Characterization of the L-form switch in the Gram-negative pathogen Streptobacillus moniliformis.

Almost all major classes of bacteria are surrounded by a peptidoglycan cell wall, which is a crucial target for antibiotics. It is now understood that many bacteria can tolerate loss of the cell wall provided that they are in an isotonic environment. Furthermore, in some cases the cells can continue to proliferate in a state known as the L-form. L-form proliferation occurs by an unusual blebbing or tubulation mechanism that is completely independent of the normally essential division machine or cell wall synthetic enzymes, and is resistant to cell wall-active antibiotics. However, the growth is limited by reactive oxygen species generated by the respiratory chain pathway. In this work, we examined the walled to L-form transition in a pathogenic Gram-negative bacterium, Streptobacillus moniliformis, which naturally lacks the respiratory chain pathway, under aerobic conditions. L-form-like cells often emerged spontaneously, but proliferation was not observed unless the cells were treated with cell wall-active antibiotics. Time-lapse imaging revealed that cell division of S. moniliformis L-forms involves unusual membrane dynamics with an apparent imbalance between outer membrane and cytoplasmic volume growth. The results suggest that outer membrane expansion may be an important general factor for L-form proliferation of diderm bacteria.

Geometric principles underlying the proliferation of a model cell system.

Many bacteria can form wall-deficient variants, or L-forms, that divide by a simple mechanism that does not require the FtsZ-based cell division machinery. Here, we use microfluidic systems to probe the growth, chromosome cycle and division mechanism of Bacillus subtilis L-forms. We find that forcing cells into a narrow linear configuration greatly improves the efficiency of cell growth and chromosome segregation. This reinforces the view that L-form division is driven by an excess accumulation of surface area over volume. Cell geometry also plays a dominant role in controlling the relative positions and movement of segregating chromosomes. Furthermore, the presence of the nucleoid appears to influence division both via a cell volume effect and by nucleoid occlusion, even in the absence of FtsZ. Our results emphasise the importance of geometric effects for a range of crucial cell functions, and are of relevance for efforts to develop artificial or minimal cell systems.

Stress-induced formation of cell wall-deficient cells in filamentous actinomycetes.

The cell wall is a shape-defining structure that envelopes almost all bacteria and protects them from environmental stresses. Bacteria can be forced to grow without a cell wall under certain conditions that interfere with cell wall synthesis, but the relevance of these wall-less cells (known as L-forms) is unclear. Here, we show that several species of filamentous actinomycetes have a natural ability to generate wall-deficient cells in response to hyperosmotic stress, which we call S-cells. This wall-deficient state is transient, as S-cells are able to switch to the normal mycelial mode of growth. However, prolonged exposure of S-cells to hyperosmotic stress yields variants that are able to proliferate indefinitely without their cell wall, similarly to L-forms. We propose that formation of wall-deficient cells in actinomycetes may serve as an adaptation to osmotic stress.

Effects of Thermoextracts of Brucella S and L Forms on Lipid Peroxidation and Antioxidant Defense in Organs of Laboratory Animals.

The dynamics of LPO marker malondialdehyde formation and peroxidase-destroying activity was studied in homogenized organs of guinea pigs, immunized with thermoextracts from S and L forms Brucella abortus I-206. The L form brucella thermoextract exhibited a lower reactogenicity and adequately activated the antioxidant system, due to which the destructive effects of ROS could be partially neutralized during the vaccinal process.

L-form bacteria, chronic diseases and the origins of life.

The peptidoglycan cell wall is widely conserved across the bacterial domain, suggesting that it appeared early in the evolution of bacteria. It is normally essential but under certain conditions wall-deficient or 'L-form' bacteria can be isolated. In Bacillus subtilis this normally requires two genetic changes. The first, exemplified by mutations shutting down wall precursor synthesis, works by increasing membrane synthesis. This promotes the unusual form of proliferation used by L-forms, involving a range of relatively disorganized membrane blebbing or vesiculation events. The secondary class of mutations probably work by relieving oxidative stress that L-forms may incur due to their unbalanced metabolism. Repression or inhibition of cell wall precursor synthesis can stimulate the L-form transition in a wide range of bacteria, of both Gram-positive and -negative lineages. L-forms are completely resistant to most antibiotics working specifically on cell wall synthesis, such as penicillins and cephalosporins, consistent with the many reports of their involvement in various chronic diseases. They are potentially important in biotechnology, because lack of a wall can be advantageous in a range of production or strain improvement applications. Finally, L-forms provide an interesting model system for studying early steps in the evolution of cellular life.This article is part of the themed issue 'The new bacteriology'.

Presence of mycobacterial L-forms in human blood: Challenge of BCG vaccination.

