Direct Transfer of a Mycoplasma mycoides Genome to Yeast Is Enhanced by Removal of the Mycoides Glycerol Uptake Factor Gene glpF.

We previously discovered that intact bacterial chromosomes can be directly transferred to a yeast host cell where they can propagate as centromeric plasmids by fusing bacterial cells with S accharomyces cerevisiae spheroplasts. Inside the host any desired number of genetic changes can be introduced into the yeast centromeric plasmid to produce designer genomes that can be brought to life using a genome transplantation protocol. Earlier research demonstrated that the removal of restriction-systems from donor bacteria, such as Mycoplasma mycoides, Mycoplasma capricolum, or Haemophilus influenzae increased successful genome transfers. These findings suggested that other genetic factors might also impact the bacteria-to-yeast genome transfer process. In this study, we demonstrated that the removal of a particular genetic factor, the glycerol uptake facilitator protein gene glpF from M. mycoides, significantly increased direct genome transfer by up to 21-fold. Additionally, we showed that intact bacterial cells were endocytosed by yeast spheroplasts producing organelle-like structures within these yeast cells. These might lead to the possibility of creating novel synthetic organelles.

Engineering Bacterial Shape Using Soft Matter Microchambers.

This article describes the design and fabrication of microchambers that are used for the study of bacterial cells. The design allows for the confinement and precise manipulation of bacterial cell shape. The application of fluorescent dyes and fluorescent proteins enables the precise analysis of the localization of biomolecules within confined bacterial cell. This article also outlines three methods to engineer cell shape from a filamentous cell type and from spheroplasts without a cell wall using soft lithography-based technologies. © 2018 by John Wiley & Sons, Inc.

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.

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.

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization.

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.

Application of Environmental Scanning Electron Microscope-Nanomanipulation System on Spheroplast Yeast Cells Surface Observation.

The preparation and observations of spheroplast W303 cells are described with Environmental Scanning Electron Microscope (ESEM). The spheroplasting conversion was successfully confirmed qualitatively, by the evaluation of the morphological change between the normal W303 cells and the spheroplast W303 cells, and quantitatively, by determining the spheroplast conversion percentage based on the OD800 absorbance data. From the optical microscope observations as expected, the normal cells had an oval shape whereas spheroplast cells resemble a spherical shape. This was also confirmed under four different mediums, that is, yeast peptone-dextrose (YPD), sterile water, sorbitol-EDTA-sodium citrate buffer (SCE), and sorbitol-Tris-Hcl-CaCl2 (CaS). It was also observed that the SCE and CaS mediums had a higher number of spheroplast cells as compared to the YPD and sterile water mediums. The OD800 absorbance data also showed that the whole W303 cells were fully converted to the spheroplast cells after about 15 minutes. The observations of the normal and the spheroplast W303 cells were then performed under an environmental scanning electron microscope (ESEM). The normal cells showed a smooth cell surface whereas the spheroplast cells had a bleb-like surface after the loss of its integrity when removing the cell wall.

Mode of Action of Antimicrobial Peptides on E. coli Spheroplasts.

We investigated the phenomena of antimicrobial peptides (AMPs) directly attacking the cytoplasmic membranes of Escherichia coli spheroplasts. We developed a procedure for fluorescence recovery after photobleaching to examine dye leakage through bacterial membranes as AMPs in solution bound to the membranes. We found that the AMP binding did not increase the apparent membrane area of a spheroplast, contrary to the response of a lipid-bilayer vesicle, which always showed a membrane area expansion by AMP binding. The permeability through the bacterial membrane increased in a sigmoidal fashion as the AMP binding increased in time, exhibiting a cooperative behavior of AMPs. The analysis of fluorescence recovery after photobleaching showed that the fluxes of dye molecules into and out of the cell were consistent with diffusion of molecules through a number of pores that increased with binding of AMPs and then saturated to a steady level. We discovered a new, to our knowledge, experimental parameter called the flux rate that characterizes the AMP-induced permeability of dye molecules through bacterial membranes. The phenomena observed in bacterial membranes are consistent with the pore-forming activities of AMPs previously observed in lipid bilayers. The experimental value of the flux rate per pore is much smaller than a theoretical value that assumes no friction for the dye molecule's permeation through the pore. We believe that experimental studies of the flux rate will be useful for further analysis of AMPs' permeabilization mechanisms.

Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides.

Studies attempting to characterize the membrane translocation of antimicrobial and cell-penetrating peptides are frequently limited by the resolution of conventional light microscopy. This study shows that spheroplasts provide a valuable approach to overcome these limits. Spheroplasts produce less ambiguous images and allow for more systematic analyses of localization. Data collected with spheroplasts are consistent with studies using normal bacterial cells and imply that a particular peptide may not always follow the same mechanism of action.

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.

Imaging of the Actin Cytoskeleton and Mitochondria in Fixed Budding Yeast Cells.

The budding yeast Saccharomyces cerevisiae is widely used as a model system to study the organization and function of the cytoskeleton. In the past, its small size, rounded shape, and rigid cell wall created obstacles to explore the cell biology of this model eukaryote. It is now possible to acquire and analyze high-resolution and super-resolution multidimensional images of the yeast cell. As a result, imaging of yeast has emerged as an important tool in eukaryotic cell biology. This chapter describes labeling methods and optical approaches for visualizing the cytoskeleton and interactions of the actin cytoskeleton with mitochondria in fixed yeast cells using wide-field and super-resolution fluorescence microscopy.

NP24 induces apoptosis dependent on caspase-like activity in Saccharomyces cerevisiae.

Tomato NP24 is a homolog of osmotin, a PR-5 protein from tobacco that can initiate apoptosis in yeast via PHO36 in the plasma membrane. We cloned and sequenced NP24 from tomato cv. Momotaro. Based on phylogenetic analysis, NP24 from Momotaro belonged to the Solanaceae clade. The amino acid sequence was identical to that of cv. Ailsa Craig including signal peptide, but the residues predicted to interact with the adiponectin receptor, ADIPOR, were slightly different from osmotin. Recombinant NP24 (rNP24) was expressed in a reductase-deficient mutant of Escherichia coli as host cell, and purified from cell extract by affinity chromatography. Purified rNP24 significantly inhibited growth of Saccharomyces cerevisiae wild-type spheroplasts. In contrast, growth of PHO36 deletion mutant (ΔIzh2) spheroplasts was not inhibited. Moreover, rNP24 induced significant activity of reactive oxygen species, caspase-like activity, and also nuclear fragmentation in wild-type spheroplast cells. These results demonstrated that rNP24 from Momotaro greatly influenced cell viability due to triggering apoptosis through PHO36. Notably, apoptosis induced by NP24 was caspase-like protease dependent.

Mutations in a Conserved Domain of E. coli MscS to the Most Conserved Superfamily Residue Leads to Kinetic Changes.

In Escherichia coli (E. coli) the mechanosensitive channel of small conductance, MscS, gates in response to membrane tension created from acute external hypoosmotic shock, thus rescuing the bacterium from cell lysis. E. coli MscS is the most well studied member of the MscS superfamily of channels, whose members are found throughout the bacterial and plant kingdoms. Homology to the pore lining helix and upper vestibule domain of E. coli MscS is required for inclusion into the superfamily. Although highly conserved, in the second half of the pore lining helix (TM3B), E. coli MscS has five residues significantly different from other members of the superfamily. In superfamilies such as this, it remains unclear why variations within such a homologous region occur: is it tolerance of alternate residues, or does it define functional variance within the superfamily? Point mutations (S114I/T, L118F, A120S, L123F, F127E/K/T) and patch clamp electrophysiology were used to study the effect of changing these residues in E. coli MscS on sensitivity and gating. The data indicate that variation at these locations do not consistently lead to wildtype channel phenotypes, nor do they define large changes in mechanosensation, but often appear to effect changes in the E. coli MscS channel gating kinetics.

Imaging yeast NPCs: from classical electron microscopy to Immuno-SEM.

