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.     

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.     

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.     

Envelope structure in three different L-forms ofProteus mirabilis

One A-type, stable and two different B-type, unstable L-forms were obtained from a strain ofProteus mirabilis and studied by electron microscopy and by chemical analysis for the presence of peptidoglycan. The wall of the parent bacterium is characterized by a profile of three superimposed dense lines and a content of 11.07 nmoles of muramic acid (MUR) and of 7.85 nmoles of diaminopimelic acid (DAP) per mg of dry weight. The stable, A-type L-form has completely lost the cell wall of the bacterium and is enveloped only by the plasma membrane to which very small quantities of peptidoglycan components are associated (MUR: 0.041 nmoles/mg; DAP: 0.075 nmoles/mg). The two B-type, unstable L-forms have the same wall structure in only two dense lines, but they differ in their peptidoglycan content. The first one does not contain more peptidoglycan components than the A-type, L-form (MUR: 0.022 nmoles/mg; DAP: 0.016 nmoles/mg), whereas the peptidoglycan content of the second one (MUR: 2.6 nmoles/mg; DAP: 1.65 nmoles/mg) is about one fifth of the content of muramic acid and diaminopimelic acid of the bacterial cell wall.     

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。     

The analysis of cell division and cell wall synthesis genes reveals mutationally inactivated ftsQ and mraY in a protoplast-type L-form of Escherichia coli

Cell division and cell wall synthesis are tightly linked cellular processes for bacterial growth. A protoplast-type L-form Escherichia coli, strain LW1655F+, indicated that bacteria can divide without assembling a cell wall. However, the molecular basis of its phenotype remained unknown. To establish a first phenotype-genotype correlation, we analyzed its dcw locus, and other genes involved in division of E. coli. The analysis revealed defective ftsQ and mraY genes, truncated by a nonsense and a frame-shift mutation, respectively. Missense mutations were determined in the ftsA and ftsW products yielding amino-acid replacements at conserved positions. FtsQ and MraY, obviously nonfunctional in the L-form, are essential for cell division and cell wall synthesis, respectively, in all bacteria with a peptidoglycan-based cell wall. LW1655F+ is able to survive their loss-of-functions. This points to compensatory mechanisms for cell division in the absence of murein sacculus formation. Hence, this L-form represents an interesting model to investigate the plasticity of cell division in E. coli, and to demonstrate how concepts fundamental for bacterial life can be bypassed.     

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.     

Glycerol Uptake Is Important for L-Form Formation and Persistence in Staphylococcus aureus

S. aureus is a significant human pathogen and has previously been shown to form cell wall deficient forms or L-forms in vitro and in vivo during infection. Despite many previous studies on S. aureus L-forms, the mechanisms of L-form formation in this organism remain unknown. Here we established the L-form model in S. aureus and constructed a transposon mutant library to identify genes involved in L-form formation. Screening of the library for mutants defective in L-form formation identified glpF involved in glycerol uptake being important for L-form formation in S. aureus. Consistent with this observation, glpF was found to be highly expressed in L-form S. aureus but hardly expressed in normal walled form. In addition, glpF mutant was found to be defective in antibiotic persistence. The defect in L-form formation and antibiotic persistence of the glpF mutant could be complemented by the wild type glpF gene. These findings provide new insight into the mechanisms of L-form formation and persistence in S. aureus and may have implications for development of new drugs targeting persisters for improved treatment.     

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.     

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.     

Induction of Enterococcal L-Forms by the Action of Lysozyme

Suspensions of enterococci were treated with lysozyme in the presence of osmotic stabilizers. The resulting osmotically fragile bodies prepared from Streptococcus faecium strain F24 and S. faecalis strain E1 gave rise to L-forms under optimal osmotic and nutritional conditions for treatment and subsequent growth. The most critical component of the growth medium, to obtain maximum yields, was the nature and concentration of the added salt. The two most effective salts were sodium chloride and ammonium chloride in the range of 2 to 3% (w/v) added to a suitable agar base. Ammonium chloride was more versatile, because it could be used with either sucrose or polyethylene glycol 4000 as the osmotic stabilizer for preparation and dilution of the osmotically fragile bodies. Sodium chloride would not consistently support growth of S. faecium F24 as L-forms when polyethylene glycol 4000 was used as the osmotic stabilizer during lysozyme treatment. Time-course studies of concurrent cell wall removal and L-form induction suggested that maximal induction required only cell wall damage rather than complete wall removal. This method for induction of L-forms from a suspension of enterococci is a significant improvement over other presently known methods.     

STREPTOCOCCAL L-FORMS IV. : Comparison of the Metabolic Rates of a Streptococcus and Derived L-Form.

Panos, Charles (University of Illinois College of Medicine, Chicago, and Albert Einstein Medical Center, Philadelphia, Pa.). Streptococcal L-forms. IV. Comparison of the metabolic rates of a Streptococcus and derived L-form. J. Bacteriol. 84:921-928. 1962.-Glycolytic rates of hexoses, amino sugars, pentoses, two-carbon compounds, and certain intermediates of glycolysis and the adaptive response to glucose of a group A Streptococcus and its derived L-form were compared. It was found that removal of the streptococcal cell wall did not result in the loss of the homolactic characteristic of the parent coccus or in a marked increase in the metabolism of certain glycolytic intermediates by the L-form. It was shown that (i) a major difference exists between the coccus and its L-form in the metabolism of glucosamine and N-acetylglucosamine; (ii) apparently, a loss of selectivity and internal control occurred in the transformation to the L-form; and (iii) this form, unlike the parent coccus, displayed an adaptive response to glucose. These data were not the result of an internal loss of essential cofactors or enzymes by diffusion from within the L-form. Nor could they be accounted for by dry-weight differences due to loss of the streptococcal cell wall. Finally, it was observed that the sonically disintegrated L-form in 0.5 m NaCl was capable of a glycolytic activity of 46% of that of the total intact culture. These data suggest that the conversion of a streptococcus to the L-form is accompanied by an alteration in carbohydrate metabolism as well as the loss of the cell wall. Previously reported data are in agreement with these findings and support the conclusion that the resulting form is not merely a bacterial cell without a rigid cell wall.     

