Journal: bioRxiv

Article Title: Programmable domestication of thermophilic bacteria through removal of non-canonical defense systems

doi: 10.64898/2026.03.21.713436

Figure Lengend Snippet: Plasmid artificial modification–assisted conjugative engineering (PACE). Methylation-matched plasmids produced in engineered E. coli donor strains are transferred to Geobacillus recipients via pRK24-mediated conjugation. While host-specific methylation enables restriction evasion, intracellular defense systems limit plasmid establishment in wild strains. b, Architecture of the native Type II-C CRISPR–Cas system (GeoCas9EF) adapted for genome editing. A 21-bp spacer sgRNA cassette targets genomic loci adjacent to a protospacer-adjacent motif (PAM). c, Sequence logo of the preferred PAM recognized by GeoCas9EF in G. stearothermophilus EF60045, indicating a consensus 5’-NNNNCAAA- 3’ motif. d, CRISPR-mediated genome editing strategy. A conjugative plasmid carrying GeoCas9EF, sgRNA, and homologous repair arms enables genome modification via Cas9-induced cleavage and homologous recombination. Single-crossover (SCO) intermediates are resolved into double-crossover (DCO) mutants or false positives. Promotor optimization for Cas9 expression in strain SJEF4-2 is shown. e, PCR validation of representative gene deletions generated by CRISPR editing in SJEF4-2 and EF60045, targeting pyrimidine biosynthesis loci (e.g., pyrR and pyrFE ). Expected fragment sizes are indicated. f, Transformation efficiencies following sequential removal of endogenous defense systems. In EF60045 and SJEF4-2, deletion of native plasmids and non-canonical defense modules, including Wadjet II, BREX, CBASS, Gabija, Abi, and SspBCDE, dramatically increased electroporation efficiency (CFU µg ¹ DNA) and conjugation efficiency (transconjugants per recipient), revealing these systems as dominant barriers to genetic domestication.

Article Snippet: The broad-host-range conjugative plasmid pRK24 (Addgene plasmid # 51950, courtesy of Farren Isaacs) was first introduced into E. coli MC variants and selected on tetracycline (10 μg/mL).

Techniques: Plasmid Preparation, Modification, Methylation, Produced, Conjugation Assay, CRISPR, Sequencing, Homologous Recombination, Expressing, Biomarker Discovery, Generated, Transformation Assay, Electroporation

Journal: bioRxiv

Article Title: Advancing Vibrio genetics: A platform for efficient genomic manipulation

doi: 10.64898/2026.03.10.710856

Figure Lengend Snippet: A. pVFI plasmids are suitable for conjugative transfer and can be customized with unique targeting, promoter, and coding sequences. A schematic map of pVFI is shown. pVFI plasmids contain the following elements that are flanked by restriction enzyme sites to facilitate modification: 1) a sequence encoding GFP; 2) a promoter sequence, such as the E. coli lac promoter; 3) oriV R6Kγ , a conditionally replicative R6K origin that requires pir expression for maintenance; 4) an origin of transfer ( ori T RP4 ) to facilitate conjugative transfer; 4) a targeting sequence that directs plasmid integration; and 5) the chloramphenicol resistance gene ( cat ). Total plasmid size varies based on the promoter and coding sequences. B. The integration site does not disrupt known coding or regulatory sequences. The genomic context of the pVFI integration sequences in V. diazotrophicus is shown (red). Neighboring gene sequences are indicated in gray and labeled with NCBI accession numbers. C. Promoter sequences of endogenous V. diazotrophicus genes drive variable levels of transcript expression. pVFI plasmids were generated in which the E. coli lac promoter was replaced with predicted V. diazotrophicus promoter sequences for the following genes: arcA, ftsZ, gyrA, gyrB, recA, rpoD, rpsL, and tusBC . The expression of gfp transcripts was measured using RT-PCR. To ensure that amplification was not due to genomic DNA contamination, samples lacking reverse transcriptase (-RT) are shown. D. Predicted promoter sequences exhibit differential levels of sequence conservation. Sequences upstream of the start codon (500 nt or the distance to a neighboring gene) were isolated from 20 representative Vibrio spp. (Supplemental Table 4). Sequence diversity was calculated as Shannon’s entropy for each region such that higher values correspond to increased sequence variation.

Article Snippet: The conjugative E. coli strain RHO3 was a gift from Herbert Schwizer (Addgene bacterial strain #124700) and was cultured at 37 °C in LB supplemented with 2,6-diaminopimelic acid (DAP; 400 μg/mL) to accommodate its Δ asd auxotrophy.

