Ultrastructure of the unbalanced growth forms of Salmonellae obtained by the action of various factors

The unbalanced growth forms (UGF), also known as heteromorphous or pleomorphous forms, of salmonellae were obtained under the action of penicillin, homologous antiserum in combination with complement and lysozyme, as well as ultraviolet radiation. The effect of these factors was studied after their momentaneous action and in successive passages. The viability of UGF was determined in terms of colony-forming units. The appearance of UGF was induced by penicillin, a complex of immune substances and ultraviolet radiation, yet the action of two latter factors irreparably damaged most of the cells. The formation of UGF, showing stability in prolonged passages and capable of transformation into L-forms, occurred only under the influence of penicillin. Elementary bodies appeared in UGF, both capable and incapable of transformation into L-forms after successive passages. In the viable part of the UGF population obtained under the action of all the above factors damages in the peptidoglycan layer of the cell wall and, partly, in the cytoplasmic membrane, as well as disturbances in the process of cell division, linked with these cell elements, were observed.     

紫外诱变改良柑橘采后病害拮抗菌柠檬形克勒克酵母研究

以柠檬形克勒克酵母(Kloeckera apiculata)为出发菌株,采用紫外线(UV)、UV LiCl等方法对其进行诱变改良.确定了处理的最佳剂量:紫外处理15 W 30 cm照射20 s,UV LiCl处理UV照射20 s并在平板中加入LiCl 0.3%(W/V).选育到一株生理特性有明显改善的变异菌株UV20-13,果实试验中,7 d后柑橘青、绿霉病的发病率分别比出发菌株降低25.56%和10.00%.生长动态测定和传代试验表明,该菌株生长特性优于出发菌株,连续传代10代,该菌株没有出现退化、回复突变等,在遗传上是稳定的.     

紫外诱变改良柑橘采后病害拮抗菌柠檬形克勒克酵母研究

以柠檬形克勒克酵母(Kloeckera apiculata)为出发菌株, 采用紫外线(UV)、UV+LiCl等方法对其进行诱变改良。确定了处理的最佳剂量：紫外处理15 W 30 cm照射20 s, UV+LiCl处理UV照射20 s并在平板中加入LiCl 0.3% (W/V)。选育到一株生理特性有明显改善的变异菌株UV20-13, 果实试验中, 7 d后柑橘青、绿霉病的发病率分别比出发菌株降低25.56%和10.00%。生长动态测定和传代试验表明, 该菌株生长特性优于出发菌株, 连续传代10代, 该菌株没有出现退化、回复突变等, 在遗传上是稳定的。     

Study of the L-transforming action on Brucella cells of tetracycline, streptomycin, rifampicin and their combinations with penicillin in in vitro experiments]

The in vitro study of the possible formation of L-forms of Brucella under the effect of tetracycline, streptomycin, rifampicin or their combinations with penicillin showed that passages of various Brucella species on media containing increasing concentrations of the antibiotics resulted in insignificant morphological changes in the cells. Passages of the same cultures on media containing combinations of the above antibiotics with penicillin resulted in more significant changes in the cell morphology, up to formation of spheroplasts. No L-transformation was observed under the experimental conditions.     

Stable L-forms of Clostridium perfringens and their growth on glass surfaces.

L-forms of Clostridium perfringens were induced in brain heart infusion broth containing 10% sucrose and 2 units of penicillin. After a few hours of growth, spheroplasts, granules, and elongated bacilli were apparent. At 24-h intervals, serial subcultures were made in the above medium which resulted in a culture composed entirely of spheroplasts (or protoplasts) and granules. Upon the withdrawal of penicillin these L-form cultures grew well and, after 100 passages, there was no reversion to the bacillary form. Sucrose could also be withdrawn from the medium. The effects of centrifugation, osmotic stabilizer, ultraviolet light, temperature, pH, and lyophilization upon stable L-forms were examined. L-forms were found to attach to the walls of culture tubes during trowth and sheets of L-form growth were obtained on cover slips in Leighton tubes and on the sides of medicine bottles.     

Biological properties and ultrastructure of brucellae during L-transformation and reversion]

There was shown a difference in the biological properties and the ultrastructure of two strains of brucellae, spheroplasts obtained from them under the action of penicillin, L-form and revertants obtained from the L-form. Spheroplasts formation was characterized by a change of brucellae into R-form and some virulence reduction. The cells had an outer and a cytoplasmic membranes, and usually lost their capacity to binary division. L-forms were obtained during the 9th and the 35th passage on a medium with penicillin; their formation was accompanied by the change in serological properties of the culture and significant reduction of the virulence; the cells were characterized by a marked polymorphism and the capacity to budding; they had 2 membranes on the cell surface and an intensively developed system of intracytoplasmic membranes. The revertants formed on the medium without penicillin during the 16th-30th passage or spontaneously on the medium with penicillin. They differed from the initial strains of brucella culture by a marked increase in penicillin-resistance, by the changes in serological properties, and also by polymorphism of cells, capable, however, of binary division.     

