Effects of magnesium, calcium, and serum on reversion of stable L-forms.

The L-form of Agromyces ramosus was stable in the absence of penicillin when transferred on heart infusion agar containing NaCl and serum. It reverted to its bacterial form, however, when magnesium replaced the serum in this medium. On a dilute medium containing NaCl but lacking serum, the L-form died out unless calcium, magnesium, or serum was added. It grew as the L-form in the presence of calcium of serum but reverted to the bacterial form in the presence of magnesium. Reversion also occurred when magnesium was added to the dilute medium containing serum. Calcium interfered with or prevented the magnesium-induced reversion. The revertant bacterial form resulting from these studies was not NaCl sensitive, as was the case of the bacterial revertant of this organism produced in soil (A. H. Horwitz and L. E. Casida, Jr., Can. J. Microbiol, 24:50--55, 1978).     

Effect of the composition of reversion medium on change of Staphylococcus aureus lysostaphin protoplasts to coccal forms and L-forms

The experimental conditions under which protoplasts of Staphylococcus aureus strain MS353 (pCp) are converted to the coccal or L-form were investigated. Protoplasts prepared by treating coccal MS353 (pCp) strain with Lysostaphin formed various types of colonies (coccal form, L-form and mixed types) in about 50% yield when they were plated on reversion (R) medium consisting of 2% brain heart infusion, 0.5M sodium succinate, 0.01% bovine serum albumin, 20 mM MgCl2 and 0.6% agar. The L-form type colonies with a typical fried-egg appearance that developed on the R medium at an early stage gradually reverted to the coccal form through a mixed type stage in which a high density area first appeared in the periphery of the colony and then spread throughout the colony. The use of modified R medium without MgCl2 or R medium in which 0.5M sodium succinate as an osmotic stabilizer was replaced by 7.5% NaCl resulted in marked delay in the appearance of reverted cells. R medium without bovine serum albumin yielded atypical L-form type colonies, which contained masses of coccal cells with very irregular margins. On the other hand, R medium without MgCl2 but with penicillin G supported development of L-form type colonies at high rate (13-15%) from the inoculated protoplasts.     

Induction and cultivation of a stable L-form of Bacillus subtilis

The induction of L-forms of Bacillus subtilis from protoplasts is described. The method involved the frequent subculture of the unstable L-form on a growth medium supplemented with lysozyme and horse serum. A stable culture, which did not revert when lysozyme and horse serum were omitted from the medium, was obtained after 13 subcultures. This culture could be grown on solid and in liquid medium by routine microbiological methods. Long-term storage of these cells was achieved by freeze drying and maintenance in glycerol at −70°C. The cultural adaptability of the L-form is described and discussed with respect to methods of cultivation and growth.     

Induction and cultivation of a stable L-form of Bacillus subtilis

The induction of L-forms of Bacillus subtilis from protoplasts is described. The method involved the frequent subculture of the unstable L-form on a growth medium supplemented with lysozyme and horse serum. A stable culture, which did not revert when lysozyme and horse serum were omitted from the medium, was obtained after 13 subcultures. This culture could be grown on solid and in liquid medium by routine microbiological methods. Long-term storage of these cells was achieved by freeze drying and maintenance in glycerol at -70 degrees C. The cultural adaptability of the L-form is described and discussed with respect to methods of cultivation and growth.     

Morphology, growth and reversion in a stable L-form of Escherichia coli K12

An L-form isolated from Escherichia coli K12 by sequential treatment with N-methyl-N'-nitro-N-nitrosoguanidine and lysozyme was adapted to grow in hyperosmolar liquid cultures. It was stable in the absence of antibiotic when cultured in brain heart infusion (BHI) broth containing NaCl and CaCl2, the optimal concentrations being 0.34 M and 1 mM, respectively. No growth of the L-form was observed when CaCl2 was not added to BHI medium containing 0.34 M-NaCl. On the other hand, when KCl replaced NaCl as the osmotic stabilizer, growth of the L-form was repressed in the presence of CaCl2. Electron microscopy of the L-form confirmed the absence of a cell wall. A revertant strain derived from the L-form grew as a stable bacillary form in BHI medium without osmotic stabilizer. The growth characteristics of the revertant strain resembled those of the parent strain. The revertant strain produced L-forms in the presence of NaCl.     

Adaptation of a stable L-form of Bacillus subtilis to minimal salts medium without osmotic stabilizers.

