Spheroplast formation of Mycobacterium smegmatis and morphological aspects of their reversion to the bacillary form.

Cell wall-deficient forms (spheroplasts) of Mycobacterium smegmatis strain P53 were prepared by combined treatment with glycine, lysozyme, and lytic enzyme no. 2 as the spheroplasting agents. Quantitative mass conversion to spherical forms was effected by pretreatment of the intact cells with 1.2% glycine in nutrient broth, followed by transfer to spheroplasting medium containing the above agents. Two apparent modes of reversion to the bacillary form were observed under electron microscopy. The first one was initiated by budding from the spheroplasts. The buds gradually elongated to become the mycelial form, which showed branching, septation, and fragmentation. The second resulted from the intracellular formation of tiny cells, possibly the elementary bodies, and their release from the spheroplasts.     

An improved method for the preparation of mycobacterial spheroplasts and the mechanism involved in the reversion to bacillary form: electron microscopic and physiological study

An efficient method is described for preparing spheroplasts and protoplasts by treating bacillary cells of Mycobacterium smegmatis with precise concentrations of L-glycine (followed by lysozyme). This improved procedure was widely applicable to many rapidly growing mycobacteria by selecting the concentrations of glycine suitable for the individual strains used. The process of reversion of spheroplasts to original bacillary form on solid and in liquid media, as revealed by electron microscopy, appeared to involve the formation of an internal elementary or initial body with subsequent budding from the spheroplast. The internal membrane systems appeared to function in the induction of initial bodies and in the maturation of elementary bodies to become dividing forms. Possible mechanisms involved in the development of bacilli from spheroplasts are discussed.     

Morphological study of the reversion to bacillary form, of Bacillus megaterium protoplasts.

Protoplasts of Bacillus megaterium readily reverted to bacillary form in liquid media and when plated in a soft-agar layer onto the surface of appropriate agar media. Three phases of the reversion sequence could be differentiated by phase contrast microscopy: (i) increase in size of the individual protoplasts, (ii) non oriented division of the protoplasts and (iii) outgrowth of the bacillary forms. With time-lapse photomicrography, reversion sequences of single protoplasts were demonstrated.     

Stable L-forms of Clostridium perfringens and their growth on glass surfaces.

L-forms of Clostridium perfringens were induced in brain heart infusion broth containing 10% sucrose and 2 units of penicillin. After a few hours of growth, spheroplasts, granules, and elongated bacilli were apparent. At 24-h intervals, serial subcultures were made in the above medium which resulted in a culture composed entirely of spheroplasts (or protoplasts) and granules. Upon the withdrawal of penicillin these L-form cultures grew well and, after 100 passages, there was no reversion to the bacillary form. Sucrose could also be withdrawn from the medium. The effects of centrifugation, osmotic stabilizer, ultraviolet light, temperature, pH, and lyophilization upon stable L-forms were examined. L-forms were found to attach to the walls of culture tubes during trowth and sheets of L-form growth were obtained on cover slips in Leighton tubes and on the sides of medicine bottles.     

Observations on cell-wall deficient forms of Pseudomonas maltophilia

Pseudomonas maltophilia cell-wall deficient forms were induced using a medium containing carbenicillin and polyvinyl pyrrolidone. Scanning electron microscopy proved to be a very effective method of demonstrating the various phases. It is suggested that the rapid rate of reversion to bacterial forms could have been due to plasmids.     

Properties of rod cells reversed from penicillin spheroplasts in Escherichia coli

Hirokawa, Hideo (The University of Tokyo, Tokyo, Japan). Properties of rod cells reversed from penicillin spheroplasts in Escherichia coli. J. Bacteriol. 91:125-128. 1966.-Spheroplasts of Escherichia coli B-054 were formed by penicillin, and the properties of rod cells reversed from the spheroplasts were examined. It was found that the rod cells were not capable of forming colonies when placed in a minimal medium immediately after the reversion, the postaddition of the nutrient to this medium being without effect. In this respect, the so-called residual cells which did not convert to spheroplasts by penicillin treatment behaved just as the reversed rod cells. These two types of cells, the reversed and residual rod cells, however, could grow up to form colonies when they were cultivated in a complex medium immediately after the reversion. The nutrient requirement and penicillin sensitivity of the progeny of these cells were found to be quite similar to those of the original untreated cells. Accordingly, it was concluded that the nutritional defect induced by penicillin treatment was not a hereditary change.     

Protoplast formation and regeneration of a thermophilic Clostridium sp.

Abstract Protoplasts of a thermophilic Clostridium sp. were prepared by lysozyme treatment using lactose as osmotic stabilizer. High frequency reversion (3–29.8%) to the bacillary form was obtained on hypertonic rich medium.     

