Online Monitoring of Escherichia coli Ghost Production

Abstract

Controlled expression of cloned X174 gene E in gram-negative bacteria results in lysis of the bacteria by the formation of a transmembrane tunnel structure built through the cell envelope complex. Production of bacterial ghosts is routinely monitored by classical microbiological procedures. These include determination of the turbidity of the culture and the total number of cells and the number of reproductive cells present during the time course of growth and lysis. Although conceptually simple, these methods are labor intensive and time consuming, providing a complete set of results after the determination of viable cell counts. To avoid culturing methods for bacterial growth, an alternative flow cytometric procedure is presented for the quantification of ghosts and polarized, as well as depolarized, nonlysed cells within a culture. For this method, which is based on the discriminatory power of the membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol, a staining protocol was developed and optimized for the maximum discrepancy in fluorescence between bacterial ghosts and viable cells. The total quantitative analysis procedure takes less than 2 min. The results derived from classical or cytometric analyses correlate with respect to the total cell numbers and the viability of the culture.The formation of bacterial ghosts of Escherichia coli is a well-characterized process (19,24). Initiated by the expression of cloned phage X174 lysis gene E, a transmembrane tunnel structure is formed in consequence of the oligomerization of protein E. Driven by the high osmotic pressure inside the cell, the cytoplasmic content is expelled into the surrounding medium, thus giving rise to empty bacterial cell envelopes. Except for the lysis hole, the morphology of the bacteria, including all cell surface structures and the cell membranes, is not affected by the lysis event (23). As this procedure is applicable to a diverse spectrum of gram-negative bacteria, the ghosts are under investigation as genetically inactivated candidate vaccines (4, 9) and as carriers of foreign antigens (3, 10).The procedures for monitoring and characterizing the lysis process are based mainly on classical methods. The lysis of the first bacterial cells is associated with a decrease in the turbidity of the culture, which can be detected by measuring the optical density. As a consequence, the viability of the lysing culture decreases drastically, to reach a minimum at the endpoint of lysis. For E. coli, a correlation between the decrease in optical density (OD) and the reduction of viable cell counts was determined, although it was found not to be accurate. To this day, classical microbiological procedures, like plating or the use of counting chambers, are used to determine the amount of residual viable (reproductive) bacteria and the total number of cells (including ghosts) in time course experiments. As these parameters are critical for the characterization of the growing or lysing culture, a method is desired for the onlin...

The bacterial ghost platform system

“…DiBAC-positive cells with a diminished scatter signal are identified as BGs. The general procedure for online monitoring E-lysis of E. coli by flow cytometry has been developed by Haidinger et al 39,40 and was adapted recently. The flow cytometry result for a given sample is available in less than 10 min after sampling.…”

“…Flow cytometry has been established as a reliable real-time tool for the assessment of E-lysis onset and the progress of BG formation. 39 For flow cytometry diluted samples of the culture broth are stained with two fluorescent dyes and run through a CyFlow analyzer (Partec, Münster, Germany). The first dye (RH414) stains phospholipid membranes and its fluorescence signal defines a gate for the exclusion of all non-cellular background.…”

Escherichia coli Ghost Production by Expression of Lysis Gene E and Staphylococcal Nuclease

“…In previous studies, when green fluorescent protein-derived fluorescence was used for microscopic discrimination between ghosts and nonlysed cells, the percentage of nonlysed reproductive and inactivated E. coli cells was determined as 1.3% and 1.2%, respectively (13). The population of nonlysed cells was cytometrically subdivided into polarized and depolarized cells, and the detected populations were successfully quantified during the time course of lysis (14). The minimum ratios of nonlysed cells within the ghost preparations were cytometrically ascertained to be 4% for the polarized and 1% for the depolarized population, which correlated well with the results derived from classical microbiological procedures, indicating a minimum of 1% reproductive bacteria within the ghost preparations.…”

Autotransporter-Based Antigen Display in Bacterial Ghosts

“…1C). Both the forward and side scatters of ghosts and control cells were monitored, which allowed comparing their size and internal complexity, respectively (32)(33)(34). The size (forward scatter) of a large fraction of the ghosts resembled the size of the control cells (Fig.…”