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Efficient Robust Yield Method for Preparing Bacterial Ghosts by Escherichia coli Phage ID52 Lysis Protein E.

ABSTRACT: Bacterial ghosts (BGs) are nonliving empty bacterial shells without cytoplasm retaining original morphology and identical antigenicity of natural bacteria, making them high potential and promising vaccine candidates and delivery vehicles. However, the low yield of commonly used BGs preparation methods limits its mass production and widely application. In order to improve BGs production, E. coli phage ID52 lysis protein E was introduced to generating BGs for the first time. Above all, we compared the lysis activity of lysis protein of E. coli phage φX174 and E. coli phage ID52 as well as the effects of promoters on the lysis activity of ID52-E, which shown that the lysis activity and BGs formation rate of protein ID52-E was significantly higher than protein φX174-E. Further, the lysis activity of ID52-E was significantly improved under the control of L-arabinose inducible promoter which initial induction OD₆₀₀ reached as high as 2.0. The applicability of lysis protein ID52-E induced by L-arabinose was proved by preparing probiotic E. coli Nissle 1917 BGs and pathogenic Salmonella typhimurium BGs in mass production. This paper introduced a novel and highly efficient method for BGs preparation depending on recombinant expression of E. coli phage ID52-E under eco-friendly and reasonable price inducer L-arabinose.

SUBMITTER: Ma Y

PROVIDER: S-EPMC9311611 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The L protein of the single-stranded RNA phage MS2 causes lysis of Escherichia coli without inducing bacteriolytic activity or inhibiting net peptidoglycan (PG) synthesis. To find host genes required for L-mediated lysis, spontaneous Ill (insensitivity to Llysis) mutants were selected as survivors of L expression and shown to have a missense change of the highly conserved proline (P330Q) in the C-terminal domain of DnaJ. In the dnaJP330Q mutant host, L-mediated lysis is completely blocked at 30°C without affecting the intracellular levels of L. At higher temperatures (37°C and 42°C), both lysis and L accumulation are delayed. The lysis block at 30°C in the dnaJP330Q mutant was recessive and could be suppressed by Lovercomes dnaJ (Lodj ) alleles selected for restoration of lysis. All three Lodj alleles lack the highly basic N-terminal half of the lysis protein and cause lysis ?20 min earlier than full-length L. DnaJ was found to form a complex with full-length L. This complex was abrogated by the P330Q mutation and was absent with the Lodj truncations. These results suggest that, in the absence of interaction with DnaJ, the N-terminal domain of L interferes with its ability to bind to its unknown target. The lysis retardation and DnaJ chaperone dependency conferred by the nonessential, highly basic N-terminal domain of L resembles the SlyD chaperone dependency conferred by the highly basic C-terminal domain of the E lysis protein of ?X174, suggesting a common theme where single-gene lysis can be modulated by host factors influenced by physiological conditions.IMPORTANCE Small single-stranded nucleic acid lytic phages (Microviridae and Leviviridae) lyse their host by expressing a single "protein antibiotic." The protein antibiotics from two out of three prototypic small lytic viruses have been shown to inhibit two different steps in the conserved PG biosynthesis pathway. However, the molecular basis of lysis caused by L, the lysis protein of the third prototypic virus, MS2, is unknown. The significance of our research lies in the identification of DnaJ as a chaperone in the MS2 L lysis pathway and the identification of the minimal lytic domain of MS2 L. Additionally, our research highlights the importance of the highly conserved P330 residue in the C-terminal domain of DnaJ for specific protein interactions.

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Efficient Robust Yield Method for Preparing Bacterial Ghosts by Escherichia coli Phage ID52 Lysis Protein E.

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