Rhs1 and Rhs2 depend exclusively on VgrG2 for their delivery into target cells.

<p>(A-B) Recovery of a target strain lacking <i>rhsI1</i> (<i>S</i>. <i>marcescens</i> Db10 Δ<i>tssH</i>Δ<i>rhsI1</i>), part A, or lacking <i>rhsI2</i> (<i>S</i>. <i>marcescens</i> Db10 Δ<i>rhs2</i>Δ<i>rhsI2</i>), part B, following co-culture with wild type (WT) or mutant (Δ<i>tssE</i>, Δ<i>rhs1</i>, Δ<i>rhs2</i>, Δ<i>vgrG1</i>, Δ<i>vgrG2</i> and Δ<i>vgrG1</i>Δ<i>vgrG2</i>) strains of Db10 as attacker, as indicated. Points show mean ± SEM (n = 4).</p

VgrG1 and VgrG2 require specific PAAR proteins to assemble a functional T6SS.

<p>(A) Immunoblot detection of Hcp1 in cellular and secreted fractions of wild type (WT) or mutant (Δ<i>tssE</i>, Δ<i>paar1</i>, Δ<i>rhs1</i>Δ<i>rhs2</i>, Δ<i>paar</i>Δ<i>rhs1</i>Δ<i>rhs2</i>, Δ<i>vgrG1</i>Δ<i>paar1</i>, Δ<i>vgrG2</i>Δ<i>rhs1</i>Δ<i>rhs2</i>, Δ<i>vgrG1</i>Δ<i>rhs1</i>Δ<i>rhs2</i> and Δ<i>vgrG2</i>Δ<i>paar1</i>) strains of <i>S</i>. <i>marcescens</i> Db10. (B-C) Recovery of target organisms <i>P</i>. <i>fluorescens</i> 55, part B, or <i>S</i>. <i>marcescens</i> Db10 Δ<i>ssp4</i>Δ<i>sip4</i>, part C, following co-culture with wild type or mutant strains of Db10 as attacker (mutants as part A, with also Δ<i>vgrG1</i>, Δ<i>vgrG2</i>, Δ<i>vgrG1</i>Δ<i>rhs1</i> and Δ<i>vgrG1</i>Δ<i>rhs2</i>). Points show mean +/- SEM (n = 4). (D) Hcp1 secretion by wild type or mutant strains carrying either the vector control plasmid (+VC) or plasmids directing expression of Paar1 (+Paar1, pSC734), Rhs1 and RhsI1 (+Rhs1, pSC791), Rhs2 and RhsI2 (+Rhs2, pSC788), or VgrG1 (+VgrG1, pSC622) <i>in trans</i>. The empty vector control was pSUPROM for Paar1, Rhs2 and VgrG1; for Rhs1 the empty vector was pBAD18-Kn and expression was induced with 0.0002% l-arabinose. Abbreviations: Δ3x<i>paar</i>, Δ<i>paar</i>Δ<i>rhs1</i>Δ<i>rhs2</i>; Δ2x<i>rhs</i>, Δ<i>rhs1</i>Δ<i>rhs2</i> mutant (E) Recovery of target strain <i>S</i>. <i>marcescens</i> Db10 Δ<i>0927–0929</i> following co-culture with wild type (WT) or mutant (Δ<i>tssE</i>, Δ<i>slp</i>, Δ<i>vgrG2</i>, Δ<i>rhs1</i>, Δ<i>rhs2</i> and Δ<i>rhs1</i>Δ<i>rhs2</i>) strains of Db10.</p

Isolation of native VgrG and PAAR containing complexes.

