Reversion of Bacillus subtilis Protoplasts to the Bacillary Form Induced by Exogenous Cell Wall, Bacteria and by Growth in Membrane Filters

Clive, D.; Landman, Otto E.

doi:10.1099/00221287-61-2-233

Cited by 24 publications

(11 citation statements)

References 14 publications

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“…N16-5 could not regenerate the cell wall on the modified DM3 medium, which could support the successful protoplast regeneration and transformation of alkaliphilic Bacillus pseudofirmus OF4 and Bacillus halodurans C-125 [10], [12]. According to the previous report, protoplast reversion of Bacillus subtilis was stimulated by the close physical contact between the naked protoplasts and the hard substratum provided by the regeneration medium with 25% gelatin, hard agar or membrane filters [31], [35]. So succinate medium solidified with 25% gelatin (SG25 medium), a hard gelatin regeneration medium, was used to stimulate the protoplast regeneration.…”

Section: Resultsmentioning

confidence: 79%

“…For reversion of the protoplast to the bacillary form, several modifications of SA1.2 medium had been examined, including addition of autoclaved Bacillus sp. N16-5 cells [31], substitution of bovine serum albumin (BSA) by maize starch or skim milk [32], replacement of sodium succinate by sucrose [33] or mannitol [34], addition of 2.5% gelatin [34], removal of BSA, and lowering the incubation temperature from 37°C to 25°C. However, neither L colonies nor bacillary colonies arose when sucrose or mannitol was used as the osmotic stabilizer and BSA was substituted by skim milk.…”

Section: Resultsmentioning

confidence: 99%

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Protoplast Transformation of Recalcitrant Alkaliphilic Bacillus sp. with Methylated Plasmid DNA and a Developed Hard Agar Regeneration Medium

Gao

Xue

2011

PLoS ONE

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Among the diverse alkaliphilic Bacillus strains, only a little have been reported to be genetically transformed. In this study, an efficient protoplast transformation procedure was developed for recalcitrant alkaliphilic Bacillus sp. N16-5. The procedure involved polyethylene glycol-induced DNA uptake by the protoplasts and subsequent protoplast regeneration with a developed hard agar regeneration medium. An in vivo methylation strategy was introduced to methylate the exogenous plasmid DNA for improving the transformation efficiency. The transformation efficiency reached to 1.1×105 transformants per µg plasmid DNA with methylated plasmid pHCMC04 and the developed hard agar regeneration medium. This procedure might also be applicable to the genetic transformation of other Bacillus strains.

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Section: Resultsmentioning

confidence: 79%

Section: Resultsmentioning

confidence: 99%

Protoplast Transformation of Recalcitrant Alkaliphilic Bacillus sp. with Methylated Plasmid DNA and a Developed Hard Agar Regeneration Medium

Gao

Xue

2011

PLoS ONE

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“…In earlier publications, it was reported that reversion occurred efficiently when the protoplasts were immobilized in special solid media, namely, 25% gelatin medium at 26°C and 2 or 2.5% agar (4,13,16,17). Reversion was stimulated by membrane filters and by various autoclaved bacteria and yeast (3). It was concluded that a physically solid surface was an important factor in the induction of reversion.…”

Section: Resultsmentioning

confidence: 99%

Inhibitory protein controls the reversion of protoplasts and L forms of Bacillus subtilis to the walled state

