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.     

细胞壁缺陷对白喉棒状杆菌致病性影响的研究

检测白喉棒状杆菌稳定L型对动物的致病性,探讨细胞壁缺陷对白喉棒状杆菌致病性的影响及其可能的分子机制。采用氨苄青霉素在非高渗培养基内诱导并获得产毒性白喉棒状杆菌稳定L型纯培养物。收集白喉棒状杆菌稳定L型纯培养物及其代谢产物,将收集的高于细菌型10 000倍浓度的白喉棒状杆菌稳定L型纯培养物及其代谢产物皮内注射家兔,观察局部注射部位皮肤或全身的病理改变。分别采用对流免疫电泳(CIEP)和SDS-不连续聚丙烯酰胺凝胶电泳(SDS-PAGE)检测白喉棒状杆菌稳定L型可溶性代谢产物中的白喉毒素蛋白质。结果显示,白喉棒状杆菌稳定L型不能引起动物局部或全身发生异常表现,在其可溶性代谢产物中并未检测到白喉毒素蛋白质。提示细胞壁缺陷变异可影响白喉棒状杆菌产生白喉毒素蛋白质,从而使其丧失了产生外毒素致病的作用。     

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.     

Development of protoplasts of Streptomycetes on media favoring the regeneration and formation of L-forms

The protoplasts of three Streptomyces species and their regenerative ability were studied using light microscopy. When Streptomyces lividans and S. erythraeus protoplasts are cultivated on regeneration media, their regeneration is not synchronous during the first day; some protoplasts revert to yield the mycelial form and also L-forms of these cultures are produced. If the protoplasts are transferred to a medium inducing L-forms, they grow and multiply for a long time with the production of L-form colonies. This process is maintained if S. lividans L-form cells are passaged on the medium inducing L-forms, but the protoplasts revert to yield the mycelial form on the regeneration medium.     

Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria

Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.     

Morphology, growth and reversion in a stable L-form of Escherichia coli K12

An L-form isolated from Escherichia coli K12 by sequential treatment with N-methyl-N'-nitro-N-nitrosoguanidine and lysozyme was adapted to grow in hyperosmolar liquid cultures. It was stable in the absence of antibiotic when cultured in brain heart infusion (BHI) broth containing NaCl and CaCl2, the optimal concentrations being 0.34 M and 1 mM, respectively. No growth of the L-form was observed when CaCl2 was not added to BHI medium containing 0.34 M-NaCl. On the other hand, when KCl replaced NaCl as the osmotic stabilizer, growth of the L-form was repressed in the presence of CaCl2. Electron microscopy of the L-form confirmed the absence of a cell wall. A revertant strain derived from the L-form grew as a stable bacillary form in BHI medium without osmotic stabilizer. The growth characteristics of the revertant strain resembled those of the parent strain. The revertant strain produced L-forms in the presence of NaCl.     

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.     

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.     

Antigenic characteristics of the L forms of Streptococcus group B

Stable L-forms of group B streptococci (GBS) have been obtained and their antigenic features have been studied by the serological methods (the passive hemagglutination test, the aggregate agglutination test, the gel diffusion test), as well as by using ferritin and peroxidase labels with the subsequent electron microscopy. The use of the serological methods has made it possible to reveal the antigenic differences between the stable L-forms of GBS and their bacterial forms. Specific antigenic substances can be found in the supernatant fluid obtained after the sedimentation of the ultrasonically disintegrated cellular mass of streptococcal L-forms and bacterial cultures. The use of ferritin and peroxidase labels has revealed the specificity of GBS L-form antigen and its localization on the cytoplasmic membrane of all L-form structural elements.     

The chemical composition of the membranes of protoplasts and L-forms of Staphylococcus

Membrane fractions were prepared from Staphylococcus aureus H and 100 after dissolution of the cell walls by a lytic enzyme from Streptomyces griseus. Membranes were also prepared from the L-forms derived from the same strains. The membranes were analysed for protein, lipid, carbohydrate and RNA contents, and the fatty acid composition of the lipids was determined. A branched-chain saturated C(15) acid was the major component in all samples, and the correspondence between L-forms and parent bacteria was fairly close. The lipids were separated into non-polar-lipid, glycolipid and phospholipid fractions; the L-forms contained a little more neutral lipid and much more glycolipid than the parent bacteria. In all membranes the glycolipid, which accounted for all the carbohydrate present, was a diglucosyl diglyceride. The major phospholipids of the protoplast membranes were phosphatidylglycerol and some lipoamino acids (lysine and a little alanine). On the other hand, diphosphatidylglycerol was the chief phospholipid found in L-form membranes.     