<p>(A) Identification of VgrG2-associated proteins by small-scale affinity purification. Total cellular proteins from <i>S</i>. <i>marcescens</i> Db10 encoding the VgrG2-His₆ fusion protein, in an otherwise wild type genetic background (parental) or in strains lacking Rhs1 (Δ<i>rhs1</i>) or Rhs2 (Δ<i>rhs2</i>) or both Rhs proteins (Δ<i>rhs1</i>Δ<i>rhs2</i>), or from the wild type (WT, no His₆ tag) as a negative control, were incubated with Ni²⁺ beads to co-isolate VgrG2-His₆ (VgrG2-His) with any bound proteins. Eluted proteins were resolved by SDS-PAGE and visualised by Coomassie staining. Arrows indicate the positions of two VgrG2-His specific bands, one corresponding to VgrG2-His (predicted MW 72 kDa) and one corresponding to a mixture, in the parental strain, of Rhs1 and Rhs2 (predicted MW 165 and 158 kDa, respectively); identifications were confirmed by mass spectrometry. (B) Quantitative analysis of the proteins present in samples isolated as above by affinity purification from the control strain (WT) or the strain carrying the VgrG2-His fusion (in an otherwise wild type background) by solution mass spectrometry. Proteins were identified as being associated with VgrG2 when significantly enriched in the VgrG2-His sample compared with the WT sample, with relative abundance in VgrG2-His/WT > 4-fold, p<0.05 (red diamonds) (C) Bacterial two-hybrid assay to detect interactions between the N-terminal domain of Rhs1 or Rhs2, each fused with T18 (Rhs1^NT-T18, pSC699, and Rhs2 ^NT -T18, pSC700), and full-length EagR1 or EagR2, each fused with T25 (T25-EagR1, pSC688, and T25-EagR2, pSC689). Negative controls were provided by the empty vectors, pUT18 and pT25 (-), and a positive control by the self-interaction of TssK (+ve; TssK-T18, pSC048, and T25-TssK, pSC053). Shown is β-galactosidase activity, expressed as Δ405/min/ml/OD₆₀₀, of the reporter strain transformed with the combinations of plasmids indicated. Bars show mean +/- SEM (n = 3 independent transformations) and the background level of activity is indicated by a red line. (D) As for part B, except that the VgrG2-His affinity purification was performed in the parental and Δ<i>rhs2</i> backgrounds. The abundance of each of the VgrG2-associated proteins identified in part B in the two strains is given (represented as the log2 of the label-free quantitation intensities), with the effective limit of detection indicated and points showing mean +/- SD (n = 3).</p

Differential requirement for the two VgrG proteins of <i>Serratia marcescens</i> for Type VI secretion system function and anti-bacterial activity.

<p>(A) Schematic depiction of the T6SS genetic loci in <i>S</i>. <i>marcescens</i> Db10 indicating the two VgrG homologues (red), PAAR proteins or PAAR repeat containing domains (yellow), effectors (violet), immunity proteins (pink) and other conserved T6SS components (blue; core TssA-TssM components are indicated by letter). (B) Immunoblot detection of Hcp1, Ssp1 and Ssp2 in cellular and secreted fractions of wild type (WT) or mutant (Δ<i>tssE</i>, Δ<i>vgrG1</i>, Δ<i>vgrG2</i> and Δ<i>vgrG1</i>Δ<i>vgrG2</i>) strains of <i>S</i>. <i>marcescens</i> Db10. The Δ<i>tssE</i> mutant has an inactive T6SS. (C) Immunoblot detection of secreted proteins in wild type or mutant strains carrying either the vector control plasmid (+VC, pSUPROM) or plasmids directing expression of VgrG1 (+VgrG1, pSC622) or VgrG2 (+VgrG2, pSC623) <i>in trans</i>. (D) Number of recovered cells of <i>P</i>. <i>fluorescens</i> 55 following co-culture with wild type or mutant strains of <i>S</i>. <i>marcescens</i> Db10 as attacker. Points show mean +/- SEM (n = 4).</p

VgrG2 requires at least one of its specific PAAR proteins for secretion but not stability.

<p>(A) A VgrG2-His₆ fusion protein (VgrG2-His) encoded at the normal chromosomal location is fully functional, whereas VgrG1-His₆ (VgrG1-His) is non-functional. Recovery of target organism <i>P</i>. <i>fluorescens</i> 55 following co-culture with wild type <i>S</i>. <i>marcescens</i> Db10 (WT), the Δ<i>tssE</i>, Δ<i>vgrG1</i> and Δ<i>vgrG2</i> mutants, or strains expressing VgrG2-His in the Δ<i>vgrG1</i> background or VgrG1-His in the Δ<i>vgrG2</i> background. Points show mean +/-SEM (n = 4). (B) Anti-His immunoblot of cellular and secreted protein from wild type <i>S</i>. <i>marcescens</i> Db10 or strains expressing the chromosomal VgrG2-His₆ fusion protein, either in a wild type background (VgrG2-His) or in strains lacking TssE (VgrG2-His, Δ<i>tssE</i>), Rhs1 (VgrG2-His, Δ<i>rhs1</i>), Rhs2 (VgrG2-His, Δ<i>rhs2</i>) or both Rhs proteins (VgrG2-His, Δ<i>rhs1</i>Δ<i>rhs2</i>). (C) Immunoblot detection of VgrG2-His in the cellular and secreted fractions of strains expressing VgrG2-His in a wild type or Δ<i>rhs1</i>Δ<i>rhs2</i> background and carrying either the vector control plasmid (+VC) or plasmids directing the expression of Rhs1 and RhsI1 (+Rhs1, pSC791) or Rhs2 and RhsI2 (+Rhs2, pSC788) <i>in trans</i>. The empty vector control for Rhs2 was pSUPROM; for Rhs1 it was pBAD18-Kn and expression was induced with 0.0002% l-arabinose.</p

Quantitative secretomics allows comparison of individual VgrG dependence across multiple T6SS substrates and reveals a new VgrG2-specific effector.

