Factors That Affect the Enlargement of Bacterial Protoplasts and Spheroplasts.

Cell enlargement is essential for the microinjection of various substances into bacterial cells. The cell wall (peptidoglycan) inhibits cell enlargement. Thus, bacterial protoplasts/spheroplasts are used for enlargement because they lack cell wall. Though bacterial species that are capable of gene manipulation are limited, procedure for bacterial cell enlargement does not involve any gene manipulation technique. In order to prevent cell wall resynthesis during enlargement of protoplasts/spheroplasts, incubation media are supplemented with inhibitors of peptidoglycan biosynthesis such as penicillin. Moreover, metal ion composition in the incubation medium affects the properties of the plasma membrane. Therefore, in order to generate enlarged cells that are suitable for microinjection, metal ion composition in the medium should be considered. Experiment of bacterial protoplast or spheroplast enlargement is useful for studies on bacterial plasma membrane biosynthesis. In this paper, we have summarized the factors that influence bacterial cell enlargement.

Spheroplast-Mediated Carbapenem Tolerance in Gram-Negative Pathogens.

Antibiotic tolerance, the ability to temporarily sustain viability in the presence of bactericidal antibiotics, constitutes an understudied and yet potentially widespread cause of antibiotic treatment failure. We have previously shown that the Gram-negative pathogen Vibrio cholerae can tolerate exposure to the typically bactericidal ß-lactam antibiotics by assuming a spherical morphotype devoid of detectable cell wall material. However, it is unclear how widespread ß-lactam tolerance is. Here, we tested a panel of clinically significant Gram-negative pathogens for their response to the potent, broad-spectrum carbapenem antibiotic meropenem. We show that clinical isolates of Enterobacter cloacae, Klebsiella aerogenes, and Klebsiella pneumoniae, but not Escherichia coli, exhibited moderate to high levels of tolerance of meropenem, both in laboratory growth medium and in human serum. Importantly, tolerance was mediated by cell wall-deficient spheroplasts, which readily recovered wild-type morphology and growth upon removal of antibiotic. Our results suggest that carbapenem tolerance is prevalent in clinically significant bacterial species, and we suggest that this could contribute to treatment failure associated with these organisms.

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.

Enlargement of Deinococcus grandis spheroplasts requires Mg2+ or Ca2.

While the cell wall strictly controls cell size and morphology in bacteria, spheroplasts lack cell walls and can become enlarged in growth medium under optimal conditions. Optimal conditions depend on the bacterial species. We frequently observed extreme enlargement of spheroplasts of the radiation-resistant bacterium Deinococcus grandis in Difco Marine Broth 2216, but not in TGY broth (a commonly used growth medium for Deinococcus). Thorough investigation of media components showed that the presence of Mg2+ or Ca2+ promoted extreme spheroplast enlargement, synthesizing the outer membrane. Our findings strongly suggest that Mg2+ or Ca2+ enlarges spheroplasts, which could change the lipid composition of the spheroplast membrane.

DivIVA concentrates mycobacterial cell envelope assembly for initiation and stabilization of polar growth.

In many model organisms, diffuse patterning of cell wall peptidoglycan synthesis by the actin homolog MreB enables the bacteria to maintain their characteristic rod shape. In Caulobacter crescentus and Escherichia coli, MreB is also required to sculpt this morphology de novo. Mycobacteria are rod-shaped but expand their cell wall from discrete polar or subpolar zones. In this genus, the tropomyosin-like protein DivIVA is required for the maintenance of cell morphology. DivIVA has also been proposed to direct peptidoglycan synthesis to the tips of the mycobacterial cell. The precise nature of this regulation is unclear, as is its role in creating rod shape from scratch. We find that DivIVA localizes nascent cell wall and covalently associated mycomembrane but is dispensable for the assembly process itself. Mycobacterium smegmatis rendered spherical by peptidoglycan digestion or by DivIVA depletion are able to regain rod shape at the population level in the presence of DivIVA. At the single cell level, there is a close spatiotemporal correlation between DivIVA foci, rod extrusion and concentrated cell wall synthesis. Thus, although the precise mechanistic details differ from other organisms, M. smegmatis also establish and propagate rod shape by cytoskeleton-controlled patterning of peptidoglycan. Our data further support the emerging notion that morphology is a hardwired trait of bacterial cells.