细菌L型的厌氧诱导和培养

厌氧条件下以羧卡青霉素诱导金黄色葡萄球菌、大肠杆菌和蜡样芽胞杆菌形成Ｌ型,观察细菌Ｌ型在厌氧条件下的形成、形态、生长及时渗透压的敏感性等特性。结果表明：蜡样芽胞杆菌在厌氧条件下不能形成Ｌ型或其Ｌ型在厌氧条件下亦不能返祖。金黄色葡萄球菌和大肠杆菌在厌氧条件下虽能诱生Ｌ型,但形成丝状体的构成Ｌ型菌落难以传代培养,厌氧培养未见Ｌ型圆球体和典型Ｌ型油煎蛋样菌落。金黄色葡萄球菌Ｌ型在含１％～１０％ＮａＣｌ的Ｌ型培养基上可生长形成Ｌ型菌落或非菌落形式存在的Ｌ型巨形体;大肠杆菌和蜡样芽胞杆菌的Ｌ型在含２％～６％ＮａＣｌ的Ｌ型培养基上可生长形成Ｌ型菌落或非菌落形式存在的Ｌ型巨形体。涂片染色或返祖试验证实细菌Ｌ型在含０．５％ＮａＣｌ的Ｌ型培养基或常规细菌学培养基上亦可生存。非菌落性Ｌ型巨形体和丝形体是细菌Ｌ型在琼脂培养基上广泛的存在形式。     

从医院使用的新洁尔灭溶液中分离出细菌L型的报告

本文报道,作者采用高渗和等渗牛肉汤试管及平板培养法,从某医院正在使用的新洁尔灭器械浸泡液中分离出4株细菌L型,其中金黄色葡萄球菌L型1株,表皮葡萄球菌L型2株,类白喉杆菌L型1株。上述细菌经形态观察,细胞壁染色,返祖鉴定等一系列细菌L型鉴定程序;并通过电镜观察,菌体细胞图像分析。其结果均提示,细菌L型与原菌之间存在明显差异。作者认为,新洁尔灭器械浸泡液,消毒灭菌的不彻底性,是造成术后感染的重要因素。     

Polyacrylamide Gel Identification of Bacterial L-Forms and Mycoplasma Species of Human Origin

Polyacrylamide gel electrophoretic patterns of acidified phenol extracts prepared from whole cells can be used for the identification of bacterial L-forms and Mycoplasma species of human origin. Ten human Mycoplasma serotypes and eight L-forms belonging to five different genera were studied. The gel patterns were sufficiently distinct and reproducible that it was possible not only to identify L-forms at the genus level (group with streptococci) and different Mycoplasma serotypes but also to differentiate between the two of them. The parentage of L-forms of Streptobacillus moniliformis L1, Listeria monocytogenes, Streptococcus MG, and Staphylococcus aureus Smith strain was established by relating their gel patterns directly to parent bacteria. It was found that an L-form designated S. moniliformis An (ATCC 14220) was actually an L-form of Proteus. In addition, it was shown electrophoretically that no relationship existed between the Streptococcus MG L-form and M. pneumoniae. The applicability of this method as a diagnostic and taxonomic tool for the differentiation of L-forms and mycoplasmas is discussed.     

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.     

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.     

慢性肾盂肾炎L型细菌感染及耐药分析

目的了解L型细菌在慢性肾盂肾炎的感染及耐药状况。方法对71例患者清洁中段尿做普通细菌培养(B型)、L型细菌培养(L型)及耐药分析。结果细菌阳性率为77.5%,其中单独L型阳性率为49.3%、B型与L型混合感染为15.5%,而B型阳性率仅为12.7%。主要是大肠埃希菌,其次是葡萄球菌;青霉素及头孢噻肟均有较高的耐药率(88.9%及73.6%)。结论L型细菌在慢性肾盂肾炎感染中占主导,β-内酰胺类药物有较高的耐药性,临床治疗应据药敏结果合理选择及时调整抗生素。     

Growth and adhesion of Enterococcus faecium L-forms

Abstract 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.     

CCD detection of lux-marked Pseudomonas syringae pv. phaseolicola L-forms associated with Chinese cabbage and the resulting disease protection against Xanthomonas campestris

R.N. WATERHOUSE, H. BUHARIWALLA, D. BOURN, E.J. RATTRAY AND L.A. GLOVER. 1996. CCD image-enhancement was used to detect lux -marked Pseudomonas syringae pv. phaseolicola L-form bacteria in association with Chinese cabbage. Bioluminescence from colonizing bacteria was maximal 8 d after association. Bioluminescing L-forms were reisolated from 22 d post-association tissue. L-forms but not parental, cell-walled forms, were effective at inducing protection against a heterologous pathogen ( Xanthomonas campestris ).     

Survival of Escherichia coli under lethal heat stress by L-form conversion

Transition of bacteria to cell wall deficient L-forms in response to stress factors has been assumed as a potential mechanism for survival of microbes under unfavorable conditions. In this article, we provide evidence of paradoxal survival through L-form conversion of E. coli high cell density population after lethal treatments (boiling or autoclaving). Light and transmission electron microscopy demonstrated conversion from classical rod to polymorphic L-form shape morphology and atypical growths of E. coli. Microcrystal formations observed at this stage were interpreted as being closely linked to the processes of L-form conversion and probably involved in the general phenomenon of protection against lethal environment. Identity of the morphologically modified L-forms as E. coli was verified by species specific DNA-based test. Our study might contribute to a better understanding of the L-form phenomenon and its importance for bacterial survival, as well as provoke reexamination of the traditional view of killing strategies against bacteria.