Transformation of Tn7 insertion elements across strains of Vibrio fischeri

Andrew G. Cecere; Chris Muriel-Mundo; Derek J. Fisher; Tim I. Miyashiro

doi:10.1371/journal.pone.0338632

Abstract

Animals typically form symbiotic relationships with bacteria that contribute to their physiology and behaviors. The ability to genetically modify these bacterial symbionts is important for investigating the molecular mechanisms that promote symbiosis establishment and maintenance. However, the molecular tools developed for laboratory-adapted strains may fail when applied to non-canonical strains. Here, we report a method to expand the use of Tn7 site-specific transposon-insertion mutagenesis in Vibrio fischeri, which is the bioluminescent bacterial symbiont of the Hawaiian bobtail squid Euprymna scolopes. In this protocol, the laboratory-adapted strain ES114 is used as a surrogate strain for introducing genetic information into the attTn7 insertion site. Genomic DNA extracted from the resulting strain is used as template for transformation of another strain, in which natural transformation is induced. As a proof of principle, this approach is used to complement an rpoN mutant with an IPTG-inducible rpoN construct in trans.

Citation: Cecere AG, Muriel-Mundo C, Fisher DJ, Miyashiro TI (2025) Transformation of Tn7 insertion elements across strains of Vibrio fischeri. PLoS One 20(12): e0338632. https://doi.org/10.1371/journal.pone.0338632

Editor: Claudia Isabella Pogoreutz, Université de Perpignan: Universite de Perpignan Via Domitia, FRANCE

Received: September 19, 2025; Accepted: November 25, 2025; Published: December 30, 2025

Copyright: © 2025 Cecere et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The sequencing data for this project are available at the Sequence Read Archive (SRA) of NCBI under BioProject accession PRJNA1322440 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA1322440). BioSample accession numbers are as follows: SAMN51247791 (TIM313): https://www.ncbi.nlm.nih.gov/sra/SRX30438874[accn] SAMN51247792 (FQ-A001): https://www.ncbi.nlm.nih.gov/sra/SRX30438875[accn] SAMN51247793 (AGC020): https://www.ncbi.nlm.nih.gov/sra/SRX30438876[accn] SAMN51247794 (AGC028): https://www.ncbi.nlm.nih.gov/sra/SRX30438877[accn] SAMN51247795 (AGC029): https://www.ncbi.nlm.nih.gov/sra/SRX30438878[accn] SAMN51247796 (AGC030): https://www.ncbi.nlm.nih.gov/sra/SRX30438879[accn] Other data are provided in S1 Data Set.

Funding: This work was supported by the National Institute of General Medical Sciences Grant R35 GM152259 (to T.I.M.). The funder did not and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Vibrio fischeri (aka, Aliivibrio fischeri) is a Gram-negative marine bacterium that is capable of establishing symbiosis with various squid and fish [1]. Of these symbioses, the one V. fischeri forms with the Hawaiian bobtail squid Euprymna scolopes is the best understood [2]. From within a symbiotic light organ, V. fischeri populations produce bioluminescence that enables E. scolopes to disrupt its shadow while swimming in the water column at night [3]. The symbiosis forms in hatchlings, following the acquisition of environmental V. fischeri cells that rapidly colonize the nascent light organ and grow into light-emitting populations. For more than 30 years, researchers have used the genetically manipulatable strain ES114 to investigate the general mechanisms by which V. fischeri colonizes the light organ [4]. However, wild-caught animals feature multiple strains of V. fischeri [5], and recent research efforts have investigated how different strains interact while colonizing the nascent light organ [6]. For example, strain FQ-A001 has a type 6 secretion system (T6SS) that facilitates interference competition among V. fischeri strains in vivo [7]. Notably, this T6SS is not encoded by ES114, indicating that the development of approaches for manipulating the genomes of non-canonical strains are of significance for elucidating the molecular mechanisms underlying the interactions between symbiotic V. fischeri strains.

Like most Vibrionaceae spp., V. fischeri encodes for the alternative sigma factor RpoN (aka, σ⁵⁴), which promotes transcription of genes involved in bioluminescence production, biofilm formation, type 6 secretion, and motility [8–11]. Furthermore, σ⁵⁴ is necessary for V. fischeri to colonize E. scolopes hatchlings [8,9], highlighting its significant regulatory role during the initial stages of symbiosis establishment. Despite its importance in these cellular processes, rpoN is typically nonessential for growth in standard laboratory medium, which has made it a useful control for testing new techniques [12]. Of relevance to the protocol described here, rpoN mutants are amotile due to their inability to activate transcription of flagellar biosynthesis genes [13], thereby permitting simple motility assays to distinguish between wild-type and rpoN alleles.