Opportunities and challenges in the development of Cutaneotrichosporon oleaginosus ATCC 20509 as a new cell factory for custom tailored microbial oils.

Cutaneotrichosporon oleaginosus ATCC 20509, previously known as Trichosporon oleaginosus, Cryptococcus curvatus, Apiotrichum curvatum or Candida curvata D is an oleaginous yeast with several favorable qualities: it is fast growing, accumulates high amounts of lipid and has a very broad substrate spectrum. Its resistance to hydrolysis byproducts and genetic accessibility make it a promising cell factory for custom tailored microbial oils. However, literature about this organism is of varying degree of quality. Moreover, due to numerous changes of the species name, reports are highly scattered and poorly cited. This led to a poor integration of the findings into a unified body of knowledge. Particularly, errors in strain name usage and consequently citation are found even in most recent literature. To simplify future work, this review provides an overview of published studies and main findings regarding the metabolic capacities of C. oleaginosus.

Endonucleolytic cleavage in the expansion segment 7 of 25S rRNA is an early marker of low-level oxidative stress in yeast.

The ability to detect and respond to oxidative stress is crucial to the survival of living organisms. In cells, sensing of increased levels of reactive oxygen species (ROS) activates many defensive mechanisms that limit or repair damage to cell components. The ROS-signaling responses necessary for cell survival under oxidative stress conditions remain incompletely understood, especially for the translational machinery. Here, we found that drug treatments or a genetic deficiency in the thioredoxin system that increase levels of endogenous hydrogen peroxide in the yeast Saccharomyces cerevisiae promote site-specific endonucleolytic cleavage in 25S ribosomal RNA (rRNA) adjacent to the c loop of the expansion segment 7 (ES7), a putative regulatory region located on the surface of the 60S ribosomal subunit. Our data also show that ES7c is cleaved at early stages of the gene expression program that enables cells to successfully counteract oxidative stress and is not a prerequisite or consequence of apoptosis. Moreover, the 60S subunits containing ES7c-cleaved rRNA cofractionate with intact subunits in sucrose gradients and repopulate polysomes after a short starvation-induced translational block, indicating their active role in translation. These results demonstrate that ES7c cleavage in rRNA is an early and sensitive marker of increased ROS levels in yeast cells and suggest that changes in ribosomes may be involved in the adaptive response to oxidative stress.

Requirement of dark culture condition for enlargement of spheroplasts of the aerobic anoxygenic photosynthetic marine bacterium Erythrobacter litoralis.

In the present study, spheroplasts from the aerobic anoxygenic photosynthetic marine bacterium Erythrobacter litoralis were generated and cultivated. In the presence of penicillin, the spheroplasts grew and enlarged in marine broth without undergoing cell division. However, continuous light inhibited their enlargement, and they were therefore cultivated in the dark. Cellular DNA was quantified at various time points (0, 24, and 48 h) and temperatures (20°C, 25°C, and 30°C) using real-time quantitative PCR. The DNA content was highest at 30°C in the absence of penicillin, whereas there was no observable change with exposure to penicillin at all evaluated temperatures. During growth, larger spheroplasts were more frequently observed at 25°C in the presence of penicillin. These results demonstrate that the optimal culture conditions for the enlargement of spheroplasts in E. litoralis differ from those required for cell division.

Quantitative analysis of chromosomal and plasmid DNA during the growth of spheroplasts of Escherichia coli.

We generated spheroplasts from Escherichia coli carrying a broad-host-range plasmid. In the presence of penicillin, the spheroplasts did not divide but grew and enlarged in marine broth, whereas, in the absence of penicillin, they elongated. We quantified cellular DNA at different time points by using real-time quantitative PCR. Both chromosomal and plasmid DNA had replicated during spheroplast growth not only in the absence but also in the presence of penicillin. Thus, plasmid DNA and chromosomal DNA replication might be regulated synchronously during the growth of spheroplasts.

A stable hybrid containing haploid genomes of two obligate diploid Candida species.