An L-form isolated from Bacillus subtilis 168 was adapted to growth in a 340 mOsm minimal salts medium without the addition of osmotically protective solutes. This L-form had no chemically detectable peptidoglycan residues on its surface, but 0.8% of the dry weight of washed membranes was hexosamine. The osmotic stability and susceptibility to bacitracin and vancomycin of the L-form adapted to growth in 340 mOsm osmotically unprotected medium was twice that of the L-form grown in 2,680 mOsm medium supplemented with 1.2 M NaCl.     

Induction of L-Phase Variant from Protoplast of Staphylococcus aureus

Protoplasts of Staphylococcus aureus 209P and Cowan 1 were induced by treatment with lysostaphin. These protoplasts were sensitive to detergent, a low concentration of sodium chloride and low temperature. Almost all protoplast cells spread on CLYS agar medium (casein hydrolysate, yeast extract, Na-lactate, and NaCl) formed typical L-form colonies. Horse serum (0.25%) and Mg²⁺ (109 mm) are essential factors for formation of the L-form colonies of 209P. In the case of Cowan 1, Mg²⁺ was not required. The active factor(s) in horse serum was heat-resistant and protein in nature.     

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.     

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.     

An L-Form of Staphylococcus aureus Adapted to a Brain Heart Infusion Medium without Osmotic Stabilizers

An L-form derived from halotolerant Staphylococcus aureus Tasaki was adapted to growth in a brain heart infusion medium without any supplemental osmotically protective solutes (360 mOsm/kg). This L-form had no chemically detectable peptidoglycan residues on its surface. Electron microscopic observations confirmed morphologically the absence of the structures and also of other osmotically protective polymers within or exterior to the cytoplasmic membrane. The osmotic stability and susceptibility to bacitracin, d-cycloserine, and vancomycin of the L-form adapted to growth in 360 mOsm osmotically unprotective medium was higher than that of the L-form grown in 1,950 mOsm supplemented with 4.5% NaCl. The adapted L-form tended to be more sensitive to almost all of the antibiotics examined, other than the inhibitors for cell wall-synthesis, than the original L-form strain requiring osmotic protection for growth. Chemical analysis of the membrane of the adapted L-form indicated 16.3% total lipids and 20.6% proteins by dry weight of the membrane, and it contained larger amounts of lipid phosphorus (20.0 μ/mg).     

假结核耶氏菌在实验条件下细菌L型的观察

细菌L型广泛分布在生物体内外,并为细菌生命周期的一相。本文应用假结核耶氏菌(Yersinia pseudotuberculosis)作为研究材料,在L型培养基中无抗生素存在情况下连续传代,观察到:正常型(杆状)假结核菌出现圆球体、丝状体,继续传代又返祖为正常型。实验表明,细菌L型是细菌生命周期中的一相。     

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。     

Exhibition of persistent and drug-tolerant L-form habit of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> during infection in rats

A model for studying mycobacterial L-form formation in vivo was established to demonstrate the ability of M. tuberculosis to behave as a drug-tolerant L-form persister. Rats were infected by intranasal (i.n.) and intraperitoneal (i.p.) routes with 1×108 cells/ml of M. tuberculosis. At weekly intervals during a period of five weeks, samples from lung, spleen, liver, kidney, mesenterial and inguinal lymph nodes, broncho-alveolar and peritoneal lavage liquid were plated simultaneously on Löwenstein-Jensen (LJ) medium or inoculated into specially supplemented for L-forms Dubos broth (drug-free and drug-containing variants). The use of liquid media enabled isolation of mycobacterial L-form cultures during the whole period of experiment including the last two weeks, when tubercle bacilli were not isolated on LJ medium. An unique feature of mycobacterial L-forms was their ability to grow faster than the classical tubercle bacilli. Isolation and growth of L-form cultures in primary drug-containing media demonstrated their drug-tolerant properties. Electron microscopy of liquid media isolates showed that they consisted of morphologically heterogenous populations of membrane-bound and of variable sized L-bodies that completely lack cell walls. The identity of the isolated non-acid fast and morphologically modified L-forms as M. tuberculosis was verified by specific spoligotyping test. The results contribute to special aspects concerning the importance of mycobacterial L-form phenomenon for persistence and latency in tuberculosis, phenotypic drug tolerance, as well as for diagnosis of difficult to identify morphologically changed tubercle bacilli which are often mistaken for contaminants.     

Transformation of a Bacillus subtilis L-form with bacteriophage deoxyribonucleic acid.