Tn7 is a transposon that inserts into a specific site within the chromosome, known as attTn7 (3’ to the glmS terminator), of many bacterial species including V. fischeri [14,15], which has led to an efficient way to introduce genetic information into the chromosome of a strain without affecting its fitness. Unlike most plasmids, Tn7 chromosomal insertions are stable without selection, thereby eliminating the need to introduce antibiotics in other assays, e.g., squid-colonization assays [16]. This method has proven valuable for experiments designed to test for genetic complementation during studies of ES114 [17,18]. Tn7 insertions have also facilitated competition assays, in which strains can be distinguished by the marker(s) inserted at the Tn7 site [17].

Like many taxa within the Vibrionaceae family, V. fischeri is capable of natural transformation, which describes the ability of a bacterium to incorporate exogenous DNA into its genome [19]. In V. fischeri, this process is controlled by environmental and genetic factors, and investigations of these factors have led to the development of molecular tools to enhance the rates of natural transformation. For instance, pLosTfoX is a plasmid that overexpresses the TfoX regulator that promotes transcription of genes that facilitate natural transformation [20]. The goal of the protocol described here is to use pLosTfoX-dependent natural transformation to introduce genetic elements associated with the Tn7-insertion site of one V. fischeri strain into another. Consequently, this protocol has the potential to overcome technical barriers for inserting genetic content into the Tn7 site of strains recalcitrant to standard transposition approaches. Conditions that may limit the utility of this protocol include the necessity for homology associated with the Tn7 site between strains and the introduction of additional genetic information linked to the Tn7 site by homologous recombination.

Materials and methods

The protocol described in this peer-reviewed article is published on protocols.io DOI: dx.doi.org/10.17504/protocols.io.kqdg31k11l25/v1 (S1 File).

Media and growth conditions

V. fischeri strains (Table 1) were grown aerobically in LBS medium [1% (wt/vol) tryptone, 0.5% (wt/vol) yeast extract, 2% (wt/vol) NaCl, 50 mM Tris–HCl (pH 7.5)]. When appropriate, LBS was supplemented with chloramphenicol at a final concentration of 2.5 μg ml^-1 and/or erythromycin (Erm) at a final concentration of 5 μg ml^-1.

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Table 1. Bacterial strains and plasmids used in this study.

https://doi.org/10.1371/journal.pone.0338632.t001

Tn7 integration assay

Chromosomal integration at the attTn7 site in V. fischeri was performed as previously reported using mini-Tn7 delivery plasmids derived from pEVS107, which carries an erythromycin resistance marker (erm^R) [14]. To initiate transposition, 0.1-mL cultures of E. coli donor strains harboring the pEVS107-derived plasmid, the helper plasmid pEVS104, and the transposase-encoding plasmid pUX-BF13 were washed in LBS and then combined with the V. fischeri recipient strain in a tetra-parental mating mixture. The mixture was spotted onto LBS agar and incubated at 28 °C for approximately 20 hours. Agar plugs with the mating mixtures were excised using flame-sterilized forceps, resuspended in 1 mL of LBS, and vortexed for 10 seconds.

To select for Tn7 integrants, 0.2 mL of the resuspension was plated onto LBS agar supplemented with erythromycin (LBS-erm) and incubated at 28°C. Together, the higher salt concentration of LBS and lower temperature promote the growth of V. fischeri over E. coli. To quantify total viable cells, the remaining suspension was serially diluted 10-fold to 10 ⁻ ⁷, and 0.1 mL aliquots from the 10 ⁻ ⁵ to 10 ⁻ ⁷ dilutions were plated onto LBS agar. Integration frequency was calculated as the ratio of erm^R colony-forming units (CFU) to total CFU.

Molecular Biology

In this protocol, DNA templates for pLosTfoX-dependent natural transformation consisted of either purified PCR products or genomic DNA. For the PCR product, a 3.5-kb amplicon featuring the Tn7::erm region with 0.5-kb flanking homology was amplified from TIM313 genomic DNA by PCR using PFU Ultra Polymerase (Agilent) and primers ES_Tn7-NT-u1 & -l1 (Table 2). The PCR product was purified using the E.Z.N.A. Cycle Pure Kit (Omega Bio-tek, Inc., Norcross, GA, USA).

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Table 2. Primers used in this study.

https://doi.org/10.1371/journal.pone.0338632.t002

Plasmid pCAM003 features VF_0387 (rpoN), which encodes σ⁵⁴ in ES114, downstream of the IPTG-inducible P_trc promoter. To construct pCAM003, a 1.5-kb fragment containing rpoN was amplified from ES114 genomic DNA by PCR using PFU Ultra Polymerase (Agilent) and primers rpoN-Ptrc-KpnI-u1 & -SalI-l1 and cloned into pCR-Blunt (Invitrogen). Following whole-plasmid sequencing via Plasmidsaurus, the rpoN gene was excised using KpnI/SalI DNA restriction enzymes (New England Biolabs) and subcloned into a KpnI/SalI vector fragment of pTM214 downstream of the Isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible trc promoter (P_trc).

