Factors That Affect the Enlargement of Bacterial Protoplasts and Spheroplasts

Abstract

Cell enlargement is essential for the microinjection of various substances into bacterial cells. The cell wall (peptidoglycan) inhibits cell enlargement. Thus, bacterial protoplasts/spheroplasts are used for enlargement because they lack cell wall. Though bacterial species that are capable of gene manipulation are limited, procedure for bacterial cell enlargement does not involve any gene manipulation technique. In order to prevent cell wall resynthesis during enlargement of protoplasts/spheroplasts, incubation media are supplemented with inhibitors of peptidoglycan biosynthesis such as penicillin. Moreover, metal ion composition in the incubation medium affects the properties of the plasma membrane. Therefore, in order to generate enlarged cells that are suitable for microinjection, metal ion composition in the medium should be considered. Experiment of bacterial protoplast or spheroplast enlargement is useful for studies on bacterial plasma membrane biosynthesis. In this paper, we have summarized the factors that influence bacterial cell enlargement.

Borrelia burgdorferi, the Lyme disease spirochete, possesses genetically-encoded responses to doxycycline, but not to amoxicillin

“…Instead, the observed membrane swellings were probably results of water diffusing into the cytoplasm and expanding the inner membrane that was no longer constrained by an intact cell wall. Similar osmotically-induced spheroplasts can be generated in other bacterial species through β-lactam induced weakening of their cell walls [19][20][21][22]. Although β-lactam derived spheroplasts of B. burgdorferi are evidently not biologically relevant to these bacteria in nature, such experimentally-derived structures can be useful for investigations of membrane functions [21,22].…”

“…Similar osmotically-induced spheroplasts can be generated in other bacterial species through β-lactam induced weakening of their cell walls [19][20][21][22]. Although β-lactam derived spheroplasts of B. burgdorferi are evidently not biologically relevant to these bacteria in nature, such experimentally-derived structures can be useful for investigations of membrane functions [21,22].…”

“…Exposing B. burgdorferi to sub-lethal levels of β-lactams may result in the spirochetes producing membrane protrusions or acquiring a spherical shape [13][14][15][16][17][18]. In other bacterial species, treatment with low levels of β-lactam antibiotics leads to weakening of the cell wall and cytoplasmic distortion due to osmotic influx of water [19][20][21][22]. However, there is a pervading hypothesis in the literature and among some physicians that β-lactam-induced "round bodies" are a genetically-encoded response by B. burgdorferi to avoid antibiotic killing [16][17][18][23][24][25][26][27][28][29][30][31][32][33][34].…”

DNA uptake by cell wall-deficient bacteria reveals a putative ancient macromolecule uptake mechanism

“…An imbalance in the cell surface to volume ratio due to excess membrane synthesis can lead to internal vesicle formation in spherical E. coli and B. subtilis shape mutants (Bendezu and de Boer, 2008;Mercier et al, 2013). Internal vesicles or vacuoles can also be formed in enlarged protoplasts and spheroplasts (containing an outer membrane) which are maintained in conditions that allow cell membrane expansion (Nishida, 2020;Takahashi et al, 2020). Indeed, a lack of excess membrane production may also explain why we did not observe consistent DNA uptake in protoplasts and S-cells, both of which are unable to proliferate without their wall.…”

Cell wall damage increases macromolecular crowding effects in the Escherichia coli cytoplasm

“…One study showed that these cells have the same biopolymer volume fraction as exponentially growing cells 1 . In contrast, other groups measured an increase in biomacromolecule content (DNA, RNA, and protein) 25,26,[28][29][30][31][32] : Spheroplasts continue to synthesize molecules and can remain viable and regain their usual rod-like shape after spheroplasting 25,[33][34][35] . Cell wall disruption can lead to a host of downstream effects.…”