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.     

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.     

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.     

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 ).     

A novel method for differentiating L-form bacteria from their parental form using the Hucker Gram staining technique

L-forms of Enterococcus faecium, Bacillus subtilis and Pseudomonas syringae pv. phaseolicola were differentiated from their parent, cell-walled forms by a modified Gram staining technique. The addition of glutaraldehyde to the culture medium fixed the cells to prevent lysis of the L-forms. The cell-walled forms exhibited typical Gram staining reactions whereas the L-forms remained red due to the counterstain. L-forms were easily differentiated from cell-walled forms by their size and morphology which was made more obvious by the staining procedure. This is a very rapid and easy technique which distinguishes L-form bacteria from cell-walled organisms.     

Bacterial L-forms on tap: an improved methodology to generate Bacillus subtilis L-forms heralds a new era of research

Bacterial L-forms are cell wall-less forms of bacteria that usually grow with a conventional cell wall. Despite being important for research, L-forms are difficult to generate reproducibly and research in this area is challenging. Domínguez-Cuevas et al. (2011) report a method to rapidly, quantitatively and reproducibly generate populations of L-forms in Bacillus subtilis. Importantly, the methodology may be applicable to other bacteria heralding a new era of L-form research. Moreover, the genetic requirements of this method provide insights into how Lipid II synthesis and autolysin expression/activity are normally balanced and the central role of the WalRK two-component system in this process.     

聚合酶链反应-单链构型多态性检测细胞壁缺陷型淋病奈瑟菌cppB基因变异

目的：检测与分析淋病奈瑟菌L型的cppB基因,探讨细胞壁缺陷对淋病奈瑟菌cppB基因的影响。方法：用青霉素诱导淋病奈瑟菌成为L型并获得稳定L型纯培养物,用cppB基因特异性引物以聚合酶链反应(PCR)检测稳定L型纯培养物的cppB基因和进行单链构型多态性(SSCP)分析。结果：淋病奈瑟菌的细菌型及其L型都具有cppB基因扩增产物,但PCR—SSCP分析可见异常泳动DNA带型(细菌型有2条带、L型有3条带)。结论：细胞壁缺陷淋病奈瑟菌仍然具有cppB基因,但其碱基序列可以发生改变。     

尿路感染患者尿中细菌L型培养的意义

目的通过对尿路感染患者尿标本中的细菌（普通型和L型）检测,以确定L型菌检测的临床价值。方法采用一般培养法和特殊（L型菌）培养法对临床标本检测。结果364份标本中一般培养法检出细菌112株,阳性率为30．8％,而特殊培养法共培养细菌及L型菌共162株,细菌总检出率为44．5％,其中单独检出L型菌50株。结论细菌在一定条件下可演变成L型菌,增加临床标本中L型菌的培养可明显提高尿路感染的细菌阳性检出率。     

Use of transformation for studying the nature of formation of stable bacterial L forms]

The authors obtained a stable variant of the L-forms of Bacillus subtilis capable of exponential growth of the minimal and synthetic medium. An electron-microscopic study of different stages of the L-form formation was carried out by the method of ultra-thin sections. A possibility was shown of the transfer of the L-form formation sign by the method of transformation. DNA isolation from the L-forms by soft lysis considerably facilitated and simplified the genetic analysis of the L-form formation by the transformation method.     

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。     

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。     

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.     

农杆菌介导的玉米原位转化方法改良

原位转化是一种简便的植物转基因方法,在拟南芥中已经应用较为成熟,在玉米上的应用并不多见.本文在玉米开花期间,将含有目标载体pCAMBIA1301的农杆菌菌株LBA4404的细胞悬浮液直接涂抹到事先授粉约8小时后并去除掉苞叶和花丝的幼穗上.转化后种子的幼苗,经过gus活性染色和潮霉素基因(hpt)的PCR扩增,证实有部分的种子T-DNA整合进入了基因组.在潮霉素筛选后,有2.6 %抗性苗存活.其中57.7 %抗性植株表现出GUS阳性,相当于全部检测幼苗的1.5 %.这一结果通过潮霉素基因的PCR扩增,得到进一步的证实.     

VEGF、KDR和细菌L型感染在卵巢肿瘤中的表达及临床相关性研究

目的探讨血管内皮生长因子（VEGF）及其受体VEGFR-2（KDR）和细菌L型感染在卵巢肿瘤中的表达及临床相关性研究。方法应用免疫组化、原位杂交和革兰染色等方法检测了120例卵巢肿瘤中的VEGF、KDR蛋白及mRNA的表达,以及细菌L型的检出率。并对97例卵巢乳头状癌和23例卵巢乳头状瘤主要临床资料和病理分级参数进行比较,用χ^2检验进行统计学处理。结果VEGF、KDR蛋白及mRNA阳性表达率恶性肿瘤明显高于良性肿瘤（P〈0．005）。细菌L型检出阳性率与卵巢良、恶性肿瘤差异无显著性（P〉0．5）。VEGF、KDR蛋白及mRNA阳性表达以及细菌L型检出阳性率与卵巢乳头状癌的临床分期、病理分级和腹腔淋巴结有转移、腹水差异有显著性（P〈0．001—0．05）。细菌L型阳性患者中VEGF、KDR阳性明显高于L型阴性患者中阳性表达,2组差异具有非常显著性（P〈0．0001）。结论VEGF、KDR蛋白及mRNA在卵巢肿瘤中有不同程度的异常表达,与卵巢癌的临床分期、病理分级和浸润、转移呈正相关,L型感染极有可能成为诱发肿瘤因素之一,它们可能有协同致瘤作用。研究L型感染与卵巢肿瘤的关系,具有重要的临床应用价值。     

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.     

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.     

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.     

Artificially induced symbiotic associations of L-form bacteria and plants

A loysius , S. K. D. & P aton , A.M. 1984. Artificially induced symbiotic associations of L-form bacteria and plants. Journal of Applied Bacteriology 56 , 465–477.
L-forms of a small but varied selection of bacteria were found to enter viable plant cells and establish novel symbiotic relationships. Evidence is presented for the ability of the L-forms to spread and to express at least some of their metabolic functions within plant tissue.     

Antimicrobial activities of daunorubicin and adriamycin derivatives on bacterial and protoplast type L-form cells of Bacillus subtilis 170, Escherichia coli B, and Proteus mirabilis VI. Structure--activity relationship

The antibacterial activity of ten N-alkylated derivatives of daunorubicin and adriamycin as well as of 5-iminodaunorubicin has been tested by using Bacillus subtilis 170, Escherichia coli B, and Proteus mirabilis VI and their stable protoplast type L-forms in an agar diffusion test. Eight of the substances showed similar activities against B. subtilis and the L-forms of all test organisms, but no activity against the bacterial forms of E. coli and P. mirabilis. The cell wall of these gram-negative bacteria is responsible for this resistance by not allowing the antibiotics to enter the cells. The piperidino compound N-(CH2)5 daunorubicin shows 2-4 times higher activity against B. subtilis and all L-forms in comparison to daunorubicin and the other derivatives. Five of the substances were inactive against all test strains. Their inactivity seems to be associated with the larger substituents at the C-3' position. Relations between molecular structure and activity are discussed considering data about the interaction with DNA and the antitumor activity. Stable protoplast type L-forms and their bacterial forms represent a suitable and effective test system to screen for more effective substances and to get more information about their mode of action.