Plasmid pCAM004 contains the IPTG-inducible rpoN construct that can be inserted into the attTn7 site. To construct pCAM004, a 1.7-kb fragment containing rpoN was excised from pCAM003 using KpnI/BsaI and correspondingly subcloned into a 6.8-kb vector fragment of pTM318, which is a Tn7-insertion shuttle vector that contains lacI^q P_trc::mCherry [26], thereby replacing mCherry with rpoN.

Strain CAM001 was constructed by inserting the lacI^q P_trc::rpoN erm cassette of pCAM004 into the attTn7 site of KRG003 (ES114 ΔrpoN) using helper plasmids pEVS104 and pUX-BF13.

Genome sequencing and analysis

To sequence the genome of a strain, genomic DNA was extracted from 0.5 mL LBS starter cultures using the MasterPure Complete DNA and RNA Purification Kit (EpiCentre) and submitted to SeqCenter (Pittsburgh, PA) for Illumina Sequencing. Illumina sequencing libraries were prepared using the tagmentation-based and PCR-based Illumina DNA Prep kit and custom IDT 10-bp unique dual indices (UDI) with a target insert size of 280 bp. No additional DNA fragmentation or size selection steps were performed. Illumina sequencing was performed on an Illumina NovaSeq X Plus sequencer in one or more multiplexed shared-flow-cell runs, producing 2x151-bp paired-end reads. Demultiplexing, quality control and adapter trimming was performed with bcl-convert (v4.2.4). De novo genome assembly was performed using SPAdes genome assembler v3.13.1 and annotated using prokka v1.14.6. The data for this project are available at the Sequence Read Archive (SRA) of NCBI under BioProject accession PRJNA1322440 with BioSample accession numbers SAMN51247791 (TIM313), SAMN51247792 (FQ-A001), SAMN51247793 (AGC020), SAMN51247794 (AGC028), SAMN51247795 (AGC029), and SAMN51247796 (AGC030).

Regions of homology involved in natural transformation events at the Tn7 site of FQ-A001-derived strains were determined using Clone Manager 9 Professional Edition. For each genome, the contig containing the glmS region was identified by scanning the contigs for the sequence 5’-GGAACGGATGTCGATCAGCCTCGT-3’. Then for each glmS-containing contig, the Multi-Way Alignment tool was used to align to the corresponding contigs of TIM313 and FQ-A001. Crossover sites for homologous recombination were defined as the SNPs corresponding to TIM313 that were detected in the transformant at the greatest distance from the attTn7 site.

Motility assay

For each strain, a starter culture was initiated by inoculating LBS medium with an isolated colony and incubating it at 28°C overnight. Starter cultures were diluted 1:100 into fresh LBS medium ± 1 mM IPTG and incubated at 28°C until OD₆₀₀ ~ 0.2. To initiate the motility assay, a 5-µL volume of the culture was injected into a soft-agar motility plate [0.5% (wt/vol) tryptone, 0.3% (wt/vol) yeast extract, 0.25% (wt/vol) agar, 50% (vol/vol) Instant Ocean] ± 1 mM IPTG and incubated at 28°C. Images of the motility plates were taken over time, and motility rates were calculated from measurements of the motility ring diameter.

Statistical analysis

Statistical analyses were performed using Prism v. 10.5.0 (GraphPad Software, LLC).

Expected results

Overview of the pLosTfoX-dependent natural transformation protocol

Certain strains of V. fischeri, e.g., FQ-A001, are recalcitrant to approaches that facilitate Tn7 insertion in ES114 (S1 Fig and S1 Data Set). This protocol provides an alternative strategy for introducing genetic information at the Tn7 site into a recipient strain using pLosTfoX-dependent natural transformation. ES114 is used as a surrogate strain to insert genetic information at the Tn7 site. Genomic DNA (gDNA) is then harvested from the resulting strain for use as template for pLosTfoX-dependent natural transformation of the recipient strain.

Transformation of FQ-A001 with a Tn7 insertion from ES114

The genomes of ES114 and FQ-A001 exhibit significant homology near the Tn7 insertion site (Fig 1A). TIM313 is an ES114-derived strain with an erm marker inserted at the attTn7 site [17]. A PCR product featuring this Tn7 insertion with ~500 bp of genomic sequence flanking either side (Tn7::erm PCR) can be used as donor DNA for introducing the erm marker into ES114 (Fig 1B and S1 Data Set). Transformation frequency increases 100-fold when TIM313 gDNA is used in place of the PCR product (Fig 1B). FQ-A001 could be transformed with TIM313 gDNA but not the PCR product derived from TIM313. However, FQ-A001 could also be transformed using a PCR product containing the erm marker that had been integrated into the rpoN locus of FQ-A001 (rpoN::erm PCR), suggesting that the use of a PCR product as a template was not the underlying reason for failed transformation with the Tn7::erm cassette. Whole genome sequencing of four FQ-A001-derived strains transformed with TIM313 gDNA showed the presence of the erm marker at the attTn7 site, with homology facilitating homologous recombination ranging from 12–30 kb (Fig 1A and Table 3).

