L-forms bacteria, protoplast and spheroplasts Bacteria

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1. L-FORMS, PROTOPLAST AND SPHEROPLASTS Prepared by Amjad Khan Afridi .

2. 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 .

3. 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.

4. While some L-forms form spontaneously (e.g Streptobacillus monoliformis ) others are inducible. Since they lack cell wall, they don’t 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-egg colony” . 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.

5. 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.

6. L-Form Bacteria

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

8. 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.

9. The etymology of this designation of the “L-forms” is not widely known and it is possible to encounter the opinion that the term has been derived from the roll- or L-shape cells . L-forms of bacteria are characterized by a complete or partial loss of cell wall components and accordingly, the term ”CWD-forms” started to be used in recent literature; that is more general and gives a true picture of them (Domingue and Woody, 1997; Markova et al., 1997; Mattman, 2001; Huber, 2002; Hines and Styer, 2003).

10. Bacterial L-form conversion , i.e. existence without rigid walls, is universal but difficultly recognized 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).

11. L-Form Bacteria

12. Basic classification of CWD-forms of bacteria

13. According to their ability to spontaneously revert to cells with a standard cell wall, CWD-forms have been classified as follows: (i) stable (spontaneously non-reverting) and (ii) non-stable (spontaneously reverting). According to the degree of loss of cell wall components, CWD-forms have been described as two basic types ( Kapralek , 2000; Mattman, 2001): 1. protoplasts and 2. spheroplasts.

14. (1) Protoplasts

15. Protoplasts have completely lost the cell wall and therefore, they are lined only with the surface membrane that separates them from external environment. Protoplasts of bacteria can be confused with mycoplasmas 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 subsequently divide (Mattman, 2001).

16. Protoplast

17. (2) Spheroplasts

18. 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 ).

19. Spheroplasting Spheroplasting involves removal of the cell wall through treatment with lysozyme and EDTA

20. Spheroplast

21. Immobilizing Spheroplasts Pretreat mica with aminopropyltriethoxysilane and glutaraldehyde (APTES/glut) − Previously shown to successfully immobilize chromatin on mica ( Wang, H.D., et al., Biophysical Journal, 2002. 83(6): 3619-3625) • Incubate the spheroplast suspension on the treated mica − Immobilization results from interactions between the proteins and the substrate − Conceivably, only the surface in contact with the substrate is affected, leaving the exposed surface in its native state and accessible to the tip • Rinse and image in sucrose buffer

22. The End Thank You …. Prepared by Amjad Khan Afridi