Alterations of the L-forms of a sporebearing bacillus

A large pleomorphic gram-negative bacillus developed as a contaminant on blood-agar. Spores were formed in one culture. L-forms were produced with penicillin on blood-agar with 2.5% NaCl; they grew well when transplanted to agar with 0.5% NaCl. After several transplants and long incubation of the L-forms without penicillin, in three transplants small gram-negative pleomorphic bacilli grew, but no L-forms. This occurred once on blood-agar and twice on 30% gelatin. The growth obtained from these small bacilli was similar in morphology and in the physical properties of the organisms to the altered L-forms of Proteus and Salmonella. Multiplication of the pleomorphic organisms and development of branching filaments from the round forms was apparent. The original large gram-negative bacillus was regularly recovered from the L-forms, and was recovered several times from the descendants of the small bacilli. These observations are essentially similar to those made with L-forms of Proteus and with an L-form studied in 1952, indicating alterations in L-forms of bacteria which do not produce B type L-forms.     

L-Phase variants ofAgromyces ramosus

Agromyces ramosus, which is a numerically prevalent bacterium in soil, was easily induced into the L-phase by growing it on agar media containing low levels of penicillin or glycine. The L-forms were stable after initial contact with the inducing agent and could not be reverted to the bacterial form by any of the procedures tried. These results are discussed in relation to a possible natural occurrence of L-forms of this bacterium in soil.     

高产Monacolin K红曲菌株的复合诱变选育

利用紫外线和氯化锂对一株紫红曲Monascus purpureus菌株进行复合诱变试验。在最佳紫外照射时间为45s和最佳氯化锂浓度为1.0‰的情况下,获得了一株MonacolinK高产突变菌株Monascus purpureus ZT32且连续传接5代产量稳定,用紫外分光光度法检测其MonacolinK的含量为219.9μg/mL,较原始菌株高出2倍多,然后将其用于固态发酵,结果表明:红曲米中的MonacolinK的含量达到8.33mg/g,是原始菌株的3.3倍。     

Ultrastructure of Brucella abortus L-forms induced by penicillin in a liquid and in a semisolid medium

Brucella abortus L-forms were induced by 5.0 or 10.0 mug of penicillin/ml in a broth medium containing 0.3 m sucrose, and in a semisolid medium containing 10% calf serum and 20.0, 40.0, or 60.0 mug of penicillin/ml. After 96 hr of incubation, L-forms of various sizes and shapes were observed. Basic structures of the L-forms were similar whether induced in liquid or semisolid medium. L-forms had two "unit" membranes, each consisting of two outer dense layers separated by a lucent layer. A few large, irregularly shaped organisms in penicillin-treated broth cultures had additional surface material and were referred to as "transitional" forms. In contrast with L-forms, the bacterial cells were fairly uniform in size and shape, were smaller, and had a more complex cell wall structure. Small bodies limited by a "unit" membrane were present within and around numerous L-forms from liquid and semisolid medium cultures. Other internal membranous structures were also seen in some L-forms. Most Brucella L-forms described in this paper reverted to bacteria in the absence of penicillin and were structurally characteristic of unstable L-forms.     

Fatty Acid Composition of L-Forms of Streptococcus faecalis Cultured at Different Osmolalities

The fatty acid composition of the membranes of three different penicillin-produced L-forms of Streptococcus faecalis was determined: (i) a stable (nonreverting) L-form (T(53)) cultured in brain heart infusion (BHI) with 0.5 M sucrose; (ii) a stable L-form (T(531)) cultured in BHI without sucrose; and (iii) an unstable L-form (T(9)) cultured in BHI with 0.5 M sucrose and 1,000 U of penicillin per ml. L-forms were obtained by centrifugation and lysed by washing in 1 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer. The parent S. faecalis was also cultured in BHI and BHI containing 0.5 M sucrose, and washed with buffer. The fatty acid composition of L-forms of S. faecalis cultured in BHI without sucrose (370 mosmol) had higher C(18:1) and lower C(18) than L-forms cultured in the same media with added 0.5 M sucrose (950 mosmol) in both exponential and stationary cultures. In the stationary phase of growth, C(19) was reduced in the L-forms cultured without sucrose. Similar changes were seen in the parent S. faecalis cultured in the two types of media. These changes in membrane fatty acids may relate to osmo-regulation of the L-forms.     

Isolation of bacterial L forms from the joint fluid in rheumatoid arthritis in children]

In the bacteriological study of intra-articular fluid in 22 children with rheumatoid arthritis the L-forms were isolated in 11. Of these, 7 had not been treated with preparations of the penicillin series. The complete isolation of the focus of inflammation from the environment and the presence of the L-forms of bacteria in the children who had not been treated with preparations of the penicillin series gave us grounds for conclusions on the etiological and pathological significance of the L-forms of streptococci in rheumatoid arthritis.     

On the pathogenicity of nonstable Brucella L-forms and their revertants.

The pathogenicity of B. abortus 870 L-forms obtained by long-term passaging of virulent culture on media with penicillin and of revertants obtained in vitro and in vivo was studied. L-form cultures stimulated only a mild response of the reticulo-endothelial system of the animal organism, at the same time displaying a certain level of toxicity. In vitro revertants approximated to L-forms, while in vivo revertants stood closer to the initial virulent culture, as regards pathogenicity. This seems to be evidence of a potential danger of brucella L-forms for the human and animal organisms.     

