Journal: bioRxiv

Article Title: Suppressing Transfer of Antibiotic Resistance by a Small RNA Virus

doi: 10.64898/2026.03.25.714153

Figure Lengend Snippet: (A) Organization of the major operons in the RP4 plasmid. Tra1 and Tra2 contain the genes for the relaxosome and T4SS, respectively. The Ctl operon helps regulate gene expression and the Rep operon functions in plasmid replication through initiation at the oriV site. The Par operon encodes the toxin-antitoxin host-defense system ParDE. Lastly, the three antibiotic resistance genes are encoded on bla (β-lactamase enzyme that breaks down ampicillin), aphA (aminoglycoside-3-phosphotransferase enzyme inactivating kanamycin), and the Tet operon (encodes tetA which produces the tetracycline efflux pump). (B) The genes on the Tra1 (transfer 1) operon are shown. Genes in gray are not critical for formation of the T4SS machinery and pilus biogenesis. The origin of transfer (oriT) is highlighted in black. (C) The genes on the Tra2 (transfer 2) operon are shown. Genes in gray are not critical for formation of the T4SS machinery or pilus biogenesis. (D) Cryo-EM reconstruction of the mature PRR1 virion, showing the Coat (tan), Mat (blue), and the viral RNA (vRNA, gray) with the 3′ end of the vRNA labeled (orange). The virion diameter (292Å) and Mat prominence (35Å) are labeled. One stem of the 3’ vRNA extends 20Å outside the capsid. Left: top-down view of the intact virion from the Mat. Right: cross-sectional view (rotated 90°), half of the Coat shell is removed to show the 3′ vRNA as well as the rest of the vRNA. (E) Secondary structure topology of the Mat PRR1 , highlighting its two major components: the α-helical region (α-region) and β-sheet region (β-region). Two important β-sheet sub-regions, β4-α1 loop and the tip region, are denoted by arrows. (F) The cryo-EM map of the “Mat-less” PRR1 with the Coat shown in pink and the vRNA shown in gray. The lack of Mat density is shown in the inset (viewing angle indicated by the eye cartoon). This class was composed of 67,975 particles (29%) from the PRR1 data-set. (G) The Coats (tan) immediately surrounding the Mat PRR1 (blue), with the surface contacts between the two labeled red. The surface area of the contacts (803Å ) is reported below. (H) The Coats (maroon) immediately surrounding the Mat (gray) of MS2 (PDB ID: 5TC1), with the surface contacts between the two labeled red. The surface area of the contacts (1,689Å ) is reported below.

Article Snippet: The RP4 plasmid and bacterial genome extracted were sequenced by Plasmidsaurus.

Techniques: Plasmid Preparation, Gene Expression, Cryo-EM Sample Prep, Labeling

Journal: bioRxiv

Article Title: Suppressing Transfer of Antibiotic Resistance by a Small RNA Virus

doi: 10.64898/2026.03.25.714153

Figure Lengend Snippet: (A) Model of a single TrbC pilin monomer with surface exposed residues that possibly interact with Mat PRR1 shown in stick representations. (B) The change in phage infectivity (titer) of each TrbC mutant is shown as the efficiency of plaquing (EOP). Each bar in the infectivity assay represents the mean ± SD of n=3 biological replicates. The lack of a bar indicates a lack of infection (e.g. S72A). (C) The percent change in conjugation efficiencies of TrbC variants carrying mutations in binding residues. Each bar in the conjugation assay represents the mean ± SD of n=4 biological replicates. The dashed red line represents a transfer efficiency of zero. (D) The surface representation of the PRR1 virion (tan and blue) bound to the RP4 pilus (gray), using the Mat of the highest-scored docking model of the RP4-Mat complex as an anchor. The axis of the RP4 pilus is shown as a black line with another black line perpendicular to it. A two-fold axis of PRR1 is shown as a red dashed line. The measured tilt angle of binding (between the lines) is 5.6°. (E) Side view of the Mat PRR1 footprint (blue) on the RP4 pilus (gray). Two Mat regions participate in pilus binding; the β4-α1 loop is bound two TrbC pilin monomers (orchid and orange), and the tip region engages one pilin monomer (light pink). The rest of the pilin monomers (gray) are shown in surface representations. The β-region of Mat PRR1 spans three turns on the pilus. The inset shows a 90° rotation of the full Mat bound to the RP4 pilus. (F) The tip region (blue shading) from Panel E is zoomed in here to show the detailed interactions between the pilus (light pink) and the tip region of the Mat (blue). The gray bond indicates a π-stacking interaction, while the blue line indicates a hydrogen bond. (G) The β4-α1 loop (blue shading) from panel E is zoomed in here to show the detailed interactions between the pilus (orchid and orange) and the β4-α1 loop of the Mat (blue).

