Parameters governing bacterial regeneration and genetic recombination after fusion of Bacillus subtilis protoplasts.

Bacterial protoplast fusion, induced by polyethylene glycol, has been made more regular and convenient by further specification and improvement of various steps in the previously used procedure. These have made it possible to obtain regularly 100% regeneration of Bacillus subtilis cells from protoplasts before treatment with polyethylene glycol and yields of 10 to 75% from polyethylene glycol-treated protoplasts. Genetic recombination frequencies do not increase correspondingly. Also, when regeneration is reduced by various experimental conditions, recombination does not decrease in proportion. It is concluded that regeneration of recombinant-forming cells is independently determined and not closely related to the average regeneration for the population. Kinetic studies with varying individual parental or total protoplast concentrations strongly indicate that protoplast collision and contact is not the limiting factor determining the number of genetic recombinants obtained. Recombination approximates a linear, rather than quadratic, function of the total or of the majority protoplast population present, from which it is concluded that fusion events are always adequate to produce substantially more potential recombinants than are registered. The strong effect of the majority/minority ratio upon the number of minority cells that become recombinant is independent of which parent is in excess. This shows in a direct and physiological way that both parents are equivalent partners in their genetic contributions.     

Rheological properties of highly cross-linked waxy maize starch in aqueous suspensions of skim milk components. Effects of the concentration of starch and skim milk components

The storage modulus of various concentrations of highly cross-linked waxy maize starch was measured in water, a salt solution, a salt solution with lactose, whey, and skim milk. The influence of the skim milk components was deduced from the differences in moduli between these systems. Salts appeared to have no direct influence on the modulus of the starch. Lactose increased the storage modulus, probably due to an increase in the particle rigidity of the swollen starch granules. The whey proteins had only a small effect on the modulus of the starch. However, the concentration of these proteins was very low. Casein micelles increased the modulus of the starch because the swollen starch granules are not accessible to the casein. A model based on the mutual exclusion of swollen starch granules and milk proteins was applied to predict the storage modulus of starch-milk systems as a weighed sum of the moduli of the starch and protein phases. Weight factors were derived from the hydration capacity of starch granules and casein micelles. Although the behaviour of aqueous starch-skim milk systems fell within the boundaries imposed by the model, the predictive power of this model was rather poor.     

Release from protoplasts of the Bacillus subtilis protease inhibitor

Abstract Conversion of Bacillus subtilis to protoplasts resulted in the release of 70–80% of the total protease inhibitor activity. Inhibitor fractions contained a polypeptide of approx. 15 kDa which reacted with inhibitor antibody. There was no release of protease inhibitor into the medium by sporulating cells, by osmotic shock of cells nor by washing with high concentrations of salt. The release of inhibitor activity was selective in that only 10–20% of the total protein, and < 10% of the glutamine synthetase activity was found in the protoplast supernatant. The inhibitor could be localized near the cell surface and function in cell protection.     

Synthesis of teichoic acid by Bacillus subtilis protoplasts

Protoplasts of Bacillus subtilis W23 readily synthesized ribitol teichoic acid from nucleotide precursors in the surrounding medium. With cytidine diphosphate-ribitol they made poly(ribitol phosphate), presumably attached to lipoteichoic acid carrier; when cytidine diphosphate-glycerol and uridine diphosphate-N-acetylglucosamine were also present a 10-fold increase in the rate of polymer synthesis occurred, and the product contained both the main chain and the linkage unit. Synthesis was inhibited by trypsin or p-chloromercuribenzenesulfonate in the medium, and we concluded that it occurred at the outer surface of the membrane. During synthesis, which was also achieved readily by whole cells after a brief period of wall lysis, the cytidine phosphate portion of the nucleotide precursors did not pass through the membrane. No evidence could be obtained for a transphosphorylation mechanism for the translocation process. It is suggested that reaction with exogenous substrates was due to temporary exposure of a protein component of the enzyme complex at the outer surface of the membrane during the normal biosynthetic cycle.     

