Bacterial L-forms.

L-forms are "cell wall-deficient" bacteria which are able to grow as spheroplasts or protoplasts. They can be differentiated into four types depending on their ability to revert to the parental, cell-walled form and to the extent of their cell-wall modification. L-forms are significant in modern science because of their contributions to an improved understanding of principal questions and their interactions with eukaryotes. This review particularly focuses on research using stable protoplast-type L-forms which have contributed to a better understanding of the structural and functional organisation of the cytoplasmic membrane and of cell division. These L-forms, which have only a single surrounding bilayer membrane, also represent a unique expression system for production of recombinant proteins. A large proportion of L-form publications concern their putative role in human disease and its therapy, a topic which is discussed briefly. L-forms have also been used to form intracellular associations with plant cells and have been shown to elicit induced disease resistance offering a novel method for plant protection. The recent decline in active research on L-forms is a concern as knowledge and experience, as well as unique L-form strains which have been maintained for decades, are being lost.

Effect of left-sided valvular regurgitation on mortality and causes of death among a population of middle-aged and older horses.

The effect of left-sided valvular regurgitation (LSVR) on the mortality of middle-aged and older horses was investigated in a prospective cohort study involving 19 yards and 1153 horses. The horses were examined to determine whether they had a cardiac murmur and its type, and their age, sex, breed type and occupation were recorded. They were followed up at intervals of two years by postal questionnaire, and after four years information on 773 horses was available. There was no significant difference in the mortality of the horses with and without LSVR, but small horses had a significantly higher risk of having LSVR than small ponies (odds ratio [OR] 2.33), and older horses were slightly more likely to have LSVR than young horses (OR 1.07). Twenty-nine per cent of the deaths reported by the owners were due to orthopaedic problems, 23.3 per cent to gastrointestinal problems, and only 7.9 per cent to cardiovascular problems. Orthopaedic problems were the main cause of death in the horses, and gastrointestinal problems were the main cause of death in the ponies.

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.

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.

The potential biocontrol agent Pseudomonas antimicrobica inhibits germination of conidia and outgrowth of Botrytis cinerea.

AIMS: Antifungal metabolites of Pseudomonas antimicrobica have previously been shown to inhibit conidial germination of the grey mould pathogen Botrytis cinerea. In this study, metabolites of the bacterium have been tested at different stages of Botrytis germination to determine their effects on germ tube production and extension. METHODS AND RESULTS: Metabolites were added to conidia that had been pre-incubated for either 120 or 255 min. Pseudomonas antimicrobica inhibited B. cinerea conidial germination and caused a significant reduction in germ tube extension, irrespective of the stage of germination. Abnormal germination and a reduction in the frequency of lateral branching of the germ tubes in the presence of the metabolites were also reported, suggesting interference with normal hyphal development. CONCLUSION: The bacterium can inhibit germination of conidia and extension of germ tubes at different stages of Botrytis development. SIGNIFICANCE AND IMPACT OF THE STUDY: The antagonistic activity of the bacterium has promising implications for its use as a biocontrol agent.

Induction, growth and antibiotic production of Streptomyces viridifaciens L-form bacteria.

AIMS: To induce, cultivate and investigate the characteristics of L-form bacteria derived from the filamentous actinomycete Streptomyces viridifaciens. METHODS AND RESULTS: L-forms were induced in a liquid medium supplemented with lysozyme and penicillin. A stable culture which no longer required inducing agents but could still revert, was obtained by the twelfth subculture. The specific growth rate of stable L-forms was faster (0.751) than unstable L-forms (0.361). After the exponential growth phase, the cell diameter continued to increase, as did the percentage of vacuoles. Morphologically, the L-forms appeared as spherical bodies with no signs of differentiation and were sensitive to osmotic stress, indicating removal of the cell wall. The L-forms produced secondary metabolites although much lower levels of antibiotic were assayed in the L-forms compared with the cell walled forms. CONCLUSION: Stable L-form bacteria were induced from S. viridifaciens and their growth characterized. The L-forms produced secondary metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Stable Streptomyces L-forms were induced and have potential as biocontrol agents.

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.

A standard procedure for the micropropagation of the neem tree (Azadirachta indica A. Juss).

