Using Methyl Bromide for Interspecies Cell–Cell Signaling and As a Reporter in a Model Soil Consortium

Kim, Jiwoo; Lu, Li Chieh; Gao, Xiaodong; Hofmockel, Kirsten S.; Masiello, Caroline A.; Silberg, Jonathan J.

doi:10.1021/acssynbio.3c00559

Cited by 2 publications

(2 citation statements)

References 55 publications

(148 reference statements)

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“…We next sought to investigate if ribozyme-mediated gene-fragment complementation can report on gene transfer when conjugation is performed within a soil, which can present a background CH 3 Br signal. 24 Conjugation measurements were performed in a previously described soil (Figure 4A) that had been hydrated to ∼20% water holding capacity (WHC). 23 Prior to performing conjugation experiments, we evaluated donor cell viability upon adding different DAP concentrations (Figure S7).…”

Section: Resultsmentioning

confidence: 99%

“…20 MHT reporters have been used for in soils to monitor the bioavailability of microbe-microbe signals 21 , microbe-plant signals 22 , and sugars. 23 In addition, they have been used to monitor the conditional activities of soil microbe promoters 24 and coded into mobile DNA to report on conjugation in soil. 25 Unfortunately, MHT reporters remain limited in their ability to report on HGT in soils, because a background signal accumulates with time in the absence of HGT due to leaky MHT expression.…”

Section: Introductionmentioning

confidence: 99%

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Ribozyme-mediated gene-fragment complementation for non-destructive reporting of DNA transfer within soil

Selinidis,

Corliss,

Chappell

et al. 2024

Preprint

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Enzymes that produce volatile metabolites can be coded into genetic circuits to report non-disruptively on microbial behaviors in hard-to-image soils. However, these enzyme reporters remain challenging to apply in gene transfer studies due to leaky off states that can lead to false positives. To overcome this problem, we designed a reporter that uses ribozyme-mediated gene-fragment complementation of a methyl halide transferase (MHT) to regulate the synthesis of methyl halides. We split themhtgene into two non-functional fragments and attached these to a pair of splicing ribozyme fragments. While the individualmht-ribozyme fragments did not produce methyl halides when transcribed alone inEscherichia coli, co-expression resulted in a spliced transcript that translated the MHT reporter. When cells containing onemht-ribozyme fragment transcribed from a mobile plasmid were mixed with cells that transcribed the secondmht-ribozyme fragment, methyl halides were only detected following rare conjugation events. When conjugation was performed in soil, it led to a 16-fold increase in methyl halides in the soil headspace. These findings show how ribozyme-mediated gene-fragment complementation can achieve tight control of protein reporter production, a level of control that will be critical for monitoring the effects of soil conditions on gene transfer and the fidelity of biocontainment measures developed for environmental applications.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Ribozyme-mediated gene-fragment complementation for non-destructive reporting of DNA transfer within soil

Selinidis,

Corliss,

Chappell

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

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Ribozyme-Mediated Gene-Fragment Complementation for Nondestructive Reporting of DNA Transfer within Soil

Selinidis,

Corliss,

Chappell

et al. 2024

ACS Synth. Biol.

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Enzymes that produce volatile metabolites can be coded into genetic circuits to report nondisruptively on microbial behaviors in hard-to-image soils. However, these enzyme reporters remain challenging to apply in gene transfer studies due to leaky off states that can lead to false positives. To overcome this problem, we designed a reporter that uses ribozyme-mediated gene-fragment complementation of a methyl halide transferase (MHT) to regulate the synthesis of methyl halide gases. We split the mht gene into two nonfunctional fragments and attached these to a pair of splicing ribozyme fragments. While the individual mht-ribozyme fragments did not produce methyl halides when transcribed alone in Escherichia coli, coexpression resulted in a spliced transcript that translated the MHT reporter. When cells containing one mht-ribozyme fragment transcribed from a mobile plasmid were mixed with cells that transcribed the second mht-ribozyme fragment, methyl halides were only detected following rare conjugation events. When conjugation was performed in soil, it led to a 16-fold increase in methyl halides in the soil headspace. These findings show how ribozyme-mediated gene-fragment complementation can achieve tight control of protein reporter production, a level of control that will be critical for monitoring the effects of soil conditions on gene transfer and the fidelity of biocontainment measures developed for environmental applications.

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Using Methyl Bromide for Interspecies Cell–Cell Signaling and As a Reporter in a Model Soil Consortium

Cited by 2 publications

References 55 publications

Ribozyme-mediated gene-fragment complementation for non-destructive reporting of DNA transfer within soil

Ribozyme-mediated gene-fragment complementation for non-destructive reporting of DNA transfer within soil

Ribozyme-Mediated Gene-Fragment Complementation for Nondestructive Reporting of DNA Transfer within Soil

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