Take a look at the following

1. PLoS One. 2013 Oct 18;8(10):e78193. doi: 10.1371/journal.pone.0078193.

eCollection 2013.

Development of a biosafety enhanced and immunogenic Salmonella enteritidis ghost

using an antibiotic resistance gene free plasmid carrying a bacteriophage lysis

system.

Jawale CV(1), Lee JH.

Author information:

(1)College of Veterinary Medicine, Chonbuk National University, Jeonju, Republic

of Korea.

In the development of genetically inactivated bacterial vaccines, plasmid

retention often requires the antibiotic resistance gene markers, the presence of

which can cause the potential biosafety hazards such as the horizontal spread of

resistance genes. The new lysis plasmid was constructed by utilizing the approach

of balanced-lethal systems based on auxotrophic gene Aspartate semialdehyde

dehydrogenase (asd). The PhiX174 lysis gene E and λPR37-cI857

temperature-sensitive regulatory system was cloned in the asd gene positive

plasmid and this novel approach allowed the production of antibiotic resistance

marker free Salmonella Enteritidis (S. Enteritidis) ghost. The immunogenic

potential of the biosafety enhanced antibiotic resistance gene free S.

Enteritidis ghost was evaluated in chickens by employing the prime-boost

vaccination strategy using a combination of oral and intramuscular routes. A

total of 75 two-week-old chickens were equally divided into five groups: group A

(non-immunized control), group B (intramuscularly primed and boosted), group C

(primed intramuscularly and boosted orally), group D (primed and boosted orally),

and group E (primed orally and boosted intramuscularly). Chickens from all

immunized groups demonstrated significant increases in plasma IgG, intestinal

secretory IgA levels, and antigen-specific lymphocyte proliferative response.

After a virulent S. Enteritidis challenge, all immunized groups showed fewer

gross lesions and decreased bacterial recovery from organs in comparison with the

non-immunized control group. Among the immunized chickens, groups B and D

chickens showed optimized protection, indicating that the prime-booster

immunization with the ghost via intramuscular or oral route is efficient. Taken

together, our results demonstrate that an antibiotic resistance gene free lysis

plasmid was successfully constructed and utilized for production of safety

enhanced S. Enteritidis ghost, which can be used as a safe and effective vaccine

against virulent S. Enteritidis infections.

PMCID: PMC3799721

PMID: 24205152 [PubMed - indexed for MEDLINE]

2. Mol Biotechnol. 2013 Jun;54(2):436-44. doi: 10.1007/s12033-012-9581-0.

A novel dual vector coexpressing PhiX174 lysis E gene and staphylococcal nuclease

A gene on the basis of lambda promoter pR and pL, respectively.

Fu L(1), Lu C.

Author information:

(1)College of Veterinary Medicine, Nanjing Agricultural University, Nanjing,

China.

Bacterial ghost is a novel vaccine platform, and its safe and efficient

production depends largely upon a suitable and functional vector. In this study,

a series of temperature-inducible plasmids, carrying Phix174 lysis gene E and/or

staphylococcal nuclease A (SNA) gene, were constructed and evaluated in

Escherichia coli. The results showed that the direct product of SNA (pBV220-SNA)

could degrade the plasmid and genomic DNA of E. coli while the fusion product of

gene E and partial Cro gene (pKF396M-2) lost the ability to lyse the host strain.

The insertion of enhancer T7g10 elements and Shine-Dalgarno box (ESD) between

them (pKF396M-3) could resume the function of gene E. Using plasmid pKF396M-4

with gene E and SNA, respectively, under the immediate control of promoter pR and

pL, the remnant plasmids and genomic DNA of E. coli were eliminated, and the

rates of inactivation increased by two orders of magnitude over that obtained

with the exclusive use of E-mediated lysis plasmid. By substituting these two

genes with customized multiple cloning sites sequences, the plasmid could be

modified to a dual expression vector (pKF396M-5).

PMID: 22782703 [PubMed - indexed for MEDLINE]

3. Appl Environ Microbiol. 2003 Oct;69(10):6106-13.

Escherichia coli ghost production by expression of lysis gene E and

Staphylococcal nuclease.

Haidinger W(1), Mayr UB, Szostak MP, Resch S, Lubitz W.

Author information:

(1)Apovia AG, D-82152 Martinsried, Germany. [email protected]

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.

PMCID: PMC201253

PMID: 14532068 [PubMed - indexed for MEDLINE]

4. Expert Opin Biol Ther. 2001 Sep;1(5):765-71.

Bacterial ghosts as carrier and targeting systems.

Lubitz W(1).

Author information:

(1)Institute of Microbiology and Genetics, University of Vienna, Dr. Bohrgasse 9,

A-1030 Wien, Austria. [email protected]

Bacterial ghosts are empty cell envelopes originating from Gram-negative

bacteria. They have a natural outer surface make-up which provides them with the

original targeting functions of the bacteria they are derived from and are thus

able to bind to and/or are taken up by specific cells or tissues of animal, human

or plant origin. The extended bacterial ghost system represents a platform

technology for creating new qualities in non-living carriers which can be used

for the specific targeting of drugs, DNA or other compounds to overcome toxic or

non-desired obstacles. Freeze dried bacterial ghosts are stable without the

requirement of a cold chain and can be effectively administered orally and

aerogenically as drug carriers. The new system is an alternative to liposomes and

may have an advantage due to its higher specificity for targeting specific

tissues, its easy method of production and its versatility in entrapping and

packaging various compounds in different compartments of the carriers.

PMID: 11728212 [PubMed - indexed for MEDLINE]

As far as I remember PhiX174 adsorbs on some LPS component and E.coli C was the host of choice for many years. There are also E.coliK12 mutants that have a proper receptor. You can or instance look at

Alix Michel, Olivier Clermont, Erick Denamur and Olivier Tenaillon in AEM in 2010.