A stable L-form, sal-1, of Bacillus subtilis was transformed with deoxyribonucleic acid (DNA) from bacteriophages phi 25 and phi 29 to determine whether exogenous DNA can be introduced into this organism. The viral transformation (transfection) was successful with the use of polyethylene glycol. In the presence of the fusogen, bacteriophage phi 25 DNA initiated a single cycle of infection. When compared with transfection of competent cells of Bacillus subtilis, the appearance of viral particles was delayed and their production occurred over a longer time period. L-form cells were best able to support intracellular replication of phi 25 viral particles when in balanced growth in a rich medium. The addition of polyethylene glycol also induced infection of sal-1 with whole bacteriophage phi 25 particles which could not otherwise infect the L-form and enhanced infection by intact phi 29 particles. Primary recombination was shown to be required for polyethylene glycol-mediated phi 25 transfection, but not phi 29 transfection or for whole bacteriophage phi 25 infection mediated by polyethylene glycol. Successful transfection of sal-1 suggests that the L-form may be amenable to genetic modification with exogenous DNA.     

Molecular detection and beta-glucuronidase expression of gus-marked Bacillus subtilis L-form bacteria in developing Chinese cabbage seedlings

AIM: To detect L-form bacteria in developing Chinese cabbage seedlings. METHODS AND RESULTS: Stable Bacillus subtilis L-forms were genetically modified to express the gus gene (encoding beta-glucuronidase). Germinated seeds of Chinese cabbage were soaked in mannitol based suspensions of the L-form bacteria or with mannitol alone and after washing were grown in aseptic conditions on plant growth medium. Histochemical staining of beta-glucuronidase activity (X-gluc) and Polymerase Chain Reaction (PCR) detection of the gus gene were achieved in the L-form associated seedlings. beta-Glucuronidase was localized in discrete spots, mainly in the roots with staining, and was also observed in the cotyledons and base of stems. Correlation was observed between PCR detection of the gus gene and histochemical staining with detection in similar tissues. Stable L-form bacteria were non-culturable after their association with plant material. CONCLUSIONS: The gus reporter gene system with its associated histological staining for enzyme activity was used successfully for detecting B. subtilis L-form bacteria in plant material. SIGNIFICANCE AND IMPACT OF THE STUDY: These molecular marked L-forms should provide a specific and sensitive technique for detecting L-form bacteria in planta and offer a method for further understanding the L-form/plant association.     

Listeria monocytogenes L Forms I. Induction, Maintenance, and Biological Characteristics

L forms were induced from 15 of 16 strains of Listeria monocytogenes on penicillin gradient plates incubated under aerobic conditions. The culture medium for maintenance of these L forms must contain an electrolyte in a concentration of 1% or sucrose in a concentration of 10%. The electrolytes NaCl, KCl, or MgSO(4) were used in both induction and maintenance media. Induction of L forms occurred more rapidly on media containing KCl. Listeria L forms had the same fermentation reactions as the parent bacterium. The L-form growth in liquid medium was slow, not extensive, and appeared as clumps on the bottom of culture tubes. The morphology of Listeria L forms was similar to that reported for other bacterial L forms. The L forms derived from strain 10403, serotype 1, were stable after two or more passages on penicillin media. They did not revert to the bacterial form after 40 subcultures on penicillin-free media. Some L-form colonies derived from strain 10403 did revert to the bacterial form when transferred directly from induction plates to penicillin-free media. Studies of the growth characteristics for L forms derived from strain 10403 gave the following results: an optimal temperature of 30 C, high electrolyte or sucrose concentration necessary for induction and maintenance, and no requirement for serum.     