DeCastro-Costa¹,

Landman²

1977

J Bacteriol

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When the cell wall of Bacillus subtilis is removed by lysozyme and the resultant protoplasts are plated on hypertonic soft agar medium, each protoplast forms an L colony. L bodies from such L colonies again plate as L-colony-forming units (CFU). However, if protoplasts or L bodies are "conditioned" by 1 h of incubation in 0.4% casein hydrolysate medium and then incubated in 25% gelatin medium for 1 h, 60 to 100% of the formerly naked cells give rise to bacillary colonies. The present experiments largely explain the mechanism responsible for the "heritable" persistence of the wall-less state in B. subtilis. It is shown that protoplasts produce a reversion inhibitory factor (RIF) which blocks reversion when the cell concentration exceeds 5 x 105 CFU/ml. This inhibitor is nondialyzable and sensitive to trypsin, heat, and detergent. Efficient reversion at 2 x 107 CFU/ml is obtained if the protoplasts are treated with trypsin after conditioning and chloramphenicol is incorporated into the gelatin reversion medium. In the presence of 500 ,ug of trypsin per ml, the requirement for gelatin is sharply reduced, and reversion occurs rapidly in liquid medium containing only 10% gelatin. Trypsin also stimulates reversion in L colonies growing on soft agar. Latent RIF is activated by ,3-mercaptoethanol. This reagent blocks reversion of protoplast suspensions at densities of 5 x 105 CFU/ ml. Comparison of the autolytic behavior ofB. subtilis and of the RIF revealed that several of the properties ofthe two activities coincide: both are inhibited by high concentrations of gelatin, both are activated by (3-mercaptoethanol, and both have high affinity for cell wall. Going on the assumption that REF is autolysin, models for protoplast reversion and L-form stability are proposed. A role of teichoic acid in reversion is suggested by the finding that mutants with altered teichoic acid show altered reversion behavior.When the cell wall is removed from Bacillus subtilis with lysozyme and the resultant protoplasts are plated on hypertonic soft-agar medium, each protoplast gives rise to an L colony consisting of wall-less, irregularly shaped L bodies (16,24). L bodies from suspended L colonies again give rise to L colonies in further platings (16). Thus, a brief transient enzyme treatment produces an alteration in each member of the treated population-namely, walllessness-which is then passed on "heritably" through an indefinite number of cell generations. We have called these naked offspring "mass conversion stable L forms" (13) to underline the distinction between these L forms and certain kinds of gene mutants blocked in peptidoglycan or peptidoglycan precursor biosynthesis, which are also sometimes called stable L forms (see, for instance, 28). We are quite certain that the persistent change induced in mass Present address:

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“…The following media were previously described: SS medium, a solid 0.5 M succinate-containing medium for the efficient growth of L colonies by the spread plate method, was modified from an earlier version (9) by substituting Baltimore Biological Laboratories gelatin for purified pigskin gelatin. Osmotically stabilized enriched (liquid) minimal medium (SP3S) was the same as described previously (6), except that the only amino acid supplement added was L-tryptophan.…”

Section: Methodsmentioning

confidence: 99%

Interaction of protoplasts, L forms, and bacilli of Bacillus subtilis with 12 strains of bacteriophage

Jacobson¹,

Landman²

1975

J Bacteriol

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The interaction of 12 phage strains with bacilli, protoplasts, and L forms of Bacillus subtilis 168 and with eight of its mutants and two of its lysogens is described qualitatively and quantitatively. After removal of the cell wall from B. subtilis 168, 11 of the 12 phage strains can still adsorb to the protoplasts, nine kill their wall-less host cells, and five multiply in the naked bacteria, forming plaques on L form lawns. Individual gene mutations can have similarly pleiotropic effects, strongly dependent upon the plating medium. Thus, the gta A mutation, which causes loss of glucosylation of the wall teichoic acid, results in loss of wall adsorption sites for 029 (but not membrane sites) and for X105. Phages X25, SP82G and oe can still adsorb to gta A bacilli and plaque in unstabilized and sorbitol-stabilized lawns of this mutant, but they can not plaque in sucrosestabilized lawns. The lysogenized wild type, B. subtilis 168 (SP02), also exhibits a pleiotropic pattern, showing different levels of resistance to phages SP02, X1, 4e, and 025. Its resistance pattern is very similar to that of wild-type protoplasts. On the basis of such patterns, the bacterial mutants and strain B. subtilis 168 strain 4)1

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Reversion of Bacillus subtilis Protoplasts to the Bacillary Form Induced by Exogenous Cell Wall, Bacteria and by Growth in Membrane Filters

Cited by 24 publications

References 14 publications

Protoplast Transformation of Recalcitrant Alkaliphilic Bacillus sp. with Methylated Plasmid DNA and a Developed Hard Agar Regeneration Medium

Protoplast Transformation of Recalcitrant Alkaliphilic Bacillus sp. with Methylated Plasmid DNA and a Developed Hard Agar Regeneration Medium

Inhibitory protein controls the reversion of protoplasts and L forms of Bacillus subtilis to the walled state

Interaction of protoplasts, L forms, and bacilli of Bacillus subtilis with 12 strains of bacteriophage

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