18. Cell Wall-less Forms. Protoplasts. Spheroplasts. L-forms of the bacterium. Mycoplasma

Protoplast - is all of the cell from the plasma membrane inward (i.e., the plasma membrane plus the cytoplasm). Spheroplast - The gram-negative equivalent of protoplasts. In spheroplasts cell wall is partially preserved. Specially prepared giant spheroplasts of Gram-negative bacteria can be used to study the function of bacterial ion channels through a technique called patch clamp, which was originally designed for characterizing the behavior of neurons and other excitable cells. To prepare giant spheroplasts, bacteria are grown in a medium containing chemicals that prevent the cells from dividing completely. This causes bacteria to form long "snakes" that share a single membrane and cytoplasm. After a period of time, the cell walls of the "snakes" are digested, and the bacteria collapse into very large spheres surrounded by a single lipid bilayer. The membrane can then be analyzed on a patch clamp apparatus to determine the phenotype of the ion channels embedded in it. It is also common to overexpress a particular channel to amplify its effect and make it easier to characterize.The technique of patch clamping giant E. coli spheroplast have been used extensively for studying the native mechanosensitive channels (MscL, MscS, and MscM) of E. coli since 1987.Recently, it has been extended to study other heterologously expressed ion channels and has been shown that the giant E. coli spheroplast can be used as an ion-channel expression system comparable to Xenopus oocyte. Yeast cells are normally protected by a thick cell wall which makes extraction of cellular proteins difficult. Enzymatic digestion of the cell wall with zymolyase, creating spheroplasts, renders the cells vulnerable to easy lysis with detergents or rapid osmolar pressure changes.Bacterial spheroplasts, with suitable recombinant DNA inserted into it, can be used to transfect animal cells. Spheroplasts with recombinant DNA are introduced into the media containing animal cells and are fused by polyethylene glycol (PEG). With this methodology, nearly 100% of the animal cells may take up the foreign DNA. Occasionally wall-less bacteria that can replicate are generated by the treatments (L forms).

Two types of L-forms are distinguished:

1)unstable L-forms, that are capable of dividing, but can revert to the original morphology

2)stable L-forms, that are unable to revert to the original bacteria

Transmission electron micrograph of L-form Bacillus subtilis, showing a range of sizes. Scale bar is 10micrometers. Appearance and cell division.Bacterial morphology is determined by the cell wall. Since the L-form has no cell wall, its morphology is different from that of the strain of bacteria from which it is derived. Typical L-form cells are spheres or spheroids. For example, L-forms of the rod-shaped bacterium Bacillus subtilis appear round when viewed by phase contrast microscopy or by transmission electron microscopy. Although L-forms can develop from Gram-positive as well as from Gram-negative bacteria, in a Gram stain test, the L-forms always colour Gram-negative, due to the lack of a cell wall. The cell wall is important for cell division, which, in most bacteria, occurs by binary fission. The lack of cell wall in L-forms means that division is disorganised, giving rise to a variety of cell sizes, from very tiny to very big. Phase contrast image of L-form cells fromBacillus subtilis showing a range of sizes. Scale bar is 5 micrometers.In bacteria, cell division usually requires a cell wall and components of the bacterial cytoskeleton such as FtsZ. The ability of L-form bacteria to grow and divide in the absence of both of these structures is highly unusual, and may represent a form of cell division that was important in early forms of life. This novel mode of division seems to involve the extension of thin protrusions from the cell's surface and these protrusions then pinching off to form new cells. L-forms can be generated in the laboratory from many bacterial species that usually have cell walls, such as Bacillus subtilis or Escherichia coli. This is done by inhibiting peptidoglycan synthesis with antibiotics or treating the cells with lysozyme, an enzyme that digests cell walls. The L-forms are generated in a culture medium that is the same osmolarity as the bacterial cytosol (an isotonic solution), which prevents cell lysis by osmotic shock. L-form strains can be unstable, tending to revert to the normal form of the bacteria by regrowing a cell wall, but this can be prevented by long-term culture of the cells under the same conditions that were used to produce them. Some studies have identified mutations that occur, as these strains are derived from normal bacteria.^[1][2] One such point mutation is in an enzyme involved in the mevalonate pathway of lipid metabolism that increased the frequency of L-form formation 1,000-fold.^[1] The reason for this effect is not known, but it is presumed that the increase is related to this enzyme's role in making a lipid important in peptidoglycan synthesis. Another methodology of induction relies on nanotechnology and landscape ecology. Microfluidics devices can be built in order to challengepeptidoglycan synthesis by extreme spatial confinement. After biological dispersal through a constricted (sub-micrometre scale) biological corridorconnecting adjacent micro habitat patches, L-form-like cells can be derived. The mycoplasmas are a group of bacteria that lack a cell wall. Mycoplasmas have sterol-like molecules incorporated into their membranes and they are usually inhabitants of osmotically-protected environments. An older name for Mycoplasma was Pleuropneumonia-Like Organisms (PPLO). Characteristics There are over 100 recognized species of the genus Mycoplasma, one of several genera within the bacterial class Mollicutes. Mollicutes are parasites or commensals of humans, other animals (including insects), and plants; the genus Mycoplasma is by definition restricted to vertebrate hosts. Cholesterol is required for the growth of species of the genus Mycoplasma as well as certain other genera of mollicutes. Their optimum growth temperature is often the temperature of their host if warmbodied (e. g. 37° C in humans) or ambient temperature if the host is unable to regulate its own internal temperature. Analysis of 16S ribosomal RNA sequences as well as gene content strongly suggest that the mollicutes, including the mycoplasmas, are closely related to either theLactobacillus or the Clostridium branch of the phylogenetic tree (Firmicutes sensu stricto).Cell morphology The bacteria of the genus Mycoplasma (trivial name: mycoplasmas) and their close relatives are characterized by lack of a cell wall. Despite this, the cells often present a certain shape, with a characteristic small size, with typically about 10% of the volume of an Escherichia coli cell. These cell shapes presumably contribute to the ability of mycoplasmas to thrive in their respective environments. Most are pseudococcoidal, but there are notable exceptions. Species of the M. fastidiosum cluster are rod-shaped. Species of the M. pneumoniae cluster, including M. pneumoniae, possess a polar extension protruding from the pseudococcoidal cell body. This tip structure, designated an attachment organelle or terminal organelle, is essential for adherence to host cells and for movement along solid surfaces (gliding motility), and is implicated in normal cell division. M. pneumoniae cells are pleomorphic, with an attachment organelle of regular dimensions at one pole and a trailing filament of variable length and uncertain function at the other end, whereas other species in the cluster typically lack the trailing filament. Other species like M. mobile and M. pulmonis have similar structures with similar functions. Mycoplasmas are unusual among bacteria in that most require sterols for the stability of their cytoplasmic membrane. Sterols are acquired from the environment, usually as cholesterol from the animal host. Mycoplasmas generally possess a relatively small genome of 0.58-1.38 megabases, which results in drastically reduced biosynthetic capabilities and explains their dependence on a host. Additionally they use an alternate genetic code in which the codon UGA encodes the amino acid tryptophan instead of the usual stop codon. They have a low GC-content (23-40 mol %).