Utilization of IncP-1 plasmids as vectors for transposon mutagenesis in myxobacteria

No free plasmid has ever been found in the myxobacterium Myxococcus xanthus, but IncP-1 plasmids are able to integrate into the chromosome of this bacterium. The frequency of integration depends greatly upon the structure of the IncP-1 plasmid used. This property has been used to devise new delivery systems for transposon mutagenesis in this species. Plasmids with low integration efficiencies have proved to be efficient donors of Tn5, while plasmids with very high frequencies of integration could be used directly to generate mutations. These vectors have also proved efficient for Tn5 transfer into other species of myxobacteria, which have not so far been susceptible to genetic analysis.     

Transfer of plasmid RP4 to Myxococcus xanthus and evidence for its integration into the chromosome.

The broad-host-range plasmid RP4 and its derivative R68.45 were transferred to Myxococcus xanthus DK101 and DZ1; RP4 was maintained integrated in the chromosome. Loss of plasmid markers occurred during the growth of the transconjugants, which could be prevented by selective pressure with oxytetracycline. The integrated plasmid was transferred back to Escherichia coli often as RP4-prime plasmids carrying various segments of the M. xanthus chromosome. It also mediated chromosomal transfer between M. xanthus strains.     

Characterization of a novel cry2Aa-type gene from Bacillus thuringiensis subsp. kurstaki

A 4 kb BamHI-HindIII fragment, corresponding to the cry2A operon of Bacillus thuringiensis subsp. kurstaki strain BNS3, was cloned. The sequencing of the corresponding cry2Aa-type gene, termed crybns3-4, revealed an open reading frame of 1902 bp, encoding a protein of 633 amino-acid residues. Both nucleotide and amino-acid sequences similarity analysis revealed that crybns3-4 is a new cry2Aa-type gene which has several differences from the reported cry2Aa-type genes. The transfer of the cloned operon to an acrystalliferous mutant of BNS3, revealed an expression of the new cry2Aa-type gene and a production of parasporal crystal inclusions in the transformants.     

Identification of salt stress-induced transcripts in potato leaves by cDNA-AFLP

Potato (Solanum tuberosum L.) is highly sensitive to salt stress, which is one of the most important factors limiting plant cultivation. The investigation of plant response to high salinity was envisaged in this report using cDNA-amplified fragment length polymorphism (AFLP). This technique was applied to salt- stressed and control potato plants (cv. Nicola). The expression profiles showed approx 5000 bands. Of these, 154 were upregulated and 120 were repressed by salt stress. In this study we have only considered cDNA fragments that seem to be originated from salt-induced mRNA. Eighteen fragments were then reamplified, cloned, and sequenced. Sequence comparison of these cDNA, identified in response to salt stress in potato, revealed that some of them present homologies with proteins in other species that are involved in cell wall structure and turnover such as proline-rich proteins and beta-galactosidase. A number of identified clones encoded putative stress response proteins such as NADP-dependant glyceraldehyde dehydrogenase and wound-induced protein. In addition, some of them encode proteins related to hypersensitive response against pathogens such as putative late blight and nematode as well as putative pathogenesis-related proteins. These cDNA seem to be differentially expressed in the presence of salt stress as shown by Northern blot or reverse Northern hybridization experiments.     

Evidence of DNA rearrangements in the 128-kilobase pBtoxis plasmid of Bacillus thuringiensis israelensis

BUPM97 is a novel Tunisian isolate of Bacillus thuringiensis israelensis presenting insecticidal activity against Culex pipiens larvae. The δ-endotoxins pattern of this strain was different from that of the reference strain B. thuringiensis israelensis H14. Therefore, the study of its cry genes content was carried out by restriction-fragment-length-polymorphism (RFLP) using specific cry genes probes and by DNA sequencing. It was clearly demonstrated that in the strain BUPM97 the cry4A and cry10A genes were deleted from the B. thuringiensis israelensis 128-kb pBtoxis plasmid. In addition, a strong DNA sequence polymorphism was evidenced in the same plasmid downstream from the cry4B gene. This very particular DNA dynamic evidenced in this new strain of B. thuringiensis israelensis should be taken into consideration, regarding the strain stability during the industrial production of B. thuringiensis bioinsecticides.     

Improvement of Photorhabdus temperata bioinsecticides production in low‐cost media through adequate fermentation technology

To develop a cost effective process for bioinsecticides production by Photorhabdus temperata, dissolved oxygen (DO) requirements were investigated in both the complex and the optimized media using diluted seawater as a source of micronutrients. By varying DO concentrations, tolerance to hydrogen peroxide was shown to be medium dependant. Indeed, P. temperata cells grown in the complex medium, exhibited higher tolerance than cells grown in the optimized medium (OM). Tolerance to H₂O₂ was shown to be related to intracellular reactive oxygen species (ROS) accumulation during soya bean meal or glucose assimilation, as shown by flow cytometry analysis. To avoid oxidative stress damages in P. temperata cells cultured in the OM, DO concentration should be constant 50% saturation throughout the fermentation. However, a DO‐shift control strategy was demonstrated to be beneficial for P. temperata bioinsecticide production in the complex medium. By using such a strategy biomass, culturability, and oral toxicity reached 16.5 × 10⁸, 1.15 × 10⁸ cells/mL and 64.2%, respectively, thus was 16.19, 26.37, and 12.2% more than in the cultures carried out at a constant 50% saturation. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Study of the δ-endotoxins produced by three recently isolated strains of Bacillus thuringiensis