Candida albicans and Candida dubliniensis are diploid, predominantly asexual human-pathogenic yeasts. In this study, we constructed tetraploid (4n) strains of C. albicans of the same or different lineages by spheroplast fusion. Induction of chromosome loss in the tetraploid C. albicans generated diploid or near-diploid progeny strains but did not produce any haploid progeny. We also constructed stable heterotetraploid somatic hybrid strains (2n + 2n) of C. albicans and C. dubliniensis by spheroplast fusion. Heterodiploid (n + n) progeny hybrids were obtained after inducing chromosome loss in a stable heterotetraploid hybrid. To identify a subset of hybrid heterodiploid progeny strains carrying at least one copy of all chromosomes of both species, unique centromere sequences of various chromosomes of each species were used as markers in PCR analysis. The reduction of chromosome content was confirmed by a comparative genome hybridization (CGH) assay. The hybrid strains were found to be stably propagated. Chromatin immunoprecipitation (ChIP) assays with antibodies against centromere-specific histones (C. albicans Cse4/C. dubliniensis Cse4) revealed that the centromere identity of chromosomes of each species is maintained in the hybrid genomes of the heterotetraploid and heterodiploid strains. Thus, our results suggest that the diploid genome content is not obligatory for the survival of either C. albicans or C. dubliniensis. In keeping with the recent discovery of the existence of haploid C. albicans strains, the heterodiploid strains of our study can be excellent tools for further species-specific genome elimination, yielding true haploid progeny of C. albicans or C. dubliniensis in future.

A simple method for culture and stable maintenance of giant spheroplasts from the yeast Saccharomyces cerevisiae.

When spheroplasts of the yeast Saccharomyces cerevisiae are cultured in liquid medium containing osmotic stabilizer, they undergo nuclear division and growth without cell division, resulting in the formation of giant spheroplasts with multinuclei. In this study, we report a simple method for the culture and stable maintenance of giant spheroplasts. The selection of culture media and cell concentration was found to be important for the growth and maintenance of giant spheroplasts. Among the conditions that we tested, static culture in a synthetic Burkholder's medium in 96-well U-bottomed culture plates was most effective. Under appropriate conditions, we could maintain giant spheroplasts for more than 6 days without proliferation of whole cells or marked lysis. The average diameter of spheroplasts can vary from 16 to 53µm, depending on their initial concentration.

Novel method for preparing spheroplasts from cells with an internal cellulosic cell wall.

Protoplast and spheroplast preparations allow the transfer of macromolecules into cells and provide the basis for the generation of engineered organisms. Crypthecodinium cohnii cells harvested from polyethylene glycol-containing agar plates possessed significantly lower levels of cellulose in their cortical layers, which facilitated the delivery of fluorescence-labeled oligonucleotides into these cells.

[Application of Bacillus-antagonists for biocontrol of fungi degrading raw wood].

Species composition of micromycete complexes colonizing aspen, birch, and pine wood was studied. Calculation of the Sorens-Tchekanovsky similarity coefficients showed that these complexes shared a high degree of similarity. They were dominated by the representatives of the genera Penicillium, Paecilomyces, Trichoderma, and Rhizopus. Some antagonistic bacilli inhibited growth and development of wood-decay fungi in vitro and led to the formation of spheroplasts on growing hyphae. A study of possible involvement of bacillary mycolytic enzymes in the inhibition of wood-decay fungi demonstrated selectivity of their lytic effect, which was determined by the genus and species of micromycetes and did not correlate with their relative resistance to antagonists.

Efficiency of procedures for induction and cultivation of Pseudomonas syringae pv. pisi L-form.

The L-form of Pseudomonas syringae pv. phaseolicola has been proved to induce resistance to bean halo blight. Various procedures were tested to induce the L-form of Pseudomonas syringae pv. pisi for its potential use as biocontrol agent of pea bacterial blight. Cell-wall deficient cells were induced in a liquid medium with penicillin following a protocol described for P. s. pv. phaseolicola. Cell growth on solid induction medium developed as typical granular and vacuolated structures, and characteristic colonies were observed in the first transfer. However, there was poor growth in subsequent transfers and some reversion to the parental type. To improve the induction procedure, the following new procedures were applied: (1) viability of cells was monitored during induction. The optimum induction time in liquid medium with penicillin was lower for pv. pisi than for pv. phaseolicola. Viability of L-forms in solid induction medium with penicillin was low and decreased in time. (2) the inducer ticarcillin was combined with clavulanic acid, which prevented the reversion to the parental type and (3) a range of concentrations of penicillin and ticarcillin/clavulanic acid was applied by the spiral gradient endpoint method for calculation of minimum inhibitory concentrations (MIC). Based on the results from these tests an induction method for P. s. pv. pisi L-form is proposed and the relevance of L-form is discussed for practice.

