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We investigated the production and regeneration of Thiobacillus ferrooxidans spheroplasts. Regeneration frequencies of up to 90% reversion were obtained. Attempts to transform T. ferrooxidans spheroplasts with a recombinant T. ferrooxidans plasmid were unsuccessful.
Intergeneric protoplast fusion between Escherichia coli HB101 with pBR322 carrying the cloned o-(carboxymethyl)cellulase (CMCase) gene of Ruminococcus albus (Pro Leu Ap Km) and an anaerobic mutant strain, FEM29 (Trp His Ap Km), with dehydrodivanillin-degrading activity was performed in the presence of 40% polyvinyl alcohol 300 under aerobic and anaerobic conditions to transfer the cloned cellulase gene into the mutant. The mutant FEM29 had a unique property. When it was incubated in liquid medium with 1% glucose and sucrose, protoplasts could be produced autogenously and regenerated on the agar slant. E. coli spheroplasts formed from a plasmid-amplified overnight culture after 10 min of treatment with lysozyme (20 mug/ml) in a hypertonic solution (0.01 M Tris hydrochloride [pH 7.5], 0.4 M mannitol). Protoplast regeneration rates of FEM29 and HB101 were 30 and 83%, respectively, on the agar-yeast extract medium. Ap Km fusants were obtained at high frequency: 1.7 x 10 anaerobically and 8.2 x 10 aerobically. These fusants showed 23 to 57% of CMCase and dehydrodivanillin-degrading activities, respectively, as compared with parental strains. All the fusants isolated were gram-negative rods with main phenotypes such as urease and catalase activities as in HB101 and esterase and chymotrypsin activities as in FEM29. Southern hybridization experiments suggested that pBR322 with the cloned CMCase gene existed autonomously in the fusant cells. This is the first report describing transfer of pBR322 with a cloned cellulase gene into an anaerobic mutant by polyvinyl alcohol-mediated fusion with an E. coli spheroplast.
Penetration of substrates into marine bacteria as influenced by cations has been demonstrated by the effects of increased osmotic pressure in spheroplasts of these cells. Spheroplasts of Pseudomonas natriegens, stabilized with lactose, underwent a metabolic swelling in the presence of a substrate to which they had been induced. Maximal and persistent swelling was achieved only by addition of catabolizable substrate and both Na(+) and K(+). Addition, along with substrate, of Na(+) alone or K(+) alone did not stimulate swelling; no metabolic swelling occurred in the presence of a sugar to which the cells had not been induced. Confirmation of rapid uptake by induced cells of the inducer sugar, l-arabinose, but not the d-isomer, was obtained with (14)C-labeled substrate. Addition of NaN(3) completely inhibited swelling, and 2, 4-dinitrophenol and ouabain each suppressed it by 50%, indicating requirement for energy metabolism and involvement of an adenosine triphosphatase in the penetration phenomena of these cells.
Tabor, Celia W. (National Institute of Arthritis and Metabolic Diseases, Bethesda, Md.). Stabilization of protoplasts and spheroplasts by spermine and other polyamines. J. Bacteriol. 83:1101-1111. 1962.-Spermine (10(-3)m) or spermidine prevents lysis of lysozyme-produced protoplasts of Escherichia coli W, E. coli B, and Micrococcus lysodeikticus in hypotonic media. Spheroplasts prepared by the action of penicillin are also stabilized by these concentrations of spermine and spermidine, but the protection is not as complete. Streptomycin, polylysine, and Ca(++) are also effective or partially effective stabilizers, but 1,4-diaminobutane, 1,5-diaminopentane, ornithine, Mg(++), and monovalent cations have no protective action at 10(-3)m concentration, and only a slight effect at higher concentrations. The osmotic stability conferred on protoplasts by spermine is irreversible. However, the protective effect of polyamines against lysis is not accompanied by restoration of viability to lysozyme protoplasts. There is a marked reduction in the loss of ultra-violet-absorbing material from the protoplasts to the medium when 10(-3)m spermine is present.
Lark, Cynthia (Saint Louis University, St. Louis, Mo.) and Robert Schichtel. Comparison of spheroplast induction in Alcaligenes faecalis by three different agents. J. Bacteriol. 84:1241-1244. 1962.-Alcaligenes faecalis strain LB was exposed to different concentrations of cycloserine, d-methionine, or penicillin. The time course of spheroplast induction by these agents was measured as a function of their concentration. The results were consistent with models in which cycloserine reversibly inhibited an intracellular enzyme, and d-methionine was built into a defective cell-wall unit. Penicillin simulated cycloserine. This was taken as further evidence that penicillin inhibits an enzyme associated with cell-wall synthesis.
Spheroplasts of Vibrio cholerae types Inaba, Hikojima, and Ogawa were produced in serum. An excess of lysozyme was added to expedite transformation, and CaCl(2) was used as the stabilizing agent. At the optimal time for each strain, when less than 1% classical rod forms were observed microscopically, samples were plated on both conventional and modified L-form media. No growth occurred on conventional media; L-form type colonies appeared on L-form media after 2 to 6 days of incubation. L-form colonies could be subcultured on conventional media and required from four to six passages for complete reversion to classical parent forms. Reaction mixtures of Hikojima and Inaba types were passed through membrane filters (0.45 mu); L-form colonies were grown from both strains after spheroplast transformation. Appropriate controls were negative. It is suggested that the replicative particle may be filtrable.