Micropropagated shoots were initiated from leaf explants of the neem tree, Azadirachta indica A. Juss. Regardless of their origin, shoots were successfully produced by culturing leaf explants on Murashige and Skoog medium containing benzylaminopurine (1 mg l-1), kinetin (0.8 mg l-1) and adenine sulphate (6 mg l-1) in complete darkness. These shoots were further multiplied on Murashige and Skoog medium containing benzylaminopurine (0.1 mg l-1), kinetin (0.08 g l-l) and adenine sulphate (0.6 mg l-1). Within 32 weeks, 80 shoots could be produced from a single leaf explant (10 mm×10 mm). Fifty-five percent of these shoots rooted on Murashige and Skoog medium containing indolebutyric acid (1 mg l-1) and all of these grew on transfer to soil.

Bioassay methods for the detection of antifungal activity by Pseudomonas antimicrobica against the grey mould pathogen Botrytis cinerea.

Antagonism against the grey mould pathogen Botrytis cinerea by Pseudomonas antimicrobica was demonstrated in vitro and in vivo. Cell-free filtrates showed activity against B. cinerea growing on Potato Dextrose Agar (PDA) in a media-dependent manner with the most distinct antagonism being produced in Czapek Dox Broth (CDB). Cell-free filtrates of CDB-grown cultures also significantly reduced conidial germination of B. cinerea. An assay based on the inhibition of conidial germination was compared with two assays measuring the antagonism of mycelial growth on PDA. The conidial germination bioassay was more sensitive in the detection of this antifungal activity than the Petri dish bioassay while a bioassay using Microdetection plates did not detect antagonism due to the small loading capacity of the latter. The conidial germination bioassay was modified for detection of antibiosis on the surface of strawberry leaves. Significant reductions in percentage conidial germination were recorded on the surface of leaves of both micropropagated and glasshouse grown strawberry plants when the antifungal compounds of Ps. antimicrobica were applied to the leaf tissue with the conidia. In addition, antifungal compounds were also detectable when conidia were applied to leaf tissue which had previously been sprayed with cells of Ps. antimicrobica. These tests indicate that Ps. antimicrobica would be a suitable biocontrol agent for the control of B. cinerea.

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

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.

Growth and adhesion of Enterococcus faecium L-forms.

Comparisons of growth and surface colonisation of Enterococcus faecium L-forms and their cell-walled forms were undertaken to produce information about their ability to form sessile cells. The growth of L-forms in liquid culture was slower than that of the parent. This was reflected in their longer lag phase and slower specific growth rates: 0.16 h-1 for the L-form and 0.81 h-1 for the parent. Although E. faecium L-forms attached to a silastic rubber surface, the attached population density was 10-100-fold less than that of the parent. Confluent biofilms on the silastic surfaces were not observed for either bacterial form. Comparison of the attachment of E. faecium L-form and parent may provide important information on how bacteria overcome host defence mechanisms and antibiotic treatment.

Growth and physiological characteristics of Bacillus subtilis L-forms.

The specific growth rate of stable Bacillus subtilis L-forms was slower (mu = 0.127) than the cell-walled form (mu = 0.219) when measured by optical density (O.D.). However, the L-form growth rate increased (mu = 0.288) when determined by viable cell counts. L-forms of B. subtilis appear to enter a phase of rapid cell division, followed by a period of cell enlargement which is associated with an increase in the number of cells with vacuoles and granules. Thus, maximum viable L-form numbers and DNA content were attained at approx. 30h, before maximum protein content was achieved (46h) and before maximum O.D. was observed at 71 h. Measurements showed that L-form cell size increased even after cell division had stopped. O.D. was therefore inaccurate for assessment of L-form growth. L-forms were sensitive to osmotic shock and unlike the cell-walled organisms from which they were derived, were resistant to penicillin, indicating a loss of peptidoglycan. The L-forms were similar to cell-walled forms in that antibiotic(s) and proteases were produced.

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.

Production of the triterpenoid quassin in callus and cell suspension cultures of Picrasma quassioides Bennett.

Plant cell and suspension cultures have been established from stem cuttings of Picrasma quassioides Bennett. The effect of 244 different types/concentrations of plant growth regulators on growth and quassin accumulation in callus tissue was investigated. Best growth, in terms of wet/dry weight after four weeks growth, was obtained on B5 media supplemented with 2% glucose, 10% coconut milk, 0.5 mg.l(-1) zeatin riboside and 1.5 mg.l(-1) IBA. The highest yields of quassin (0.014-0.018%) were detected on this same media supplemented with 1.0 mg.l(-1) IBA and varying concentrations of zeatin riboside. Suspension cultures were easily established on B5 media supplemented with 2% glucose, 1.0 mg.l(-1) 2,4-D and 0.5 mg.l(-1) kinetin. The carbon source had a marked effect on quassin accumulation with 0.32% quassin being detected when cells were grown in 2% galactose. This is comparable to the highest reported quassin yield for the whole plant.