Genetic manipulations in auto-induced protoplasts of Bacillus thuringiensis.
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Bacillus thuringiensis↗
Biomedical subjects
C Sanchez-Rivas
Publications and source records attributed to C Sanchez-Rivas.
Further studies on recombination in diploid clones from Bacillus subtilis protoplast fusion.
Diploid prototrophs were obtained from protoplast fusion of Bacillus subtilis strains. They are unstable but upon further cultivation they stabilize retaining diploidy but are genetically inactive. It has been suggested that recombination between the parental chromosomes is involved in the production of stable prototrophs and recombinants. In this work the occurrence of this recombination was searched for by determining genetic linkages in transformation experiments. In prototrophs two alleles: hisH2 and trpE8 carried originally on each parental chromosome, were shown to be 48% co-transformable in a stable clone whereas they were only cotransformed in 10% of the unstable colonies. For Trp- recombinants (the most frequent type of a Leu- Met- Thr- x Ade- Ura- Trp- fusion pair) lysed protoplasts were used as donor DNA for the transformations. High values of co-transfer for Ura+ Met+ were obtained. These results confirm the occurrence of recombination in stable diploid clones, prototrophs or recombinants.
Alleles↗
Bacterial fusion assayed by a prophage complementation test.
In previous studies of bacterial protoplast fusion, only the frequencies of cell wall regeneration and of bacterial recombination were determined. In this work the frequency of the heterozygous fusion products is measured by prophage complementation. Two multiply marked nonsuppressing strains of Bacillus subtilis, each lysogenic for a different Sus mutant of the phage phi 105, were induced by mitomycin C, protoplasted, fused, and, after dilution in hypertonic broth, incubated until plating with phi 105-sensitive indicator bacteria. When cell lysis was avoided, the frequency of the heterozygous fused cells could be determined from the number of infectious centers produced. The very high frequencies observed are in good agreement with those determined directly, with nonlysogenic strains, by electron microscopic examination of the fused protoplasts (C. Frehel, A. M. Lheritier, C. Sanchez-Rivas, and P. Schaeffer, J. Bacteriol. 137:1354--1361, 1979). Evidence is presented that fusion occurs in two steps, one polyethylene glycol dependent, the other energy requiring. The bacterial growth medium affects the ability of the protoplasts to fuse and to regenerate a cell wall. When experiments using different growth media were compared, an inverse relationship between these abilities was observed, and a direct relationship appeared between the heterozygotes (corrected for wall regeneration) and the recombinant bacteria that were found.
Bacillus subtilis↗
Electron microscopic study of Bacillus subtilis protoplast fusion.
When protoplasts derived from sporulating cells of Bacillus subtilis were fused by exposure to polyethylene glycol (PEG) and fixed immediately thereafter, protoplasts with two enclosed prespores could be seen by electron microscope. The number of fusion events was greatly increased, and multiply fused protoplasts appeared, when the PEG-treated suspension was diluted in hypertonic broth and reincubated before fixation. This post-PEG incubation effect is taken to indicate a fusion mechanism of two steps: a short, PEG-dependent step of membrane activation, followed by a slow, metabolism-requiring step completing fusion. When prespore-bearing protoplasts from two genetically different strains were mixed and fused, the extent of fusion could also be followed by counting clones of recombinant bacteria. Maximal from the start, their number (1% of each parent type protoplast present) was unaffected by post-PEG incubation. Fusion in this case is apparently completed after plating on the wall-regeneration medium. After optimal post-PEG incubation, the majority of the protoplasts were seen to participate in fusion, and the cytological fusion observed, corrected for wall-regeneration frequency, accounted quantitatively for the prototrophic bacteria eventually recovered. These results are in good agreement with those obtained independently by Sanchez-Rivas and Garro (J. Bacteriol. 137:1340--1345, 1979).
Bacillus subtilis↗
Expression and maintenance of plasmid resistance in regenerating protoplasts of Bacillus subtilis.
Expression of plasmid-encoded resistances in regenerating protoplasts of Bacillus subtilis occurs only after wall synthesis has been resumed. This is observed for protoplasts obtained from cells already containing plasmids (pC194, pT127, pK545) or for plasmid-bearing cells coming from a PEG-mediated transformation. Recovery of expression needs a 2-h incubation of protoplasts, previously washed to get rid of their lysozyme content, in rich hypertonic medium (SMMP). A longer incubation (24-h) results in the obtention of regenerants; however, most of them have lost their resistant phenotype in contrast to those obtained from the usual solid regeneration plates. This finding suggests either a high curing effect or some kind of gene inactivation phenomenon. Discussion is focused on the critical points that have to be considered when polyethylenglycol-mediated transformation of protoplasts is applied to recombinant DNA technology.
Bacillus subtilis↗