Peptide-Mediated Gene Transfer into Marine Purple Photosynthetic Bacteria.

Mieko Higuchi-Takeuchi,Choon Pin Foong,Takaaki Miyamoto,Kumiko Morisaki,Keiji Numata,Mami Goto

doi:10.3390/ijms21228625

Abstract

Use of photosynthetic organisms is one of the sustainable ways to produce high-value products. Marine purple photosynthetic bacteria are one of the research focuses as microbial production hosts. Genetic transformation is indispensable as a biotechnology technique. However, only conjugation has been determined to be an applicable method for the transformation of marine purple photosynthetic bacteria so far. In this study, for the first time, a dual peptide-based transformation method combining cell penetrating peptide (CPP), cationic peptide and Tat-derived peptide (dTat-Sar-EED) (containing D-amino acids of Tat and endosomal escape domain (EED) connected by sarcosine linkers) successfully delivered plasmid DNA into Rhodovulum sulfidophilum, a marine purple photosynthetic bacterium. The plasmid delivery efficiency was greatly improved by dTat-Sar-EED. The concentrations of dTat-Sar-EED, cell growth stage and recovery duration affected the efficiency of plasmid DNA delivery. The delivery was inhibited at 4 °C and by A22, which is an inhibitor of the actin homolog MreB. This suggests that the plasmid DNA delivery occurred via MreB-mediated energy dependent process. Additionally, this peptide-mediated delivery method was also applicable for E. coli cells. Thus, a wide range of bacteria could be genetically transformed by using this novel peptide-based transformation method.

Highlights

Genetic transformation is the process of introducing exogeneous DNA into cells and is an important method in bioengineering technology
Method of chemically competent E. coli cells using calcium chloride has been developed by Hanahan [5] and is shown to be applicable for a lot of bacterial species [6,7]
BP100-(KH)9 was used for the delivery into marine purple photosynthetic bacteria

Summary

Introduction

Genetic transformation is the process of introducing exogeneous DNA into cells and is an important method in bioengineering technology. Method of chemically competent E. coli cells using calcium chloride has been developed by Hanahan [5] and is shown to be applicable for a lot of bacterial species [6,7]. The precise mechanism of the transformation induced by calcium chloride has not been solved yet, it is thought that divalent cations interact with negatively charged DNA and cell membranes, leading to DNA internalization into the cells due to alteration in membrane permeability [8]. Another method for induction of competence in bacteria is the electroporation. Competent cells need to be prepared in both calcium chloride and electroporation methods, and the preparation steps of competent cells are time consuming

Methods

Results

Conclusion