Escherichia coli ghost production by expression of lysis gene E and Staphylococcal nuclease

Abstract

The production of bacterial ghosts from Escherichia coli is accomplished by the controlled expression of phage phiX174 lysis gene E and, in contrast to other gram-negative bacterial species, is accompanied by the rare detection of nonlysed, reproductive cells within the ghost preparation. To overcome this problem, the expression of a secondary killing gene was suggested to give rise to the complete genetic inactivation of the bacterial samples. The expression of staphylococcal nuclease A in E. coli resulted in intracellular accumulation of the protein and degradation of the host DNA into fragments shorter than 100 bp. Two expression systems for the nuclease are presented and were combined with the protein E-mediated lysis system. Under optimized conditions for the coexpression of gene E and the staphylococcal nuclease, the concentration of viable cells fell below the lower limit of detection, whereas the rates of ghost formation were not affected. With regard to the absence of reproductive cells from the ghost fractions, the reduction of viability could be determined as being at least 7 to 8 orders of magnitude. The lysis process was characterized by electrophoretic analysis and absolute quantification of the genetic material within the cells and the culture supernatant via real-time PCR. The ongoing degradation of the bacterial nucleic acids resulted in a continuous quantitative clearance of the genetic material associated with the lysing cells until the concentrations fell below the detection limits of either assay. No functional, released genetic units (genes) were detected within the supernatant during the lysis process, including nuclease expression.

FIG. 1.

Expression plasmids for lysis gene E and the staphylococcal nuclease (SNUC). The vectors are presented as plasmid combinations utilized for the coexpression of gene E and SNUC in E. coli NM522. The expression of the killing genes was triggered by either the addition of an inducer (+IPTG) or a thermal upshift from 28°C to 42°C (28°C ⇒ 42°C). Amp, ampicillin resistance gene; Kan, kanamycin resistance gene; Tet, tetracycline resistance gene; colE1 and p15A, origins of replication; A1-O4/O3, synthetic, chemically inducible promoter; p_RM/p_R, rightward “maintenance” and rightward promoters of bacteriophage lambda; cI857, gene encoding the thermosensitive repressor for the lambda p_R promoter; EcoRI, restrictions sites used for the construction of pHSSNUC.

FIG. 2.

Growth kinetics of E. coli NM522 harboring pSNUC1. The cells were grown at 37°C in the presence of added Mg and Ca until mid-log phase. The culture was split, and one part was induced by the addition of IPTG at a final concentration of 5 mM (◊), whereas the other part served as a noninduced control (⧫). OD600, optical density measured at 600 nm; the arrow indicates the time of induction.

FIG. 3.

Growth and lysis kinetics and electrophoretic analysis of the total DNA content of E. coli NM522 harboring either pML1 plus pSNUC1 (a to c) or only pML1 (d to f). Diagrams a and d focus on the growth and lysis kinetics, which were monitored by measurement of the optical density at 600 nm (OD600; ○) and the determination of the number of CFU per milliliter (•). For practical reasons, the concentration of viable cells that fell below the lower limit of detection (grey lines; d.l.) is illustrated as corresponding to the detection limit, although it does not refer to a certain concentration of cells. The time points of supplementation with IPTG and Mg and Ca and the temperature upshift to 42°C are indicated by arrows. The total DNA contents within the pellets (b, e) and the supernatants (c, f) were analyzed by electrophoretic separation with 0.6% agarose gels. The time points of sampling are given in minutes relative to the time of lysis induction (0 min). Lane m1, BstEII-digested lambda DNA; the sizes of prominent fragments (indicated by arrowheads) are given in kilobases.

FIG. 4.

Quantitative analysis of the total DNA content within the pellets (⧫) and the supernatants (◊) of E. coli NM522 harboring either pML1 plus pSNUC1 (a, b) or only pML1 (c). The data are presented in nanograms of DNA per milliliter of culture taken at various time points during the production of ghosts. The time points of sampling are given in minutes relative to the induction of lysis (0 min). The quantitative results were obtained via real-time PCR with regard to amplification of either the amp^r gene (a) or the kan^r gene (b, c) encoded by pSNUC1 and pML1, respectively. For practical reasons, the DNA concentrations that fell below the detection limits (grey lines; d.l.) are illustrated as corresponding to the limits of detection, although they do not refer to certain concentrations. The time points of supplementation with IPTG and Mg and Ca and the temperature upshift to 42°C are indicated by arrows.