瑞拉菌素产生菌RL-2的诱变育种

本研究以委内瑞拉链霉菌秦岭变种RL-2(Streptomyces venezuelaevar. qinlingensis RL-2)为出发菌株, 分别采用紫外线、氯化锂及紫外线加氯化锂的复合诱变方式对其孢子悬液进行诱变处理。在复合诱变的紫外线照射时间为45 s和氯化锂浓度为0.4%的情况下, 获得一株瑞拉菌素的高产突变菌株UVL-108且连续传接6代其遗传特性较稳定。采用双向培养及生长速率法对其进行初筛和复筛, 结果表明: UVL-108的拮抗性能较出发菌株提高了77%, 其发酵液对稻瘟病菌的抑菌毒力是出     

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.     

Ultrastructure of Salmonella typhimurium forms with unbalanced growth induced by lithium chloride

The action of 1.0 and 1.5 M LiCl on S. typhimurium induces the appearance of unbalanced growth forms capable of growing and multiplication, when subcultured in a medium with this preparation. In this culture the prevalence of cells differing in their structure from the initial Salmonella cells and from stable L-form cultures is observed. Cells characteristic of the initial culture and cells resembling the L-forms occur in lesser numbers. LiCl seems to affect peptidoglycan and the cytoplasmic membrane, which brings about disturbances in the permeability of the surface structures of the cell.     

L-Form Induction, Morphology, and Development in Two Related Strains of Erysipelothrix rhusiopathiae

Two related strains of Erysipelothrix rhusiopathiae, one the parent and the other an L-form revertant, were studied for their propensity or ability to produce L-forms under the influence of penicillin. The parent strain produced L-forms in nutrient solid media in an osmolarity range between 0.85 and 5.0% NaCl concentration whereas the revertant strain did so between 0.5 and 3.0% NaCl concentration. When various hyperosmolar media were tried without penicillin, recovery of L-forms from the revertant strain was optimal at a salt concentration of 2.0%, whereas the parent strain occasionally produced a few L-forms on 3.0% salt medium only. The process of penicillin-induced transformation from bacteria to L-form followed an unusual morphological sequence, beginning with beading of the bacterial body, followed by disintegration into granules from which the L-form colony derived. No large bodies were seen during the initial process of L-form induction, but they evolved later from the original granules and had the potential to reproduce L-type growth. The spontaneous development of L-forms in hyperosmolar media had a different morphological sequence starting with elongation of the bacteria into filaments which later developed polar and central dilatations from which granules and L-type growth developed. The differences in biological behavior between these related bacterial strains suggest that the revertant strain developed new properties, probably of genetic origin. Consequently, the assumption that L-forms revert to the "parent" bacteria may not always be justified. It can be made only after the biological properties of the parent and the revertant organisms have been properly identified.     

Fine Structure of Protoplasts and L-Forms of Clostridium botulinum Types A and E

Spherical bodies, that are obtained by adding penicillin and lysozyme to Clostridium botulinum types E and A cultures which are growing in an osmotically stabilized medium, are shown to be protoplasts by electron microscopy. The L-forms of these two culture types have morphologically different inclusion bodies.     

Comparative characteristics of antigenic peculiarities and several other properties of stable long-term cultures of salmonella L-forms]

Enzymatic properties, sensitivity to antibiotics and peculiarities of the antigenic structure of stable L-forms of S. paratyphi B (L-115), S. typhimurium (L-71a-16) and S. typhi (L-5761) were studied. In difference from the initial strains, the L-form under study possessed a high penicillin and ampicillin sensitivity, and also a marked sensitivity to polymyxin and detergents; this characterized them as L-forms of protoplastic type. All the L-variants differed considerably by the enzymatic properties from the initial strains, and, in the majority of cases, retained the antigenic components and the species-specificity characteristic of the initial parental cultures. Despite the loss of the cell wall the synthesis of O-antigen in the L-forms under study was undisturbed. The 6-forms contained an antigenic component not found in the initial cultures and apparently causing the neutralizing activity of the L-antiser.     

Ultrastructure of L-forms. II. L-forms of Listeria monocytogenes]

A study was made of ultrathin sections of the stable l-forms of listeria obtained under the action of penicillin in meat-peptone-liver broth. A marked cellular polymorphism was found in the L-form culture: within the same colony cells differed in size, shape and fine structure. It is supposed that polymorphism could be partially explained by a different plasticity and premeability of cytoplasmic membrane in different types of cells of the same L-colony. The three-layer structure of the membrane does not always display the same distinctness in various L-colony cells and also in different areas of the cell surface. Structureless material of low electron density, possibly a defective murein or its precursor, was revealed on the membrane surface. Electrondense inclusion bodies, mesosomes of ring-shaped or more complicated structure and two-contour vesicles were found in the cytoplasm. The cells multiplied by budding, by binary and anomalies division participation of mesosomes in this process was not proved by the L-forms.