Possible persistence of bacteria in human blood as cell wall deficient forms (L-forms) represents a top research priority for microbiologists. Application of live BCG vaccine and L-form transformation of vaccine strain may display a new intriguing aspect concerning the opportunity for occurrence of unpredictable colonization inside the human body by unusual microbial life forms. L-form cultures were isolated from 141 blood samples of people previously vaccinated with BCG, none with a history of exposure to tuberculosis. Innovative methodology to access the unusual L-form elements derived from human blood was developed. The methodology outlines the path of transformation of non- cultivable L-form element to cultivable bacteria and their adaptation for growth in vitro. All isolates showed typical L-forms growth features ("fried eggs" colonies and biofilm). Electron microscopy revealed morphology evidencing peculiar characteristics of bacterial L-form population (cell wall deficient polymorphic elements of variable shape and size). Regular detection of acid fast bacteria in smears of isolated blood L-form cultures, led us to start their identification by using specific Mycobactrium spp. genetic tests. Forty five of 97 genetically tested blood cultures provided specific positive signals for mycobacteria, confirmed by at least one of the 3 specific assays (16S rRNA PCR; IS6110 Real Time PCR and spoligotyping). In conclusion, the obtained genetic evidence suggests that these L-forms are of mycobacterial origin. As the investigated people had been vaccinated with BCG, we can assume that the identified mycobacterial L-forms may be produced by persisting live BCG vaccine.

Stress-induced L-forms of Mycobacterium bovis: a challenge to survivability.

This study addressed the ability of Mycobacterium bovis to produce unusual extreme morphologic forms (cell wall-deficient or L-forms) under stress conditions. Models using nutrient starvation and cryogenic stress treatments of Mycobacterium bovis, as well as the filtration technique followed by cultivation in semisolid medium, were used for isolation of L-form variants. Morphological transformations and developmental stages, typical for the bacterial L-cycle were observed by electron microscopy. Of special interest was the formation of giant filaments and common extremely thick membranous structures enveloping the entire L-form population. Following collapse of giant filamentous structures small viable cell elements, mainly granules and coccobacilli, were released and proved able to grow into large bodies or multiply by fission or budding. Derivation of viable filterable forms from L-form cultures and parental strain and their identification as Mycobacterium bovis based on specific IS6110 PCR was noteworthy. We suggest that formation of giant filaments and thick common membranous envelopes, observed under stress conditions, may serve a twofold purpose - protection against an unfavourable environment, and a role in reproduction of Mycobacterium bovis L-forms. The observed L-form conversion phenomenon in Mycobacterium bovis seems to be associated with an adaptive strategy of this pathogen for survival and reproduction in an unfavorable environment.

Formation and characterization of non-growth states in Clostridium thermocellum: spores and L-forms.

BACKGROUND: Clostridium thermocellum is an anaerobic thermophilic bacterium that exhibits high levels of cellulose solublization and produces ethanol as an end product of its metabolism. Using cellulosic biomass as a feedstock for fuel production is an attractive prospect, however, growth arrest can negatively impact ethanol production by fermentative microorganisms such as C. thermocellum. Understanding conditions that lead to non-growth states in C. thermocellum can positively influence process design and culturing conditions in order to optimize ethanol production in an industrial setting. RESULTS: We report here that Clostridium thermocellum ATCC 27405 enters non-growth states in response to specific growth conditions. Non-growth states include the formation of spores and a L-form-like state in which the cells cease to grow or produce the normal end products of metabolism. Unlike other sporulating organisms, we did not observe sporulation of C. thermocellum in low carbon or nitrogen environments. However, sporulation did occur in response to transfers between soluble and insoluble substrates, resulting in approximately 7% mature spores. Exposure to oxygen caused a similar sporulation response. Starvation conditions during continuous culture did not result in spore formation, but caused the majority of cells to transition to a L-form state. Both spores and L-forms were determined to be viable. Spores exhibited enhanced survival in response to high temperature and prolonged storage compared to L-forms and vegetative cells. However, L-forms exhibited faster recovery compared to both spores and stationary phase cells when cultured in rich media. CONCLUSIONS: Both spores and L-forms cease to produce ethanol, but provide other advantages for C. thermocellum including enhanced survival for spores and faster recovery for L-forms. Understanding the conditions that give rise to these two different non-growth states, and the implications that each has for enabling or enhancing C. thermocellum survival may promote the efficient cultivation of this organism and aid in its development as an industrial microorganism.

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.

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.

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.

Persistence of Staphylococcus aureus L-form during experimental lung infection in rats.

The course of pulmonary infection in rats infected by intranasal inoculation with a Staphylococcus aureus stable protoplast L-form was studied. Blood and bronchoalveolar samples were taken on days 3, 7, 14 and 30 after challenge and were investigated by microbiological, electron-microscopic, cytochemical and cytometric methods. The electron microscopic data and isolation of L-form cultures from bronchoalveolar samples at all experimental times demonstrated the ability of S. aureus L-form cells to internalize, replicate and persist in the lungs of infected rats to the end of the observation period, in contrast to the S. aureus parental form. It was found that persisting L-form evoked ineffectual phagocytose by alveolar macrophages and low but long-lasting inflammatory reaction in rats. The experimental model of pulmonary infection with S. aureus L-form suggests that the cell-wall-deficient bacterial forms may be involved in the pathogenesis of chronic and latent lung infections.