Improvements in the transformation of Bacillus subtilis protoplasts with plasmid DNA

Abstract The transformation system currently used for Bacillus subtilis protoplasts has been improved. Special emphasis was made on three parameters of practical importance:
(a) conditions for direct selection of transformants, (b) optimization of the transformation system for Rec⁻ mutants, and (c) conservation of protoplast suspensions for further use.
Selective regeneration was efficiently achieved for kanamycin or neomycin. Chloramphenicol, tetracycline and erythromycin were only expressed when low concentrations of the antibiotics were used to select transformants during regeneration.     

Transfer of phospholipids between mesosomes and protoplasts from Bacillus subtilis

1. Phospholipids were more intensively labelled from ammonium [1-14C]glycerophosphate in mesosomes than in protoplasts isolated from Bacillus subtilis. 2. When mesosomes, containing phospholipids labelled from sodium-[32P] phosphate were incubated with non radioactive protoplasts, labelled phospholipids were recovered in protoplasts after incubation. 3. This transfer of phospholipids from mesosomes toward protoplasts is time-dependent. 4. Soluble proteins obtained from Bacillus subtilis after ammonium sulphate precipitation were separated on a Sephadex G-100 column. 5. The two main fractions were able to accelerate the transfer of phospholipids from mesosomes toward protoplasts. 6. The first peak stimulated more actively the transfer of phosphatidylethanolamine whereas the second one preferentially accelerated the transfer of phosphatidylglycerol and diphosphatidylglycerol.     

Complementation of succinate dehydrogenase mutants in fused Bacillus subtilis protoplasts

Abstract Succinate dehydrogenase (SDH) and cytochrome b ₅₅₈ are present in Bacillus subtilis cytoplasmic membranes as a complex consisting of three different subunits. Complementation of membrane-bound SDH activity was studied in fused protoplasts of two SDH-negative mutants mutated in different subunits of the complex. The complementation was found to be rapid, temperature dependent and not sensitive to inhibitors of respiration. It is concluded that the complementation predominantly results from the assembly of preformed SDH subunits.     

Properties of regeneration mutants of Bacillus subtilis

Abstract Higher regeneration mutants were isolated from Bacillus subtilis . Protoplasts from two out of four mutants regenerated at a 100% frequency on a semi-synthetic hypertonic medium. They conferred less autolytic productivity, and a revertant regained the parental levels of regeneration frequency and autolytic activities. This mutation ( rgn -1) expressed the other pleiotropic properties, i.e., nonmotility, phage PBS1 resistance and different cell morphology.     

Biofilm formation onto starch fibres by Bacillus subtilis governs its successful adaptation to chickpea milk

Beneficial biofilms may confer effective adaptation to food matrices that assist bacteria in enduring hostile environmental conditions. The matrices, for instance, dietary fibres of various food products, might serve as a natural scaffold for bacterial cells to adhere and grow as biofilms. Here, we report on a unique interaction of Bacillus subtilis cells with the resistant starch fibresof chickpea milk (CPM), herein CPM fibres, along with the production of a reddish-pink pigment. Genetic analysis identified the pigment as pulcherrimin, and also revealed the involvement of Spo0A/SinI pathway in modulating the observed phenotypes. Besides, through successful colonization of the CPM fibres, the wild-type cells of B. subtilis displayed enhanced survivability and resilience to environmental stress, such as heat and in vitro gastrointestinal treatments. In total, we infer that the biofilm formation on CPM fibres is an adaptation response of B. subtilis for strategic survival.     

Regeneration of protoplasts of Bacillus subtilis 168 and closely related strains

Regeneration of protoplasts to bacilli was attempted in several strains of Bacillus closely related to Bacillus subtilis 168. On DM3 and similar media using succinate as osmotic support, only B. subtilis 168 and Bacillus natto ATCC 15245 were able to regenerate. Media containing mannitol as osmotic support, and agar as gelling agent gave rise to L-form colonies with Bacillus licheniformis NCTC 6346. Many of the L-form colonies were able to regenerate to the bacillary form when plated on the mannitol medium solidified with gelatin. All of the Bacillus species tested were able to regenerate on the latter medium at rates sufficient to allow protoplast transformation and fusion experiments.     

