A study on induction of minicell formation by FTSZ overproducing L-form E. coli


Thesis Type: Postgraduate

Institution Of The Thesis: Orta Doğu Teknik Üniversitesi, Faculty of Arts and Sciences, Department of Biology, Turkey

Approval Date: 2014

Student: İSMAİL CEM YILMAZ

Co-Consultant: GÜLAY ÖZCENGİZ, MAYDA GÜRSEL

Abstract:

L-form organisms (or L-phase, L-variants) are cell wall-deficient (CWD) bacteria consisting of protoplasts and spheroplasts that have the ability to grow and divide. The mutant strain LW1655F+ (also known as L-form E. coli) was originally derived from the parental E. coli K-12 parental strain and is known to be deficient in cell wall, periplasmic space, flagella, fimbriae and outer membrane. Minicells are small cell-like structures that do not have genomic DNA and are produced during the logarithmic phase of growth during which division event does not occur. Minicell production in E. coli K-12 parental strain can be induced by introducing a mutation at the minB locus of the bacterial genome. Moreover, previous studies have shown that overproducing FtsZ to two to seven folds led to minicell production in rod-shaped E. coli K-12. The present study originally intended to investigate whether spherical; cell-wall deficient L-form E. coli is also capable of producing minicells upon overproduction of FtsZ. To achieve this, two types of model organisms were used: a) L-form E. coli, b) L-form E. coli overproducing 2.25-fold more FtsZ as constructed in the present study. Electron microscopy analyses of the organism demonstrated that L-form E. coli cells spontaneously produced vesicle-like structures. Such spontaneous vesicle production was previously reported for other L-form bacteria such as Listeria monocytogenes, yet the current study is the first to demonstrate this event in L-form E. coli. Furthermore, flow cytometric analyses revealed that overproduction of FtsZ in L-form E. coli significantly increased the number of vesicles released into the growth medium when compared to L-form E. coli. Although minicell formation by both L-form E. coli and its FtsZ overproducing derivative could not be verified and remained ambiguous, the present study demonstrated two new findings: (i) L-form E. coli spontaneously produces vesicle-like structures without any modification, and (ii) FtsZ overproduction in such cells leads to a significantly increased vesicle secretion.