Induction of L-form-like cell shape change of Bacillus subtilis under microculture conditions.

A remarkable cell shape change was observed in Bacillus subtilis strain 168 under microculture conditions on CI agar medium (Spizizen's minimal medium supplemented with a trace amount of yeast extract and Casamino acids). Cells cultured under a cover glass changed in form from rod-shaped to spherical, large and irregular shapes that closely resembled L-form cells. The cell shape change was observed only with CI medium, not with Spizizen's minimum medium alone or other rich media. The whole-cell protein profile of cells grown under cover glass and cells grown on CI agar plates differed in several respects. Tandem mass analysis of nine gel bands which differed in protein expression between the two conditions showed that proteins related to nitrate respiration and fermentation were expressed in the shape-changed cells grown under cover glass. The cell shape change of CI cultures was repressed when excess KNO3 was added to the medium. Whole-cell protein analysis of the normal rod-shaped cells grown with 0.1% KNO3 and the shape-changed cells grown without KNO3 revealed that the expression of the branched-chain alpha-keto acid dehydrogenase complex (coded by the bfmB gene locus) was elevated in the shape-changed cells. Inactivation of the bfmB locus resulted in the repression of cell shape change, and cells in which bfmB expression was induced by IPTG did show changes in shape. Transmission electron microscopy of ultrathin sections demonstrated that the shape-changed cells had thin walls, and plasmolysis of cells fixed with a solution including 0.1 M sucrose was observed. Clarifying the mechanism of thinning of the cell wall may lead to the development of a new type of cell wall biosynthetic inhibitor.

Hanseníase indeterminada: evoluçao clínico-patológica / ?

Foi estudada, em um período de 2 meses a 24 anos, a evoluçao de 51 doentes de hanseníase indeterminada, para formas pauci ou multibacilares da moléstia. O objetivo do estudo foi o de procurar critérios para facilitar a comprovaçao diagnóstica das formas iniciais e o de buscar parâmetros - clínicos, histopatológicos e imunohistoquímicos - que permitissem inferir, desde o início dos sintomas, o sentido evolutivo da doença. Foram examinados os prontuários dos doentes, analisando-se, em relaçao ao quadro inicial da moléstia, o número e características morfológicas das lesoes, a história pregressa, a reaçao de Mitsuda e o tratamento recebido. Nos exames histopatológicos, na coloraçao pela hematoxilina-eosina, foram estudadas as alteraçoes epidérmicas e o infiltrado dermico - sua intensidade, constituiçao e localizaçao

Nocardia asteroides recovery from a dog with steroid- and antibiotic-unresponsive idiopathic polyarthritis.

This report describes a fatal case of idiopathic polyarthritis in a dog that was partially responsive to vigorous immunosuppressive treatment. Synovial fluids were cultured for L-forms at the following stages of disease: (i) acute arthritic relapse, (ii) incomplete remission, and (iii) death. Nocardia asteroides UCD 1-581 was recovered from the L-form broth culture of the specimen taken during acute relapse, 5 weeks after inoculation, but not at any other stage of disease. Numerous conventional microbiological cultures were unproductive during all phases. Changes occurring in L-form plates included the formation of large irregular mineral deposits and many transferable bodies resembling pseudocolonies. Microscopic examination revealed the presence of many intracellular golden-brown granules and acid-fast bodies in macrophages of the lung and bronchial lymph node tissues. The granules are believed to be the variants embedded in calcium deposits similar to those which developed in the L-form cultures in vitro. Fluorescence of these acid-fast bodies with antibody specific for superoxide dismutase of N. asteroides GUH-2 and labeled anti-immunoglobulin G established their relationship to the isolate. The unrelenting course of disease and the persistence of N. asteroides as an L-form in this animal despite vigorous immunosuppression suggest that this organism plays a direct role in the etiology of this disease.

Isolation of L-forms from the spleens of Brucella suis-infected, penicillin-treated mice.

Previous attempts to obtain in vitro wall-deficient stable L-forms of various strains of Brucella have failed because the obtained spheroplasts revert quickly to bacterial form. Here, we report the isolation of L-forms from mice infected with a B. suis strain type 1 and treated with penicillin. In defined experimental conditions, L-type microcolonies associated with tissue debris were observed in primary spleen cultures, even on antibiotic free media. After several transfers on penicillin-containing medium. typical, tissue-free L colonies were obtained. At first, when cultivated on antibiotic-free medium, these colonies reverted to the bacterial form (identified as B suis, biotype 1). Later, after approximately fifteen transfers on penicillin-supplemented medium, they no longer reverted even after several subcultures on antibiotic-free medium. The L-forms' ultrastructural features included many giant empty bodies, considerable variation in size, shape and density of the wall-deficient cells, and many multilayered membranes. The stabilized L-forms were propagated in vitro and inoculated into mice, and then recovered from their spleens as tissue associated L-microcolonies. An occasional in vivo revertant was identified as B. suis, biotype 1. These data provide one possible explanation for earlier failures to detect the presence of atypical bacteria in clinical or experimental Brucella infections.

