Cross-Genus "Boot-Up" of Synthetic Bacteriophage in Staphylococcus aureus by Using a New and Efficient DNA Transformation Method

Abstract

Staphylococcus aureus is an opportunistic pathogen that causes a wide range of infections and food poisoning in humans with antibiotic resistance, specifically to methicillin, compounding the problem. Bacteriophages (phages) provide an alternative treatment strategy, but these only infect a limited number of circulating strains and may quickly become ineffective due to bacterial resistance. To overcome these obstacles, engineered phages have been proposed, but new methods are needed for the efficient transformation of large DNA molecules into S. aureus to "boot-up" (i.e., rescue) infectious phages. We presented a new, efficient, and reproducible DNA transformation method, NEST (non-electroporation Staphylococcus transformation), for S. aureus to boot-up purified phage genomic DNA (at least 150 kb in length) and whole yeast-assembled synthetic phage genomes. This method was a powerful new tool for the transformation of DNA in S. aureus and will enable the rapid development of engineered therapeutic phages and phage cocktails against Gram-positive pathogens. IMPORTANCE The continued emergence of antibiotic-resistant bacterial pathogens has heightened the urgency for alternative antibacterial strategies. Phages provide an alternative treatment strategy but are difficult to optimize. Synthetic biology approaches have been successfully used to construct and rescue genomes of model phages but only in a limited number of highly transformable host species. In this study, we used a new, reproducible, and efficient transformation method to reconstitute a functional nonmodel Siphophage from a constructed synthetic genome. This method will facilitate the engineering of Staphylococcus and Enterococcus phages for therapeutic applications and the engineering of Staphylococcus strains by enabling transformation of higher molecular weight DNA to introduce more complex modifications.

Keywords: bacteriophage assembly; bacteriophage genetics; phage engineering; synthetic biology; transformation.

FIG 1

Overview of NEST. (A) The basic steps needed to make stocks of S. aureus competent cells by NEST. (B) Frozen competent cells are thawed, incubated with either plasmid or phage DNA and PEG before plating as indicated. See Material and Methods for details.

FIG 2

Comparison of NEST versus electroporation transformation methods. The effects of DNA amount and size on the efficiency of transformation between NEST and electroporation were determined using different amounts (10 ng, 100 ng, 500 ng, and 1,000 ng) of plasmids pCM28, pCAS9counter, and pFG25 purified from E. coli DH10B. For the NEST method, plasmids were transformed using a 20% PEG solution to the S. aureus RN4220 competent cells prepared using HI seed media. For electroporation, the electrocompetent cells prepared with B2 media were electroporated (2.3 kV, 100 Ω, 25 μF) with the indicated plasmids. Cells were plated on NYE media plates with 12.5 μg/mL chloramphenicol for pCM28 and 10 μg/mL erythromycin for pCAS9counter and pGF35 and incubated at 30°C for 24 to 48 h. Each data point represents the mean from three independent experiments (except for pGF35 electroporation, which had two independent experiments), and the error bars indicate standard error.

FIG 3

Transfection of S. aureus phage genomic DNA using NEST. (A) Workflow of the boot-up of S. aureus phage genomic DNA using NEST method. Phenol-chloroform extracted gDNA was transformed into S. aureus competent cells using 20% PEG solution. After a brief incubation, PEG solution was removed using centrifugation, and pellets were dissolved in HI seed media. Samples were incubated at RT for 24 h followed by the top agar overlay method and plates were incubated at 30°C for 24 to 48 h to visualize the plaques. (B) phage SA75 and phage K were booted-up using 100 ng of gDNA and between 1.13 × 10⁸ to 6.13 × 10⁸ CFU of S. aureus competent cells prepared by the NEST method. S. aureus MRSN 7983 was used as an indicator strain for phage K. (C) Efficiency of boot-up was determined by calculating the plaques per 10⁸ CFU using different amounts of gDNA of phage SA75 and phage K. Each data point represents the mean from three independent experiments, and the error bars indicate standard error.

FIG 4

Boot-up of yeast-assembled synthetic S. aureus phage genomes using NEST. (A) The boot-up of either the whole yeast cloned (YC) or yeast assembled (YA) genomes of phage SA75 is depicted. To make SA75YC, cloned SA75 DNA, a PCR-generated repeat fragment, and a linear Ycp/BAC vector harboring terminal homology to the phage DNA and unique restriction site (ISce-I) were transformed into S. cerevisiae (yeast). Similarly, to make SA75YA, TAR-cloned fragments are transformed into yeast with the linear Ycp/BAC vector. DNA from positive clones was transformed into E. coli DH10B cells to produce high concentration plasmid stocks. These plasmids were further transfected/transformed into S. aureus competent cells using the NEST method, incubated at RT for 24 h, and plated using the agar overlay method to observe plaques. (B) To determine the efficiency of boot-up of SA75 DNA isolated from packaged genomes and synthetic genomes, 100 ng of DNA was incubated with 1.13 × 10⁸ CFU of NEST competent cells for 30 min before plating. The efficiency of boot-up was computed by calculating the number of plaques formed per 10⁸ CFU. Each data point represents the mean from three independent experiments, and the error bars indicate standard error.

FIG 5

Cross-genus transfection of E. faecalis phage genomic DNA using NEST. Appropriate amount (100 to 200 ng) of E. faecalis phage ɸ5.1, ɸ5.2, ɸ5.3, ɸ5.4 and ɸ6.4 gDNA was transformed into S. aureus cells (∼9 × 10⁸ CFU) using the NEST method and incubated for 24 h at 23°C. (A) Transformants were mixed with the corresponding indicator strain (E. faecalis AH5 for ɸ5.1, ɸ5.2, ɸ5.3, and ɸ5.4, or AH6 for ɸ6.4) and plated on BHI media using the top agar overlay method and incubated at 30°C for 24 to 48 h to observe the plaques. The control plate (Cntl) was a lawn of S. aureus RN4220 cells mixed with E. faecalis AH5 lacking phage DNA. (B) The efficiency of boot-up was determined by calculating the number of plaques per 10⁸ CFU of NEST competent cells of each E. faecalis phage and SA75 for comparison. Each data point represents the mean from three independent experiments, and the error bars indicate standard error.