Article Snippet: The RP4 plasmid and bacterial genome extracted were sequenced by Plasmidsaurus.

Techniques: Infection, Mutagenesis, Conjugation Assay, Binding Assay

Journal: bioRxiv

Article Title: Suppressing Transfer of Antibiotic Resistance by a Small RNA Virus

doi: 10.64898/2026.03.25.714153

Figure Lengend Snippet: (A) The effect of PRR1 (red), UV-PRR1 (blue), PP7 (green), and buffer (gray) on RP4 conjugation in P. aeruginosa PAO1Δ pilA (RP4). Each point represents the average of n≥4 replicates. (B) A model of the RP4 T4SS. The arrangement of the core proteins within the transfer complex is shown. The protein names are colored to indicate that they contain one of the nine mutations. Those left black do not contain a mutation. This structural model of the RP4 system is derived from its homology to the R388 system (PDB: 7OIU, 7O43, 8RT4, 8RT6, 8RT9, 8RTD). (C) The conjugation ability of the nine PRR1 resistant mutants. Data represent mean ± SD of n ≥8 independent replicates. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test; all comparisons shown were highly significant (p < 0.0001). (D) Conjugation efficiencies of the four transfer-positive mutants in the absence (left) or presence (right) of PRR1 (MOI=10). Data represent mean ± SD of n = 4 biological replicates. Statistical significance was determined using an unpaired two-tailed t-test for each mutant; p-values are: traF 1= 0.0003, trbE 1< 0.0001, trbH 1< 0.0001, trbJ 2= 0.0002.

Article Snippet: The RP4 plasmid and bacterial genome extracted were sequenced by Plasmidsaurus.

Techniques: Conjugation Assay, Mutagenesis, Derivative Assay, Two Tailed Test

Journal: ACS environmental Au

Article Title: Bacterial Concentrations and Water Turbulence Influence the Importance of Conjugation Versus Phage-Mediated Antibiotic Resistance Gene Transfer in Suspended Growth Systems.

doi: 10.1021/acsenvironau.1c00027

Figure Lengend Snippet: Figure 2. Lower frequency but higher relative importance of genR transfer via phage delivery (relative to conjugation) occurs at lower bacterial concentration. (a) Transfer frequency was determined after 1 h of conjugation and phage delivery assays in minimal medium with 3.0 g/L glucose and shaken at an Re of 5 × 104. The genR donor (E. coli DH5α harboring plasmid RP4 for conjugation assays or λ phage for phage delivery assays) and recipient (E. coli MG1655) were mixed at a 1:1 ratio. Frequency determined by the plate counting method is presented as bars with the detection limit (10−6) indicated by the horizontal dashed line; orange dots represent the frequency determined by qPCR analysis. Asterisks (*) indicate significant differences (p < 0.05) between conjugation and phage delivery frequency based on Student’s t-test. “N.D.” refers to “not detected”. (b) Upregulation of conjugation-related genes (traI, traJ, and trbAp) and phage delivery-related genes (lamB and malT) at lower bacterial concentration (twofold gene expression change). The top X-axis depicts decreasing bacterial concentration (CFU/mL). The expression level at a bacterial concentration of 108 CFU/mL was used as the control. Error bars depict ± one standard deviation from the mean of at least three independent replicates.

Article Snippet: Conjugative plasmid RP4 was obtained from Addgene (plasmid 79813).

Techniques: Conjugation Assay, Concentration Assay, Plasmid Preparation, Gene Expression, Expressing, Control, Standard Deviation

Journal: ACS environmental Au

Article Title: Bacterial Concentrations and Water Turbulence Influence the Importance of Conjugation Versus Phage-Mediated Antibiotic Resistance Gene Transfer in Suspended Growth Systems.

doi: 10.1021/acsenvironau.1c00027

Figure Lengend Snippet: Figure 3. Turbulence (represented by Reynolds number) significantly impacts the relative frequency of conjugation vs phage delivery for the horizontal transfer of genR in a bell-shaped fashion. The transfer frequency was determined after 1 h of conjugation and phage delivery assays in minimal medium containing 3.0 g/L glucose with bacterial concentration at 107 CFU/mL. The genR donor (E. coli DH5α harboring plasmid RP4 for conjugation assays or λ phage for phage delivery assays) and recipient (E. coli MG1655) were mixed at a 1:1 ratio. Asterisks (*) indicate significant differences (p < 0.05) between conjugation and phage delivery frequency based on Student’s t-test. Error bars depict ± one standard deviation from the mean of at least three independent replicates.

Article Snippet: Conjugative plasmid RP4 was obtained from Addgene (plasmid 79813).

Techniques: Conjugation Assay, Concentration Assay, Plasmid Preparation, Standard Deviation