Gelatin-induced Reversion of Protoplasts of Bacillus subtilis to the Bacillary Form: Electron-microscopic and Physical Study

Protoplasts of Bacillus subtilis plated on SD medium form L colonies in quantitative yield and propagate in the L form indefinitely. L bodies or protoplasts placed in 25% gelatin medium form bacillary colonies. Details of the reversion of naked bodies to the walled form are reported. In 25% gelatin medium, reversion begins earlier (about 50% reversion in 4 hr) than the multiplication of bacilli. Thus, virtually all the observed bacillary forms are themselves revertants and not the offspring of a few growing clones. The optimal temperature for reversion is 26 C in 25% gelatin. When cells reverting at 26 C are warmed to 40 C for 3 min, reversion is delayed markedly, whereas viability is unaffected. For electron microscopy, a dense protoplast inoculum was placed on a gelatin surface, incubated, and then fixed in situ. There was no multiplication, but crowding delayed reversion markedly. Successive events of reversion are as follows. The loose nucleoid of the protoplasts condenses in response to the gelatin medium and condenses further and further as reversion proceeds. A thin coat of wall develops around the bodies of various sizes and shapes and then increases uniformly in thickness until a wall of normal aspect is formed. Rod-shaped cells grow out from these bodies-sometimes in several directions at once. A few mesosomes begin to appear only after a thin coat of wall has been formed. These are dense, atypical structures compartmented by membranes. They are located at the cell periphery and do not seem to be in contact with the nucleoids. Quantitative estimates showed that only 20 to 25% of revertant cells or cells grown on gelatin contain even a single mesosome. The others have no mesosome at all. Mesosomes thus do not appear to play a significant role in reversion, and normal mesosome functions must presumably be performed elsewhere in the cell in gelatin-grown bacilli. The role of cell wall, its synthesis, and its chemical nature in successive steps in reversion are discussed.     

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

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 rist 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 mug 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 beta-mercaptoethanol. This reagent blocks reversion of protoplast suspensions at densities of 5 x 105 CFU/ml. Comparison of the autolytic behavior of B. subtilis and of the RIF revealed that several or the properties of the two activities coincide: both are inhibited by high concentrations of gelatin, both are activated by beta-mercaptoethanol, and both have high affinity for cell wall. Going on the assumption that RIF is autolysin, models for protoplast reversion is suggested by the finding that mutants with altered teichoic acid show altered reversion behavior.     

Die Bildung und die Reversion von Sphäroplasten einer Diaminopimelinsäure-auxotrophen Mutante von Escherichia coli

1. The formation and reversion of spheroplasts of the diaminopimelic acid-auxotrophic mutant Escherichia coli K 12, 335, dap ⁻, R⁺TEM in a medium lacking diaminopimelic acid have been investigated by microphotography: During their development from rod form cells to spheroplasts cells on slide-surface-agar preparations underwent two successive cell divisions in the course of which the cells retained their rod form. The cells formed by these divisions partitioned into a varying number of spheroplasts of different size. The reversion of spheroplasts to rod form cells, started by the addition of diaminopimelic acid showed two characteristic steps: Each spheroplast partitioned again into several spheroplast-like cell bodies; most of them reverted directly to rod form cells.
2. The release of the R-factor mediated periplasmic TEM-β-lactamase, E. C. 3.4.2.6., into the growth medium during the development of spheroplasts attained more than 50% of the entire TEM-β-lactamase activity.

The spheroplasts showed a multiple enhancement of TEM-β-lactamase activity per mg cell protein compared with rod form cells.     

Susceptibility to Polymyxin B of Penicillin G-induced Proteus mirabilis L Forms and Spheroplasts

A polymyxin B-resistant strain of Proteus mirabilis was converted into L forms and spheroplasts in the presence of penicillin G. This treatment caused a 400-fold increase in polymyxin B susceptibility. The acquired susceptibility was in the range of the natural susceptibility reported for susceptible gram-negative bacteria ( approximately 1 mug/ml). The high susceptibility to polymyxin B was lost as soon as the spheroplasts and L forms were allowed to reconvert into the bacillary form in penicillin-free media. This behavior is strong evidence that the natural resistance of Proteus strains to polymyxins is due to the impermeability of the outer cell wall structures to these antibiotic substances.     

Formation of cell wall polymers by reverting protoplasts of Bacillus licheniformis.

