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.     

Selective association of the chromosome with membrane in a stable L-form of Bacillus subtilis.

A stalbe L-form (Sal-1) of Bacillus subtilis was found to have retained a markedly modified chromosome-membrane association when compared to intact cells. The membrane-deoxyribonucleic acid complex of the L-form was similar to that of its parental strain in quantity and stability. Genetic analysis of the L-form membrane-deoxyribonucleic acid complex revealed enrichment for markers close to the replication origin, but not for internal markers, indicating preferential attachment of the origin of chromosomal replication to the membrane. These results are in close agreement with those found for the parental bacterial form. In contrast, the replication termius region was not preferentially attached to the membrane of the L-form, even though it is enriched in the bacterial form. The association of the chromosome with the membrane at the replication terminus does not appear to be necessary for cell growth and separation, but because the L-form divides aberrantly, it may be one of the factors required for normal deoxyribonucleic acid segregation and septation.     

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.     

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.     

An investigation of enumeration and DNA partitioning in Bacillus subtilis L-form bacteria

R.N. WATERHOUSE, E.J. ALLAN, F. AMIJEE, V.J. UNDRILL AND L.A. GLOVER. 1994. Cell numbers of two morphogenic forms of Bacillus subtilis (the cell-walled parental and the derived stable cell wall-deficient L-form) have been compared by two methods: DNA hybridization (i.e. deduced genome numbers) and viable cell counts (i.e. number of colony-forming units (cfu)). The DNA hybridization method was shown to be a reliable and reproducible method for estimating genome numbers. Comparison of different L-form populations showed that the two methods of enumeration gave different values, with the deduced genome numbers much higher (by several orders of magnitude) than cell numbers deduced from viable cell counts. In contrast, when a culture of the cell-walled form was enumerated, the discrepancy between the two methods was low (by a factor of about 6) The combination of a high number of L-form genomes detected by DNA hybridization and a relatively low number of cfu was thought to be a consequence of a diminished co-ordination between the DNA replication and cell division processes in L-form bacteria. This suggestion was further substantiated by assessing the stability of plasmid pPL608 in a transformed B. subtilis L-form cell line, where even in the presence of continued kanamycin selection, 25% of the population lost kanamycin resistance. The results are discussed with particular reference to cell division in cell wall-deficient, stable L-form bacteria.     

Structurally stable Bacillus subtilis cloning vectors

Cloning of long DNA segments (greater than 5 kb) in Bacillus subtilis is often unsuccessful when naturally occurring small (less than 10 kb) plasmids are used as vectors. In this work we show that vectors derived from the large (26.5 kb) plasmids pAM beta 1 and pTB19 allow efficient cloning and stable maintenance of long DNA segments (up to 33 kb). The two large plasmids differ from the small ones in several ways. First, replication of the large plasmids does not lead to accumulation of detectable amounts of ss DNA, whereas the rolling-circle replication typical for small plasmids does. In addition, the replication regions of the two large plasmids share no sequence homology with the corresponding regions of the known small plasmids, which are highly conserved. Taken together, these observations suggest that the mode of replication of the large plasmids is different from that of small plasmids. Second, short repeated sequences recombine much less frequently when carried on large than on small plasmids. This indicates that large plasmids are structurally much more stable than small ones. We suggest that the high structural stability of large plasmids is a consequence of their mode of replication and that plasmids which do not replicate as rolling circles should be used whenever it is necessary to clone and maintain long DNA segments in any organism.     

Temperature-Sensitive Induction of Bacteriophage in Bacillus subtilis 168

In a temperature-sensitive mutant of Bacillus subtilis 168, induction of the defective phage PBSX occurred at 48 C. Cell lysis began after 90 min of growth at 48 C, and cell viability began to decrease after 10 to 30 min. The loss in viability at the nonpermissive temperature was prevented by azide or cyanide. Deoxyribonucleic acid (DNA), ribonucleic acid, and protein synthesis were not inhibited at 48 C. Temperature induction of the temperate phage SPO2 also occurred in this mutant. The temperature-sensitive mutation, designated tsi-23, was linked by transduction to purB6 and pig, the order being purB6 pig tsi-23. Mutation tsi-23 was transformable to wild type by B. subtilis 168 DNA but not by DNA from the closely related strains W23 or S31. DNA from the latter two strains transformed auxotrophic markers of strain 168 at frequencies close to those found with 168 donor DNA. Upon temperature induction, cellular DNA was broken to a size of 22S, characteristic of DNA in PBSX particles. The DNA isolated from temperature-induced PBSX did not give an increased Ade(+)/Met(+) transformant ratio relative to cellular DNA nor contain preferential break points as determined by transformation of four closely linked markers.     

Small stable RNA maturation and turnover in Bacillus subtilis

Stable RNA maturation is a key process in the generation of functional RNAs, and failure to correctly process these RNAs can lead to their elimination through quality control mechanisms. Studies of the maturation pathways of ribosomal RNA and transfer RNA in Bacillus subtilis showed they were radically different from Escherichia coli and led to the identification of new B. subtilis‐specific enzymes. We noticed that, despite their important roles in translation, a number of B. subtilis small stable RNAs still did not have characterised maturation pathways, notably the tmRNA, involved in ribosome rescue, and the RNase P RNA, involved in tRNA maturation. Here, we show that tmRNA is matured by RNase P and RNase Z at its 5′ and 3′ extremities, respectively, whereas the RNase P RNA is matured on its 3′ side by RNase Y. Recent evidence that several RNases are not essential in B. subtilis prompted us to revisit maturation of the scRNA, a component of the signal recognition particle involved in co‐translational insertion of specific proteins into the membrane. We show that RNase Y is also involved in 3′ processing of scRNA. Lastly, we identified some of the enzymes involved in the turnover of these three stable RNAs.     

