Subtractive modification of bacterial consortium using antisense peptide nucleic acids

Abstract

Microbiome engineering is an emerging research field that aims to design an artificial microbiome and modulate its function. In particular, subtractive modification of the microbiome allows us to create an artificial microbiome without the microorganism of interest and to evaluate its functions and interactions with other constituent bacteria. However, few techniques that can specifically remove only a single species from a large number of microorganisms and can be applied universally to a variety of microorganisms have been developed. Antisense peptide nucleic acid (PNA) is a potent designable antimicrobial agent that can be delivered into microbial cells by conjugating with a cell-penetrating peptide (CPP). Here, we tested the efficacy of the conjugate of CPP and PNA (CPP-PNA) as microbiome modifiers. The addition of CPP-PNA specifically inhibited the growth of Escherichia coli and Pseudomonas putida in an artificial bacterial consortium comprising E. coli, P. putida, Pseudomonas fluorescens, and Lactiplantibacillus plantarum. Moreover, the growth inhibition of P. putida promoted the growth of P. fluorescens and inhibited the growth of L. plantarum. These results indicate that CPP-PNA can be used not only for precise microbiome engineering but also for analyzing the growth relationships among constituent microorganisms in the microbiome.

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

“…They were used as host strains to isolate bacteriophages. A chloramphenicol-resistant E. coli MG 1655 derivative ( E. coli Cm R ), a neomycin-resistant derivative of P. putida NBRC 14164 ( P. putida Neo R ) ( Hizume et al, 2024 ), a spontaneous mutant of B. subtilis NBRC 111470 with streptomycin resistance ( B. subtilis Str R ), and a wild-type L. plantarum NCIMB 8826 were used as artificial microbiome components in the microbiome engineering experiments. B. subtilis Str R was obtained by directly spreading wild-type B. subtilis culture on Luria-Bertani (LB) agar medium with 50 μg/mL streptomycin.…”

“…Targeted antimicrobial agents are potential tools to modify the composition of microbiomes in a subtractive manner ( Chou et al, 2022 ). By specifically reducing target microorganisms in microbiomes and evaluating their functional and structural changes, the role of target microorganisms could be elucidated ( Hsu et al, 2019 ; Hizume et al, 2024 ). Antibiotics are the most frequently used antimicrobial agents.…”

“…Some antimicrobial peptides such as bacteriocins show a relatively narrow spectrum compared to antibiotics but still act on a variety of microbes across classes and orders ( Rea et al, 2010 ). To precisely manipulate microbiomes, we previously reported a microbiome engineering technique using peptide nucleic acid (PNA) conjugated with cell-penetrating peptide (CPP) ( Hizume et al, 2024 ). PNA is a DNA analog with a peptide backbone and high biostability.…”