<p>(A) Volcano plot summarising the secretomic comparison between the wild type (WT) and the T6SS-inactive double VgrG mutant (Δ<i>vgrG1</i>Δ<i>vgrG2</i>) strains of <i>S</i>. <i>marcescens</i> Db10. The log2 of the ratios of peptide intensities between the wild type and the Δ<i>vgrG1</i>Δ<i>vgrG2</i> mutant are plotted against the p-values for label-free quantitation data intensities. Red squares correspond to proteins significantly more abundant in the wild type than the Δ<i>vgrG1</i>Δ<i>vgrG2</i> mutant (WT/Δ<i>vgrG1</i>Δ<i>vgrG2</i> > 4; p < 0.05), green triangles to those significantly more abundant in the mutant (WT/Δ<i>vgrG1</i>Δ<i>vgrG2</i> < 0.25; p < 0.05) and blue diamonds to those without significant changes. (B) Heat map illustrating individual VgrG dependences for each of the proteins significantly decreased in abundance in the secretome of the Δ<i>vgrG1</i>Δ<i>vgrG2</i> mutant (criteria above and significant ANOVA score overall). Note that proteins significantly increased in the double mutant are not included. (C) Immunoblot detection of secreted Hcp1 after 5 h or 7 h growth for wild type or mutant (Δ<i>tssE</i>, Δ<i>vgrG1</i>, Δ<i>vgrG2</i> and Δ<i>vgrG1</i>Δ<i>vgrG2</i>) strains of <i>S</i>. <i>marcescens</i> Db10. (D) Recovery of a target strain lacking <i>SMDB11_0927–0929</i> (<i>S</i>. <i>marcescens</i> Db10 Δ<i>0927–0929</i>), following co-culture with wild type or mutant (Δ<i>tssE</i>, Δ<i>slp</i>, Δ<i>vgrG1</i>, Δ<i>vgrG2</i> and Δ<i>vgrG1</i>Δ<i>vgrG2</i>) strains of Db10 as attacker. Points show mean ± SEM (n = 4). Inset: genetic arrangement of <i>SMDB11_0927–0929</i>, where <i>SMDB11_0927</i> encodes the new lipase-like effector, Slp.</p

VgrG-dependent secreted proteins in <i>Serratia marcescens</i> analysed using label-free mass spectrometry.

<p>VgrG-dependent secreted proteins in <i>Serratia marcescens</i> analysed using label-free mass spectrometry.</p

Hcp-dependent cargo effectors Ssp2 and Ssp4 display a preference for delivery by VgrG2, even in the presence of excess VgrG1.

<p>(A-B) Recovery of a target strain lacking <i>rap2a</i> (<i>S</i>. <i>marcescens</i> Db10 Δ<i>ssp2</i>Δ<i>rap2a</i>), part A, or lacking <i>sip4</i> (<i>S</i>. <i>marcescens</i> Db10 Δ<i>ssp4</i>Δ<i>sip4</i>), part B, following co-culture with wild type (WT) or mutant (Δ<i>tssE</i>, Δ<i>vgrG1</i>, Δ<i>vgrG2</i> and Δ<i>vgrG1</i>Δ<i>vgrG2</i>) strains of Db10 as attacker. Points show mean ± SEM (n = 4). (C) Anti-HA and anti-RNAP immunoblot of total cellular protein from wild type <i>S</i>. <i>marcescens</i> Db10 or strains expressing an Ssp4-HA fusion protein from the normal chromosomal location, either in a wild type background (Ssp4-HA) or in strains lacking TssE (Ssp4-HA, Δ<i>tssE</i>) or TssE and Hcp1 (Ssp4-HA, Δ<i>tssE</i>Δ<i>hcp1</i>). (D-E) Recovery of target organisms <i>P</i>. <i>fluorescens</i> 55, part D, or <i>S</i>. <i>marcescens</i> Db10 Δ<i>ssp4</i>Δ<i>sip4</i>, part E, following co-culture with wild type or mutant strains carrying either the vector control plasmid (+VC, pSUPROM) or a plasmid directing the expression of VgrG1 (+VgrG1, pSC622) <i>in trans</i>.</p