Methionine regeneration and aminotransferases in Bacillus subtilis,Bacillus cereus,and Bacillus anthracis

The conversion of ketomethiobutyrate to methionine has been previously examined in a number of organisms, wherein the aminotransferases responsible for the reaction have been found to be members of the Ia subfamily (L. C. Berger, J. Wilson, P. Wood, and B. J. Berger, J. Bacteriol. 183:4421-4434, 2001). The genome of Bacillus subtilis has been found to contain no subfamily Ia aminotransferase sequences. Instead, the analogous enzymes in B. subtilis were found to be members of the If subfamily. These putative aspartate aminotransferases, the yugH, ywfG, ykrV, aspB, and patA gene products, have been cloned, expressed, and characterized for methionine regeneration activity. Only YkrV was able to convert ketomethiobutyrate to methionine, and it catalyzed the reaction only when glutamine was used as amino donor. In contrast, subcellular homogenates of B. subtilis and Bacillus cereus utilized leucine, isoleucine, valine, alanine, phenylalanine, and tyrosine as effective amino donors. The two putative branched-chain aminotransferase genes in B. subtilis, ybgE and ywaA, were also cloned, expressed, and characterized. Both gene products effectively transaminated branched-chain amino acids and ketoglutarate, but only YbgE converted ketomethiobutyrate to methionine. The amino donor preference for methionine regeneration by YbgE was found to be leucine, isoleucine, valine, phenylalanine, and tyrosine. The B. subtilis ybgE gene is a member of the family III of aminotransferases and falls in a subfamily designated here IIIa. Examination of B. cereus and Bacillus anthracis genome data found that there were no subfamily IIIa homologues in these organisms. In both B. cereus and B. anthracis, two putative branched-chain aminotransferases and two putative D-amino acid aminotransferases were discovered as members of subfamily IIIb. These four sequences were cloned from B. cereus, expressed, and characterized. Only the gene product from the sequence designated Bc-BCAT2 was found to convert ketomethiobutyrate to methionine, with an amino donor preference of leucine, isoleucine, valine, phenylalanine, and tyrosine. The B. anthracis homologue of Bc-BCAT2 was also cloned, expressed, and characterized and was found to be identical in activity. The aminooxy compound canaline was found to be an uncompetitive inhibitor of B. subtilis YbgE and also inhibited growth of B. subtilis and B. cereus in culture.     

Studies on transfection and transformation of protoplasts of Bacillus larvae, Bacillus subtilis, and Bacillus popilliae

Protoplasts of Bacillus larvae NRRL b-3555 and Bacillus subtilis RM125 (restrictionless, modificationless mutant) were transfected with DNA from the B. larvae bacteriophage PBL1c in the presence of polyethylene glycol. B. subtilis 168 and Bacillus popilliae NRRL B-2309M protoplasts could not be transfected with PBL1c DNA. Protoplasts of B larvae NRRL B-3555 were transformed with plasmids pC194 and pHV33 in the presence of polyethylene glycol. The frequency of transformation was much higher when the plasmids were isolated from B. larvae NRRL B-3555 transformants than when they were isolated from B. subtilis 168. These results indicate that the restriction-modification systems found in B. larvae NRRL B-3555 and B. subtilis 168 may be different. Conditions for protoplast formation and cell wall regeneration were developed for B. popilliae NRRL B-2309S. However, no transformation occurred with plasmids pC194 and pHV33 (isolated from B. subtilis 168).     

Studies on transfection and transformation of protoplasts of Bacillus larvae, Bacillus subtilis, and Bacillus popilliae.

Protoplasts of Bacillus larvae NRRL b-3555 and Bacillus subtilis RM125 (restrictionless, modificationless mutant) were transfected with DNA from the B. larvae bacteriophage PBL1c in the presence of polyethylene glycol. B. subtilis 168 and Bacillus popilliae NRRL B-2309M protoplasts could not be transfected with PBL1c DNA. Protoplasts of B larvae NRRL B-3555 were transformed with plasmids pC194 and pHV33 in the presence of polyethylene glycol. The frequency of transformation was much higher when the plasmids were isolated from B. larvae NRRL B-3555 transformants than when they were isolated from B. subtilis 168. These results indicate that the restriction-modification systems found in B. larvae NRRL B-3555 and B. subtilis 168 may be different. Conditions for protoplast formation and cell wall regeneration were developed for B. popilliae NRRL B-2309S. However, no transformation occurred with plasmids pC194 and pHV33 (isolated from B. subtilis 168).