[Action of penicillin on the causative agent of paratrachoma in the dynamics of an in vitro infection]. / Deistvie penitsillina na vozbuditel' paratrakhomy v dinamike infektsii in vitro.

Monolayer cultures of L-cells (mouse fibroblasts) were inoculated with the causative agent of paratrachoma (strain LB-I). Simultaneously penicillin in doses of 0.01, 0.1, 1, 10, 25, 50, 100, 150 and 200 microgram/ml was added and its effect on the causative agent in the infection dynamics (18, 24, 48 and 72 hours after the inoculation) was studied with the light and electron microscopes. Gradual changes in the ultrastructure of the vegetative forms were observed by the 24th hour with the use of penicillin in a dose of 0.01 microgram/ml: the size of the vegetative forms increased, the cell wall membranes separated and periplasmic space significantly enlarged, the protoplast fragments disjoined into it, large forms vacuolized and were fragment with membranes, sometimes multilayer ones. When the culture was exposed to large doses of penicillin, the rate of the changes in the structure was higher and they were simultaneously of several types. Various types of the changes and possible modes of their formation were analyzed. Morphologically they are similar to the processes observed in L-transformation of bacteria. However, these structures were not infectious.

Cell wall characteristics of Pseudomonas aeruginosa and its carbenicillin-induced L-form.

L-forms of Pseudomonas aeruginosa were induced and cultured on a medium supplemented with carbenicillin. Morphological studies of the passaged variant revealed the presence of a triple-layered cell wall similar to that found in the parent species. Furthermore, the L-form was found to be more susceptible to gentamicin, kanamycin, tetracycline and colistin sulphate. Chemical analysis of the lipopolysaccharide fraction showed a difference in phosphorus content, and changes in cell wall envelope fatty acid content were also exhibited. It is suggested that these differences may influence the transport of certain antibiotics through the cell wall.

In vitro spheroplast and L-form induction within the pathogenic nocardiae.

Six strains of Nocardia asteroides, two strains of N. caviae, and two strains of N. braziliensis were grown in medium supplementted with glycine, lysozyme, D-cycloserine, glycine plus lysozyme, and glycine plus D-cycloserine. It was shown that three strains of N. asteroides, and two strains of N. caviae, readily formed spheroplasts and/or protoplasts when grown in the presence of glycine plus either lysozyme or D-cycloserine. This process was studied by both phase contrast microscopy and electron microscopy. The induced cultures were then plated on hypertonic medium for the isolation of L-forms. It was shown that the organisms differed greatly in their ability to produce spheroplasts and subsequently grew as L-forms or transitional-phase variants.

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.

Teichoic acid of a stabilized L-form of Streptococcus pyogenes.

A stabilized L-form of Streptococcus pyogenes continues to synthesize glycerol teichoic acid. This polymer was obtained from S. pyogenes and its L-form, treated in identical fashion, and compared. Highly purified glycerol teichoic acid from only the L-form was found to be devoid of d-alanine and to have a shorter chain length. Otherwise, the glycerol teichoic acid from these two organisms was found to be a 1,3-phosphodiester-linked glycerophosphate polymer substituted with d-glucose. Evidence is presented that most, if not all, of the glycerol teichoic acid in this streptococcus lies between the wall and membrane. A possible need for the continued synthesis of a minute amount of glycerol teichoic acid by this L-form for survival is discussed in terms of the known function of teichoic acids in bacteria.

Action of yeast killer factor: a resistant mutant with sensitive spheroplasts.

Yeast killer factor proteins bind to cells of both sensitive and killer-producing strains, although the latter are immune to killer action. Spheroplasts prepared from sensitive cells bind less than 1% of the killer bound to whole cells, but remain fully sensitive to killer. This finding and those obtained from binding studies of partially purified, radioactive killer protein suggest that most of the toxins remain bound to the yeast cell wall and do not function further in the killing process. A killer-resistant mutant R(18) was isolated from a sensitive strain. Whole cells of the mutant were unable to bind killer and were fully resistant. In contrast, spheroplasts of R(18) were fully sensitive to killer. These data suggest that the sites exposed to killer in spheroplasts are distinct from those on the cell wall. These wall sites appear to be necessary for killer action in whole cells.