The spheroplast lysis assay for yeast in microtiter plate format.

A yeast lysis assay in the microtiter plate format improved precision and throughput and led to an improved algorithm for estimating lag time. The assay reproducibly revealed differences of 10% or greater in the maximal lysis rate and 50% or greater in the lag time. Clonal differences were determined to be the major source of variation. Microtiter-based assays should be useful for screening for drug susceptibility and for analyzing mutant phenotypes.

A spheroplast rate assay for determination of cell wall integrity in yeast.

The rate of formation of spheroplasts of yeast can be used as an assay to study the structural integrity of cell walls. Lysis can be measured spectrophotometrically in hypotonic solution in the presence of Zymolyase, a mixture of cell wall-digesting enzymes. The optical density of the cell suspension decreases as the cells lyse. We optimized this assay with respect to enzyme concentration, temperature, pH, and growth conditions for several strains of Saccharomyces cerevisiae. The level of variability (standard deviation) was 1-5% between trials where the replications were performed on the same culture using enzyme prepared from the same lot, and 5-15% for different cultures of the same strain. This assay can quantitate differences in cell wall structure (1) between exponentially growing and stationary phase cells, (2) among different S. cerevisiae strains, (3) between S. cerevisiae and Candida albicans, (4) between parental and mutated lines, and (5) between drug- or chemically-treated cells and controls.

Glucosylation of cell wall proteins in regenerating spheroplasts of Candida albicans.

The cell wall of Candida albicans contains mannoproteins that are covalently associated with beta-1,6-glucan. When spheroplasts were allowed to regenerate a new cell wall, initially non-glucosylated cell wall proteins accumulated in the medium. While the spheroplasts became osmotically stable, beta-1,6-glucosylated proteins could be identified in their cell wall by SDS-extraction or beta-1,3-glucanase digestion. At later stages of regeneration, beta-1,3-glucosylated proteins were also found. Hence, incorporation of proteins into the cell wall is accompanied by extracellular coupling to beta-1,6-/beta-1,3-glucan. The SDS-extractable glucosylated proteins probably represent degradation products of wall proteins rather than their precursors. Tunicamycin delayed, but did not prevent the formation of beta-1,6-glucosylated proteins, demonstrating that beta-1,6-glucan is not attached to N-glycosidic side-chains of wall proteins.

Sphaeroplast formation process in the fungus humicola sp

A formaçäo de esferoplastos foi estudada um Humicola sp. Amostras foram retiradas a cada 30 minutos, e determinado o número de esferoplastos/ml, número de núcleos/célula e freqüências de regeneraçäo. Esta última diminuiu com tempo de tratamento, mas o número de esferoplastos/ml e de núcleos/esferoplasto aumentou com o tempo.

[Various data concerning the ultrastructure of Proteus spheroplasts]. / Nekotorye dannye po ul'trastrukture sferoplastov u protea

Spheroplasts were obtained under the action of penicillin on the same medium which was later used to obtain the L-forms. Spheroplast formation started 15 to 20 minutes after the addition of penicillin and reached the maximum in 2 hours. The first dividing forms were revealed at that time, and this division continued for at least 24 hours. The majority of the cells represented spheroplasts surrounded by outer and cytoplasmic membrane, and only some--true protoplasts--had cytoplasmic membrane alone. Division was anomalous in comparison with the bacterial forms with the cell wall: it was noted that either both daughter cells were surrounded by a common outer membrane, or one daughter cell had two membranes serving as a spheroplast and the other--one membrane, serving as a true protoplast. Individual vesicles and myelin-like structures were found to be extruded into the periplasmic space or directly into the surrounding environment. In the latter case pearl-like structures described by Ryter in the formation of protoplasts in bacilli were observed. However, in the Proteus such structures were largely formed by the material of the plastic layer of the cell wall, and to a lesser extent--by the lipoproteid membrane.