Precise microbiome engineering using natural and synthetic bacteriophages targeting an artificial bacterial consortium

Abstract

In natural microbiomes, microorganisms interact with each other and exhibit diverse functions. Microbiome engineering, which enables bacterial knockdown, is a promising method to elucidate the functions of targeted bacteria in microbiomes. However, few methods to selectively kill target microorganisms in the microbiome without affecting the growth of nontarget microorganisms are available. In this study, we focused on the host-specific lytic ability of virulent phages and validated their potency for precise microbiome engineering. In an artificial microbiome consisting of Escherichia coli, Pseudomonas putida, Bacillus subtilis, and Lactiplantibacillus plantarum, the addition of bacteriophages infecting their respective host strains specifically reduced the number of these bacteria more than 10² orders. Remarkably, the reduction in target bacteria did not affect the growth of nontarget bacteria, indicating that bacteriophages were effective tools for precise microbiome engineering. Moreover, a virulent derivative of the λ phage was synthesized from prophage DNA in the genome of λ lysogen by in vivo DNA assembly and phage-rebooting techniques, and E. coli-targeted microbiome engineering was achieved. These results propose a novel approach for precise microbiome engineering using bacteriophages, in which virulent phages are synthesized from prophage DNA in lysogenic strains without isolating phages from environmental samples.

Keywords: bacteriophage; microbiome engineering; rebooting; synthetic phage; virulent conversion.

Graphical abstract

Figure 1

Examination of the dose dependency of the phages for inhibiting the growth of the host bacterium. T7 phage, ΦPpMK2-1, ΦBsOK1-1, and ΦLpTT2 were added to the cultures of their respective host bacterium, E. coli, P. putida, B. subtilis, and L. plantarum at different multiplicity of infection (MOI) values. After 10 h of cultivation, the viable cell counts of each bacterium were measured. The dotted line represents the initial viable cell counts (1.00 × 10⁵ CFU/mL). Data bars show the mean ± standard deviation of three independent experiments. The viable cell counts of each bacterium with its respective bacteriophage were compared to those in the no-phage conditions. Asterisks indicate p values less than 0.05 in the t-test.

Figure 2

Evaluation of the host specificity of the bacteriophages. Each of the four phages was added to the culture of E. coli, P. putida, B. subtilis, and L. plantarum. T7 and ΦBsOK1-1 phages were added to the bacterial cultures at MOI of 0.001, whereas ΦPpMK2-1 and ΦLpTT2 were added at MOI values of 0.01 and 1, respectively. The dotted line represents the initial viable cell counts (1.00 × 10⁵ CFU/mL). Data bars show the mean ± standard deviation of three independent experiments. For statistical analyses, the viable cell counts of each bacterium with and without the addition of the bacteriophage indicated were compared. Asterisks indicate p values less than 0.05 in the t-test.

Figure 3

Microbiome engineering of the artificial microbiome by using bacteriophages. E. coli (green circles), P. putida (blue triangles), B. subtilis (brown diamonds), and L. plantarum (pink squares) were cocultivated for 24 h without phage (A), with T7 phage at MOI 0.001 (B), with ΦPpMK2-1 at MOI 0.01 (C), with ΦBsOK1-1 at MOI 0.001 (D), and with ΦLpTT2 at MOI 1 (E). The samples were collected at 0, 4, 10, 16, and 24 h, and the viable cell numbers were determined. Data points show the mean ± standard deviation of three independent experiments. For statistical analyses, the viable cell counts of the target bacteria with their respective bacteriophages were compared with their initial cell concentrations. Asterisks and triple asterisks indicate p values less than 0.05 and 0.005 in the t-test.

Figure 4

Evaluation of the host specificity of the bacteriophages in the coculture system. The viable cell counts of E. coli (green bars), P. putida (blue bars), B. subtilis (brown bars), and L. plantarum (pink bars) at 10 h with and without the addition of the bacteriophage indicated were compared. Data bars show the mean ± standard deviation of three independent experiments. Asterisks indicate p values less than 0.05 in the t-test.

Figure 5

Synthesis of the artificial λ phage from the prophage region of a λ lysogen. (A) Design of the virulent derivative of the λ phage. Five regions except for attL, int, cI, and attR genes were amplified from the prophage region of the E. coli NBRC 3301 genome and assembled. The numbers at the top of the panel show the location in the NBRC 3301 genome (Genbank, BJLE01000002.1), while the numbers in parentheses show the location in the λ phage (GenBank, J02459.1). (B) Schematic illustration of in vivo DNA assembly and rebooting of the synthetic λ phage. (C) Results of agarose gel electrophoresis of the five λ fragments amplified by PCR. M, marker; 1–5, λ fragments 1 to 5. (D) Results of the plaque assay using the synthetic virulent λ. (E) Confirmation of gene assembly by amplifying the 600-bp joint regions of each fragment. M, marker; 1–5, joint regions of fragments 1 & 2, 2 & 3, 3 & 4, 4 & 5, and 5 & 1, respectively.

Figure 6

Microbiome engineering of the artificial microbiome by using the synthetic λ Δint ΔcI. E. coli (green circles), P. putida (blue triangles), B. subtilis (brown diamonds), and L. plantarum (pink squares) were cocultivated for 24 h with λ Δint ΔcI at MOI of 0.1. The samples were collected at 0, 4, 10, 16, and 24 h, and viable cell numbers were determined. Data dots show the mean ± standard deviation of three independent experiments. For statistical analyses, the viable cell counts of E. coli Cm^R were compared with its initial cell concentration. Triple asterisks indicate p values less than 0.005 in the t-test.