Exploring the synthetic biology potential of bacteriophages for engineering non-model bacteria

Lammens, Eveline-Marie; Nikel, Pablo I.; Lavigne, Rob

doi:10.1038/s41467-020-19124-x

Cited by 56 publications

(47 citation statements)

References 176 publications

(213 reference statements)

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“…73 reported that the frequency of occurrence of Rif R mutants in a Δ)mutS derivative of P. putida KT2440 was 1,000- fold higher than that in the wild-type strain. Since mutation rates must be precisely controlled to avoid extensive accumulation of deleterious mutations and to prevent genomic instability, the overexpression of mutator alleles should be driven from tightly-regulated expression systems (which is always challenging, irrespective of the bacterial host 74 ) or during short periods of time. Thus, the easy-to-cure mutator plasmids developed in this study, which can be rapidly removed from isolated clones displaying the phenotype of interest, offer a clear advantage over conventional mutator strains—where the mutator phenotype is elicited by genomic (hence, essentially irreversible) modifications, as epitomized by the emergence of mutator phenotypes of P. aeruginosa in clinically-relevant setups 75-77 .…”

Section: Resultsmentioning

confidence: 99%

“…In addition to their application for the accelerated evolution of phenotypes that depend on multiple mutations across the bacterial genome, the use of these devices also revealed an important feature of the MMR system relevant for synthetic biology. A number of genome modification approaches rely on specifically interfering with the bacterial MMR system to enable strand invasion 51,53,74,83 . Besides the intended modifications (e.g.…”

Section: Resultsmentioning

confidence: 99%

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Spatiotemporal manipulation of the mismatch repair system ofPseudomonas putidaaccelerates phenotype emergence

Fernández-Cabezón

Cros

Nikel

2021

Preprint

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Developing complex phenotypes in industrially-relevant bacteria is a major goal of metabolic engineering, which encompasses the implementation of both rational and random approaches. In the latter case, several tools have been developed towards increasing mutation frequencies—yet the precise spatiotemporal control of mutagenesis processes continues to represent a significant technical challenge. Pseudomonas species are endowed with one of the most efficient DNA mismatch repair (MMR) systems found in bacteria. Here, we investigated if the endogenous MMR system could be manipulated as a general strategy to artificially alter mutation rates in Pseudomonas species. To bestow a conditional mutator phenotype in the platform bacterium Pseudomonas putida, we constructed inducible mutator devices to modulate the expression of the dominant-negative mutLE36K allele. Regulatable overexpression of mutLE36K in a broad-host-range, easy-to-cure plasmid format resulted in a transitory inhibition of the MMR machinery, leading to a significant increase (up to 438-fold) in mutation frequencies and a heritable fixation of genome mutations. Following such accelerated mutagenesis-followed-by selection approach, three phenotypes were successfully evolved: resistance to antibiotics streptomycin and rifampicin and reversion of a synthetic uracil auxotrophy. Thus, these mutator devices could be applied to accelerate evolution of metabolic pathways in long-term evolutionary experiments, alternating cycles of (inducible) mutagenesis coupled to selection schemes.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Spatiotemporal manipulation of the mismatch repair system ofPseudomonas putidaaccelerates phenotype emergence

Fernández-Cabezón

Cros

Nikel

2021

Preprint

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“…While our results provide insight into the limits and capabilities of phage regulatory strategies, our approach may also prove useful for synthetic biology applications in phages [43, 44]. Synthetic genetic circuits are typically designed for free-living species where they must operate in variable cellular contexts over comparatively long time-scales [45, 46].…”

Section: Discussionmentioning

confidence: 99%

Generating complex patterns of gene expression without regulatory circuits

Hill

et al. 2020

Preprint

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Synthetic biology has successfully advanced our ability to design complex, time-varying genetic circuits executing precisely specified gene expression patterns. However, such circuits usually require regulatory genes whose only purpose is to regulate the expression of other genes. When designing very small genetic constructs, such as viral genomes, we may want to avoid introducing such auxiliary gene products. To this end, here we demonstrate that varying only the placement and strengths of promoters, terminators, and RNase cleavage sites in a computational model of a bacteriophage genome is sufficient to achieve solutions to a variety of basic expression patterns. We discover these solutions by computationally evolving genomes to reproduce desired target expression patterns. Our approach shows non-trivial patterns can be evolved, including patterns in which the relative ordering of genes by abundance changes over time. We find that some patterns are easier to evolve than others, and different genomes that express comparable expression patterns may differ in their genetic architecture. Our work opens up a novel avenue to genome engineering via fine-tuning the balance of gene expression and gene degradation rates.

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“…Bacteriophage genome engineering offers the possibility of streamlining the process for determining the phage sensitivity of a target bacteria by making the bacterial isolation step redundant [ 16 , 17 ]. In this approach, currently being implemented primarily in the food industry to detect the presence of microbial contamination, engineered bioluminescence-based reporter bacteriophage assays offer the best available method for determining the presence of specific bacteria with a fast turnaround time and without the need for bacterial isolation [ 18 , 19 ].…”

Section: Introductionmentioning

confidence: 99%

Luminescent Phage-Based Detection of Klebsiella pneumoniae: From Engineering to Diagnostics

et al. 2021

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Bacteriophages (“phages”) infect and multiply within specific bacterial strains, causing lysis of their target. Due to the specific nature of these interactions, phages allow a high-precision approach for therapy which can also be exploited for the detection of phage-sensitive pathogens associated with chronic diseases due to gut microbiome imbalance. As rapid phage-mediated detection assays becoming standard-of-care diagnostic tools, they will advance the more widespread application of phage therapy in a precision approach. Using a conventional method and a new cloning approach to develop luminescent phages, we engineered two phages that specifically detect a disease-associated microbial strain. We performed phage sensitivity assays in liquid culture and in fecal matrices and tested the stability of spiked fecal samples stored under different conditions. Different reporter gene structures and genome insertion sites were required to successfully develop the two nluc-reporter phages. The reporter phages detected spiked bacteria in five fecal samples with high specificity. Fecal samples stored under different conditions for up to 30 days did not display major losses in reporter-phage-based detection. Luminescent phage-based diagnostics can provide a rapid co-diagnostic tool to guide the growing field of phage therapy, particularly for a precision-based approach to chronic diseases treatment.

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Exploring the synthetic biology potential of bacteriophages for engineering non-model bacteria

Cited by 56 publications

References 176 publications

Spatiotemporal manipulation of the mismatch repair system ofPseudomonas putidaaccelerates phenotype emergence

Spatiotemporal manipulation of the mismatch repair system ofPseudomonas putidaaccelerates phenotype emergence

Generating complex patterns of gene expression without regulatory circuits

Luminescent Phage-Based Detection of Klebsiella pneumoniae: From Engineering to Diagnostics

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