[Salmonella typhimurium population in water environment under the influence of temperature]. / Sostoianie pipuliatsii Salmonella typhimurium v vodnoi srede pod vliianiem temperatury.

The strategy of the adaptation of S. typhimurium population to water environment under the influence of temperature factor was studied by scanning electron microscopy. Salmonellae were found to adhere to the surface of the Daphnia chitin covering. The study revealed that S. typhimurium population existed in water in the form of covered microcolonies as well as in the form of spheroplast-type cells and small cells in the L-form, joined with bands. The viability of salmonellae in water environment was studied without interaction and following interaction with Daphnia.

Cell wall-deficient (CWD) bacterial pathogens: could amylotrophic lateral sclerosis (ALS) be due to one?

Recently, a number of diseases that had been thought previously to be caused by something other than an infectious agent are now known to be caused by bacteria. It now appears that it is not uncommon that bacteria, viruses, or fungi can cause diseases even when these organisms have not been detected or cultured. The most recent, well-publicized case is that of stomach ulcers; these are largely due to Helicobacter pylori infections. Here, the possibility is explored that amylotrophic lateral sclerosis (ALS) is caused by a cell wall-deficient microorganism.

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.

[Observations of properties of the L-form of M. tuberculosis induced by the antituberculosis drugs].

OBJECTIVES: To investigate the mechanism of generation of L-form of M. tuberculosis and its significance on the development, diagnosis and treatment of tuberculosis. METHODS: M. tuberculosis was inoculated into the non-high osmotic medium with rifampin, isoniazid or ethambutol and then the L-form was observed by microscopy daily. The cultures were filtrated to get the pure cultures of stable L-form by subculture with the non-high osmotic medium and characteristics of morphology, growth, susceptibilities to the antibacterial drugs and the special gene of M. tuberculosis were observed when the pure subcultures of the L-form were isolated. RESULTS: L-form of M. tuberculosis was induced by the concentrations of routine inhibition test of rifampin, isoniazid or ethambutol. The L-form would not be susceptible to the above mentioned antituberculosis drugs but susceptible to streptomycin, erythromycin, ofloxacin, norfloxacin and others. The morphologies of L-form were irregular or spherical with single, paired or chain form, and growth under the bottom of the medium but not movement or adhere to the glass. The L-form was negative by acid-fast stain and negative or positive by Gram stain. The gene of L-form reacted with the PCR kit for the M. tuberculosis and showed the same band. CONCLUSIONS: M. tuberculosis could be killed by rifampin, isoniazid or ethambutol but also could be induced to become the L-form by these antituberculosis drugs, and it is one of the important factor that affecting the effect of treatment of the tuberculosis. The cell wall deficient variants of M. tuberculosis could be determined by the PCR of M. tuberculosis. It is recommended that the L-forms should be noticed during the treatment with the antituberculosis drugs and combination treatment with antituberculous drugs to which the L-forms were susceptible, is also very important.

Atypical behaviour and survival of Streptococcus pyogenes L forms during intraperitoneal infection in rats.

Groups of rats were injected intraperitoneally with cell wall-deficient (L) forms of Streptococcus pyogenes, with their parental (S) forms, as well as with a combined inoculum of both forms (S+L). Peritoneal exudate samples were harvested on days 1, 3, 7, 15 and 30 after challenge and were investigated by microbiological, electron microscopic, cytometric and biochemical methods. Parental S forms were isolated from peritoneal exudate samples up to day 15 post infection, while L form cultures were isolated until the end of the examined interval. Electron microscopic examination revealed continuous adhesion of L forms on the macrophage surface as well as intracellular persistence inside them. It was demonstrated that the intraperitoneal inflammatory response to L form infection was higher than to the other infections and the monocyte-macrophage populations were predominant. The established atypical behaviour and long survival of S. pyogenes L forms in the rat's peritoneum could explain some of the mechanisms of the pathogens' persistence as well as the reasons for chronic streptococcal infections.

Cell wall-deficient forms (L-forms) of Listeria monocytogenes in experimentally infected rats.

Experimental infections were induced with different bacterial forms of Listeria monocytogenes: parental (S-forms), protoplastic (L-forms) and combined inoculum of both forms by i.p. injection of rats. The parental bacterial forms (S-forms) were isolated up to 7 days after challenge from the peritoneal cavity and the liver, while the L-forms were isolated up to 60 days from the peritoneal cavity. Continuous adhesion of L-forms on the peritoneal macrophage surface was found by scanning-electron microscopy. Erythrocyte and leucocyte count as well as some clinical chemistry parameters were measured during infections. They showed different dynamics in the three experimental groups. Histomorphological changes in the liver (microabscesses and mononuclear cellular granulomas) of infected animals were observed. They were less intensive and appeared later in rats infected with L-forms. The experiments demonstrated that infections caused by parental bacterial forms and by combined inoculum took an acute course, while the infection caused by L-forms could be distinguished as a prolonged and persistent one.