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 X174 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.Controlled expression of the cloned X174 lysis gene E results in the formation of empty bacterial cell envelopes (ghosts) (28, 33). Applicable in a broad range of gram-negative bacterial species, the protein E-mediated lysis procedure gives rise to a new class of genetically inactivated candidate vaccines (11,16).In contrast to the majority of tested bacterial species, which are completely inactivated by the lysis process (9, 16), the production of bacterial ghosts from Escherichia coli K-12 species is accompanied by the rare detection of nonlysed inactivated or reproductive cells within the ghost preparation. 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 bacteri...

Functional display of ice nucleation protein InaZ on the surface of bacterial ghosts

“…Briefly, 2 E-lysis plasmids have been used for this study (Fig. 1), pGLysivb which carries lysis gene E fused with an in vivo biotinylation sequence under transcriptional control of a cI857 repressor gene and mutated -λp Rmut -operator system 40,41 and pGLMivb which represents a modification of pGLysivb where Emediated cell lysis is controlled by LacIq / P TAC repressor / promoter system. To express ice nucleation protein InaZ, in the follow called INP, its full length sequence was fused to a C-terminal His-tag sequence located in plasmid pBINP downstream of the arabinose inducible P BAD promoter (Fig.…”

“…The final concentration of antibiotics was as follows: ampicillin, 100 mg ml ¡1 ; gentamycin, 20 mg ml ¡1 . The plasmids pBAD24 63 and pGLysivb 40,41 used in this study have been described previously. Lysis plasmid pGLMivb was obtained from Bird-C plasmid collection and represents a modification of lysis plasmid pGLysivb, where the temperature inducible λpR-cI857 promoter repressor system is exchanged by a chemical inducible LaqIq-repressor-P tac -promoter system.…”

The bacterial ghost platform system

“…In applications where nucleic acid-free BGs are produced, inactivation can be accomplished by the expression of an additional "kill gene" in the host cells in combination with E-lysis. 41 For this, the staphylococcal nuclease A (SNUC) is used, which reduces the DNA content below the detection limit of real-time PCR. SNUC activity is also responsible for cleaning up residual DNA in BGs and can lead to complete inactivation of the culture as it degrades the host DNA into fragments no longer than 100 base pairs.…”

Generation of safety enhancedEdwardsiella tardaghost vaccine

“…These changes improve the safety of ETG when used in vaccines. The production of bacterial ghosts by expression of both E and SNA genes have recently been described in Escherichia coli K12 strain NM522 (Haidinger et al 2003) and a pathogenic strain, E. coli O157:H7 (Mayr et al 2005). These studies used cotransformation with 2 plasmids: one expressing E gene (induced by a temperature shift to 42°C), and the other expressing the SNA gene (induced by addition of IPTG).…”