Under conditions that permitted continued protein synthesis, spheroplasts of Escherichia coli were unable to form active alkaline phosphatase, although they synthesized protein that was antigenically related to alkaline phosphatase subunits. This cross-reacting protein was primarily detected in the medium of the spheroplast culture, and it had properties that closely resembled those of the alkaline phosphatase subunit. These results suggest that formation of the active alkaline phosphatase dimer by intact E. coli cells proceeds by a pathway in which inactive subunits released from polyribosomes diffuse through the bacterial cell membrane to a periplasmic space where subsequent dimerization to active enzyme occurs. This pathway provides a possible mechanism for the specific localization of this enzyme to the E. coli periplasmic space.
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.
Gene transfer using yeast artificial chromosome (YAC) clones provides an opportunity to study the expression of several linked genes within an environment more closely approximating their normal chromosomal context. A YAC clone spanning 330 kb of the HLA class II region from centromeric of TAP 1 to telomeric of HLA-DQA1 was retrofitted by homologous recombination with a neomycin plasmid targeted either to an Alu repeat sequence within the YAC genomic insert or to the Ura-3 gene within the right arm of the YAC vector. The modified YAC clones were transferred to Chinese hamster ovary or L cells by spheroplast fusion. Eight of 14 Alu-retrofitted and 10 of 15 right arm-retrofitted neomycin clones retained the six human loci known to be encoded by the YAC as well as portions of the left and right YAC vector arms. All tested L cell transformants showed IFN-gamma-inducible TAP 1 and TAP 2 mRNA expression. Two of eight analyzed clones expressed HLA-DQB mRNA and one of four expressed HLA-DQA. Cells expressing both the HLA-DQA and -DQB mRNA showed HLA-DQ cell surface expression. These studies establish the feasibility of introducing groups of functional genes into mammalian cells by spheroplast fusion with a single YAC clone.
The rate of formation of spheroplasts of yeast can be used as an assay to study the structural integrity of cell walls. Lysis can be measured spectrophotometrically in hypotonic solution in the presence of Zymolyase, a mixture of cell wall-digesting enzymes. The optical density of the cell suspension decreases as the cells lyse. We optimized this assay with respect to enzyme concentration, temperature, pH, and growth conditions for several strains of Saccharomyces cerevisiae. The level of variability (standard deviation) was 1-5% between trials where the replications were performed on the same culture using enzyme prepared from the same lot, and 5-15% for different cultures of the same strain. This assay can quantitate differences in cell wall structure (1) between exponentially growing and stationary phase cells, (2) among different S. cerevisiae strains, (3) between S. cerevisiae and Candida albicans, (4) between parental and mutated lines, and (5) between drug- or chemically-treated cells and controls.
As clinical persistence of Borrelia burgdorferi in patients with active Lyme borreliosis occurs despite obviously adequate antibiotic therapy, in vitro investigations of morphological variants and atypical forms of B. burgdorferi were undertaken. In an attempt to learn more about the variation of B. burgdorferi and the role of atypical forms in Lyme borreliosis, borreliae isolated from antibiotically treated and untreated patients with the clinical diagnosis of definite and probable Lyme borreliosis and from patient specimens contaminated with bacteria were investigated. Furthermore, the degeneration of the isolates during exposure to penicillin G in vitro was analysed. Morphological analysis by darkfield microscopy and scanning electron microscopy revealed diverse alterations. Persisters isolated from a great number of patients (60-80%) after treatment with antibiotics had an atypical form. The morphological alterations in culture with penicillin G developed gradually and increased with duration of incubation. Pleomorphism, the presence of elongated forms and spherical structures, the inability of cells to replicate, the long period of adaptation to growth in MKP-medium and the mycoplasma-like colonies after growth in solid medium (PMR agar) suggest that B. burgdorferi produce spheroplast-L-form variants. With regard to the polyphasic course of Lyme borreliosis, these forms without cell walls can be a possible reason why Borrelia survive in the organism for a long time (probably with all beta-lactam antibiotics) [corrected] and the cell-wall-dependent antibody titers disappear and emerge after reversion.
A system relying on the use of Neurospora crassa spheroplasts has been developed for the assay of cytotoxicity and mutagenicity of chemical compounds. Mutagenicity was assayed by using reversion of alleles in the am gene selected to recognize certain specified transitions and also undefined point mutations. Cytotoxicity was quantified by measuring a 'cytotoxicity parameter', m, which appears in the exponential function that fits the survival/dose curve for each compound (under standard incubation conditions). Of the compounds tested, nitrogen mustard (Cl(CH2)2 NMe(CH2)2Cl) was cytotoxic and non-mutagenic, and ethyl nitrosourea was highly mutagenic but not cytotoxic. Of the remaining compounds tested, methyl nitrosourea, butadiene diepoxide, and cis platin (cis diammonia platinum II chloride) all showed comparable mutagenicity per survivor, although the values of m covered a wide range. Differences were found between the different compounds in the effects of the uvs-2 allele on survival and on the preponderance of G to A transitions.