Electron microscopy (EM) has been used extensively for the study of nuclear transport as well as the structure of the nuclear pore complex (NPC) and nuclear envelope. However, there are specific challenges faced when carrying out EM in one of the main model organisms used: the yeast, Saccharomyces cerevisiae. These are due to the presence of a cell wall, vacuoles, and a densely packed cytoplasm which, for transmission EM (TEM), make fixation, embedding, and imaging difficult. These also present problems for scanning EM (SEM) because cell wall removal and isolation of nuclei can easily damage the relatively fragile NPCs. We present some of the protocols we use to prepare samples for TEM and SEM to provide information about yeast NPC ultrastructure and the location of nucleoporins and transport factors by immunogold labeling within that ultrastructure.

The Rcs stress response and accessory envelope proteins are required for de novo generation of cell shape in Escherichia coli.

Interactions with immune responses or exposure to certain antibiotics can remove the peptidoglycan wall of many Gram-negative bacteria. Though the spheroplasts thus created usually lyse, some may survive by resynthesizing their walls and shapes. Normally, bacterial morphology is generated by synthetic complexes directed by FtsZ and MreBCD or their homologues, but whether these classic systems can recreate morphology in the absence of a preexisting template is unknown. To address this question, we treated Escherichia coli with lysozyme to remove the peptidoglycan wall while leaving intact the inner and outer membranes and periplasm. The resulting lysozyme-induced (LI) spheroplasts recovered a rod shape after four to six generations. Recovery proceeded via a series of cell divisions that produced misshapen and branched intermediates before later progeny assumed a normal rod shape. Importantly, mutants defective in mounting the Rcs stress response and those lacking penicillin binding protein 1B (PBP1B) or LpoB could not divide or recover their cell shape but instead enlarged until they lysed. LI spheroplasts from mutants lacking the Lpp lipoprotein or PBP6 produced spherical daughter cells that did not recover a normal rod shape or that did so only after a significant delay. Thus, to regenerate normal morphology de novo, E. coli must supplement the classic FtsZ- and MreBCD-directed cell wall systems with activities that are otherwise dispensable for growth under normal laboratory conditions. The existence of these auxiliary mechanisms implies that they may be required for survival in natural environments, where bacterial walls can be damaged extensively or removed altogether.

Analysis of nucleosome positioning using a nucleosome-scanning assay.

The nucleosome-scanning assay (NuSA) couples isolation of mononucleosomal DNA after micrococcal nuclease (MNase) digestion with quantitative real-time PCR (qPCR) to map nucleosome positions in chromatin. It is a relatively simple, rapid procedure that can produce a high-resolution map of nucleosome location and occupancy and thus is suitable for analyzing individual promoters in great detail. The analysis can also quantify the protection of DNA sequences due to interaction with proteins other than nucleosomes and show how this protection varies when conditions change. When coupled with chromatin immunoprecipitation (ChIP), NuSA can identify histone variants and modifications associated with specific nucleosomes.

De novo growth zone formation from fission yeast spheroplasts.

Eukaryotic cells often form polarized growth zones in response to internal or external cues. To understand the establishment of growth zones with specific dimensions we used fission yeast, which grows as a rod-shaped cell of near-constant width from growth zones located at the cell tips. Removing the cell wall creates a round spheroplast with a disorganized cytoskeleton and depolarized growth proteins. As spheroplasts recover, new growth zones form that resemble normal growing cell tips in shape and width, and polarized growth resumes. Regulators of the GTPase Cdc42, which control width in exponentially growing cells, also control spheroplast growth zone width. During recovery the Cdc42 scaffold Scd2 forms a polarized patch in the rounded spheroplast, demonstrating that a growth zone protein can organize independent of cell shape. Rga4, a Cdc42 GTPase activating protein (GAP) that is excluded from cell tips, is initially distributed throughout the spheroplast membrane, but is excluded from the growth zone after a stable patch of Scd2 forms. These results provide evidence that growth zones with normal width and protein localization can form de novo through sequential organization of cellular domains, and that the size of these growth zones is genetically controlled, independent of preexisting cell shape.