Cell fusion between L-forms and protoplasts of Staphylococcus aureus

Mixtures of various combinations of Lysostaphin protoplasts and stable L-forms of Staphylococcus aureus, which have different markers for drug resistance, were treated with polyethylene glycol (PEG) to examine the development of doubly resistant fusion products (fusants). To recover doubly resistant colonies as L-forms, they were incubated in 4.5% NaCl-brain heart infusion (BHI) broth containing penicillin G (PCG) for enrichment culture and cultured in PCG-4.5% NaCl-BHI agar medium (method 1), while to recover doubly resistant fusants as L-forms and coccal forms, they were grown on reversion medium (R medium) which causes reversion of protoplasts or fusants to parent type cells, and then cultured on assay media, i.e., R medium, BHI agar medium or PCG-4.5% NaCl-BHI agar medium (method 2). Under both experimental conditions, doubly resistant fusants developed as L-form cells by PEG treatment of pairs of protoplasts carrying the chloramphenicol (CP)-resistance plasmid and L-forms having chromosomal resistance to streptomycin (SM). In the reverse combinations, i.e., protoplasts showing chromosomal SM-resistance and L-form cells carrying the CP-resistance plasmid, the first method gave no doubly resistant colonies. By the second method, without enrichment culture on R medium, the latter combination gave doubly resistant fusants as L-form, coccal-type and mixed-type colonial forms, while when the PEG-treated mixture was enriched on R medium, fusants were obtained exclusively as the coccal type on either R medium or BHI agar assay medium. Neither of the methods yielded colonies of doubly resistant fusants on PEG-treatment of pairs of protoplasts and L-forms both of which were chromosomal, but with different drug resistances. These results show that PEG-induced cell fusion between protoplasts and L-forms of S. aureus, unlike the fusion between protoplasts or between L-forms, resulted in transfer of the drug resistance controlled by the plasmid to the fusion products. The fusants obtained were L-forms in method 1, and coccal type in the method 2.     

Use of transformation for studying the nature of formation of stable bacterial L forms]

The authors obtained a stable variant of the L-forms of Bacillus subtilis capable of exponential growth of the minimal and synthetic medium. An electron-microscopic study of different stages of the L-form formation was carried out by the method of ultra-thin sections. A possibility was shown of the transfer of the L-form formation sign by the method of transformation. DNA isolation from the L-forms by soft lysis considerably facilitated and simplified the genetic analysis of the L-form formation by the transformation method.     

Intracellular production of Brucella L forms . II. Induction and survival of Brucella abortus L forms in tissue culture

Hatten, Betty A. (The University of Texas Southwestern Medical School, Dallas), and S. Edward Sulkin. Intracellular production of Brucella L forms. II. Induction and survival of Brucella abortus L forms in tissue culture. J. Bacteriol. 91:14-20. 1966.-Intracellular survival of altered brucellae, possibly L forms, was not greatly affected by penicillin or streptomycin in concentrations ranging from 5.0 to 40 mug/ml, but a combination of these two antibiotics (2.5 to 20 mug/ml each) reduced the number of positive L-form cultures. Tetracycline (2.0 mug/ml) decreased the number of positive L-form cultures at about the same rate as combinations of the higher concentrations of penicillin and streptomycin. Various concentrations of tetracycline (0.1 to 2.0 mug/ml) with 5.0 mug/ml of penicillin or streptomycin significantly reduced the number of positive L-form cultures. L forms were recovered for several days after elimination of bacteria from the cultures by all of the antibiotics tested. L-form production was not dependent upon the presence of antibiotics in the culture medium, but they were recovered in greater numbers when bacteria were still present in the hamster kidney cells. Addition of thallium acetate to infected cells (at varying intervals of time after infection) to control bacterial growth and conversion to the L phase during cellular disintegration decreased the number of positive L-form cultures obtained over a 10-day period. Comparison of the antibiotic sensitivity of bacteria recovered from infected tissue culture cells with the stock strain of Brucella abortus indicated that some resistance to penicillin and tetracycline had developed. A marked resistance to streptomycin was observed in those bacteria recovered from cells maintained in the presence of this antibiotic.     

The symbiosis of Bacillus subtilis L-forms with Chinese cabbage seedlings inhibits conidial germination of Botrytis cinerea

AIMS: To establish whether germination of Botrytis cinerea was affected by the symbiosis of Bacillus subtilis L-form bacteria with Chinese cabbage. METHODS AND RESULTS: Germinating seeds of Chinese cabbage were co-cultivated with either L-forms of Bacillus subtilis or 5% (w/v) mannitol by soaking for 3 h. Seeds were then washed in sterile water, sown on a minimal medium and incubated in controlled conditions. L-form symbiosis was detected over a time course by ELISA. Conidial germination of Botrytis cinerea was significantly reduced on cotyledonous leaves of L-form-treated plants compared with controls. CONCLUSIONS: Symbiosis of B. subtilis L-form bacteria during seed germination of Chinese cabbage inhibits conidial germination in plants on subsequent exposure to Botrytis cinerea. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first account of plant symbiosis with L-form bacteria showing antagonism to a fungal plant pathogen. This has promising implications for the use of this L-form as a biocontrol agent.