The biosynthesis of peptidoglycan and teichoic acid by reverting protoplasts of Bacillus licheniformis 6346 His-, in cubated at 35 C on medium containing 2.5% agar, is detectable after 40 min. The amount of N-acetyl-[1-14C]glucosamine incorporated into peptidoglycan and teichoic acid on continued incubation doubles at the same rate as the incorporation of [3H]tryptophan into protein. At the early stages of reversion the average glycan chain length, measured by the ratio of free reducing groups of muramic acid and glucosamine to total muramic acid present, is very short. As reversion proceeds, the average chain length increases to a value similar to the found in the wall of the parent bacillus. The extent of cross-linkage found in the peptide side chains of the peptidoglycan also increases as reversion proceeds. At the completion of reversion the wall material synthesized has similar characteristics to those of the walls of the parent bacilli, containing peptidoglycan and teichoic and teichuronic acids in about the same proportions. Soluble peptidoglycan can be isolated from the reversion medium, amounting to 30% of the total formed after 3 h of incubation and 8% after 12 h. This amount was reduced by the presence in the medium of the walls of an autolysin-deficient mutant; they were not formed at all by reverting protoplasts of the autolysin-deficient mutant itself. Analysis of the soluble material provided additional evidence for their being autolytic products rather than small unchanged molecules. When protoplasts were incubated on medium containing only 0.8% agar, 53 to 67% of the peptidoglycan formed after 3 h of incubation was soluble, and 21% after 12 h. Fibers that appeared to be sheared from the protoplasts at intermediate stages of reversion on medium containing 2.5% agar were similar in composition to the bacillary walls.     

Reversion of Bacillus megaterium protoplasts to the bacillary form.

Photomicrographic evidence of reversion of Bacillus megaterium protoplasts to the bacillary form on soft agar plates hypertonic medium is demonstrated.     

A novel method for regenerating the protoplasts of thermophilic bacilli

A range of Bacillus thermophiles was tested for the ability to be converted to protoplasts with lysozyme and subsequently, to regenerate to bacillary form. Protoplast formation was straightforward but many of the strains failed to regenerate on commonly-used media. Two medium components were found to be causing the inhibition. Growth of protoplasts as L-forms only occurred if the medium lacked phosphate. However, reversion of L-forms to bacilli was asynchronous and infrequent. Regeneration of cell walls by protoplasts/L-forms to re-establish bacillary form was greatly improved when the medium was gelled with pluronic polyol F127 in place of agar or similar polysaccharides.     

Genetic recombination in fused spheroplasts of Providence alcalifaciens.

Spheroplasts of Providence alcalifaciens strain P29 auxotrophs were prepared by combined treatment with glycine and lysozyme-EDTA. About 15% of spheroplasts had areas of cytoplasmic membrane exposed where cell wall was absent. The spheroplasts of different auxotrophs were mixed pairwise and fusion was attempted with polyethylene glycol or nascent calcium phosphate. After spheroplasts had regenerated to bacterial forms selection was made for recombinants. Recombinants arose at frequencies of 3.8 X 10(-6) to 1.7 X 10(-7) per spheroplast initially present, by both methods of fusion. The frequency was strongly dependent on the number of chromosomal loci used in selection. The possible order of five loci was determined and this corresponded to that on the closely related Proteus mirabilis chromosome. Control experiments excluded possibilities of auxotrophic reversion, conjugation, transformation, transfection or transduction as explanations of the results. Analysis of prototrophic clones yielded stable prototrophs or mixtures of stable prototrophs and stable recombinants. Parental types were not encountered. Unselected markers segregated among recombinants. It was concluded that the formation of recombinant bacteria was due to spheroplast fusion and that only stable products of the very temporary heteroploid state were haploid recombinants. The low frequency of recombination was ascribed to the limited number of spheroplasts with areas of exposed cytoplasmic membrane.     

细菌L型的厌氧诱导和培养

厌氧条件下以羧卡青霉素诱导金黄色葡萄球菌、大肠杆菌和蜡样芽胞杆菌形成Ｌ型,观察细菌Ｌ型在厌氧条件下的形成、形态、生长及时渗透压的敏感性等特性。结果表明：蜡样芽胞杆菌在厌氧条件下不能形成Ｌ型或其Ｌ型在厌氧条件下亦不能返祖。金黄色葡萄球菌和大肠杆菌在厌氧条件下虽能诱生Ｌ型,但形成丝状体的构成Ｌ型菌落难以传代培养,厌氧培养未见Ｌ型圆球体和典型Ｌ型油煎蛋样菌落。金黄色葡萄球菌Ｌ型在含１％～１０％ＮａＣｌ的Ｌ型培养基上可生长形成Ｌ型菌落或非菌落形式存在的Ｌ型巨形体;大肠杆菌和蜡样芽胞杆菌的Ｌ型在含２％～６％ＮａＣｌ的Ｌ型培养基上可生长形成Ｌ型菌落或非菌落形式存在的Ｌ型巨形体。涂片染色或返祖试验证实细菌Ｌ型在含０．５％ＮａＣｌ的Ｌ型培养基或常规细菌学培养基上亦可生存。非菌落性Ｌ型巨形体和丝形体是细菌Ｌ型在琼脂培养基上广泛的存在形式。     

