coding for the 29-kDa cytolytic toxin from B. thuringiensis subsp. kyushuensis. This gene, designated cyt2Ba1, maps upstream of cry4B coding for the 130-kDa crystal toxin, on the 72-MDa plasmid of strain 4Q2-72. Sequence analysis revealed, as a remarkable feature, a 5' mRNA stabilizing region similar to those described for some cry genes. PCR amplification and Southern analysis confirmed the presence of this gene in other mosquitocidal subspecies. Interestingly, anticoleopteran B. thuringiensis subsp. tenebrionis belonging to the morrisoni serovar also showed this gene. On the other hand, negative results were obtained with the anti-lepidopteran strains B. thuringiensis subsp. kurstaki HD-1 and subsp. aizawai HD-137. Western analysis failed to reveal Cyt2A-related polypeptides in B. thuringiensis subsp. israelensis 4Q2-72. However, B. thuringiensis subsp. israelensis 1884 and B. thuringiensis subsp. tenebrionis did show cross-reactive products, although in very small amounts.

Introduction and expression of an anti-dipteran toxin gene from B. thuringiensis in nodulating Rhizobia.

Rhizobium-based inoculants are widely used in leguminous crops. Improved strains with dual functions (i.e, nitrogen fixing and insecticidal) could be successfully applied to organic pest management. These could also be used as seed protectants, reducing the use of chemical insecticides. The cryIVB gene from B. thuringiensis israelensis coding for an anti-dipteran toxin was used as a model for expression. A highly stable plasmid vector (pTR101) was chosen in order to carry out nodulation experiments in the absence of selective pressure. Constructions were introduced into Rhizobium fredii 191 and meliloti 1021, through triparental matings with a helper plasmid. Expression was monitored by Western blotting of crude extracts using a specific antibody raised against recombinant CryIVB protein. Although rearrangements leading to deletions were observed, an immunoreactive polypeptide was reproducibly detected in both Rhizobia. The presence of the recombinant plasmids did not affect the nodulative capacity nor the growth of the inoculated plants.

Anti-SOS effects induced in Bacillus subtilis by a phi 105 mutant prophage.

The presence of the mutant prophage phi 105cts23 in Bacillus subtilis strains strongly affected several biological parameters including the viability of protoplasts and the establishment of plasmid pC194. A defective inducibility of the prophage after treatments that de-repress the SOS-like response were also observed. Although these alterations suggested a Rec-deficient phenotype, homologous recombination was not impaired in these lysogenic derivatives. In fact, chromosomal DNA transformation in these competent cells was more efficient than in cells carrying the wild type prophage: cell death due to prophage induction upon competence development was lower than expected. Alterations in the response to SOS-inducing agents and to osmotic stress correlated with the presence of this particular mutant prophage or the cloned thermosensitive repressor at the permissive temperature. The induction of an anti-SOS effect is discussed.

Cyclic nucleotide phosphodiesterase activity in Neurospora crassa. Purification by immunoaffinity chromatography and characterization.

Monoclonal antibodies to Neurospora crassa cyclic nucleotide phosphodiesterase (PDE I) were selected by their capacity to inhibit the enzyme activity. The monoclonal immunoglobulin, coupled to Sepharose 4B, was used for the affinity purification of PDE I activity. After SDS-polyacrylamide gel electrophoresis the affinity purified PDE I fractions showed a single polypeptide band of about 41 kDa. This band reacted in Western blots with the above mentioned monoclonal immunoglobulin.

Trypanosoma cruzi adenylate cyclase activity. Purification and characterization.

Adenylate cyclase activity associated with Trypanosoma cruzi sedimentable fractions was solubilized by treatment with the non-ionic detergent Lubrol PX and 0.5 M-(NH4)2SO4. The following hydrodynamic and molecular parameters were established for a partially purified enzyme-detergent complex: sedimentation coefficient 6.2 S; Stokes radius 5.65 nm; partial specific volume 0.83 ml/g; Mr 244 000; frictional ratio 1.33. A Mr of about 124 000 was calculated for the detergent-free protein from these parameters. The pI of this enzyme activity was 6.2. A monoclonal antibody to T. cruzi adenylate cyclase was obtained, which inhibited cyclase activities from several lower eukaryotic organisms. The T. cruzi adenylate cyclase was further purified by using this antibody in immunoaffinity chromatographic columns. Fractions obtained after this chromatography showed, on SDS/polyacrylamide-gel electrophoresis, a main polypeptide band with an apparent Mr of about 56 000, which specifically reacted with the monoclonal antibody.