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Table 3. Regions of homology in FQ-A001-derived strains transformed with TIM313 gDNA.

https://doi.org/10.1371/journal.pone.0338632.t003

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Fig 1. Expected results.

A) Genomic loci of attTn7 sites in FQ-A001 (top) and ES114 (bottom) aligned by progressiveMauve. attTn7 site is outlined by dotted shape. Bars above alignment indicate regions homologous to ES114 in FQ-A001-derived integrants (1 = AGC020, 2 = AGC028, 3 = AGC029, 4 = AGC030). B) Transformation frequency of V. fischeri strains ES114 and FQ-A001 with donor DNA from TIM313 (ES114 Tn7::erm) or KRG006 (FQ-A001 rpoN::erm). Each point represents transformation efficiency (calculated as erm^R CFU/total CFU) of one sample (N = 3) and bars represent group geometric mean. Open symbols indicate limit of detection as no erm^R CFU were detected. One-way ANOVA detected statistical significance among group geometric means (F_4,10 = 91.38, p < 0.0001), with Tukey’s post-hoc test with p-values corrected for multiple comparisons (different letters indicate different statistical groups).

https://doi.org/10.1371/journal.pone.0338632.g001

Proof of concept: Tn7-based genetic complementation of an rpoN mutant of FQ-A001

As a proof of concept, we tested whether in trans expression of rpoN, which encodes the alternative sigma factor σ⁵⁴, would restore motility in the amotile ΔrpoN strain of FQ-A001 [9]. pLosTfoX-dependent natural transformation was used to introduce at the attTn7 locus of ΔrpoN either an erm-linked, IPTG-inducible rpoN construct (P_trc::rpoN) from CAM001 or an erm control marker from TIM313. While erm^R transformants positive for Tn7 insertions were recovered for both cases (S2A–B and S3 Figs), only the ΔrpoN-derived strain harboring the Tn7-linked P_trc::rpoN construct exhibited motility (S2C–D Figs and S1 Data Set), demonstrating genetic complementation of rpoN in FQ-A001. Consequently, one potential application of this protocol is to test for genetic complementation in strains other than ES114.

Supporting information

S1 File. Step-by-step protocol, also available on protocols.io.

https://doi.org/10.1371/journal.pone.0338632.s001

(PDF)

S1 Fig. Tn7 integration frequency of V. fischeri strains ES114 and FQ-A001.

https://doi.org/10.1371/journal.pone.0338632.s002

(PDF)

S2 Fig. Tn7-based genetic complementation of rpoN in FQ-A001.

https://doi.org/10.1371/journal.pone.0338632.s003

(PDF)

S3 Fig. Raw image shown in Fig. S2B.

https://doi.org/10.1371/journal.pone.0338632.s004

(PDF)

S1 Data Set. Values shown in Figs. 1B, S1, and S2D.

https://doi.org/10.1371/journal.pone.0338632.s005

(XLSX)

Acknowledgments

We thank members of the Miyashiro Lab for helpful discussions regarding the protocol described here.

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[ref2] 2. Visick KL, Stabb EV, Ruby EG. A lasting symbiosis: how Vibrio fischeri finds a squid partner and persists within its natural host. Nat Rev Microbiol. 2021;19(10):654–65. pmid:34089008
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Google Scholar

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View Article
PubMed/NCBI
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[14] View Article

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View Article
PubMed/NCBI
Google Scholar

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View Article
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Figures

Abstract

Introduction

Materials and methods

Media and growth conditions

Tn7 integration assay

Molecular Biology

Genome sequencing and analysis

Motility assay

Statistical analysis

Expected results

Overview of the pLosTfoX-dependent natural transformation protocol

Transformation of FQ-A001 with a Tn7 insertion from ES114

Proof of concept: Tn7-based genetic complementation of an rpoN mutant of FQ-A001

Supporting information

S1 File. Step-by-step protocol, also available on protocols.io.

S1 Fig. Tn7 integration frequency of V. fischeri strains ES114 and FQ-A001.

S2 Fig. Tn7-based genetic complementation of rpoN in FQ-A001.

S3 Fig. Raw image shown in Fig. S2B.

S1 Data Set. Values shown in Figs. 1B, S1, and S2D.

Acknowledgments

References