Gelatin-induced Reversion of Protoplasts of Bacillus subtilis to the Bacillary Form: Biosynthesis of Macromolecules and Wall During Successive Steps

Protoplasts of Bacillus subtilis plated on SDG medium formed L colonies in quantative yield and propagated in the L-form indefinitely. Protoplasts or L bodies placed in 25% gelatin medium formed bacillary colonies. Details of the reversion of these naked bodies to the walled form are reported here. Protoplasts prepared in minimal medium reverted fairly synchronously 3 to 4 hr after inoculation into gelatin, but protoplasts preincubated in casein hydrolysate (CH)-enriched minimal medium were primed to revert within 1 hr in the gelatin. Preincubation for 1.5 hr in 0.44% CH was required for good priming. Cells must be subjected to this preincubation (step 1) in the naked state; it is effective for L bodies as well as protoplasts. Priming was blocked by chloramphenicol, puromycin, and actinomycin D but was not affected by penicillin, lysozyme, or inhibition of deoxyribonucleic acid (DNA) synthesis. It is concluded that protein and ribonucleic acid (RNA) synthesis are required during step 1, that DNA synthesis is not required, and that wall mucopeptide is not made. The reversion of well-primed protoplasts in the gelatin (step 2) proceeded undisturbed in thymine-starved cells with chromosomes arrested at the terminus. It was scarcely slowed by chloramphenicol in the gelatin but was delayed about 3 hr by both puromycin and actinomycin D. Escape from inhibition occurred while the inhibitors were still actively blocking growth. Penicillin and cycloserine inhibited and lysozyme reversed reversion. Momentary melting of the gelatin delayed reversion. It is concluded that mucopeptide synthesis occurs in step 2, that concomitant RNA, DNA, or protein synthesis is not essential, but that physical immobilization of excreted cell products at the protoplast surface is necessary early in step 2. Newly reverted cells were misshapen and osmotically sensitive. Processes which confer osmotic stability after reversion (step 3) did not occur in the presence of chloramphenicol or actinomycin D.     

Facile autoplast generation and transformation in Bacillus thuringiensis subsp. kurstaki.

We describe a method for maximizing the rate of conversion of Bacillus thuringiensis subsp. kurstaki vegetative cells to osmotically fragile forms in the absence of exogenously added enzymes. Optimal generation of autoplasts occurred in 50 mM sodium acetate buffer (pH 7.0) at 37 degrees C with 10% (wt/vol) polyethylene glycol as an osmotic stabilizer. The maximum autolytic rate resulted in a conversion of greater than 90% of bacilli to spherical autoplasts in 6 min. Autoplasts regained bacillary morphology upon plating on DM3-G regeneration medium, with reversion frequencies ranging from 1.2 x 10(-1) to 5.3 x 10(-3). The autoplasts could efficiently take up exogenously added plasmid DNA. The presence of plasmids was verified by Southern hybridization analysis.     

Coccoid Helicobacter pylori Not Culturable In Vitro Reverts in Mice

An experimental rodent model was used to demonstrate the viability of the coccoid form of Helicobacter pylori. Concentrated suspensions were prepared for the two different morphologies: at 2 days incubation for the bacillary forms and at 20 days incubation for the “dormant” forms. The strains used for incubation were two fresh isolates from humans with duodenal ulceration, and two collection strains. Five hundred microliters of culture (OD₅₅₀ = 5 Mc Farland) of Helicobacter pylori with bacillary (2-5×10⁹ CFU/ml) and coccoid (0 CFU/ml) morphology were inoculated intragastrically in BALB/c mice. The gastric mucosa of the mice was colonized by Helicobacter pylori with the administration of fresh bacillary and coccoid cultures and not with the established cultures. Helicobacter pylori was isolated at 1 week after inoculation with the administration of fresh bacillary cultures, while fresh coccoid Helicobacter pylori was recovered in mice stomachs after 2 weeks of inoculation. After colonization, histopathologic changes occurred after 1 month from inoculation; all colonized mice showed a systemic antibody response to Helicobacter pylori. These results support the thesis of the viability of coccoid Helicobacter pylori non-culturable in vitro and confirm that concentrated bacterial suspensions are able to colonize and to produce gastric alterations in this suitable animal model.     

Characterization of a Stable L-Form of Bacillus subtilis 168

A stable L-form of Bacillus subtilis 168 (sal-1) has been isolated which grows and divides logarithmically in liquid medium with a generation time of 60 min. This mutant does not synthesize cell wall as evidenced by chemical, biochemical, and morphological analyses. Antibiotics which specifically inhibit cell wall biosynthesis do not affect the growth of the L-form. Significant differences exist between the membrane proteins of the bacillary form and the L-form. The relative profile of membrane proteins varies with the salt concentration of the medium in both the L-form and the bacillary form.