Identification with the aid of various serological reactions of L forms of streptococci isolated from the live organism]

Growth inhibition, agglutination, precipitation, and passive hemagglutination tests were used for the identification of the L-forms of streptococci isolated from the organism of experimental rabbits both after the infection with the L-forms of streptococci and with the streptococci of group A. The tests were positive not only with the antiserum of homologous, but also of heterologous strains of the L-form of streptococcus, group A. The L-form cultures isolated from the experimental animals failed to differ from the laboratory strain of the L-forms of streptococcus, group A, by serological properties.     

Unique biological properties of Mycobacterium tuberculosis L-form variants: impact for survival under stress

Bacteria can, under certain conditions, enter into a cell-less state known as L-form conversion. This phenomenon is universal, but also recognized with difficultly by microbiologists. The current study addresses several aspects concerning the ability of tubercle bacilli to use L-form conversion as a unique adaptive strategy to survive and reproduce under unfavorable conditions. Nutrient starvation of M. tuberculosis in vitro followed by passages in Middlebrook 7H9 semisolid medium was used for stress induction and the selective isolation of mycobacterial L-form variants. Light and electron microscopy images evidence the peculiar characteristics of mycobacterial L-forms. For example, mycobacterial L-forms were observed to lose their acid-fastness and change their morphology. In addition, wide morphological variability, the presence of large and elementary bodies, coccoids and small granular forms, as well as the appearance of unusual modes of irregular cell division were observed. Unlike classical tubercle bacilli, L-form variants grew and developed typical "fried-egg" colonies faster. L-forms were verified as M. tuberculosis by spoligotyping. The results provide insights into the nature of L-form phenomena in M. tuberculosis and link them to the mechanisms allowing mycobacterial survival under stress.     

Characterization of a stable L-form of the fish pathogen Ameromonas salmonicida

A stable L-form of Aeromonas salmonicida , which resulted form induction with benzylpenicillin, contained more of an outer membrane protein, with an estimated molecular wight of 40 kDza but less of 47.9 and 38 kDa proteins, than did parental walled cells. In addition from Western blots, two protein bands reacted strongly with a polyclonal antiserum. The antiserum did not react demonstrably with teh band detect in the L-forms on the gels.     

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.     

Crucial role for membrane fluidity in proliferation of primitive cells

The cell wall is a defining structural feature of the bacterial subkingdom. However, most bacteria are capable of mutating into a cell-wall-deficient "L-form" state, requiring remarkable physiological and structural adaptations. L-forms proliferate by an unusual membrane deformation and scission process that is independent of the conserved and normally essential FtsZ based division machinery, and which may provide a model for the replication of primitive cells. Candidate gene screening revealed no requirement for the cytoskeletal systems that might actively drive membrane deformation or scission. Instead, we uncovered a crucial role for branched-chain fatty acid (BCFA) synthesis. BCFA-deficient mutants grow and undergo pulsating shape changes, but membrane scission fails, abolishing the separation of progeny cells. The failure in scission is associated with a reduction in membrane fluidity. The results identify a step in L-form proliferation and demonstrate that purely biophysical processes may have been sufficient for proliferation of primitive cells.     

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。     

Cell fusion between L-forms and protoplasts of Staphylococcus aureus

Mixtures of various combinations of Lysostaphin protoplasts and stable L-forms of Staphylococcus aureus, which have different markers for drug resistance, were treated with polyethylene glycol (PEG) to examine the development of doubly resistant fusion products (fusants). To recover doubly resistant colonies as L-forms, they were incubated in 4.5% NaCl-brain heart infusion (BHI) broth containing penicillin G (PCG) for enrichment culture and cultured in PCG-4.5% NaCl-BHI agar medium (method 1), while to recover doubly resistant fusants as L-forms and coccal forms, they were grown on reversion medium (R medium) which causes reversion of protoplasts or fusants to parent type cells, and then cultured on assay media, i.e., R medium, BHI agar medium or PCG-4.5% NaCl-BHI agar medium (method 2). Under both experimental conditions, doubly resistant fusants developed as L-form cells by PEG treatment of pairs of protoplasts carrying the chloramphenicol (CP)-resistance plasmid and L-forms having chromosomal resistance to streptomycin (SM). In the reverse combinations, i.e., protoplasts showing chromosomal SM-resistance and L-form cells carrying the CP-resistance plasmid, the first method gave no doubly resistant colonies. By the second method, without enrichment culture on R medium, the latter combination gave doubly resistant fusants as L-form, coccal-type and mixed-type colonial forms, while when the PEG-treated mixture was enriched on R medium, fusants were obtained exclusively as the coccal type on either R medium or BHI agar assay medium. Neither of the methods yielded colonies of doubly resistant fusants on PEG-treatment of pairs of protoplasts and L-forms both of which were chromosomal, but with different drug resistances. These results show that PEG-induced cell fusion between protoplasts and L-forms of S. aureus, unlike the fusion between protoplasts or between L-forms, resulted in transfer of the drug resistance controlled by the plasmid to the fusion products. The fusants obtained were L-forms in method 1, and coccal type in the method 2.     

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。