Induction of cold shock proteins in Bacillus subtilis

The cold shock response in the Gram-positive soil bacterium Bacillus subtilis is described. Cells were exposed to sudden decreases in temperature from their optimal growth temperature of 37°C. The B. subtilis cells were cold shocked at 25°C, 20°C, 15°C, and 10°C. A total of 53 polypeptides were induced at the various cold shock temperatures and were revealed by two-dimensional gel electrophoresis. General stress proteins were identified by a comparative analysis with the heat shock response of B. subtilis. Some unique, prominent cold shock proteins such as the 115 kDa, 97 kDa, and 21 kDa polypeptides were microsequenced. Sequence comparison demonstrated that the 115-kDa protein had homology to the TCA cycle enzyme, aconitase.     

Induction of cell autolysis of Bacillus subtilis with lysophosphatidylcholine

Lysophosphatidylcholine (LPC) was found to cause autolysis of Bacillus subtilis 168 cells growing logarithmically at concentrations higher than 20 m, by inducing the activity of autolytic enzymes. The lytic activity depended upon the carbon-chain length of the acyl moiety in the LPC molecule, being most effective with palmitoyl LPC. Lysophosphatidylethanolamine also caused cell lysis but to a lesser extent, whereas lysophosphatidylglycerol did not. LPC stimulated cell autolysis in TRIS-KCl buffer and potassium phosphate buffer but was ineffective in distilled water. LPC had no influence on the activity in vitro of partially purified autolytic enzymes.Correspondence to: T. Tsuchido     

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.     

An ELISA for the detection of Bacillus subtilis L-form bacteria confirms their symbiosis in strawberry

AIMS: To develop an ELISA for the detection of antigens derived from stable Bacillus subtilis L-form bacteria and to detect these in plants injected with L-form bacteria. METHODS AND RESULTS: A sandwich ELISA was developed and its specificity was investigated using L-forms and cell-walled forms of B. subtilis, different Bacillus species and a range of bacteria isolated from glasshouse-grown strawberry plants. The detection limits of the ELISA were approximately 10(3) viable cells ml(-1) for L-forms compared with 10(7) viable cells ml(-1) for cell-walled forms. Results showed that L-forms survived and moved within strawberry tissues injected with L-form bacteria. CONCLUSION: An ELISA that selectively detects B. subtilis L-form bacteria was developed and shown to confirm the presence of L-forms in plants. SIGNIFICANCE AND IMPACT OF THE STUDY: This will be a valuable rapid method to further studies on L-form plant interactions.     

Induction and Propagation of a Bacillus subtilis L Form in Natural and Synthetic Media

The L form of Bacillus subtilis NRRL B-3275 was induced in a 7% NaCl broth medium and subsequently propagated in natural and synthetic media. The L form grew readily in tryptone broth supplemented with glucose, NaCl, and phosphate buffer, and in a synthetic medium containing only glucose and biotin, in addition to the required salts. Successive transfers from the bacillus inoculum and subsequent large bodies in the tryptone broth with 7% NaCl resulted in gradual selection or transition from the bacillary form to a stable L form without the addition of an antibiotic. The number of viable granules attained in the broth culture exceeded 9 x 10(7) per ml, and numerous large bodies were always present in rapidly growing cultures.     

Microtubular structures in a stable staphylococcal L-form.

Microtubular structures, which were demonstrated as straight, dense-walled cylinders attached to cell membranes, were found in a stable staphylococcal L-form grown in the absence of antibiotic.     

Corelike structures in a stable L-form ofStreptococcus pyogenes

Corelike structures, which were interpreted as straight, large cylinders containing ribosome-like particles surrounded by an amorphous substance of low electron density, were found in a stable L-form ofStreptococcus pyogenes grown in the absence of antibiotics.     

Induction of sporulation in developmental mutants of Bacillus subtilis.

Summary Eight alleles of spineless-aristapedia (ss ^a ) were analysed for penetrance and expressivity at 18° C and 25° C. All alleles are recessive and none exhibits a maternal effect. Both ss ^a40a and ss ^aB are temperature sensitive with a higher penetrance at 18° C than 25° C. ss ^aUCI , ss ^ak , ss ^a , ss ^ax and ss ^a are viable alleles with variable penetrance and expressivity whereas ss ^aCam is a lethal allele. All mutants were tested as hemizygotes using the Df(3R) bxd ¹⁰⁰ deletion. The viable alleles showed a more extreme penetrance and expressivity when hemizygous, several becoming lethal near eclosion. Each of the eight alleles was examined in heterozygous combinations with each of the others. The lethal allele ss ^aCam was viable in combination with all other alleles. The temperature sensitivity (t.s.) of ss ^aB and ss ^a40a when heterozygous with the non-t.s. alleles was variable; some combinations were t.s. with respect to penetrance and others with respect to expressivity, whilst some heterozygotes completely lost their sensitivity to temperature.It seems, therefore, that various aspects of the spineless-aristapedia phenotype, such as the temperature sensitivity, can be separated from the expressivity and penetrance in certain allelic combinations. This suggests a very complex gene function which is, however, experimentally separable into its components.     

Induction of autolysis in Bacillus subtilis by ochratoxin A.

Ochratoxin A (OTA) added during the exponential growth phase at a concentration higher than 12 microgram/ml caused autolysis of Bacillus subtilis. Optical density of cultures decreased, and at higher concentrations the cultures became sterile. Optimum OTA-induced lysis was about pH 5. At concentrations below 10 microgram/ml, protein synthesis was inhibited more strongly than RNA synthesis. Cell wall synthesis was also strongly inhibited. A fraction extracted from the lysates had the property of a lysis inhibitor. The relevance of this fraction in respect to autolysis is discussed.