L-Form Bacteria

L-FORMS, PROTOPLAST AND SPHEROPLASTS

Prepared by Amjad Khan Afridi.

When bacteria are treated with enzymes that hydrolyze the cell wall (e.g. lysozyme) or antibiotics that interfere with biosynthesis of peptidoglycan (penicillin), wall-less bacteria are often produced. Such a treatment of bacteria in osmotically protective medium liberates protoplasts from gram positive bacteria and spheroplasts from gram negative bacteria. Spheroplasts retain the outer membrane. Usually these treatments generate wall-less non-viable organisms that do not multiply. However, if such cells can grow and divide, they are called L forms.

L forms were first reported by Klieneberger Nobel in cultures of Streptobacillus monoliformis. They are named L forms after Lister Institute, where they were discovered. They are produced more readily with penicillin than with lysozyme, suggesting that need for residual peptidoglycan. Some L forms are stable and some are unstable. Unstable forms are those which revert back to cell wall containing state when inducing stimulus (penicillin) is removed. Such forms usually have small amounts of residual peptidoglycan that serves as primer for building cell wall. Stable forms do not revert back to normal form since they completely lack peptidoglycan.

While some L-forms form spontaneously(e.g Streptobacillus monoliformis) others are inducible. Since they lack cell wall, they dont have a definite shape. L forms are difficult to cultivate and require medium that has right osmotic strength and low concentration of agar, inactivated serum and sucrose. L forms resemble mycoplasma in morphology, type of growth on agar fried-eggcolony. While mycoplasma lack cell wall and have sterols in their membrane, the L forms may have reminiscent of cell wall but do not have sterols in their membrane.

Significance of L forms: L forms may produce chronic infections in the host. They may persist in protective regions of the body. Since L forms are relatively resistant to antibiotics, they are difficult to treat. Their reversion to normal form can result in relapse of infection.

L-Form Bacteria

Protoplasts or spheroplasts capable ofgrowth and propagation in a medium with appropriateosmotic qualities have been designated as CWD forms,or L-forms of bacteria (Kapralek, 2000).

The term L-forms of bacteria was used for the first time by Klieneberger in 1935 (Klieneberger,1935 as quoted by Domingue and Woody, 1997).This designation (L before the word forms) was used according to the Lister Institute in London where the above mentioned investigator worked.

The etymology of this designation of the L-formsis not widely known and it is possible to encounterthe opinion that the term has been derived from the roll- or L-shape cells. L-forms of bacteriaare characterized by a complete or partial lossof cell wall components and accordingly, the termCWD-forms started to be used in recent literature;that is more general and gives a true picture of them (Domingue and Woody, 1997; Markova etal., 1997; Mattman, 2001; Huber, 2002; Hines andStyer, 2003).

Bacterial L-form conversion, i.e. existence without rigid walls, is universal but difficultlyrecognized phenomenon in nature (Domingue, 1982; Mattman, 2001; Prozorovski et al., 1981). The term cell wall deficiency implies alterations in the constitution of bacterial cell wall, resulting from deletion and faulty synthesis of wall components (Mattman, 2001).

L-Form Bacteria

Basic classification of CWD-formsof bacteria

According to their ability to spontaneously revertto cells with a standard cell wall, CWD-forms havebeen classified as follows: (i) stable (spontaneouslynon-reverting) and (ii) non-stable (spontaneouslyreverting). According to the degree of loss of cell wallcomponents, CWD-forms have been described astwo basic types (Kapralek, 2000; Mattman, 2001):1. protoplasts and2. spheroplasts.

(1) Protoplasts

Protoplasts have completely lost the cell wall andtherefore, they are lined only with the surface membrane that separates them from external environment.Protoplasts of bacteria can be confused withmycoplasmas as they also have no cell wall (Krieg and Holt, 1984). From the reason of complete absence of all constituents of the cell wall, bacterial or yeast protoplasts cannot regenerate to the cells with a normally developed wall and subsequentlydivide (Mattman, 2001).

Protoplast

(2) Spheroplasts

The cell wall is usually partly maintained in spheroplasts.Their surface is considerably disturbed and therefore allows increased penetration of substances into the cells in comparison with cells with a normal cell wall (Cover et al., 1991). Spheroplasts may divide and they are more likely to form and survive under both in vivo and in vitro conditions than protoplasts (Murty and Venkitasubramanian,1983).

SpheroplastingSpheroplasting involves removalof the cell wall through treatmentwith lysozyme and EDTA

Spheroplast

Immobilizing SpheroplastsPretreat mica withaminopropyltriethoxysilane andglutaraldehyde (APTES/glut) Previously shown to successfullyimmobilize chromatin on mica (Wang,H.D., et al., Biophysical Journal, 2002.83(6): 3619-3625) Incubate the spheroplast suspension on thetreated mica Immobilization results from interactionsbetween the proteins and the substrate Conceivably, only the surface in contactwith the substrate is affected, leavingthe exposed surface in its native stateand accessible to the tip Rinse and image in sucrose buffer

The EndThank You.Prepared by Amjad Khan Afridi