Abstract Three different strains of Bacillus thuringiensis , subsp. toumanoffi, sotto and kurstaki , producing parasporal inclusion crystals, have recently been isolated in Tunisia. The δ-endotoxins produced by the different strains gave distinct patterns on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polymerase chain reaction screening of these three strains, using oligonucleotides specific for the genes cryIA, cryIII and cryIV , did not generate amplified fragment profiles characteristic of these genes. For each of the strains, the presence of one or more δ-endotoxin coding genes having partial sequence similarities to one or more genes of these three groups was found.     

Investigation of the steps involved in the difference of susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Bacillus thuringiensis Vip3Aa16 toxin

BUPM95 is a Bacillusthuringiensis subsp. kurstaki strain producing the Vip3Aa16 toxin with an interesting insecticidal activity against the Lepidopteran larvae Ephestia kuehniella. Study of different steps in the mode of action of this Vegetative Insecticidal Protein on the Mediterranean flour moth (E. kuehniella) was carried out in the aim to investigate the origin of the higher susceptibility of this insect to Vip3Aa16 toxin compared to that of the Egyptian cotton leaf worm Spodoptera littoralis. Using E. kuehniella gut juice, protoxin proteolysis generated a major band corresponding to the active toxin and another band of about 22 kDa, whereas the activation of Vip3Aa16 by S. littoralis gut juice proteases generated less amount of the 62 kDa active form and three other proteolysis products. As demonstrated by zymogram analysis, the difference in proteolysis products was due to the variability of proteases in the two gut juices larvae. The study of the interaction of E. kuehniella BBMV with biotinylated Vip3Aa16 showed that this toxin bound to a putative receptor of 65 kDa compared to the 55 and 100 kDa receptors recognized in S. littoralis BBMV. The histopathological observations demonstrated similar damage caused by the toxin in the two larvae midguts. These results demonstrate that the step of activation, mainly, is at the origin of the difference of susceptibility of these two larvae towards B. thuringiensis Vip3Aa16 toxin.     

Correlation between delta-endotoxin and proteolytic activities produced by Bacillus thuringiensis var. kurstaki growing in an economic production medium

Abstract

Bacillus thuringiensis is a Gram positive bacterium that produces an insecticidal crystalline protein making it one of the most important biocontrol agents for pest management. Bioinsecticides based on B. thuringiensis were produced by fermentation processes in liquid media. Cultural conditions controlling proteolytic activities in different culture media were investigated to study the possible correlations between B. thuringiensis production of proteases and delta-endotoxins in a low-cost complex medium. Aeration appeared to play an important role in delta-endotoxin production. The correlation between proteolytic activity and aeration does not seem to be reliable. A negative correlation (correlation coefficient =? 0.774) was established between protease activity and delta-endotoxin production. In order to prove this correlation, protease hypo-producing and overproducing mutants were isolated through random mutagenesis of two wild strains, BUPM13 and BUPM5, by using nitrous acid. Interestingly, delta-endotoxin production of BUPM13-1, BUPM13-2 and BUPM13-3 was markedly improved when compared to the wild strain BUPM 13, reaching 2.1-fold, 3.69-fold and 8.13-fold, respectively. Maximal protease activity (540-2468 UI) obtained by BUPM5-1 and BUPM5-2 was 2.34-fold and 10.7-fold, respectively, more than that obtained by the wild strain BUPM5 with a drastic decrease of their delta-endotoxin production. Study of delta-endotoxin production by the selected mutants confirmed that insecticidal crystal protein stability in the culture strongly depends on the level of endogenous protease activity. This was also confirmed by bioassays measuring the LC₅₀ using larvae of Ephestia kuehniella. Determining protease activity in fermentation culture could be useful in indirectly predicting the potency of B. thuringiensis strains with high insecticidal activities. This would allow low-cost selection of overproducing wild isolates or mutants in the screening programmes for the reduction of production cost, which is important from a practical point of view.     

Mechanism of integration of the broad-host-range plasmid RP4 into the chromosome of Myxococcus xanthus

The site-specific recombination mechanism through which the plasmid RP4 has been previously shown to integrate into the chromosome of Myxococcus xanthus has been investigated further. Once integrated in one of the numerous chromosomal sites from two different strains, through a precise site on the plasmid, the latter can be excised either precisely or after a definite 14.5-kb deletion. In some cases, the integration is followed by different DNA rearrangements that yield a higher rate of excision and integration. A model for the site-specific integration and excision of the plasmid is proposed.