Techniques: Modification, Sequencing, Expressing, Plasmid Preparation, Labeling, Generated, Reverse Transcription Polymerase Chain Reaction, Amplification, Reverse Transcription, Isolation

Journal: bioRxiv

Article Title: Advancing Vibrio genetics: A platform for efficient genomic manipulation

doi: 10.64898/2026.03.10.710856

Figure Lengend Snippet: A schematic of the two-step allelic exchange process mediated by pVRPSL or pVDOG is shown. Integration can occur with roughly equal probabilities on either side of the homology arm that directs recombination around the target gene; for simplicity, the upstream arm is illustrated here. Integrants are identified by antibiotic resistance, and integration is confirmed using a combination of integration-specific (p17 F/R) and genomic primers (the upstream flanking F and p17-R primers are depicted). Successful excision and mutant recovery require recombination through the second homology arm. Only strains that have lost the pVRPSL or pVDOG backbone can survive on streptomycin- or DOG-2-supplemented media. Although plasmid loss theoretically yields both wild-type and mutant colonies in equal proportions, inclusion of chloramphenicol selects exclusively for mutant isolates. If desired, the chloramphenicol cassette can subsequently be removed using the pVCM- flp shuttle vector . B. The pVDOG vector encodes galK to enable DOG-2 mediated counterselection. The vector map is shown. This plasmid contains the sequencing encoding the E. coli galK enzyme that phosphorylates DOG-2 to form the metabolically inert 2-deoxy-D-galactose-1-phosphate, which is toxic at high concentrations in Gram-negative bacteria ( , ). C. The pVRPSL vector encodes streptomycin-susceptible rpsL to mediate counterselection. The vector map is shown. D. Motility assays were used to verify the loss of movement-associated genes in V. diazotrophicus . The initial inoculum (4 mm) is indicated by a dashed gray line. All assays were performed in biological triplicates; error bars indicate the standard deviation. Statistical significance was calculated compared to wild type strains by Students t-test (***, p < 0.001; **, p < 0.01; * p < 0.05; ns, p > 0.05). E. RT-PCR confirms loss of transcription in deletion strains. Expression was quantified in wildtype (WT) and single-locus mutants of V. diazotrophicus generated using pVDOG or pVRPSL (Δ pomAB , Δ hcp , Δ motAB , Δ flaCD , Δ flaFDAGIfilDS ; see Supplemental Table 1 for strain information). Primer sequences are listed in Supplemental Table 3.

Article Snippet: The conjugative E. coli strain RHO3 was a gift from Herbert Schwizer (Addgene bacterial strain #124700) and was cultured at 37 °C in LB supplemented with 2,6-diaminopimelic acid (DAP; 400 μg/mL) to accommodate its Δ asd auxotrophy.

Techniques: Mutagenesis, Plasmid Preparation, Sequencing, Metabolic Labelling, Bacteria, Standard Deviation, Reverse Transcription Polymerase Chain Reaction, Expressing, Generated

Journal: bioRxiv

Article Title: Advancing Vibrio genetics: A platform for efficient genomic manipulation

doi: 10.64898/2026.03.10.710856

Figure Lengend Snippet: A. The phylogenetic relationships among Vibrio species were inferred using single-copy orthologs. A maximum-likelihood tree was constructed using the amino acid sequences of the 25 longest orthologs identified across all Vibrio genomes analyzed (Supplemental Table 4). This topology is consistent with that generated using eight commonly used orthologs (Supplemental Figure 2). Vibrio species successfully edited using the pVDOG/pVRPSL plasmids are highlighted in bold; the laboratory strain of V. mediterranei used is denoted with an asterisk. Outgroup species include Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Serratia marcescens, and Yersinia pestis. Bootstrap values shown were calculated using 1,000 replicates; only values greater than 75 are shown. B. The rpoD promoter dives high-level expression from integrated pVDOG backbones. RT-PCR was performed on V. diazotrophicus , V. paucivorans , V. mediterranei , V. coralliilyticus , and V. splendidus strains carrying single pVDOG integrations using primers specific to the E. coli galK gene. RT loading controls are depicted below each sample. C. Genomic PCR confirms deletion of pomAB across multiple Vibrio species. The chromosomal regions flanking the pomAB loci were amplified from wild-type (WT) and mutant (Δ cat ) strains of V. diazotrophicus , V. paucivorans , V. mediterranei , V. coralliilyticus , and V. splendidus . D. Motility assays demonstrate the broad applicability of this system across Vibrio strains. In each species tested, deletion of pomAB genes reduced motility. Colony diameters were measured, with the initial inoculum (4 mm) indicated by the dashed line. All assays were performed in biological triplicates; error bars indicate standard deviation. Statistical significance was calculated compared to wild type strains using Students t-test (***, p < 0.001; **, p < 0.01; * p < 0.05; ns, p > 0.05).

Article Snippet: The conjugative E. coli strain RHO3 was a gift from Herbert Schwizer (Addgene bacterial strain #124700) and was cultured at 37 °C in LB supplemented with 2,6-diaminopimelic acid (DAP; 400 μg/mL) to accommodate its Δ asd auxotrophy.

Techniques: Construct, Generated, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Mutagenesis, Standard Deviation