The occurrence of candidiasis in cancer patients who undergo chemotherapy requires the interrelation of Candida albicans and the antimitotic drug Adriamycin (ADM) which is well known as an intercalating agent. The whole yeasts were not affected by 2 h of contact with the drug at 10(-4) M neither for their growth curve nor for their ultrastructure, despite the presence of free ADM on their surface. Spheroplasts displayed a delay in their growth and exhibited altered nucleoli with segregation of their granular and fibrillar components. The modified emission spectrum of ADM, determined by spectrofluorometry, corresponded neither to the free ADM nor to the DNA-bound drug, but it could be related to a metabolite of the drug. The cell wall appeared to be one of the main sites for ADM resistance of Candida albicans in vitro.
A method was developed for preparation of spheroplasts used for transfection with phi X174 RF DNA. This method had a high-level competence and retained the competence for up to one year of storage in 7% DMSO at -70 degrees C.
Cell wall deficient forms (CWD, spheroplasts) genetically indistinguishable from M. avium subsp. paratuberculosis (MAP) have been isolated from patients with Crohn's disease and sarcoidosis. These MAP CWD may be important in the pathogenesis of these diseases and in Johne's Disease in other animal species. CWD forms are extremely difficult to isolate and generally revert to cell wall competent forms (CWC) when cultured in vitro. Cultured MAP strain 19698 were chemically treated to generate sufficient CWD to compare to CWC organisms by electron microscopy, chemotype profile (matrix solid-phase dispersion and thin layer chromatography), silver-stained SDS-PAGE gels with and without periodic acid treatment and Western blots with antigen recognition by sera from confirmed Johne's positive and Johne's negative cattle. On electron microscopy, CWD organisms were larger and rounder than cell wall competent forms and had lost the majority of their cell walls, being bounded only by a plasma membrane. Chemotype profiles of CWD lacked bands generally associated with cell wall glycolipids. Silver-stained SDS-PAGE gels of CWD demonstrated loss of bands that migrate in the same region as lipoarabinomannan (LAM) and some bands likely representing proteins and weakening of bands that migrate similarly to phosphatidylinositol mannosides (PIM). Western blots of CWD demonstrated bands with loss or attenuation of signal that migrate similarly to LAM and other constituents. In summary, CWD and CWC forms of MAP 19698 had marked differences in morphology, chemotype profile, cell wall constituents, and antigens recognized by Johne's disease positive and negative bovine sera.
The breakdown and renewal of phospholipids and glycerides from spheroplasts of Mycobacterium smegmatis ATCC 14468 were studied by means of pulse labelling techniques using 1-14C-palmitate. Most of the radioactivity incorporated into total lipids was recovered from phospholipids. Among the phospholipids studied, cardiolipin had the highest radioactivity and specific activity, suggesting its higher pool size as compared to other fractions. The appreciable loss of radioactivity for cardiolipin in pulse chase experiments within growing cells suggests its metabolically active and dynamic nature. Glycerides exhibited no appreciable turnover rates.
Spheroplast fusion using polyethylene glycol 6000 resulted in mycobacterial recombinants with a frequency of about 10(-3). The parental strains were two carotenoid pigment mutants of Mycobacterium aurum which were obtained by chemical mutagenesis of the wild-type strain with N-methyl-N'-nitro-N-nitrosoguanidine. Isolation of recombinant clones was based on their pigmentation on Löwenstein-Jensen medium, and genetically stable recombinants were analysed for their carotenoid pigments and their alpha- and beta-mycolic acids. They were then submitted to 14 biochemical tests as well as determination of their susceptibility to 13 drugs and their ability to grow at 30 and 42 degrees C. Recombinants were found to be positive for nitrate reductase (100%), gamma-glutamyl transferase (94%), urease (53%) and arylsulphatase (21%). Out of 34 recombinants tested, 5 grew both at 30 degrees and 42 degrees C, whereas 1 grew only at 37 degrees C. The results of drug susceptibility testing were inconclusive.
In order to directly prove that the periplasmic glutamine binding protein is an essential component of the osmotic shock sensitive active transport system for glutamine in Escherichia coli, we demonstrated the reconstitution of binding protein dependent glutamine transport in spheroplasts of that organism. It was shown by arsenate inhibition that the reconstituted transport system was energy dependent, and the use of azaserine, an inhibitor of glutamine-utilizing enzymes, indicated that the restoration of transport by binding protein did not require the metabolizing of the transport substrate. Furthermore, the binding protein dependent transport of glutamine was shown to require at least one other macromolecular component, presumably membrane bound, which was absent in a strain containing a deletion of the genes coding for the glutamine transport system but was present in a strain carrying a mutation only in the structural gene for the glutamine binding protein.