Presentation Information
[P03-394]Species-specific microbiome engineering using bacteriophages to control the function of microbiomes
○Tomoki Tanaka1, Ryota Ueki1, Hiroaki Iwaki1, Kenji Okano1 (1. Kansai University (Japan))
Keywords:
Microbiome engineering,Bacteriophage,Syhthetic phage,Virulent conversion,Gut microbiome
In environments, microbiomes exhibit higher-order functions that cannot be achieved by single bacterial species. To control the functions of microbiomes, modulating the abundance of specific bacterial species is necessary. However, a versatile platform enabling species-specific removal of target bacteria from microbiomes has not yet been established. Although bacteriophages exhibit host-specific lytic ability, it remains unclear whether phages can achieve species-level selective manipulation within microbial communities. In addition, the establishment of phage-mediated microbiome engineering requires the rapid acquisition of phages infecting target bacteria, yet such phages are not always readily isolated from environmental samples.
In this study, therefore, we first evaluated whether phages enable species-level selective killing of target bacteria in microbiomes using natural phages. We then developed a phage synthesis approach for the rapid generation of lytic phages and assessed its applicability for microbiome engineering.
As an initial proof-of-concept for phage-mediated microbiome engineering, we constructed an artificial microbiome consisting of four phylogenetically distant species; Escherichia coli, Pseudomonas putida, Bacillus subtilis, and Lactiplantibacillus plantarum. Individual addition of four phages targeting each species selectively reduced the viable cells of the target bacteria. To further examine the robustness selective killing under more stringent conditions, microbiome engineering was performed using an artificial microbiome consisting of Bacteroides species and closely related species (Bacteroides caccae, Bacteroides finegoldii, Bacteroides intestinalis, Bacteroides ovatus, Bacteroides uniformis, Phocaeicola dorei, and Phocaeicola vulgatus). When two bacteriophages infecting B. caccae and B. uniformis were individually added to the microbiome, the abundance of the target bacteria was reduced. Notably, the abundance of B. finegoldii increased upon addition of the B. caccae phage, suggesting that the removal of B. caccae promoted the growth of B. finegoldii. These results demonstrate that phages enable species-level engineering of microbiomes and provide a useful approach for detecting interspecies growth interactions.
To establish a method for rapid acquisition of lytic phages, a lytic λ phage was generated from the prophage sequence in a λ-lysogenic E. coli strain. DNA regions excluding lysogeny-related genes were amplified by PCR into five fragments of approximately 10 kbp each, with 50-bp overlapping sequences. These fragments were introduced into E. coli expressing λ-Red recombinase, where they were assembled and phage particles were generated. Addition of the resulting phage to a four-species microbiome selectively reduced the viable cells of E. coli. These results demonstrate that lytic phages can be generated from prophage sequences without isolation from environmental samples, providing a novel approach for microbiome engineering.
In this study, therefore, we first evaluated whether phages enable species-level selective killing of target bacteria in microbiomes using natural phages. We then developed a phage synthesis approach for the rapid generation of lytic phages and assessed its applicability for microbiome engineering.
As an initial proof-of-concept for phage-mediated microbiome engineering, we constructed an artificial microbiome consisting of four phylogenetically distant species; Escherichia coli, Pseudomonas putida, Bacillus subtilis, and Lactiplantibacillus plantarum. Individual addition of four phages targeting each species selectively reduced the viable cells of the target bacteria. To further examine the robustness selective killing under more stringent conditions, microbiome engineering was performed using an artificial microbiome consisting of Bacteroides species and closely related species (Bacteroides caccae, Bacteroides finegoldii, Bacteroides intestinalis, Bacteroides ovatus, Bacteroides uniformis, Phocaeicola dorei, and Phocaeicola vulgatus). When two bacteriophages infecting B. caccae and B. uniformis were individually added to the microbiome, the abundance of the target bacteria was reduced. Notably, the abundance of B. finegoldii increased upon addition of the B. caccae phage, suggesting that the removal of B. caccae promoted the growth of B. finegoldii. These results demonstrate that phages enable species-level engineering of microbiomes and provide a useful approach for detecting interspecies growth interactions.
To establish a method for rapid acquisition of lytic phages, a lytic λ phage was generated from the prophage sequence in a λ-lysogenic E. coli strain. DNA regions excluding lysogeny-related genes were amplified by PCR into five fragments of approximately 10 kbp each, with 50-bp overlapping sequences. These fragments were introduced into E. coli expressing λ-Red recombinase, where they were assembled and phage particles were generated. Addition of the resulting phage to a four-species microbiome selectively reduced the viable cells of E. coli. These results demonstrate that lytic phages can be generated from prophage sequences without isolation from environmental samples, providing a novel approach for microbiome engineering.
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